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B-Cell Development
10
M
illions of B lymphocytes are generated in the
bone marrow every day and exported to the
periphery. The rapid and unceasing
generation of new B cells occurs in a carefully
regulated sequence of events. Cell transfer experiments, in B cells at different stages of development
which genetically marked donor hematopoietic stem cells seek contact with stromal cells expressing
(HSCs) are injected into an unmarked recipient, have CXCL12 (pre-pro-B cells, left) or IL-7 (pro-B
indicated that B-cell development from HSC to mature B cells, right). [Tokoyoda et al. 2004. Cellular Niches
cell takes from 1 to 2 weeks; donor-derived mature B cells
Controlling B Lymphocyte Behavior within Bone
can be detected in the recipient by 2 weeks following
Marrow during Development. Immunity Vol. 20, Issue
transfer of HSCs into recipient mice.
6, 707–718. © 2004 Elsevier Ltd. All rights reserved.]
B-cell development begins in the bone marrow with the
asymmetric division of an HSC and continues through a
■ The Site of Hematopoiesis
series of progressively more differentiated progenitor stages
to the production of common lymphoid progenitors ■ B-Cell Development in the Bone Marrow
(CLPs), which can give rise to either B cells or T cells (see
■ The Development of B-1 and Marginal-Zone
Overview Figure 10-1). Progenitor cells destined to
become T cells migrate to the thymus where they complete B Cells
their maturation (see Chapter 9); the majority of those that ■ Comparison of B- and T-Cell Development
remain in the bone marrow become B cells. As
differentiation proceeds, the developing B cell expresses on
its cell surface a precisely calibrated sequence of cell-surface heavy-chain D to JH gene-segment rearrangement,
receptor and adhesion molecules. Some of the signals followed by the stitching together of the ␮ heavy-chain
received from these receptors induce the differentiation of VH and DJH segments. These rearrangements culminate
the developing B cell; others trigger its proliferation at in the cell-surface expression of the pre-B-cell receptor
particular stages of development and yet others direct its during the pre-B-cell stage, in which the rearranged
movements within the bone marrow environment. These heavy chain is expressed in combination with the
signals collectively allow differentiation of the CLP through surrogate light chain. Rearrangement of the light chain
the early B-cell stages to form the immature B cell that is initiated after several rounds of division of cells
leaves the marrow to complete its differentiation in the bearing the pre-BCR.
spleen. For the investigator, the expression of different cell- Like T cells, developing B cells must solve the
surface molecules at each stage of B-cell maturation problem of creating a repertoire of receptors capable of
provides an invaluable experimental tool with which to recognizing an extensive array of antigens, while
recognize and isolate B cells poised at discrete points in ensuring that self-reactive B cells are either eliminated
their development. by apoptosis or rendered functionally unreactive or
The primary function of mature B cells is to secrete anergic. However, unlike T-cell receptors, B-cell
antibodies that protect the host against pathogens, and receptors are not constrained by the need to be MHC
so one major focus of those studying B-cell restricted. Again, unlike T-cell maturation, B-cell
differentiation is the analysis of the timing and order of development is almost complete by the time the B cell
rearrangement and expression of immunoglobulin receptor leaves the bone marrow; in mammalian systems there is
heavy- and light-chain genes. Recall from Chapter 7 that no thymic equivalent in which B-cell development is
immunoglobulin gene rearrangements begin with accomplished. Instead, immature B cells are released to
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330 PA R T I V | Adaptive Immunity: Development
OVERVIEW FIGURE 10-1
B-Cell Development Begins in the Bone Marrow and Is

Completed in the Periphery
Early ProB ProB or preB Immature B
• DJ H chain • Complete • Deletion
recombination VDJ H chain Negative selection • Receptor
• Start of VDJ H chain recombination editing
recombination • Clonal expansion
• VJ L chain
recombination
Endogenous
antigen
Bone
endosteum
Bone marrow Central
sinus
Transitional-2
Mature B Transitional-1
IgM
IgD Spleen
B-cell development begins with a hematopoietic stem cell (HSC) recombination of the immunoglobulin genes begins. Once the
and passes through progressively more delimited progenitor-cell completed immunoglobulin is expressed on the cell surface, the
stages until it reaches the pro-B cell stage. At this stage, the precur- immature B cell, now a transitional B cell, leaves the bone marrow
sor cell is irreversibly committed to the B-cell lineage and the to complete its maturation in the spleen.
the periphery, where they complete their developmental lymphoid lineages (Chapter 2). Various non-hematopoietic
program in the spleen. cells in the bone marrow express cell-surface molecules
In this chapter, we will follow B-cell development from and secrete hormones that guide hematopoietic cell develop-
its earliest stages in the primary lymphoid organs to the ment. Developing lymphocytes move within the bone mar-
generation of fully mature B cells in the secondary row as they mature, thus interacting with different
populations of cells and signals at various developmental
lymphoid tissues. As for T cells, multiple B-cell subsets
stages. However, fetal animals face particular challenges to
exist, and we will briefly address how the process of
their developing immune systems; how can they generate
differentiation of the minority B-1 and marginal-zone blood cells when their bones are still not yet fully developed?
(MZ) B-cell subsets differs from the developmental
program followed by the predominant B-2 B-cell subset. The Site of B-Cell Generation Changes
We will conclude with a brief comparison of the
during Gestation
maturational processes of T and B lymphocytes.
Hematopoiesis is a complex process in the adult animal, and
during fetal maturation additional challenges must be met.
The Site of Hematopoiesis Red blood cells must be quickly generated de novo in order to
provide the embryo with sufficient oxygen, and HSCs must
In adult animals, hematopoiesis, the generation of blood proliferate at a rate sufficient to populate the adult as well as
cells, occurs in the bone marrow; the HSCs in the marrow provide for the hematopoietic needs of the maturing fetus.
are the source of all blood cells of the erythroid, myeloid, and Furthermore, since the bone marrow appears relatively late in
B-Cell Development | CHAPTER 10 331
Mouse Human
Placenta
Yolk sac
Fetal liver
AGM
Specification Emergence Maturation Expansion Quiescence/

self-renewal
Mesoderm Pre-HSC HSC HSC

HSC
Mouse
7.5 10.5 12.5 15.5 days Birth

Human
21 28 40 70 days Birth
Placenta
Fetal liver
AGM Bone marrow
Primitive Yolk sac

streak
FIGURE 10-2 The anatomy and timing of the earliest become blood cells emerge first as rapidly proliferating pre-HSCs
stages in hematopoiesis. (a) Blood-cell precursors are initially and eventually mature into relatively quiescent hematopoietic stem
found in the yolk sac (yellow), then spread to the placenta fetal liver cells that populate the bone marrow. The colored bars in the time-
(pink), and aorta-gonad-mesonephros (AGM) region (green), before line illustrate the ages at which the various murine and human
finding their adult home in the bone marrow. The mouse embryo is hematopoietic sites are active. Mesoderm (gray); generation of fetal
shown at 11 days of gestation; the human embryo at the equivalent HSC (yellow); active hematopoietic differentiation (red); emergence
5 weeks of gestation. (b) In the embryo, cells within the primitive of functional, adult-type HSCs (blue). [Adapted from H. K. Mikkola and S. H.
streak mesodermal tissue adopt either hematopoietic (blood-cell Orkin, 2006, The journey of developing hematopoietic stem cells, Development
forming) or vascular (blood-vessel forming) fates. Those destined to 133:3733–3744, Figures 1 and 2. ]
development, the whole process of blood-cell generation must fetal liver. Between days 11.5 and 12.5, there is rapid expansion
shift location several times before moving into its final home. of the placental HSC pool, at the end of which time the pla-
The gestation period for mice is 19 to 21 days. Hematopoi- centa holds more HSCs than either the AGM or the yolk sac.
esis begins, in the mouse, around 7 days post fertilization By day 13.5, the number of HSCs in the placenta begins to
(Figure 10-2) when precursor cells in the yolk sac begin dif- decrease while the HSC pool in the liver continues to expand.
ferentiating to form primitive, nucleated, erythroid cells that As the mouse embryo completes its development, the
carry the oxygen the embryo needs for early development. predominant site of HSC generation remains within the fetal
Fetal HSCs capable of generating all blood-cell types can be liver, but some hematopoiesis can be detected in the spleen
detected in the early aorta-gonad-mesonephros (AGM) region in the perinatal (around the time of birth) period. The num-
on day 8, when the fetal heart starts beating. On day 10, mature ber of fetal liver HSCs in mice reaches a maximum of
HSCs capable of completely repopulating the hematopoietic approximately 1000 by days 15.5 to 16.5 of embryonic
system of irradiated adult mice can be isolated from the AGM, development, after which it starts to decline. Within the
and by day 11 they can be found in the yolk sac, placenta, and fetal liver, HSCs differentiate to form progenitor cells. At the
earliest time points, hematopoiesis in the fetal liver is reside. These daughter cells use the same receptors as their
dominated by erythroid progenitors that give rise to the parents, and no new V(D)J recombinase activity is required.
true, enucleated mature erythrocytes, in order to ensure a In humans, the sequence of events is similar to that
steady oxygen supply to the growing embryo, and myeloid described for the mouse, but the time frame is obviously
and lymphoid progenitors gradually emerge. Pre-B cells somewhat elongated. Blood-cell precursors first appear in the
(precursor-B cells), defined as cells that express immuno- yolk sac in the third week of embryonic development, but
globulin in their cytoplasm but not on their surfaces, are these cells, like their analogues in the mouse provide primar-
first observed at day 13 of gestation, and surface IgM-positive ily erythroid progenitors and are not capable of generating all
B cells are present in detectable numbers by day 17. HSCs subsets of blood cells. The first cells capable of entirely repop-
first seed the bone marrow at approximately day 15, and ulating an adult human hematopoietic system arise in the
over a period of a few weeks, the bone marrow takes over as AGM region of the embryo and/or the yolk sac. By the third
the main site of B-cell development, remaining so through- month of pregnancy, these HSCs migrate to the fetal liver,
out post-natal life. which then becomes responsible for the majority of hemato-
poiesis in the fetus. By the fourth month of pregnancy, HSCs
Hematopoiesis in the Fetal Liver Differs from migrate to the bone marrow, which gradually assumes the
hematopoietic role from the fetal liver until, by the time of
That in the Adult Bone Marrow birth, it is the primary generative organ for blood cells. Prior
Developing B cells in the fetal liver differ in important to puberty in humans, most of the bones of the skeleton are
ways from their counterparts in adult bone marrow. The hematopoietically active, but by the age of 18 years only the
liver is the primary site of B-cell generation in the fetus, vertebrae, ribs, sternum, skull, pelvis, and parts of the
and provides the neonatal animal with the cells it needs to humerus and femur retain hematopoietic potential.
populate its nascent immune system. In order to accom- Just as B-cell development in the fetus and neonate differs
plish this, hematopoietic stem cells and their progeny from that in the adult, so does B-cell hematopoiesis in the
must undergo a phase of rapid proliferation, and fetal liver aging animal. Clinical Focus Box 10-1 describes some
HSCs, as well as their daughter cells, undergo several aspects of B-cell development that alter as humans age.
rounds of cell division over a short time. In contrast, HSCs
derived from the bone marrow of a healthy adult animal
are relatively quiescent. B-Cell Development in the
B cells generated from fetal liver precursors are predomi- Bone Marrow
nantly B-1 B cells, which will be described more fully in
Chapter 12. Briefly, B-1 B cells are primarily located in the In Chapter 2 (Figure 2-5), we presented the structure of bone
body (specifically the peritoneal and pleural) cavities. They and bone marrow. The bone marrow microenvironment is a
are therefore well-positioned to protect the gut and the lungs, complex, three-dimensional structure with distinctive cel-
which are the major ports of entry of microbes in the fetus lular niches which are specialized to influence the develop-
and neonate. Antibodies secreted by B-1 B cells are broadly ment of the cell populations that mature there. A dense
cross-reactive; many bind to carbohydrate antigens expressed network of fenestrated (leaky) thin-walled blood vessels—
by a number of microbial species. Since terminal deoxynucle- the bone marrow sinusoids—permeates the marrow, allow-
otidyl transferase (TdT) is minimally expressed at this point ing the passage of newly formed blood cells to the periphery
in ontogeny, and the RAG1/2 recombinase proteins appear and facilitating blood circulation through the marrow.
not to use the full range of V, D, and J region gene segments In addition to serving as a source of hematopoietic stem
at this stage in embryonic development, the immunoglobulin cells, bone marrow also contains stem cells that can differen-
receptors of B-1 B cells express minimal receptor diversity. In tiate into adipocytes (fat cells), chondrocytes (cartilage
expressing an oligoclonal (few, as opposed to many, clones) cells), osteocytes (bone cells), myocytes (muscle cells), and
repertoire of B-cell receptors that bind to a limited number of potentially other types of cells as well. Each of these different
carbohydrate antigens shared among many microbes, B-1 B classes of stem cells requires specific sets of factors, secreted
cells occupy a functional niche that bridges the innate and by particular bone marrow stromal cells to enable their
adaptive immune systems. We will describe B-1 B-cell devel- proper differentiation.
opment further at the end of this chapter. What are bone marrow stromal cells? The term stroma
Over a period of 2 to 4 weeks after birth, the process of derives from the Greek for mattress, and a stromal cell is a
hematopoiesis in mice shifts from the fetal liver and spleen to general term that describes a large adherent cell that sup-
the bone marrow, where it continues throughout adulthood. ports the growth of other cells. During B-cell development,
The B-1 B-cell population represents an exception to this gen- bone marrow stromal cells fulfill two functions. First, by
eral rule, as it is self-renewing in the periphery. This means interacting with adhesion molecules on the surfaces of HSCs
that new daughter B-1 B cells are generated continually from and progenitor cells, stromal cells retain the developing cell
preexisting B-1 B cells in the peritoneal and pleural cavities, populations in the specific bone marrow niches where they
and in those other parts of the body in which B-1 B cells can receive the appropriate molecular signals required for
CLINICAL FOCUS Box 10-1

B-Cell Development in the Aging Individual
People of retirement age and older viduals start at the very beginning of in repertoire diversity correlates with a
represent a greater segment of the popu- their developmental program. The epi- reduction in the health of the aging
lation than they used to, and these older genetic regulation of HSC genes in aging patient.
individuals expect to remain active and mice is compromised, resulting in dimin- The mechanisms for this age-related
productive members of society. However, ished levels of HSC self-renewal. Further- repertoire truncation appear to be com-
physicians and immunologists have long more, the balance between the plex. A decrease in output of immature B2
known that the elderly are more suscep- production of myeloid versus lymphoid cells from the bone marrow could provide
tible to infection than are young men and progenitors is shifted in older individuals, the opportunity for B-1 B cells to increase
women, and that vaccinations are less with down-regulation of genes associ- their share of the peripheral B-cell niche,
effective in older individuals. In this fea- ated with lymphoid specification and a and as is well appreciated, B-1 B cells have
ture, we explore the differences in B-cell correspondingly enhanced expression of a less diverse receptor repertoire than do
development between younger and genes specifying myeloid development. B-2 B cells. A lifetime of generating mem-
older vertebrates, which may account for The net effect of these changes in the ory cells may also result in an individual
some of these immunological disparities HSC population is a reduction with age having less room in B-cell follicles for
between adult and older individuals. in the numbers of early B-cell progeni- newly formed B cells to enter, and a
Aging individuals display deficiencies tors, which is reflected in a decrease in decreased concentration of the homeo-
in many aspects of B-cell function, includ- the numbers of pro- and pre-B-cell pre- static regulatory cytokines may make it
ing a poor antibody response to vaccina- cursors at all stages of development. more difficult for primary B cells to com-
tion, inefficient generation of memory B Detailed studies of the expression of pete with their more robust memory
cells, and an increase in the expression of particular genes important in B-cell devel- counterparts. Clearly, this is an area of
autoimmune disorders. Does this reflect opment demonstrate that the expression increasing clinical interest as the average
defective functioning only in the mature of important transcription factors, such as age of the population of the developed
antigen-responsive B-cell population, or those encoded by the E2A gene, is world continues to increase.
does it result from problems manifested reduced in older animals. Furthermore,
during earlier stages in B-cell develop- the Rag genes, as well as the gene encod-
ment? Current research demonstrates ing the surrogate light-chain component, REFERENCES
that aging individuals display a range of ␭5, are down-regulated in older animals Cancro, M. P., et al. 2009. B cells and aging: Mol-
shortcomings in developing B cells. compared with young adults, resulting in ecules and mechanisms. Trends in Immunol-
Experiments employing reciprocal a reduction in the bone marrow output of ogy 30:313–318.
bone marrow chimeras—in which aging immature B cells. Dorshkin, K., E. Montecino-Rodriguez, and R. A.
Signer. 2009. The ageing immune system: Is
HSCs were transplanted into young recip- Multiple mechanisms therefore help
it ever too old to become young again?
ients or HSCs from young mice were to explain why the numbers of B cells Nature Reviews Immunology 9:57–62.
injected into aging recipients—have released from the bone marrow are
Goodnow, C. C. 1992. Transgenic mice and
shown that the suboptimal process of smaller in aging than in younger individu- analysis of B-cell tolerance. Annual Reviews
B-cell development in aging individuals als. But is the antigen recognition capacity— Immunology 10:489–518.
results from deficiencies in both the aging the quality—as well as the quantity of B Labrie, J. E., 3rd, A. P. Sah, D. M. Allman, M. P.
stem cells and in the supporting stromal cells different between the two popula- Cancro, and R. M. Gerstein. 2004. Bone mar-
cells. For example, bone marrow stromal tions? In particular, do B cells from aging row microenvironmental changes underlie
reduced RAG-mediated recombination and
cells from aging mice secrete lower levels mice express a repertoire of receptors
B cell generation in aged mice. Journal of
of IL-7 than do stromal cells from younger similar to those obtained from younger Experimental Medicine 200:411–423.
animals, suggesting an environmental animals? The answer to this question has
Nemazee, D. A., and K. Bürki. 1989, February.
defect in the aging bone marrow. How- come from the development of tech- Clonal deletion of B lymphocytes in a
ever, study of isolated, aging B-cell pro- niques that enable a global assessment of transgenic mouse bearing anti-MHC class I
genitors reveals that they also respond repertoire diversity. Study of the sizes and antibody genes. Nature 337:562–566.
less efficiently to IL-7 than do B cells from sequences of CDR3 regions from large doi:10.1038/337562a0
younger mice, and so the IL-7 response in numbers of human B cells suggests that Van der Put, E., E. M. Sherwood, B. B. Blom-
berg, and R. L. Riley. 2003. Aged mice
aging individuals is affected at both the in aging individuals the size of the reper-
exhibit distinct B cell precursor pheno-
secretory and recipient-cell levels. toire (the number of different B-cell types differing in activation, proliferation
Indeed, the problems encountered receptors an individual expresses) is dras- and apoptosis. Experimental Gerontology
by developing B cells from aging indi- tically diminished, and that this decrease 38:1137–1147.
Endothelial Bone
cell
Osteoblast IL-7 expressing cell
Pro-B cell IL-7
Pre-pro- Pre-B cell

B cell
Immature
HSC B cell
Medullary CXCL12
Plasma cell
vascular
sinus CXCL12
reticular cell
FIGURE 10-3 HSCs and B-cell progenitors make contact tion has progressed to the pro-B-cell stage, the developing cell has
with different sets of bone marrow cells as they progress moved to receive signals from IL-7-producing stromal cells. After
through their developmental program. HSCs begin their devel- leaving the IL-7-expressing stromal cell, the pre-B cell completes its
opmental program close to the osteoblasts (top). An HSC is also differentiation and leaves the bone marrow as an immature B cell.
shown entering from the blood (left-hand side), illustrating the fact CXCL-12 is shown in purple; IL-7 in blue. [Adapted from T. Nagasawa,
that HSCs are capable of recirculation in the adult animal. Progenitor 2006, February, Microenvironmental niches in the bone marrow required for
cells then move to gain contact with CXCL12-expressing stromal B-cell development, Nature Reviews Immunology 6:107–116. doi:10.1038/
cells, where they mature into pre-pro B cells. By the time differentia- nri1780]
their further differentiation. Second, diverse populations of characteristics of each cell type in that pathway, as well as
stromal cells express different cytokines. At various points in the molecular signals and transcription factors that drive
their development, progenitor and precursor B cells must differentiation at each stage. Cells at particular stages of
interact with stromal cells secreting particular cytokines, differentiation can be characterized by their surface mol-
and thus the developing B cells move in an orderly progres- ecules, which include cell-surface antigens, adhesion mol-
sion from location to location within the bone marrow. This ecules, and receptors for chemokines and cytokines. They
progression is guided by chemokines secreted by particular are also defined by the array of active transcription factors
stromal cell populations. For example, HSCs begin their life that determine which genes are expressed at each step in
in close contact with osteoblasts located close to the lining of the developmental process. Finally, in the case of B cells,
the endosteal (bone marrow) cavity. Once differentiated to the developmental stages are defined by the status of the
the pre-pro-B-cell stage, the developing B cells require sig- rearranging heavy- and light-chain immunoglobulin
nals from the chemokine CXCL12, which is secreted by a genes. B-cell development is not yet completely under-
specialized set of stromal cells, in order to progress to the stood; however, most of the important cellular intermedi-
pro-B-cell stage. Pro-B cells then require signaling from the ates have been defined, and developmental immunologists
cytokine IL-7, which is secreted by yet another stromal cell are gradually filling in the gaps in our knowledge.
subset (Figure 10-3). Many of these stromal cell factors serve Investigators delineating the path of B-lymphocyte dif-
to induce the expression of specialized transcription factors ferentiation employed three general experimental strategies.
important in B-cell development. First, they generated antibodies against molecules (antigens or
markers) present on the surface of bone marrow cells. They
then determined which of these molecules were present at
The Stages of Hematopoiesis Are Defined by the same time as other antigens, and which combinations of
Cell-Surface Markers, Transcription-Factor antigens appeared to define unique cell types (Figure 10-4a).
Expression, and Immunoglobulin Gene In addition, culturing cells in vitro that bear known cell-
surface antigens, followed by flow cytometric analysis of the
Rearrangements daughter cells generated in culture, enabled them to describe
Full characterization of a developmental pathway requires the sequential expression of particular combinations of cell-
that scientists understand the phenotypic and functional surface molecules (Figure 10-4b).
(a) (d)
Characterization of progenitors bearing Knocking out particular transcription factors

different sets of cell surface molecules. (TFs) stops development at particular points.
Knocking out TF1 leaves only this population. Therefore

TF1 is required to progress to VHD recombination.
(b)
Determining sequence of marker expression by culturing cells

from each stage. Culturing cells with the red antigen gives rise to
daughter cells of both types. Culturing cells with only the blue Knocking out TF2 leaves these two populations. Therefore
antigen gives rise to the cells bearing both blue and green TF2 is required for progression to light chain rearrangement.
antigens, but never cells bearing the red antigens. Therefore, we
can sequence the three cell types in this way.
(e)
Placing GFP under the control of the TF2 promoter reveals that TF2
(c) expression occurs in these two cell populations. Clearly, it is turned
Sequencing of different antigen-bearing cells by analyzing each on during the end of the blue stage, and is needed for progression
population for the stage of V(D)J rearrangements in heavy and to the blue and green stage of development.
light chains.
D-JH VHDJH recombination VH and VL VHDJH VH and VL

recombination completed recombination recombination recombination
only completed completed completed
FIGURE 10-4 Experimental approaches to the staging and the cell-surface expression of developmental markers and molecular
characterization of B-cell progenitors. In this figure, the differ- biology to correlate the expression of specific markers with the stage
ent icons do not represent specific antigens, but are used in order to of immunoglobulin gene rearrangement. The requirement for tran-
illustrate the principle of the experiment. Investigators delineated the scription factor activity at each step was determined using both
stage of B-cell development by using flow cytometry to characterize knockout and knockin genetic approaches. (See text for details.)
Second, by sorting cells bearing particular combinations rangement characterizes a very late stage in B-cell develop-
of cell-surface markers, and analyzing those cell populations ment. In this way, cell-surface markers were defined that
for the occurrence of immunoglobulin gene rearrangements, could serve as indicators of particular steps in B-cell dif-
scientists were able to confirm the staging of the appear- ferentiation (Figure 10-4c).
ance of particular developmental antigens. For example, an Third, investigators use the power of knockout genetics
antigen that appears on a cell in which no variable region to determine the effects on B-cell development of eliminat-
gene rearrangement has taken place is clearly expressed ing the expression of particular genes, such as those encod-
very early in B-cell differentiation. Similarly, an antigen ing particular transcription factors (Figure 10-4d). For
present on the surface of a cell that has rearranged heavy- example, knocking out a gene encoding a particular tran-
chain, but not light-chain, genes defines a stage in B-cell scription factor eliminates an animal’s ability to complete VH
differentiation later than that defined by the marker to DJH recombination while still allowing D to JH rearrange-
described above, whereas an antigen present on a cell that ment. This tells us that the particular transcription factor in
has undergone both heavy-chain and light-chain rear- question is not necessary for stages of B-cell differentiation
ADVANCES
The Role of miRNAs in the Control of B-Cell Development
Geneticists have long known that

only a small fraction of chromosomal DNA Nucleus
specifies protein sequences, and early
papers relegated the nonprotein-coding DNA
Cytoplasm
DNA segments to the somewhat igno- RNA
miniously described status of “junk DNA.” polymerase
mRNA-target
In 1993, however, scientists studying the
genome of the nematode C. elegans pri-microRNA
described groundbreaking investigations
of some of the nonprotein-coding Drosha/
sequences that they had identified as hav- DGCR8
ing been transcribed but not translated. RISC
They showed that these primary tran- pre-microRNA
scripts were processed into small pieces RISC
of RNA, 18 to 30 nucleotides in length,
that were capable of exerting control over
Dicer
the level of expression of mRNA.
The biosynthesis of these micro-RNAs PACT and TRBP
follows a similar form in eukaryotes as
diverse as C. elegans and humans (Figure 1).
Fully capped and polyadenylated RNAs FIGURE 1
The generation of functioning microRNAs (miRNAs). Just like mRNA, miRNA species are
(pri-microRNAs) are synthesized by RNA
transcribed as long, capped and polyadenylated RNA species (pri-miRNA) by RNA polymerase ll. They
polymerase II and are then cleaved into a are then cleaved by a nuclear RNase, Drosha, into a hairpin shaped nucleotide precursor molecule,
hairpin-shaped 70- to 100-nucleotide pre- termed a pre-miRNA. Drosha works in a protein complex with the protein DGCR8 (DiGeorge
microRNA by the nuclear RNAase Drosha, syndrome chromosomal region 8). Pre-miRNAs are then exported to the cytoplasm where a second
which works in tandem with a second ribonuclease, Dicer, in association with the proteins PACT and TRBP processes the pre-miRNA into a
19 to 24 nucleotide miRNA duplex, by removing the terminal loop. Next, a protein complex called RISC
double-stranded RNA-binding protein,
(RNA-Induced Silencing Complex) binds to one of the two strands of the duplex. The strand of miRNA
DGCR8. The cleaved pre-micro-RNA is that binds to RISC is the mature miRNA, and it drives the RISC enzyme to the target mRNA, resulting in
then exported to the cytoplasm, where a mRNA silencing and/or destruction. [Adapted from Vasilatou et al., 2009. The role of microRNAs in normal and
second RNAase, Dicer, acting in associa- malignant hematopoiesis. European Journal of Hematology, 84, 1 to 16. Figure 1.]
tion with two other proteins, processes it
to an 18- to 30-nucleotide miRNA duplex, The mature miRNA operates by com- geted for cleavage; the mRNA can be
consisting of the mature miRNA and its plementary binding of a so-called “seed” destabilized; or translation from the mRNA
anti-sense strand. In a final step, the region of 6 to 8 nucleotides at its 5⬘ end to can be repressed. Furthermore, we now
mature miRNA, now single stranded, asso- a region on its target mRNA. Once the know that a single miRNA can target the
ciates with a protein complex called the miRNA has bound, three things can hap- synthesis of many proteins, and each
RNA-induced silencing complex, or RISC. pen: the target mRNA can be directly tar- mRNA can be the target of more than one
prior to D to JH rearrangement, but it is required for one or the transcription factor promoters, so that every point at
more stages, starting with VH to DJH recombination. which the transcription factor is expressed can be delin-
One drawback of the knockout approach, however, is that eated. The staging of transcription factor expression is then
it only defines the first stage in differentiation at which the correlated with the expression of both cell-surface markers
transcription factor is required. More recent variations have and immunoglobulin gene rearrangement. The sequence of
exploited knockin genetics (Figure 10-4e) to generate ani- B-cell development described in the next few sections has
mals that express fluorescent markers under the control of been elucidated using a combination of these strategies.
BOX 10-2
miRNA, thus adding to both the flexibility control of the pro- to pre-B-cell transition and an important factor in the regulation of
and the complexity of this mode of con- affect both the expression of pro-apoptotic the levels of the c-Myb transcription fac-
trol over gene expression. molecules and the nature of the Ig reper- tor during B-cell development.
But does regulation by miRNAs oper- toire. As one would predict from these MiR-150 is also implicated in B-1/B-2 lin-
ate during B-cell development? Several collective results, animals with increased eage specification. B-cell-specific deletion of
lines of evidence suggest that the answer expression of miR-17~92 family members the c-myb gene stops B-cell development at
to this question is an unequivocal “yes.” express lower levels of Bim and suffer from the pro- to pre-B transition and also leads to
From a theoretical standpoint, it is clear a lympho-proliferative disorder and auto- the complete disappearance of the B-1 sub-
that the developmental changes that immune disease. set of B cells. If miR-150 is responsible for
occur as B cells mature require rapid Members of the 17~92 miRNA family down-modulating the levels of c-Myb in
changes in the concentrations of such were also found to control the levels of vivo, then it might be predicted that a defi-
important proteins as transcription factors the Pten protein, which acts as an inhibi- ciency in miR-150 would result in an oppo-
and pro- and anti-apoptotic molecules, tor of the pro-survival PI3 kinase and Akt site phenotype to that expressed by a c-Myb
among other regulatory proteins. The signaling pathway. An increase in the lev- deficient animal, and such was indeed
need for such rapid alterations in protein els of the miR-17~92 molecules allows for found to be the case. Deficiency of miR-150
concentrations can be met efficiently by greater destruction of the Pten mRNA, was found to result in an expansion of the
the type of post-transcriptional control resulting in increased cell survival and a B-1-B-cell pool, with a resulting increase in
mechanisms mediated by miRNAs. corresponding increase in the number of the levels of IgM antibody secretion.
Conditional loss of the gene encoding lymphocytes available to proliferate. The study of miRNA control of mam-
the Dicer nuclease destroys all capacity to Other investigators have addressed the malian gene expression and lymphocyte
synthesize mature miRNAs. Ablation of question of which miRNAs are expressed at development is still in its infancy, but the
Dicer in early B-cell progenitors resulted in different stages of B-cell development. A ability to manipulate and isolate cells at
a developmental block at the pro- to pre-B- combination of genomic (in silico analyses) discrete stages in the developmental
cell transition. In these experiments, the and more classical molecular biological sequence provides a particularly tractable
pro-apoptotic molecule Bim, was expressed approaches have identified several miRNA system in which to analyze the range of
at higher concentrations in Dicer-ablated species and/or families that are implicated actions of different families of miRNAs. This
than in normal B-cell progenitors. in the control of B-cell development. field will undoubtedly be one to watch.
Sequences in the 3⬘ untranslated region of Perhaps the most well studied miRNA
the Bim gene were found to be comple- is miR-150, which is highly expressed in REFERENCES
mentary to miRNAs of the 17~92 family, mature and resting B cells but not in their
Baltimore et al., 2008. MicroRNAs: new regula-
suggesting that members of this family progenitors. The miR-150 molecule has tors of immune cell development and func-
normally down-regulate Bim at this stage been shown to depress the level of tion. Nature Immunology 9:839–845.
of development, enabling B cells to pass expression of the transcription factor Koralov, S. B., et al. 2008. Dicer ablation affects
through this transition. The 17~92 family of c-Myb, known to be essential for the con- antibody diversity and cell survival in the B
miRNAs was also shown to affect expres- trol of B-cell development. As might have lymphocyte lineage. Cell 132:860–874.
sion of TdT and hence N-sequence addi- been predicted, the pattern of c-Myb Vasilatou, D., S. Papageorgiou, V. Pappa, E. Papa-
tion. Other alterations in immunoglobulin expression in lymphocyte development is georgious, and J. Dervenoulas. 2009. The
role of microRNAs in normal and malignant
gene expression were also observed in the complementary to that of miR-150, in that
hematopoiesis. European Journal of Haema-
absence of 17~92 miRNAs, including an c-Myb is highly expressed in lymphoid tology 84:1–16.
increase in the expression of sterile tran- progenitors and is down-regulated upon Xiao, C., and K. Rajewsky. 2009. MicroRNA con-
scripts. These data collectively demon- their maturation; in addition, transcrip- trol in the immune system: Basic principles.
strate that miRNAs are important in the tional analysis confirmed that miR-150 is Cell 136:26–36.
Recently, attention has begun to focus on small molecular The Earliest Steps in Lymphocyte
weight miRNA species, which have profound effects on the Differentiation Culminate in the Generation of
stability of mRNA and hence on the expression of particular
a Common Lymphoid Progenitor
proteins. In Advances Box 10-2, we describe the effects of
some of the miRNAs that have recently been shown to affect In this section, we will describe the process by which an HSC
B-cell differentiation. This is currently an extremely active in the bone marrow develops into a CLP. Unless otherwise
research area. specified, the developmental pathway we describe refers to
c-Kit sca-1 flt-3 CD34 IL-7R Rag1/2

and TDT
Hematopoietic
+ + – – – –
stem cell
Erythrocyte (HSC)
Multipotent
progenitor + + – + – –
(MPP)
Megakaryocyte
Lymphoid-primed
multipotent + + + – – –
progenitor cell
Monocyte (LMPP)
Early lymphoid
progenitor cell + + + – +/– +
T-cell progenitor (ELP)
Common low low + – + +

lymphoid
Natural killer
precursor
(NK) cell
(CLP)
Dendritic Pre-pro
cell (DC) B cell
FIGURE 10-5 Expression of cell-surface markers on HSC and lymphoid progenitor cells. The maturation of HSCs into lymphoid
progenitors, and the progressive loss of the ability to differentiate into other blood-cell lineages can be followed by the expression of the cell-
surface markers as well as by the acquisition of RAG and TdT activity.
that followed by the predominant B-2-B-cell population. Spe- chromatin remodeling complexes to particular regions in
cific aspects of development that differ among the various the DNA and ensures the accessibility of genes necessary for
B-cell subsets will be addressed toward the end of this chapter. B-cell development. PU.1 presides over a leukocytic “balanc-
ing act”; low levels of PU.1 favor lymphoid differentiation,
HSCs whereas cells expressing higher levels of PU.1 veer off to a
HSCs are both self-renewing (they can divide to create iden- myeloid fate. The level of PU.1 protein expressed is in turn
tical copies of the parent cell) and multipotential (they can regulated by the transcriptional repressor Gfi1, which down-
divide to form daughter cells that are more differentiated regulates the expression of PU.1 to the levels necessary for
than the parent cell and that can develop along distinct progression down the B-cell pathway. E2A expression con-
blood-cell lineages), and can give rise to all cells of the blood. tributes to the maintenance of the HSC pool by participating
HSCs maintain a relatively large number of genes in a so- in the regulation of cell cycle control in this population.
called “primed” state, and an individual HSC may possess As noted in Chapter 9, HSCs express the cell-surface mol-
primed genes characteristic of multiple cell lineages. Primed ecule c-Kit (CD117), which is the receptor for stem cell factor
chromatin is associated with lower-than-usual numbers of (SCF). SCF is a cytokine that exists in both membrane-bound
nucleosomes, is more accessible to enzyme activity than the and soluble forms and the SCF-c-Kit interaction is critical for
majority of chromatin in the cell, and shows histone meth- the development, in adult animals, of multipotential pro-
ylation and acetylation patterns characteristic of active chro- genitor cells (MPPs). Membrane-bound SCF plays a role in
matin. Depending on the environmental stimuli to which retaining the HSCs and its daughter progenitor cells in the
any given HSC is exposed, transcription factors may drive appropriate environmental niches in the bone marrow. HSCs
the cell down a number of possible developmental pathways. also express the stem cell associated antigen-1 (Sca-1). Both
During the differentiation process that follows, primed chro- c-Kit and Sca-1 are expressed in parallel on early progenitor
matin regions containing genes that are not needed for the cells, and the levels of their expression drop as the cells com-
selected developmental pathway are shut down. mit to a particular cell lineage (Figure 10-5). HSCs are often
In HSCs bound for a B-cell fate, the transcription factors described as being Lin⫺, a designation that refers to the fact
Ikaros, Purine box factor 1 (PU.1), and E2A participate in that they have no “lineage markers” characteristic of a particu-
the earliest stages of B-lineage development. Ikaros recruits lar blood-cell subpopulation.
MPPs in the B-cell differentiation pathway. Signaling through

MPPs generated on receipt of SCF/c-Kit signaling lose the the IL-7R occurs via pathways familiar from Chapter 4.
capacity for extensive self-renewal, but retain the potential to Specifically, an IL-7R-mediated JAK-STAT pathway
differentiate into several different hematopoietic lineages. induces the up-regulation of the anti-apoptotic molecule
MPP cells retain the expression of c-Kit and Sca-1 and tran- Mcl1. Signaling through IL-7R also results in the up-
siently express the molecule CD34. Indeed, antibodies to regulation of the C-myc and N-myc genes, which signal
CD34 are used clinically to isolate cells at this stage in hema- the cell proliferation characteristic of the later, pro-B-cell
topoiesis. MPPs also express the chemokine receptor CXCR4, stage.
which enables them to bind the stromal cell-derived chemo- CLPs are c-Kitlow, Sca-1low, and IL-7R⫹ and have lost
kine CXCL12. The interaction between CXCL12 and CXCR4 myeloid potential. However, as a CLP destined to differentiate
is important in ensuring that the progenitor cell occupies the along the B cell pathway matures, the chromatin containing
correct niche within the bone marrow (see Figure 10-3). the immunoglobulin locus becomes increasingly accessible
and the developing lymphocyte approaches the point at
LMPPs which it is irrevocably committed to the B-cell lineage.
The progenitor cell on its way to becoming a B cell then
begins to express the fms-related tyrosine kinase 3 receptor The Later Steps of B-Cell Development Result
(flt-3). Flt-3 binds to the membrane-bound flt-3 ligand on in Commitment to the B-Cell Phenotype
bone marrow stromal cells and signals the progenitor cell to Figure 10-6 illustrates the expression of cell-surface markers,
begin synthesizing the IL-7 receptor (IL-7R, CD127). Flt-3 and the patterns of rearrangement of immunoglobulin
is expressed on B-cell progenitors from this point until the heavy-chain and light-chain genes starting at the pre-pro
pro-B stage and acts synergistically with IL-7R to promote B-cell stage of development. The stages of B-cell differentia-
the growth of cells bearing flt-3 and IL-7R. The expression of tion have been defined by more than one group of scientists
flt-3 on the surface of the developing cell marks the loss of and, as a result, two systems of nomenclature are in common
the potential of the MPP cell to develop into erythrocytes or use. The first, and most widely used, is the Basel nomencla-
megakaryocytes, and therefore characterizes a new level of ture (pre-pro, pro, pre-B, immature B) developed by Melchers
cell commitment; however, this progenitor still retains the and colleagues. The second (A, B, C, C⬘, D, E) is that defined
capacity to develop along either the myeloid or the lymphoid by Hardy et al., and the process by which this system of clas-
pathways. sification of B-cell development was established is described
These cells, now c-Kit⫹, Sca-1⫹, and flt-3⫹, are termed in detail in Classic Experiment Box 10-3.
lymphoid-primed, multipotential progenitors (LMPPs)
(see Figure 10-5). As they become further committed to the Pre-Pro B Cells
lymphoid lineage, levels of the stem-cell antigens c-Kit and
With the acquisition of the B-cell lineage-specific marker
Sca-1 fall, and cells destined to become lymphocytes begin
B220 (CD45R), and the expression of increasing levels of the
to express RAG1/2 and terminal deoxynucleotidyl trans-
transcription factor EBF1, the developing cell enters the pre-
ferase (TdT) (Chapter 7). Expression of the genes encoding
pro-B-cell stage. EBF1 is an important transcription factor in
RAG1/2, TdT, IL-7R, and the B-cell-specific transcription
lymphoid development, and therefore transcription of the
factor EBF1 are all up-regulated at the end of this stage.
Ebf1 gene is itself under the control of multiple transcription
factors (Figure 10-7). These each bind at distinct promoter
ELPs
regions, and hence the level of transcription of the Ebf1 gene
Expression of RAG1/2 defines the cell as an early lymphoid can vary considerably depending on the combination of
progenitor cell (ELP). A subset of ELPs migrates out of the controlling factors present at any particular developmental
bone marrow to seed the thymus and serve as the T-cell stage. At the pre-pro-B-cell stage, EBF-1, along with E2A,
progenitors (discussed in Chapter 9). The rest of the ELPs binds to the immunoglobulin gene, promoting accessibility
remain in the bone marrow as B-cell progenitors. On these of the D-JH locus and preparing the cells for the first step of
cells, the levels of the early c-Kit and Sca-1 antigens decrease Ig gene recombination. EBF-1 is also essential to the full
as the levels of the IL-7R increase, and the ELP now develops expression of many B-cell proteins, including Ig␣,Ig␤
into a CLP. (CD79␣,␤), and the genes encoding the pre-B-cell receptor,
which will be expressed when heavy-chain VDJ recombina-
CLPs tion is complete.
At the CLP stage, the progenitor on its way to B-cell com- Pre-pro B cells remain in contact with CXCL-12-secret-
mitment still retains the potential to mature along the ing stromal cells in the bone marrow. However, the onset of
NK, conventional DC, or T-cell lineages. At this point in D to JH gene recombination classifies the cell as an early
development, signals received through IL-7R promote pro-B cell, and at this stage the developing cell moves within
cell survival and enhance the production of EBF-1 and the bone marrow, seeking contact with IL-7 secreting stro-
other transcription factors that are required for later steps mal cells.
Status Surface Ig
B220
Cell stage of Ig receptor c-Kit IL-7R CD25 CD19
(CD45R)
genes expression
Pre-pro GL2 None – lo – – +

(Fr. A)1
Early pro DJH None lo lo/+ – + +

(Fr. B)
Late pro Some None lo + +/– + +

(Fr. C) VHDJH
1st checkpoint
Large Pre VHDJH Pre-BCR – + + + + FIGURE 10-6 Immunoglobulin gene

(Fr. C')
rearrangements and expression of marker
Small Pre VHDJH Decreasing – + + + + proteins during B-cell development. The
(Fr. D) VL JL re- levels expression of selected marker proteins is corre-
arrange- of Pre-BCR lated with the extent of Ig gene rearrangement
ment during B-cell development from the pre-pro B cell
begins
to the immature T1 B-cell stage. (See text for
Immature B VHDJH IgM – + + + + details.) [Adapted from K. Samitas, J. Lötvall, and A. Boss-
(Fr. E) VL JL ios, B cells: From early development to regulating allergic
diseases, Archivum Immunologiae et Therapiae Experi-
2nd checkpoint
mentalis 58:209–225, Figure 1.]
Pre-BCR
BCR
1 Labeled fractions refer to the “Hardy nomenclature,” described in the
Classic Experiment Box 10-1.

2 GL = germ line arrangement of heavy and/or light chain V
region segments
IL-7
IL-7Rγ IL-7Rα
FIGURE 10-7 The interplay of transcrip-
tion factors during early B-cell develop-
Cytoplasm ment. Dimerization and activation of the
transcription factor STAT5 is stimulated by IL-7
P binding to its receptor. STAT5 stimulates B-cell
STAT5 STAT5
P proliferation by activating the proliferative control
proteins N-myc and C-myc. STAT5 collaborates
with E2A proteins to promote the expression of
early B-cell factor 1 (EBF1). EBF1 in turn promotes
• Transcription of B-cell
associated genes survival
the expression of PAX5, and together the E2A
N-myc E2A
• B-cell specification and proteins, EBF1, and PAX5 activate many genes
C-myc
commitment survival leading to B-cell lineage specification and com-
mitment. PAX5 and EBF1 both participate in posi-
EBF1 tive feedback loops that enhance the levels of
B-cell proliferation both EBF1 and PAX5 transcription. [Adapted from B.
L. Kee, 2009, E and ID proteins branch out, Nature Reviews
Immunology 9:175–184.]
PAX5 Notch1
D-JH recombination
Nucleus
VHDJH recombination
CLASSIC EXPERIMENT BOX 10-3
The Stages of B-Cell Development: Characterization

of the Hardy Fractions
Richard Hardy’s laboratory was
one of the first to combine flow cytome-
try and molecular biology in experiments Start with bone marrow cells
designed to analyze lymphocyte matura-
tion. In this feature, we describe what
those researchers did and how they gen-
erated a model of the sequencing of the
stages of B-cell development from their
data.
When Hardy and colleagues began
their characterization of B-cell lineage Sort for B220+CD43+
development in the early 1990s, prior work
using molecular analysis of long term bone
marrow cell lines had already established Analyzed B220+
the sequential rearrangement of heavy- CD43+ cells for
chain and light-chain immunoglobulin HSA and BP-1
genes. In addition, the expression of a expression
HSA- HSA+ HSA+
number of cell-surface markers on bone BP-1- BP-1- BP-1+
marrow cells had been measured, and Fraction A Fraction B Fraction C
several of these antigens had been shown Pre-pro-B cells Early pro-B cells
to be co-expressed with B220 (CD45R),
which had already been established as a
B-cell differentiation antigen. Hardy’s Lower levels of HSA Higher levels of HSA
approach was to characterize the sequence Fraction C Fraction C'
Late pro-B cells Large (early)
of expression of those antigenic markers
pre-B cells
that were found on the same cells as B220.
The hypothesis was that some of these FIGURE 1
The isolation of Hardy’s fractions. A, B, C, C⬘. Bone marrow cells were sorted for cells bearing
markers may be expressed on early B-cell
B220 and CD43 and then analyzed for their expression of the cell-surface markers HSA and BP-1.
progenitors and might therefore help to
generate a scheme of B-cell development. marrow cells. Both HSA and BP-1 had
In order to place cells expressing different been previously shown to be differen-
combinations of markers into a develop- tially expressed at varying stages of
mental lineage, Hardy then sorted cells lymphoid differentiation. Gated B220+ CD43–
bearing each combination of his selected The first set of experiments analyzed
markers, and placed them into co-cultures those cells bearing both B220 and CD43 for
with a bone marrow stromal cell line. After the levels of their expression of HSA and
defined times in culture, he harvested the BP-1 (Figures 1 and 2). Flow cytometry
hematopoietic cells and re-characterized plots demonstrated that the B220⫹CD43⫹
their surface marker expression. cells neatly resolved into three discrete
The markers used in these experi- subpopulations. The first, labeled A in Fig-
ments included B220 (CD45R) and CD43 ure 1, expressed neither HSA, nor BP-1. The
(leukosialin), which had previously been second, labeled B, expressed HSA, but not
shown to be expressed on granulocytes BP-1, and the third expressed both of these
and all T cells, but was not present on antigens. Analysis of Ig gene rearrange-
mature B cells, with the exception of ments in these populations revealed that FIGURE 2
Flow cytometric characterization of
plasma cells. In addition, their experi- no gene rearrangements occurred in
the early developmental stages of B
ments employed antibodies directed fraction A but that D to JH gene segment cells. (See text for details.) [Hardy et al., J. Exp. Med.
against Heat Stable Antigen, or HSA rearrangements had begun in fraction B. 173, 1213–1225. May 1991. By permission of Rockefeller
(CD24) and BP-1, an antigen on bone Subsequent work has shown that VH to DJH University Press]
(continued)
CLASSIC EXPERIMENT (continued)
rearrangements occur in fraction C

(although at the time, the method of analy-
sis that Hardy and colleagues used failed to
reveal this second type of rearrangement).
So at this point, we know that three
Start with bone marrow cells distinct types of B-cell precursors express
both B220 and CD43, and can be discrimi-
nated on the basis of their levels of the
further two antigens, HSA and BP-1. Frac-
tion A corresponds to what we now know
as pre-pro B cells, fraction B to early pro-B
cells, and fraction C to late pro-B cells.
Culture of fraction C cells yielded cells
that expressed membrane (m) IgM; simi-
larly, culture of fraction B cells also yielded
daughter cells expressing mIgM, but at a
Sort for B220+, CD43- lower frequency than fraction C cells, sug-
gesting that cells in fraction C were fur-
ther along the differentiation pathway to
mIgM⫹ B cells. Furthermore, the three dif-
ferent fractions displayed differential
dependence on the need to adhere to the
stromal cell layer. Cells from fraction A
required stromal cell contact for survival.
Fraction B cells survived best in contact
mIgMlo mIgMhi mIgMhi, mIgDhi
H chain rearranged H chain rearranged H chain rearranged with the stromal cells, but were able to
L chain rearrangement L chains rearranged L chains rearranged survive in a culture in which they were
beginning separated from the stromal cells by a
Fraction D Fraction E Fraction F
semipermeable membrane. Under these
Small pre-B cells Immature B cells Mature B cells conditions, they could still receive soluble
factors generated by the stromal cells, but
FIGURE 3
The isolation of Hardy’s fractions D, E, and F. Bone marrow cells were sorted for cells were prevented from generating adhesive
bearing B220, but not CD43 (recognized by monoclonal antibody S7) and then analyzed for cell-surface interactions with stromal cell-surface-
expression of IgM and IgD. bound growth factors. Fraction C cells
Pro-B Cells Chapter 3, as one of the components of the B-cell co-receptor.

In the early pro-B-cell stage, D to JH recombination is com- CD19 is considered a quintessential B-cell marker and is often
pleted and the cell begins to prepare for V to DJH joining. used as such in flow cytometry experiments.
However, this final recombination event awaits the expres- Once the PAX5 protein is expressed, a mutual reinforce-
sion of the quintessential B-cell transcription factor, PAX5. ment occurs between PAX5 and EBF-1 expression, as illus-
The Pax5 gene is among EBF-1’s transcriptional targets trated in Figure 10-7, with each transcription factor serving
(see Figure 10-7) and transcription of genes controlled by the to enhance the expression of the other. The PAX5 protein
PAX5 transcription factor denotes passage to the pro-B-cell continues to be expressed in mature B cells until the B cell
stage of development, at which point the expression of non-B- commits to a plasma-cell fate following antigenic stimula-
lineage genes is permanently blocked. PAX5 can act as a tran- tion (see Chapter 12). The higher levels of EBF1 expression
scriptional repressor, as well as an activator, and blocks induced by PAX5 also allow for an increase in the level of
Notch-1 gene expression, thereby terminating any residual IL-7R expression.
potential of the pro-B cell to develop along the T-cell lineage. PAX5 promotes VH to D recombination by contracting the
Many important B-cell genes are turned on at this stage, under IgH locus, thus bringing the distant VH gene segments closer
the control of PAX5 and other transcription factors. Among to the D-JH region. B cells deficient in PAX5 permit D to JH
these is the gene encoding CD19, which we first encountered in Ig gene rearrangement, but do not allow recombination of
BOX 10-3
survived and proliferated in the absence rearrangement, and most of the cells in
Gated B220+ CD43–
of stromal cell contact. Analysis of the fac- that fraction also displayed light-chain
tors secreted by the stromal cells that gene rearrangement. Fraction E cells are
were necessary for survival and prolifera- thus immature B cells ready to leave the
tion of the fraction B and C cells revealed bone marrow. Subsequent further charac-
one of them to be interleukin 7. terization of fraction F cells showed that,
Hence, using the criteria of Ig gene in addition to surface IgM, these cells also
rearrangements and phenotypic analysis bear surface IgD and therefore represent
of cultured cell populations, Hardy and fully mature B cells, presumably recirculat-
colleagues were able to place the three ing through the bone marrow.
fractions in sequence; cells of fraction A Thus, Hardy’s experiments revealed
gave rise to cells of fraction B, which in that the pool of progenitor and precursor
turn mature into cells of fraction C. B cells in the bone marrow represents a
FIGURE 4
Careful analysis of the contour graph of complex mixture of cells at different Flow cytometric characterization of
fraction C reveals that it, in turn, can be stages of development, with varying the later developmental stages of B
subdivided on the basis of the levels of requirements for stromal cell contact and cells. (See text for details.) [Hardy et al., J. Exp. Med.
expression of HSA. That population of cells interleukin support. 173, 1213–1225. May 1991. By permission of Rockefeller
bearing higher levels of HSA, as well as These elegant experiments still had University Press]
BP-1, is now defined as fraction C⬘, which one more story to tell that did not appear
corresponds to early or large pre-B cells. in the original paper, but which emerged the marrow. This is consistent with the
Hardy and colleagues next turned in later publications. Single-cell PCR analy- notion that the new heavy chain is associ-
their attention to those cells that sis of fraction C cells showed that many of ating with the surrogate light chain at the
expressed B220, but had lost CD43, and them had nonproductive rearrangements C⬘ stage and the pre-B-cell receptor com-
measured their cell-surface expression of on both heavy-chain chromosomes (see plex is expressed on the cell surface, trig-
mIgM (Figure 3). Three populations of Figure 2). In contrast, all the cells from the gering the period of clonal expansion of B
cells were again evident, which they C⬘ fraction demonstrated productive rear- cells described in this chapter.
labeled D, E, and F (Figure 4). Cells belong- rangements on one of the heavy-chain
REFERENCES
ing to fraction D expressed zero to low chromosomes. Fraction C cells therefore
levels of mIgM, showed complete heavy- represent B cells that have been unable to Hardy, R. R., et al. 1991. Resolution and charac-
terization of pro-B and pre-pro-B cell stages
chain rearrangement, and some light- productively rearrange one of their heavy-
in normal mouse bone marrow. Journal of
chain rearrangement and correspond to chain genes and that will therefore even- Experimental Medicine 173:1213–1225.
small pre-B cells. Cells belonging to frac- tually die by apoptosis. The C⬘ fraction also Hardy, R. R., P. W. Kincade, and K. Dorshkind.
tion E displayed high levels of mIgM as included the highest proportion of cells in 2007. The protean nature of cells in the B
well as of B220, complete heavy-chain cycle of any of the B220⫹ B-cell stages in lymphocyte lineage. Immunity 26:703–714.
the VH to the D Ig gene segment, indicating that PAX5 is Pre-B Cells

essential to the second step of Ig gene rearrangement. During the pre-B-cell stage, the cell expresses a pre-B-cell
Expression of the signaling components of the B-cell recep- receptor composed of the rearranged heavy chain, com-
tor, Ig␣ and Ig␤, also begins at the pro-B-cell stage and the plexed with the VpreB and ␭5 components of the surrogate
Ig␣,Ig␤ signaling complex is briefly placed on the cell surface light chain (see Figure 7-10). The appearance of this pre-B-
in complex with the chaperone protein calnexin. Although this cell receptor signals the entry of the developing B cell into
Ig␣,Ig␤ complex has been referred to as a “pro-BCR,” no ligand the large, or early pre-B-cell phase. As we learned in Chap-
has yet been established for it, nor do we yet understand the ter 7, the expression of the heavy chain at the cell surface is
importance of any signaling that may emanate from it. necessary for the termination of further heavy-chain rear-
Also during the pro-B-cell stage, c-Kit is once more rangement and ensures allelic exclusion of the Ig heavy-
turned on briefly, enabling the cell to receive signals from chain genes. Animals deficient in the expression of either the
stem cell factor. By the beginning of the pre-B-cell stage of pre-B-cell receptor, or of the signaling components Ig␣,Ig␤,
development, expression of c-Kit is irreversibly turned back fail to progress to the pre-B-cell stage.
off. By the late pro-B-cell stage, most cells have initiated VH Signaling through the pre-B-cell receptor induces a few
to DJH Ig gene segment recombination, which is completed rounds of proliferation in the pre-B cell. This proliferative
by the onset of the early pre-B-cell stage.
phase correlates in time with the expression on the pre-B- receptor. At the end of pre-BCR-signaled cell proliferation, the
cell surface of CD25, the ␣ chain of the high-affinity IL-2 pre-B-cell receptor is lost from the surface, and this signals
receptor ␣ chain (see Figure 4-8), which first appears on B entry into the small or late pre-B-cell stage. At this point,
cells at the pro-B-cell stage. Since the pre-B-cell proliferative light-chain rearrangement is initiated with the re-expression
process appears mainly to be driven by IL-7, the functional of the Rag1/2 genes. Very little TdT activity remains at this
significance of CD25 at this point is unclear. However, its stage, and therefore N region addition occurs less frequently
appearance is frequently used as a marker of the late pro-B- in light chains than in heavy chains. In the mouse, light-chain
cell to early pre-B-cell stage of development. rearrangement begins on one of the ␬ chain chromosomes,
VH gene recombination is energetically expensive to the followed by the other. If neither ␬ chain rearrangement is suc-
organism, as not all developing B cells are successful in cessful, rearrangement is then successively attempted on each
undergoing productive VH gene rearrangements, and those of the ␭ chain chromosomes. In humans, rearrangement is
that fail to do so are lost by apoptosis. It therefore seems initiated randomly at either the ␬ or the ␭ loci.
logical that those B cells that have achieved productive heavy- Once a light-chain gene rearrangement has been success-
chain expression should be allowed to proliferate. Each indi- fully completed, the IgM receptor is expressed on the cell
vidual daughter cell derived from this proliferative process is surface, signaling entry into the immature B-cell stage. If the
then free to participate in a different light-chain rearrange- attempts at light-chain immunoglobulin gene rearrangement
ment event. Most individuals will therefore have multiple B are not successful, the nascent cell is eventually lost at the
cells expressing precisely the same heavy-chain rearrange- immature B-cell (2nd) checkpoint (see Figure 10-6). However,
ment but each with a different light chain, and many different given the availability of four separate chromosomes on which
receptor specificities can thereby be generated from each suc- to attempt rearrangement, and the opportunity for light-chain
cessful heavy-chain rearrangement. Recall from Chapter 9 editing in the case of unproductive rearrangement, most
that an analogous process of ␤ chain rearrangement, followed pre-B cells that have successfully rearranged their heavy
by proliferation prior to ␣ rearrangement, occurs in T cells. chains will progress to the formation of an immature B cell.
If the pre-B-cell receptor cannot be displayed on the cell
surface because of non-productive VHDJH gene rearrange- Immature B Cells in the Bone Marrow Are
ments, B-cell development is halted and the cell is lost to
apoptosis. This stage in B-cell development is therefore
Exquisitely Sensitive to Tolerance Induction
referred to as the pre-B-cell (1st) checkpoint (see Figure 10-6). Immature B cells bear a functional receptor in the form of
Progress through this checkpoint depends on some type of membrane IgM, but have not yet begun to express any other
signaling event through the pre-B-cell receptor, and recent class of immunoglobulin. They continue to express B220,
evidence suggests that this is mediated via interactions CD25, IL-7R, and CD19.
between arginine-rich regions in the non-immunoglobulin Once the functional BCR is assembled on the B-cell
portion of the ␭5 component of the surrogate light chain, or membrane, the receptor must be tested for its ability to bind
in the CDR3 regions of some heavy chains, with negatively to self antigens, in order to ensure that as few as possible
charged molecules on the surface of the stromal cells. autoreactive B cells emerge from the bone marrow. Those
Pre-B-cell receptor signaling induces the transient down- immature B cells that are found to bear autoreactive recep-
regulation of RAG1/2 and the loss of TdT activity. Together, tors undergo one of three fates; some are lost from the rep-
these events ensure that, as soon as one heavy-chain gene has ertoire prior to leaving the bone marrow, by the BCR-mediated
successfully rearranged, no further heavy-chain recombina- apoptotic process of clonal deletion. The loss of B cells bear-
tion is possible. This results in the phenomenon of allelic ing self-reactive receptors within the bone marrow is referred
exclusion, whereby the genes of only one of the two heavy- to as central tolerance. Other autoreactive B cells reactivate
chain alleles can be expressed in a single B cell. As a result of their RAG genes to initiate the process of light-chain recep-
this pre-B-cell receptor signaling, the chromatin at the unused tor editing (see Chapter 7). Some autoreactive B cells that
heavy-chain locus undergoes a number of physical changes recognize soluble self antigens within the bone marrow may
that render it incapable of participating in further rearrange- survive to escape the bone marrow environment, but become
ment events. Recall that IL-7 provided one of the signals that anergic, or unresponsive, to any further antigenic stimuli.
brought the VH, D, and JH loci into close apposition with one The concept of negative selection of lymphocytes bearing
another at the beginning of VHDJH recombination. A reduc- autoreactive receptors should be familiar from the discussion
tion in IL-7 signaling at the pre-B-cell stage now reverses that of T-cell tolerance in Chapter 9. However, functional differ-
initial locus contraction, resulting in the physical separation of ences between T cells and B cells mean that the selection
the VH, D, and JH gene segments in the unrearranged heavy- processes against autoreactive B cells are different from those
chain locus. This decontraction is then followed by deacety- that protect against the emergence of autoreactive T cells, and
lation events that deactivate the unused heavy-chain locus and indeed can be somewhat less stringent. Since stimulation of
return it to a heterochromatic (inactive, closed) configuration. B-2 B cells requires T-cell help, an autoreactive B cell cannot
Surrogate light-chain expression is also terminated by a respond to antigen with antibody production unless there is
negative feedback round of signaling through the pre-B-cell also an autoreactive T cell that can provide the necessary
cytokines and costimulation (Chapter 12). Thus, it is quite (Figure 10-8c). Closer examination revealed that some of the
possible that most individuals carry significant numbers of residual transgene-expressing B cells in the bone marrow
autoreactive B cells within their mature B-cell repertoires that had undergone light-chain receptor editing (see Chapter 7),
are never activated. changing their antigen specificity so they no longer bound
There are also mechanistic differences between the modes the H-2Kk antigen. Recent experiments suggest that in vivo,
of negative selection among B and T cells. At this point, no a significant fraction of potentially autoimmune B cells
equivalent of the AIRE protein has been shown to exist for undergo receptor editing (or even VH gene replacement
B-cell selection, and so B-cell negative selection within the (Chapter 7), and successfully generate acceptable BCRs prior
bone marrow is more limited with respect to the available to release from the bone marrow.
tolerogenic antigenic specificities than is T-cell negative In normal animals, not all potentially autoimmune B cells
selection in the thymus. are lost to clonal deletion or altered via receptor editing or
VH gene replacement within the bone marrow, however;
Many, but Not All, Self-Reactive B Cells Are some are released to the periphery and subject to further
rounds of selection.
Deleted within the Bone Marrow
Our understanding of how the immune system eliminates or
neutralizes autoreactive threats has been facilitated by the B Cells Exported from the Bone Marrow Are
development of transgenic animals which express both Still Functionally Immature
deliberately introduced auto-antigens and the receptors that
Once the B cell expresses IgM on its membrane (mIgM), it is
recognize them. It has long been established that cross-
referred to as an immature B cell. This B cell is ready for
linking the IgM receptors of immature B cells in vitro (per-
export to the spleen, where it completes its developmental
formed experimentally by treating the cells with antibodies
program. Immature B cells have a short half-life, in part as a
against the receptor ␮ chain) results in death by apoptosis. In
result of expressing low levels of the anti-apoptotic mole-
contrast, performing the same experiments with mature B
cules Bcl-2 and Bcl-xl. They also express high levels of the
cells, bearing both IgM and IgD receptors, results in activa-
cell-surface molecule, Fas, which is capable of transmitting a
tion. David Nemazee and colleagues set out to test whether
death signal when bound by its ligand (see Chapters 4 and
the apoptotic response of immature B cells in vitro reflected
9). Immature B cells are exquisitely susceptible to tolerance
what happens in the bone marrow in vivo when an imma-
induction, and if they encounter a self antigen at this stage of
ture B cell meets a self antigen.
development, the B cells will re-express the RAG1 and RAG2
Nemazee et al.’s approach was conceptually simple,
genes and edit their light-chain genes. If receptor editing
although experimentally complex, particularly for the time
period in which the work was done (1989). They generated fails to yield a suitable receptor, the cell undergoes apoptosis.
mice transgenic for both a heavy and a light chain specific The study of B-cell development in the periphery, like that
in the bone marrow, has benefited significantly from the inge-
for the MHC molecule H-2Kk. All the B cells in this mouse
nious application of flow cytometry, which has enabled the
therefore made only anti-H-2Kk antibodies. If immature B
classification of immature B cells into two subpopulations of
cells undergo selection to prevent autoimmunity, these cells
transitional B cells (T1, T2). These transitional B cells act
would be selected against in a mouse that expresses the
sequentially as the precursors to the fully mature B cell.
H-2Kk gene for MHC. By appropriate breeding, they intro-
duced the immunoglobulin H-2Kk-specific transgenes into
mice bearing two different MHC genotypes. T1 and T2 Transitional B Cells
In the first group of mice (Figure 10-8a), which bore T1 and T2 transitional B cells were characterized initially on
H-2Kd but no H-2Kk antigens, they were able to detect the the basis of their cell-surface expression of immunoglobulin
transgenic antibody at high frequency on the surface of B receptors and membrane markers (Table 10-2). T1 cells are
cells and at high concentration in the serum (Table 10-1). mIgMhi, mIgD⫺/lo, CD21⫺, CD23⫺, CD24⫹, and CD93⫹. T2
This makes sense, as the transgenic antibody would be cells differ from T1 cells in having higher levels of mIgD and
unable to bind the H-2Kd molecules and so the B cells that in expressing CD21 (the complement receptor and B-cell
produce it would not be negatively selected. However, when co-receptor; see Figure 3-7) and CD23. T2 cells also express
they bred these animals with mice of the H-2Kk type (Figure BAFF-R, the receptor for the B-cell survival factor BAFF,
10-8b), no membrane-bound or secreted anti-H-2Kk anti- whose expression is dependent on signals received through
bodies could be detected, suggesting that all immature B the BCR. As B cells differentiate from the transitional T2
cells bearing the potentially autoimmune receptor antibod- state to full maturity, they raise their levels of mIgD still fur-
ies had been deleted in the bone marrow. This deletion ther, while reducing the expression of mIgM. They also cease
occurred via induction of apoptosis in the autoimmune cells. to express CD24 and CD93.
Interestingly, in the H-2Kk/d mice, not all B cells bearing T1 cells that have been labeled and transferred into recipi-
the autoimmune transgenes were deleted, even though all B ent mice develop into T2 cells. Similarly, both transitional
cells in this mouse should bear the anti-H-2Kk receptor B-cell subpopulations have been demonstrated to have the
(a) H-2d transgenics
Mature B cells express

anti -Kk
Kd
(b) H-2d/k transgenics

Immature B cells
No mature B cells express

anti-Kk
Anti-Kk
Kd Kk
Bone marrow
stromal cell
(c) H-2d/k transgenics
Light-chain editing
A few mature B cells with new

light chains no longer bind Kk
FIGURE 10-8 Experimental evidence for negative selection H-2d/k transgenics, many of the immature B cells that recognized the
(clonal deletion) of self-reactive B cells during maturation in self antigen Kk were deleted by negative selection. (c) More detailed
the bone marrow. The presence or absence of mature peripheral B analysis of the H-2d/k transgenics revealed a few peripheral cells that
cells expressing a transgene-encoded IgM against the H-2 class I mol- expressed the transgene-encoded ␮ chain but a different light chain.
ecule Kk was determined in H-2d mice (a) and H-2d/k mice (b) and (c). Apparently, a few immature B cells underwent light-chain editing, so
(a) In the H-2d transgenics, the immature B cells did not bind to a self they no longer bound the Kk molecule and consequently escaped
antigen and consequently went on to mature, so that splenic B cells negative selection. [Adapted from D. A. Nemazee and K. Burki, 1989, Nature
expressed the transgene-encoded anti-Kk as membrane Ig. (b) In the 337:562; S. L. Tiegs et al., 1993, Journal of Experimental Medicine 177:1009.]
TABLE 10-1 Expression of transgene encoding IgM antibody to H-2k class I MHC molecules
Expression of transgene
Experimental animal Number of animals tested As membrance Ab As secreted Ab (␮g/ml)
Nontransgenics 13 (⫺) ⬍0.3

H-2d transgenics 7 (⫹) 93.0
H-2 d/k
transgenics 6 (⫺) ⬍0.3
[Adapted from D. A. Nemazee and K. Bürki, 1989, February, Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes, Nature 337:562–566.
doi:10.1038/337562a0.]
Bone marrow
sinusoids, bloodstream
T cell zone
Endosternum
Pro and Immature B Follicle
pre B
Bone marrow Central

arteriole
T2 T1
IgM Marginal sinus
IgD Marginal zone
FIGURE 10-9 T2, but not T1, transitional immature cells There, the T2 cells complete their differentiation into mature follicular
can enter the B-cell follicles and recirculate. Immature B cells B-2 cells. Marginal zone cells have also been shown to derive from T2
leave the bone marrow as T1 transitional immature B cells. They enter cells. [Adapted from J. B. Chung, M. Silverman, and J. G. Monroe, 2003, Transi-
the spleen from the bloodstream through the marginal sinuses, per- tional B cells: Stem by step towards immune competence, Trends in Immunology
colating into the T-cell zones, and differentiating into T2 transitional B 24:343–348, Figure 1; and R. E. Mebius, and G. Kraal, 2005, Structure and function
cells, which gain the ability to enter the B-cell follicles and recirculate. of the spleen. Nature Reviews Immunology 5:606–616, Figure 1.]
capacity to differentiate into mature B cells. These experiments recirculating among the blood, lymph nodes, and spleen; and
have therefore proven that the order of the developmental T2 cells, but not T1 cells, can enter B-cell follicles.
sequence progresses from T1 to T2 to mature B cell. The time Figure 10-9 shows the path of the developing transitional
in transit of a T1 cell to a mature B cell has been measured to B cell as it leaves the bone marrow and enters the spleen
be approximately 3 to 4 days. Most T1 B cells differentiate to through the central arteriole, which deposits it in the mar-
T2 cells within the spleen, but a minority of about 25% of tran- ginal sinuses, just inside the outer marginal zone. (In
sitional B cells emerge from the bone marrow already in the humans, the anatomy of the spleen is slightly different, and
T2 state. The increased level of maturity of T2 cells correlates the cells arrive in the spleen in a peri-follicular zone.) From
with changes in the expression of chemokine and cytokine there, the T1 B cell percolates through to the T-cell zone,
receptors, such that T2 cells, but not T1 cells, are capable of where some fraction of T1 cells will mature into the T2 state.
T2 B cells are then able to enter the follicles or the marginal
zone where they complete their developmental program into
Surface marker expression on
fully mature, recirculating B lymphocytes.
TABLE 10-2 transitional T1 and T2 and
In Chapter 7, we learned that mature B cells bear on their
mature B-2 B cells
surfaces two classes of membrane-bound immunoglobulins—
Mature IgM and IgD—and that the expression of mIgD along with
Marker T1 T2 B-2 cells mIgM requires carefully regulated mRNA splicing events. It is
at the point of transit between the T1 and T2 stages of develop-
mIgM High High Intermediate
ment that we observe the onset of these splicing capabilities.
mIgD ⫺ⲐLow Intermediate High Mature B cells bear almost 10 times more mIgD than mIgM,
CD24 ⫹ ⫹ ⫺ and so mIgD expression results in significant up-regulation in
CD93 ⫹ ⫹ ⫺ the number of B-cell immunoglobulin receptors.
The effect of strong BCR engagement with a multivalent, or
CD21 ⫺ ⫹ ⫹
membrane-bound, antigen depends on the maturational status
CD23 ⫺ ⫹ ⫹ of the transitional B cell (Figure 10-10 and Table 10-3). Self-
BAFF receptor reactive T1 B cells are eliminated by apoptosis in response to a
(BAFF-R) ⫹/⫺ ⫹ ⫹ strong antigenic signal, in a process reminiscent of thymocyte
Note: CD93 is defined by the AA4 monoclonal antibody. CD24 is otherwise
negative selection, leading to peripheral tolerance; recent exper-
known as the Heat Stable Antigen (HSA). CD23 is a low-affinity receptor for iments have suggested that in healthy adults, fully 55% to 75%
IgE. CD21 is a receptor for complement and part of the B-cell co-receptor. of immature B cells are lost by this process. In contrast, once the
After D. Allman and S. Pillai. 2008. Peripheral B cell subsets. Current Opinion B cell has matured into a T2 transitional B cell, it becomes resis-
in Immunology 20:149–157, and others. tant to antigen-induced apoptosis, reminiscent of thymocytes
that have reached the single-positive stage of development. This
PALS Follicle expression at different stages of B-cell development. Animals

T1 T2 that fail to express a BCR during the immature B-cell stage
lose the capacity to make any mature B cells at all. This indi-
cates that some low level of tonic signaling through the BCR,
analogous to the positive selection signal needed by develop-
ing thymocytes, is required for continued generation and
survival of immature B cells. B cells unable to receive this
signal die at the T2 stage (see Figure 10-10). Those T2 B cells
Negative Positive able to receive the follicular signal up-regulate the expres-
selection selection sion of the receptor for the B-cell survival factor BAFF.
Given the different outcomes of signaling through the
BCR for T1 versus T2 B cells, it is clear that there must be
Death differences in the signaling pathways downstream of the BCR
in the two transitional B-cell types. Specifically, BCR-mediated
signaling of T1 B cells results in calcium release without
Death B-2 significant production of diacylglycerol, and provides an
apoptotic signal. In contrast, receipt of BCR signals by T2 B
cells induces both an increase in the concentration of intra-
FIGURE 10-10 Transitional B cells bound for a follicular cytoplasmic calcium and in diacylglycerol production. This
fate undergo positive and negative selection in the spleen. T1 combination of intracellular second messengers delivers
transitional B cells, which recognize antigen with high affinity in the both maturational and survival signals to the cell and sug-
spleen, are eliminated by negative selection, and never reach the gests the involvement of a diacylglycerol-activated protein
splenic follicles. Those T1 cells that escape negative selection enter the kinase in survival signaling (see Chapter 3).
follicles and differentiate into T2 B cells. In the follicles, their BCRs inter- But what causes this difference in the signal transduction
act with an unknown molecule(s) that deliver(s) a stimulatory survival pathways between T1 and T2 cells? A partial answer to this
signal. Transitional cells that have received this survival signal up-reg- question appears to lie in differences in the composition of
ulate their BAFF receptors (positive selection). Those T2 B cells that fail the lipid membranes of the two types of cells. Immature T1
to receive the stimulatory signal (or that fail to receive a BAFF survival B cells contain approximately half as much cholesterol as
signal) die in the spleen. Selecting antigens are shown as violet shapes; their more mature counterparts, and this reduction in cho-
T1 and T2 cells are green. White cells represent dead cells that were lesterol levels appears to prevent efficient clustering of the
either negatively selected or failed positive selection. [Adapted from B-cell receptor into lipid rafts upon BCR stimulation. This
T. T. Su et al., 2004, Signaling in transitional type 2 B cells is critical for peripheral may cause a reduction of the strength of BCR signaling in T1
B-cell development, Immunological Reviews 197:161–178, Figure 3.] versus T2 immature B cells.
The development of B cells through the transitional phase is
resistance to receptor-induced cell death results in part from absolutely dependent on signaling through the BAFF receptor
the fact that T2 B cells have increased their expression of the (BAFF-R). BAFF-R expression is first detected in T1 B cells
anti-apoptotic molecule Bcl-xl. and increases steadily thereafter. BAFF is then required consti-
Using conditional knockout genetic techniques (see tutively throughout the life of mature B cells. Signaling through
Chapter 20), animals can be generated that lack Ig receptor the BAFF/BAFF-R axis promotes survival of transitional B cells
by inducing the synthesis of anti-apoptotic factors such as Bcl-
2, Bcl-xl, and Mcl-1, as well as by interfering with the function
Responses to strong BCR of the pro-apoptotic molecule Bim (see Figure 9-12).
TABLE 10-3 signaling in T1, T2, and The discovery of a B-cell survival signal mediated by
mature B-2 B cells BAFF/BAFF-R interactions, and distinct from survival signals
emanating from the BCR, extends our thinking about how B
Mature B-2 cells are selected for survival in the periphery. In the presence
Nature of Response T1 T2 B cells
of high levels of BAFF, B cells that may not otherwise receive
Formation of lipid rafts ⫹/⫺ ⫹ ⫹ sufficient quantities of survival signals via the BCR may sur-
Increase in cytoplasmic ⫹ ⫹ ⫹
vive a selection process that would otherwise eliminate them.
Ca2⫹ ion concentrations
In this way, BAFF can provide plasticity and flexibility in the
process of B-cell deletion. However, this may be accomplished
Increase in diacylglycerol ⫺ ⫹ ⫹
at the cost of maintaining potentially autoreactive cells.
concentrations
Induction of Bcl-xl ⫺ ⫹ ⫹ T3 B Cells Are Primarily Self-Reactive and Anergic
Induction of apoptosis ⫹ ⫺ ⫺ Transitional T3 B cells were first characterized in the blood
and lymphoid organs by flow cytometry and were described
as being CD93⫹mIgMlowCD23⫹. The function of CD93 is so antigen, as the B cell half-lives were restored to normal
far unknown; CD23 is a low-affinity receptor for some lengths upon adoptive transfer of the transgene-bearing B cells
classes of immunoglobulin, and the two markers were used to an animal that was not expressing HEL. The anergic response
in these experiments only to identify the cell populations, appears to be generated in vivo when immature B cells meet
and not because of any particular reasons relevant to their a soluble self antigen.
functionality. Recent experiments have suggested that the T3 More recent experiments have focused on defining the dif-
population may represent B cells that have been rendered ferences between the signal transduction events leading to
anergic by contact with soluble self antigen but have not yet anergy versus activation. Anergic B cells show much less anti-
been eliminated from the B-cell repertoire. gen-induced tyrosine phosphorylation of signaling molecules,
A transgenic system developed by Goodnow and col- when compared with their nonanergic counterparts and anti-
leagues first placed the concept of B-cell anergy, or unrespon- gen-stimulated calcium release from storage vesicles into the
siveness, onto a firm experimental footing. Anergic cytoplasm of the anergic B cells was also dramatically reduced.
lymphocytes clearly recognize their antigens, as shown by the Anergic B cells also require higher levels of the cytokine BAFF
identification of low levels of molecular signals generated for continued survival, and it is likely that their reduced half-
within the cells after binding to antigen. However, rather than lives result from unsuccessful competition with normal B cells
being activated by antigen contact, anergic B cells fail to for limiting amounts of this survival molecule. One of the out-
divide, differentiate, or secrete antibody after stimulation, comes of BAFF signaling is a reduction in the cytoplasmic
and many die a short time after receipt of the antigenic signal. levels of the pro-apoptotic molecule BIM; as might be expected,
Goodnow et al. developed the two groups of transgenic anergic B cells show higher-than-normal levels of BIM and a
mice illustrated in Figure 10-11a. One group of mice carried correspondingly increased susceptibility to apoptosis.
a hen egg-white lysozyme (HEL) transgene linked to a The conclusion from these experiments is that mecha-
metallothionine promoter, which placed transcription of the nisms exist, even after the B cells have exited the bone mar-
HEL gene under the control of zinc levels in the animals’ diet. row and entered the periphery, that minimize the risk that B
This allowed the investigators to alter the levels of soluble cells make antibodies to soluble self proteins expressed out-
HEL expressed in the experimental animals by changing the side the bone marrow. B cells reactive to such proteins
concentration of zinc in their food. Under these experimental respond to receptor stimulation in the absence of appropri-
conditions, HEL was expressed in the periphery of the ani- ate T-cell help by anergy and eventual apoptosis.
mal, but not in the bone marrow. The other group of trans- What might be the function of these anergic cells? One pos-
genic mice carried rearranged immunoglobulin heavy-chain sibility is that they serve to absorb excess self antigens that
and light-chain transgenes encoding anti-HEL antibody; in might otherwise be able to deliver activating signals to high-
these transgenic mice, the rearranged anti-HEL transgene is affinity B cells and thus lead to autoimmune reactions. Another
expressed by 60% to 90% of the mature peripheral B cells. is that they represent cells destined for apoptosis that do not yet
Goodnow then mated the two groups of transgenics to pro- display the characteristic microanatomy of apoptotic cells. Yet
duce “double-transgenic” offspring carrying both the HEL another is that these cells will eventually develop into B regula-
and anti-HEL transgenes (Figure 10-11b) and asked what tory cells (see Chapter 12). As is so often the case in the
effect peripheral HEL expression would have on antibody immune system, it is more than possible that all of these func-
expression by B cells bearing the anti-HEL antibodies. They tions are subsumed within this intriguing cell population,
found that the double-transgenic mice continued to generate which remains the subject of intensive current investigation.
mature, peripheral B cells bearing anti-HEL membrane
immunoglobulin of both the IgM and IgD classes, indicating Mature, Primary B-2 B Cells Migrate to the
that the B cells had fully matured. However, these B cells were
Lymphoid Follicles
functionally nonresponsive, or anergic.
Flow-cytometric analysis of B cells from the double- Fully mature B cells express high levels of IgD and interme-
transgenic mice showed that, although large numbers of diate levels of IgM on their cell surfaces (see Table 10-2).
anergic anti-HEL cells were present, they expressed membrane Mature B cells recirculate between the blood and the lym-
IgM at levels about 20-fold lower than anti-HEL single trans- phoid organs, entering the B-cell follicles in the lymph nodes
genics (Figure 10-11b). When these mice were given an immu- and spleen, and responding to antigen encounter in the pres-
nizing dose of HEL, few anti-HEL plasma cells were induced ence of T-cell help with antibody production (Chapter 12).
and the serum anti-HEL titer was very low (Table 10-4). Fur- Approximately 10 million to 20 million B cells are produced
thermore, when antigen was presented to these anergic B cells in the bone marrow of the mouse each day, but only about
in the presence of T-cell help, many of the anergic B cells 10% of this number ever take up residence in the periphery
responded by undergoing apoptosis. Additional analysis of and only 1% to 3% will ever enter the recirculating follicular
the anergic B cells demonstrated that they had a shorter half- B-2 B-cell pool. Some of these cells are lost to the process of
life than normal B cells and appeared to be excluded from clonal deletion, but others are perfectly harmless B cells that
the B-cell follicles in the lymph nodes and spleen. These nonetheless fail to thrive. Experimental depletion of the
properties were dependent on the continuing presence of mature B-cell population, either chemically or by irradiation,
(a)
Transgenic Transgenic
(HEL) (anti–HEL)
Double transgenic
(carrying both HEL and anti–HEL transgenes)
(b)
Nontransgenic Anti–HEL transgenic Anti–HEL/HEL
double transgenic
HEL-binding B cells
100
10
1 10 100 1 10 100 1 10 100

IgM expression on membrane (arbitrary fluorescence units)
FIGURE 10-11 Goodnow’s experimental system for dem- fluorescence emitted from this label indicated the level of mem-
onstrating clonal anergy in mature peripheral B cells. (a) brane IgM expressed by the B cells. The nontransgenics (left) had
Production of double-transgenic mice carrying transgenes encod- many B cells that expressed high levels of surface IgM but almost no
ing HEL (hen egg-white lysozyme) and anti-HEL antibody. (b) Flow- B cells that bound HEL above the background level of 1. Both anti-
cytometric analysis of peripheral B cells that bind HEL compared HEL transgenics (middle) and anti-HEL/HEL double transgenics
with membrane IgM levels. The number of B cells binding HEL was (right) had large numbers of B cells that bound HEL (blue), although
measured by determining how many cells bound fluorescently the level of membrane IgM was about 20-fold lower in the double
labeled HEL. Levels of membrane IgM were determined by incubat- transgenics. The data in Table 10-4 indicate that the B cells express-
ing the cells with anti-mouse IgM antibody labeled with a fluores- ing anti-HEL in the double transgenics cannot mount a humoral
cent label different from that used to label HEL. Measurement of the response to HEL.
followed by in vivo reconstitution, results in rapid replenish- Experiments using conditional RAG2 knockout ani-
ment of the B-cell follicular pool. This suggests that the fol- mals, in which all new B-cell development was prevented
licular B-cell niches have a designated capacity and that once in otherwise healthy adult animals, indicate that follicular
full they turn away additional B cells. Most probably, the B-2 B cells have a half-life of approximately 4.5 months. In
mechanism for this homeostatic control of B-cell numbers contrast, since B-1 B cells can self-renew in the periphery,
relies on competition for survival factors, particularly BAFF their numbers are unaffected in this experimental knock-
and its related proteins. out animal.
Expression of anti-HEL transgene by mature peripheral B cells in single- and

TABLE 10-4 double-transgenic mice
Experimental Group HEL level Membrane anti-HEL Ig Anti-HEL PFC/spleen* Anti-HEL serum titer
Anti-HEL single transgenics None ⫹ High High

⫺9
Anti-HEL/HEL double transgenics 10 M ⫹ Low Low
* Experimental animals were immunized with hen egg-white lysozyme (HEL). Several days later hemolytic plaque assays for the number of plasma cells secreting anti-
HEL antibody were performed and the serum anti-HEL titers were determined. PFC ⫽ plaque-forming cells.
Adapted from Goodnow, C. C., 1992, Annual Review of Immunology 10:489.
The Development of B-1 and entering the spleen and, like B-1 B cells, mainly (although
not exclusively) produce broadly cross-reactive antibodies of
Marginal-Zone B Cells the IgM class. Both B-1 and marginal-zone B cells can gener-
ate antibodies in the absence of T-cell help, although the
This chapter has so far focused on the development of those addition of helper T cells enhances antibody secretion and
B cells that belong to the best characterized B-cell subpopu- allows for some degree of heavy-chain class switching.
lation, B-2 B cells (or follicular B cells). Mature B-2 B cells
recirculate between the blood and the lymphoid organs, and
can be found in large numbers in the B-cell follicles of the B-1 B Cells Are Derived from a Separate
lymph nodes and spleen. However, other subsets of B cells
Developmental Lineage
have been recognized that perform distinct functions,
occupy distinct anatomical locations, and pursue different B-1 B cells are phenotypically and functionally distinct from
developmental programs. This section of the chapter will B-2 B cells in a number of important ways. They occupy dif-
therefore address the development and function of B-1 B ferent anatomical niches from B-2 B cells, constituting 30% to
cells and of marginal-zone B cells (see Figure 10-12 for a 50% of the B cells in the pleural and peritoneal cavities of
comparison of the properties of these three cell types). mice, and representing about 1 million cells in each space. A
As described more fully in Chapter 12, B-1 B cells gener- similar number of B-1 B cells can also be found in the spleen,
ate antibodies against antigens shared by many bacterial but there they represent a much smaller fraction (around 2%)
species and may do so even in the absence of antigenic of the splenic B-cell population. B-1 B cells have only a rela-
stimulation. They are the source of the so-called natural tively limited receptor repertoire, and their receptors tend to be
antibodies: serum IgM antibodies that provide a first line of directed toward the recognition of commonly expressed
protection against invasion by many types of microorgan- microbial carbohydrate antigens. These broadly cross-reactive,
isms. Marginal-zone B cells take their name from their loca- low-affinity antigen receptors expressing minimal repertoire
tion in the outer zones of the white pulp of the spleen. They diversity are reminiscent of the pathogen-associated molecu-
are the first B cells encountered by blood-borne antigens lar pattern (PAMP) receptors of the innate immune system
Follicular (B-2) B cells B-1 B cells Marginal zone B cells
CD19/ CD19/
CD21 IgM IgM CD21 IgM
IgD
CD5 CD1
CD23
(B-1a cells only)
Attribute Follicular (B-2) B cells B-1 B cells Marginal zone B cells
Major sites Secondary lymphoid organs Peritoneal and pleural cavities Marginal zones of spleen
Source of new B cells From precursors in bone Self-renewing (division of existing Long-lived
marrow B-1 cells) May be self-renewing
V-region diversity Highly diverse Restricted diversity Somewhat restricted

Somatic hypermutation Yes No Unclear
Requirements for T-cell help Yes No Variable
Isotypes produced High levels of IgG High levels of IgM Primarily IgM; some IgG
Response to carbohydrate Possibly Yes Yes

antigens
Response to protein antigens Yes Possibly Yes
Memory Yes Very little or none Unknown

Surface IgD on mature B cells Present on naïve B cells Little or none Little or none
FIGURE 10-12 The three major populations of mature B cells in the periphery. The cell-surface properties and functions of B-2,
B-1, and marginal-zone (MZ) cells are shown. Conventional B-2 cells were so named because they develop after B-1 B cells.
(Chapter 5), and B-1 B cells are thus considered to play a role the notion that B-1 B cells are derived from a different pro-
that bridges those of the innate and adaptive immune systems. genitor cell lineage from B-2 B cells.
In contrast with the T-1 transitional B-2 B cells, which
undergo apoptosis upon antigen challenge, transitional B-1
cells undergo apoptosis unless they interact with self antigens. Marginal-Zone Cells Share Phenotypic and
Work in a number of different transgenic systems has suggested Functional Characteristics with B-1 B Cells and
that relatively strong BCR engagement by self antigens pro- Arise at the T2 Stage
vides a positive selection rather than a negative selection survival
The term marginal zone refers to the fact that marginal zone
signal for B-1 B cells. In contrast with B-2 cells and marginal-
(MZ) B cells are located in the outer regions of the white pulp
zone cells (see below), B-1 B cells do not require interaction
of the spleen (Figure 10-13). The spreading out of the fast-
with BAFF during the transitional stage of development.
moving arterial flow into the marginal sinuses results in a
For many years, the preeminent issue debated by those
decrease in the rate of blood flow and allows blood-borne
interested in the development of B-1 B cells was whether they
antigens to interact with cells resident within the marginal
constituted a separate developmental lineage, or whether they
zone. Indeed, MZ B cells appear to be specialized for recog-
derived from the same progenitors as B-2 B cells. This contro-
nizing blood-borne antigens. They are capable of responding
versy has since been resolved in favor of the assertion that B-1
to both protein and carbohydrate antigens, and evidence
and B-2 B cells derive from distinct lineages of progenitor
suggests that some, but maybe not all, MZ B cells can do so
cells. Several lines of evidence support this conjecture:
without the need for help from T cells.
• B-1 B cells appear before B-2 B cells during ontogenic MZ cells are characterized by relatively high levels of
development. B cells generated from the AGM region and membrane IgM, and the complement receptor/B-cell co-
the liver in the fetus have a cell-surface phenotype charac- receptor CD21 (see Chapters 3 and 6), but low levels of
teristic of B-1 B cells and secrete natural IgM antibodies membrane IgD and the Fc receptor CD23. They also display
without the need for deliberate immunization. B-1 B cells phospholipid receptors and adhesion molecules that enable
may be generated early in development in order to protect them to make adhesive interactions with other cells within
the fetus from commonly encountered bacterial pathogens. the marginal zone that hold them in place. MZ cells are long
• B-1 B cells display much more limited V region diversity lived and may self-renew in the periphery.
than B-2 B cells. Generated at a point in ontogeny before What drives an immature B cell down the MZ pathway?
TdT can be efficiently activated, many B-1 cells lack any Phenotypic characterization of bone marrow and peripheral
evidence of N region addition. immature B-cell populations, combined with labeling studies
that define precursor/daughter-cell relationships have sug-
• B-1 B cells populate different anatomical niches in the
gested that MZ and follicular B-2 B cells both derive from the
mouse than the later-arising B-2 B cells, in much the
T2 transitional population (see Figure 10-9). MZ cells, like
same manner that has been described for the fetally
B-2 B cells, are also reliant on the B-cell survival factor BAFF,
derived ␥␦ T cells (Chapter 9).
which binds to the MZ B cells through the receptor BR3. Like
• B-cell progenitors of the CD19⫹CD45Rlow/⫺ phenotype developing B-1 B cells, developing MZ cells appear to require
transferred into an immunodeficient mouse were able to relatively strong signaling through the BCR in order to sur-
repopulate the B-1, but not the B-2 B-cell compartments. vive. Surprisingly, unlike any other B-cell subset so far
Conversely, CD19⫹CD45Rhi B-cell progenitors gave rise to described, the differentiation of MZ B cells also requires sig-
B-2, but not to B-1, daughter cells in an immunodeficient naling through ligands of the Notch pathway (see Chapter 9).
recipient mouse, supporting the notion that the two sub- Loss of the Notch 2 receptor or deletion of the Notch 2 ligand,
classes derive from different lineages of progenitor cells. Delta-like 1 (Dl-1), results in the selective deletion of MZ B
• Whereas B-2 B cells must be constantly replenished by cells. Both MZ and B-1 B cell populations are enriched in cells
the emergence of newly generated cells from the bone that express self antigen-specific receptors, and so relatively
marrow, B-1 B cells are constantly regenerated in the strong signaling of T2 cells by binding of self antigens through
periphery of the animal. Bone marrow ablation therefore the BCR is also necessary for MZ B-cell differentiation.
leaves the mouse with a depleted B-2 pool, but with a
fully functional B-1 population.
There are very few absolutes in biology, and so it should Comparison of B- and T-Cell
be clearly stated that it is probable that not all B-1 B cells in Development
an adult animal are derived from fetal liver precursors and
that some replenishment of B-1 precursors occurs in the In closing this chapter, it is instructive to consider the many
adult bone marrow. Not all B-1 B cells are restricted to IgM points of comparison between the development of the two
production, and the antibody production of some B-1 B cells arms of the adaptive immune system, B cells and T cells
does benefit from the provision of T-cell help. But notwith- (Table 10-5). Both cell lineages have their beginnings in the
standing these variations on the general themes we have fetus and the neonate. In the neonate, ␥␦ T cells and B-1 B
elaborated, it remains the case that evidence clearly supports cells are dispatched to their own particular peripheral niches,
Central arteriole
T cell zone
Follicle
B cell zone
Marginal sinus
Marginal zone
FIGURE 10-13 The relative locations of the marginal zone and the follicles in the spleen. This figure shows a cross-section of the
spleen, displaying the anatomical relationships between the central arteriole, the T-cell and B-cell zones, and the marginal zone.
TABLE 10-5 Comparison between T-cell and B-cell development

Structure or process B cells T cells
⫹ ⫹
Development begins in the bone marrow.
Development continues in the thymus. ⫺ ⫹
Ig heavy chain or TCR␤ chain many gene rearrangement begins ⫹ ⫹
with D-J and continues with V-DJ recombination.
The H chain (BCR) or ␤ chain (TCR) is expressed with a ⫹ ⫹
surrogate form of the light chain on the cell surface. Signaling
from this pre-BCR or pre-TCR is necessary for development
to continue.
Signaling through the pre-B- or pre-TCRs results in proliferation. ⫹ ⫹
The ␬/␭ (BCR) or ␣ (TCR) chain bears only V and J segments. ⫹ ⫹
Signaling from the completed receptor is necessary for survival ⫹ ⫹
(positive selection).
Positive selection requires recognition of self components. ⫹/⫺ True for the minority B-1 ⫹ Low-affinity binding of self
B-cell subset. Ligand for B-2 MHC and self peptide in the
and MZ subsets unclear. thymus is necessary for posi-
tive selection.
Receptor editing of ␬/␭ (BCR) or ␣ (TCR) chain modifies autore- ⫹ Has been shown to occur, but
active specificities. rarely used as a mechanism
of escaping autoreactivity.
Immature cells bearing high affinity autoreactive receptors are ⫹ ⫹
eliminated by apoptosis (negative selection).
Negative selection involves ectopic expression of auto-antigens ⫺ ⫹ Auto-antigens are
in the primary lymphoid organs. expressed in the thymus
under the influence of the
AIRE transcription factor.
Negative selection involves recognition of MHC-presented ⫺ ⫹
peptides.
Heavy or TCR␤ chain allelic exclusion. ⫹ ⫹
Light (␬/␭) or TCR␣ chain allelic exclusion. ⫹ Many T cells display more
than one TCR␣ chain.
90% of lymphocytes are lost prior to export to the periphery. ⫹ ⫹
Development is completed in the periphery to allow for toler-
ance to antigens not expressed in the primary lymphoid organs ⫹ ⫹
Note: The comparisons in this table most accurately refer to the predominant ␣␤ T and follicular B-2 cell subsets, although many of the statements made are equally
valid for the minority lymphocyte subsets.
353
there to function as self-renewing populations until the death B cells and T cells must both pass through stages of posi-
of the host. In the adult animal, B-cell and T-cell develop- tive selection, in which those cells capable of receiving sur-
ment continue in the bone marrow, starting with hematopoi- vival signals are retained at the expense of those which
etic stem cells. B and T cells share the early phases of their cannot. They must also survive the process of negative selec-
developmental programs, as they pass through progressively tion, in which lymphocytes with high affinity for self anti-
more differentiated stages as MPPs, LMPPs, and ELPs. At the gens are deleted. The process of positive selection in B
ELP and CLP stages, T-cell progenitors leave the bone mar- cells—its mechanism, and the BCR ligands involved—
row, and migrate to the thymus to complete their develop- remains one of the least well characterized processes in
ment, leaving B-cell progenitors behind. In mammals, B cells B-cell development. Unlike T cells, however, B cells do not
do not have an organ analogous to the thymus in which to undergo selection with respect to their ability to bind to self
develop into mature, functioning cells, although birds do MHC antigens.
possess such an organ—the bursa of Fabricius. (Many stu- With the expression of high levels of IgD on the cell sur-
dents are unaware that it is from this organ, and not from the face and the necessary adhesion molecules to direct their
bone marrow, that the “B” in B cells originates.) recirculation, development of the mature, follicular B-2 cell
With the initiation of V(D)J recombination, the B cell is complete and, for a few weeks to months, it will recircu-
irreversibly commits to its lineage, and begins the process of late, ready for antigen contact in the context of T-cell help
receptor rearrangement, followed by B-cell selection and dif- and subsequent differentiation to antibody production. For
ferentiation that will culminate in the formation of a com- the final, antigen-stimulated stages of B-cell differentiation,
plete repertoire of functioning peripheral B cells. the reader is directed to Chapter 12.
S U M M A R Y
■ Hematopoiesis in the embryo generates rapidly dividing ranged heavy chain in combination with many different
hematopoietic stem cells that populate the blood system of light chains.
the animal, providing red blood cells that supply the oxy- ■ After light-chain rearrangement, and the expression of the
gen needs of the fetus, and generating the early precursors completed immunoglobulin receptor on the cell surface,
of other blood-cell lineages. Early sites of blood-cell devel- immature B cells specific for self antigens present in the
opment include the yolk sac and the placenta, as well as bone marrow are deleted by apoptosis.
the aorta-gonad-metanephros (AGM) region and the fetal
liver.
■ Immature B cells emerge from the bone marrow as transi-
tional 1 (T1) B cells and circulate to the spleen. Interaction
■ The earliest B-cell progenitors in the fetal liver supply B-1 with self antigens in the spleen can give rise to apoptosis.
B cells that migrate into the pleural and peritoneal cavi-
ties and remain self-renewing throughout the life of the
■ T1 B-2 B cells then enter the follicles, where the level of
animal. IgD expression increases, they become T2 cells, and then
mature into either a follicular B-2 cell (a conventional B
■ In adult animals, hematopoiesis occurs in the bone marrow. cell) or a marginal-zone (MZ) B cell.
■ Progenitor stages of the conventional B-2 subset are ■ The three major subsets of B cells differ according to their
defined by the presence of particular cell-surface markers,
site of generation, their sites of maturation, their anatomi-
which include chemokine and lymphokine receptors and
cal niches in the adult, the antigens to the which they
proteins involved in adhesive interactions.
respond, their need for T-cell help in antibody production,
■ Progenitor stages are also defined by the status of immu- the diversity of their immunoglobulin repertoires, and
noglobulin V region gene rearrangements. The heavy- their abilities to undergo somatic hypermutation and
chain V genes rearrange first, with D to JH recombination memory generation following antigenic stimulation.
occurring initially, followed by VH to DJH recombination. ■ Like T cells, developing B cells must undergo both positive
■ The heavy chain is then expressed on the cell surface in and negative selection. Unlike T cells, B cells need not be
combination with the surrogate light chain, which is made selected for their ability to recognize antigens in the con-
up of VpreB and ␭5. Together they form the pre-B-cell text of MHC antigens, nor is there a primary immune
receptor, which is expressed on the cell surface along with organ aside from the bone marrow specialized for their
the Ig␣,Ig␤ signaling complex. maturation. There is no equivalent of the AIRE protein
■ Signaling through the B-cell receptor stops VH gene rear- that has yet been discovered to provide for ectopic expres-
rangement and calls for a few rounds of cell division. This sion of antigens in the bone marrow in order to facilitate
allows multiple B cells to use the same, successfully rear- clonal deletion of self antigen-specific B cells.
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S T U D Y Q U E S T I O N S
1. You wish to study the development of B-1 B cells in the (b)

absence of the other two major B-cell subsets. You have a
recipient Rag1⫺/⫺ mouse that you have already repopu-
lated with T cells. What would you choose to be your
source of B-1 progenitors and why? Which anatomical sites
would you expect to harvest the B-1 B cells from?
2. Describe the phenotypic and functional differences
between T1 and T2 immature B cells.
3. Following expression of the pre-B-cell receptor on the pro-
genitor B-cell surface, the B cell undergoes a few rounds of
cell division. What purpose does this round of division
serve in the development of the B-cell repertoire?
4. Immature B cells bearing potentially autoimmune recep-
tors can be managed in three ways to minimize the prob-
ability of disease. Describe these three strategies, noting
whether they are shared by T-cell progenitors.
5. You suspect that a new transcription factor is expressed at
the pre-pro-B-cell stage of development. How would you
test your hypothesis? What is the status of heavy-and light-
chain rearrangement at this stage of development and how
would you test it?
6. How would you determine whether a particular stage of
B-cell development occurs in association with a stromal ANALYZE THE DATA The following figure is derived from the
cell that expresses CXCL12? same paper as those above. In this case the data are expressed
as histograms, in which the y axis represents the number of
7. Describe the order in which B-cell receptor genes undergo
cells binding the molecule shown on the x axis, Annexin V.
rearrangement, indicating at what steps you might expect
Annexin V binds to phosphatidyl serine on the outer leaflet
to see the B cell express one or both chains on the cell sur-
of cell membranes. Phosphatidyl serine is found on the outer
face. In what sense(s) does this gene rearrangement pro-
leaflet only in cells about to undergo apoptosis. The top two
cess mimic the analogous progression in ␣␤ T cells, and in
panels represent cells from a wild-type animal, and the bottom
what ways do the two processes differ?
two panels represent cells from animals in which the Dicer
ANALYZE THE DATA The two columns of data in the following gene has been knocked out.
figure below are flow cytometric plots that describe the levels of
the antigens denoted on the x and y axes. The left column rep- Pro-B cells Pre-B cells
resents the antigens present on spleen (part A) and bone mar-
row (parts B) from wild-type (genetically normal) animals. The Wild
plots represent all lymphocytes in the spleen (part A) or B-cell type
progenitor and precursor cells in the bone marrow (part B). mouse
The right column shows the same plots from animals in which
the Dicer gene has been knocked out. As you recall, the Dicer
gene is required for the maturation of controlling miRNAs. Dicer
knockout
a. For each pair of plots, describe the differences in the cell
mouse
populations, indicating whether the differences reflect
losses or gains in particular developing B-cell populations.
b. At what point(s) in B-cell development do you think
miRNAs are functioning?
Wild type Dicer knockout a. Does the presence of Dicer have an effect on the frac-
mouse mouse tion of pro-B cells undergoing apoptosis? Explain your
(a) reasoning.
b. Does the presence of Dicer have an effect on the frac-
tion of pre-B cells undergoing apoptosis? Again explain
your reasoning.
c. Describe one function that you now think miRNAs
fulfill in B-cell development.
c11T-CellActivation,Differentiation,andMemory.indd Page 357 12/20/12 5:03 PM user-t044 /Volumes/203/MHR00209/siL52070/disk1of1/0071052070
T-Cell Activation,
11
Differentiation,
and Memory
T
he interaction between a naïve T cell and an
antigen-presenting cell (APC) is the initiating
event of the adaptive immune response. Prior to
this, the innate immune system has been alerted
at the site of infection or tissue damage, and APCs,
typically dendritic cells, have been activated via their
pattern recognition receptors. These cells may have Dendritic cell (orange) interacting with T cells
engulfed extracellular (or opsonized intracellular) (green). [M. Rohde, HZI, Braunschweig, Germany.]
pathogens, or they may have been infected by an
intracellular pathogen. In either case, they have ■ T-Cell Activation and the Two Signal Hypothesis
processed and presented peptides from these pathogens
in complex with surface MHC class I and class II ■ T-Cell Differentiation
molecules, and have made their way to a local (draining) ■ T-Cell Memory
lymph node and/or the spleen. The APCs have taken up
residence in the T-cell zones of the lymph node or
spleen to join networks of other cells that are continually This chapter focuses on this event, which is critical for the
scanned by roving naïve CD8 and CD4 T cells, which development of both humoral and cell-mediated
recognize MHC class I-peptide and MHC class II- immunity, as well as the development of B-cell and CD8
peptide complexes, respectively. T-cell memory. As discussed below, CD4 T cells can
We have seen that each mature T cell expresses a differentiate into a surprising number of distinct helper
unique antigen receptor that has been assembled via subsets, each of which has a different function in
random gene rearrangement during T-cell development combating infection.
in the thymus (Chapter 9). Because developing T cells In this chapter, we briefly review the cellular and
undergo selection events within the thymus, each mature, molecular events that activate T cells and then deepen
naïve T cell is tolerant to self antigens, and restricted to your understanding of the costimulatory interactions that
self-MHC (Chapter 9). Some naïve T cells have play an important role in determining the outcome of
committed to the CD8 cytotoxic T-cell lineage, some to T cell-APC interactions. We then discuss the outcomes of
the CD4 helper T-cell lineage. When a naïve CD8 or naïve T-cell activation—the development of effector and
CD4 T cell binds tightly to an MHC-peptide complex memory T cells—focusing primarily on the different fates
expressed by an activated dendritic cell, it becomes and functions of the CD4 helper T-cell subsets that drive
activated by signals generated through the TCR (see the adaptive response.
Chapter 3). These signals, in concert with signals from Which helper subset a naïve CD4 T cell becomes
other factors that we will describe below, stimulate the T depends on the types of signals (e.g., cytokines,
cell to proliferate and differentiate into an effector cell. costimulatory signals) they receive from the dendritic
As you know, naïve CD8 T cells become cytotoxic cells they engage via their TCRs. And as described in
cells in response to engagement of MHC class I-peptide Chapter 5, the signals dendritic cells are able to deliver
combinations. Although we refer to the activation of depend in large part on the pathogen to which they have
CD8 T cells in this chapter, we will discuss their effector been exposed (see Figure 5-18). Investigators are still
functions in detail in Chapter 13. Naïve CD4 T cells working to understand all the variables involved in
become helper cells in response to engagement of MHC determining the lineage choices of T helper cells, but we
class II-peptide combinations (Overview Figure 11-1). introduce you to the current thinking.
357
358 PA R T V | Adaptive Immunity: Effector Responses
T-Cell Activation and Differentiation

Antigen Clonal Effector
Activation Differentiation
recognition expansion functions
Effector Activation of
CD4+ macrophages,
T cell B cells, other
Naïve
cells
CD4+
T cell
Memory
IL-2R CD4+
T cell
IL-2
APC Cytokines
Other cellular
sources of Effector
cyotkines CD8+ T cell
(CTL) Killing of
infected
Naïve “target cells”;
CD8+ IL-2R macrophage
T cell Memory activation
IL-2
CD8+
T cell
Lymphoid organs Peripheral tissues
Activation of a naïve T cell in a secondary lymphoid organ results in activating APC, as well as other supportive cells in the lymphoid
the generation of effector and memory T cells. Activation requires organ. Effector CD4 T cells become helper T cells (TH) and secrete
several receptor-ligand interactions between the T cell and a den- cytokines that enhance the activity of many other immune cells.
dritic cell, as well as signals through cytokines produced by the Effector CD8 T cells are cytotoxic cells (TC) that kill infected cells.
We also describe the known functions of the The Advances box describes a more recent effort to
specialized helper cells, focusing on TH1, TH2, TH17, TFH, figure out precisely how many T-cell receptors must be
and TREG cells. Finally, we close the chapter with a engaged to initiate T-cell activation. The answer was
discussion of T-cell memory, which is dependent on initially surprising, yet in hindsight may not be surprising
CD4 T cell help, and describe both what is known and at all. The final Clinical Focus box discusses how a
what is currently under investigation. disease, an “experiment of nature,” has helped us to better
A Classic Experiment box and Clinical Focus box are understand the basic biology and physiological function
offered as a pair and describe the basic research behind of the effector cells introduced in this chapter.
the discovery of the costimulatory molecule CD28, an
essential participant in naïve T-cell activation, and then
the development of a molecular therapy for autoimmune T-Cell Activation and
diseases that takes advantage of what we know about the
biology of costimulation. These boxes, together, illustrate
the Two-Signal Hypothesis
the powerful connections between basic research and CD4 and CD8 T cells leave the thymus and enter the cir-
clinical development, which underlie translational culation as resting cells in the G0 stage of the cell cycle. These
research, an effort to bring bench scientific discovery to naïve T cells are mature, but they have not yet encountered
the “bedside” that has captured the imagination of many antigen. Their chromatin is condensed, they have very little
biomedical investigators. cytoplasm, and they exhibit little transcriptional activity.
T-Cell Activation, Differentiation, and Memory | CHAPTER 11 359
However, they are mobile cells and recirculate continually a costimulatory ligand, which can only be expressed by a func-
among the blood, lymph, and secondary lymphoid tissues, tional APC. When a T cell receives both Signal 1 and Signal 2,
including lymph nodes, browsing for antigen. It is estimated it will be activated to produce cytokines that enhance entry
that each naïve T cell recirculates from blood through lymph into cell cycle and proliferation (Figure 11-3).
nodes and back again every 12 to 24 hours. Because only It is now known that Signal 2 results from an interaction
about 1 in 105 naïve T cells is likely to be specific for any between specific costimulatory receptors on T cells and
given antigen, this large-scale recirculation increases the costimulatory ligands on dendritic cells (Table 11-1). Recall
chances that a T cell will encounter appropriate antigen. from Chapter 5 that dendritic cells and other APCs become
If a naïve T cell does not bind any of the MHC-peptide activated by antigen binding to PRRs, to express costimula-
complexes encountered as it browses the surfaces of stromal tory ligands (e.g., CD80 and CD86) and produce cytokines
cells of a lymph node, it exits through the efferent lymphatics, that enhance their ability to activate T cells. CD28 is the
ultimately draining into the thoracic duct and rejoining the most commonly cited example of a costimulatory receptor,
blood (see Chapter 2). However, if a naïve T cell does encoun- but other related molecules that provide costimulatory sig-
ter an APC expressing an MHC-peptide to which it can bind, nals during T-cell activation have since been identified and
it will initiate an activation program that produces a diverse are also described below. Because these molecules enhance
array of cells that orchestrate efforts to clear infection. TCR signaling, they are collectively referred to as “positive”
Recall from Chapter 3 that a successful T cell-APC inter- costimulatory receptors and ligands.
action results in the stable organization of signaling mole- Negative costimulatory receptors, which inhibit TCR
cules into an immune synapse (Figure 11-2). The TCR/ signaling, have also been identified. Although our under-
MHC-peptide complexes and coreceptors are aggregated in standing of their specific functions is incomplete, as a group
the central part of this synapse (central supramolecular acti- these play important roles in (1) maintaining peripheral T-cell
vating complex, or cSMAC). The intrinsic affinity between tolerance and (2) reducing inflammation both after the natu-
the TCR and MHC-peptide surfaces is quite low (Kd ranges ral course of an infection and during responses to chronic
from 104 M to 107 M) and is stabilized by the activity of infection. As you can imagine, the expression and activity of
several molecules which together increase the avidity (the negative and positive costimulatory molecules must be care-
combined affinity of all cell-cell interactions) of the cellular fully regulated temporally and spatially. Naïve T cells, for
interaction. The coreceptors CD4 and CD8, which are found example, do not express negative costimulatory receptors,
in the cSMAC, stabilize the interaction between TCR and allowing them to be activated in secondary lymphoid tissue
MHC by binding MHC class II and MHC class I molecules, during the initiation of an immune response. On the other
respectively. Interactions between adhesion molecules and hand, effector T cells up-regulate negative costimulatory
their ligands (e.g., LFA-1/ICAM-1 and CD2/LFA-3) help to receptors at the end of an immune response, when prolifera-
sustain the signals generated by allowing long-term cell tion is no longer advantageous. However, these generaliza-
interactions. These molecules are organized around the cen- tions belie the complexity of regulation of this highly important
tral aggregate, forming the peripheral or “p” SMAC. costimulatory network, and investigators are still working to
However, even the increased functional avidity offered by understand the details. Below we introduce aspects of the
coreceptors and adhesion molecules is still not sufficient to structure, function, expression, and, when known, regulation
fully activate a T cell. Interactions between costimulatory of several positive and negative costimulators.
receptors on T cells (e.g., CD28) and costimulatory ligands
on dendritic cells (e.g., CD80/86) provide a second, required Positive Costimulatory Receptors: CD28
signal. In addition, as you will see below, a third set of sig- CD28, a 44 kDa glycoprotein expressed as a homodimer, was
nals, provided by local cytokines (Signal 3), directs T-cell the first costimulatory molecule to be discovered (see Classic
differentiation into distinct effector cell types. Experiment Box 11-1). Expressed by all naïve and activated
human and murine CD4 T cells, all murine CD8 T cells,
Costimulatory Signals Are Required for and, interestingly, only 50% of human CD8 T cells, it mark-
edly enhances TCR-induced proliferation and survival by
Optimal T-Cell Activation and Proliferation cooperating with T-cell receptor signals to induce expression
What evidence pointed to a requirement for a second, costim- of the pro-proliferative cytokine IL-2 and the prosurvival
ulatory signal? In 1987, Helen Quill and Ron Schwartz recog- bcl-2 family member, bcl-xL.
nized that, in the absence of functional APCs, isolated high CD28 binds to two distinct ligands of the B7 family of
affinity TCR-MHC interactions actually led to T-cell non- proteins: CD80 (B7-1) and CD86 (B7-2). These are members
responsiveness rather than activation—a phenomenon they of the immunoglobulin superfamily, which have similar
called T-cell anergy. Their studies led to the simple but pow- extracellular domains. Interestingly, their intracellular
erful notion that not one but two signals were required for full regions differ, suggesting that they might not simply act as
T-cell activation: Signal 1 is provided by antigen-specific TCR passive ligands; rather, they may have the ability to generate
engagement (which can be enhanced by coreceptors and signals that influence the APC, a view that has some experi-
adhesion molecules), and Signal 2 is provided by contact with mental support.
(a)
cSMAC pSMAC
TCR/CD3 Adhesion
Coreceptors CD4 or CD8 molecules
Costimulatory receptors LFA-1/ICAM-1
(CD28) LFA-3/CD2
(b)
CD4+ T cell Antigen- presenting cell CD8+ T cell Antigen-presenting cell
Antigen
Antigen
Peptide
pSMAC
CD2 LFA-3
CD2 LFA-3
Peptide
SS
TCR− TCR− Class I MHC

CD3 CD3
Class II
cSMAC
MHC
SS
p56lck
CD8
CD4
CD28 CD80 or CD28 CD80 or

SS
SS
CD86 CD86
pSMAC
LFA-1 ICAM-1 LFA-1 ICAM-1
FIGURE 11-2 Surface interactions responsible for T-cell dendritic cell. A dendritic cell (to the right of each diagram) can
activation. (a) A successful T-cell/dendritic-cell interaction results in engulf an antigen and present peptide associated with MHC class
the organization of signaling molecules into an immune synapse. A II to a CD4 T cell or can load internal peptides into MHC class I and
scanning electron micrograph (left) shows the binding of a T cell (ar- present the combination to a CD8 T cell. Binding of the TCR to MHC-
tificially colored yellow) and dendritic cell (artificially colored blue). A peptide complexes is enhanced by the binding of coreceptors CD4
fluorescent micrograph (right) shows a cross-section of the immune and CD8 to MHC class II and class I, respectively. CD28 interactions
synapse, where the TCR is stained with fluorescein (green) and ad- with CD80/86 provide the required costimulatory signals. Adhesion
hesion molecules (specifically LFA-1) are stained with phycoerythrin molecule interactions, two of which (LFA-1/ICAM-1, LFA-3/CD2) are
(red). Other molecules that can be found in the central part of the depicted, markedly strengthen the connection between the T cell
synapse (central supramolecular activation complex [cSMAC]) and and APC or target cell so that signals can be sustained. [(a) right:
the peripheral part of the synapse (pSMAC) are listed. (b) The interac- Michael L. Dustin, J Clin Invest. 2002; 109(2): 155, Fig.1. doi:10.1172/JCI14842. Left:
tions between a CD4 (left) or CD8 (right) T-cell and its activating Dr. Olivier Schwartz, Institut Pasteur/Photo Researchers.]
Normal stitutively express CD80/86, and macrophages and B cells

APC have the capacity to up-regulate CD80/86 after they are acti-
1 TCR signaling
vated by an encounter with pathogen (see Chapter 5).
Positive Costimulatory Receptors: ICOS

Gene
expression
Since the discovery of CD28, several other structurally
related receptors have been identified. Like CD28, the closely
related inducible costimulator (ICOS) provides positive
costimulation for T-cell activation. However, rather than
2 Costimulatory binding CD80 and CD86, ICOS binds to another member of
interaction
Autocrine the growing B7 family, ICOS-ligand (ICOS-L), which is also
(e.g., IL-2) Paracrine expressed on a subset of activated APCs.
(e.g., IL-12) Differences in patterns of expression of CD28 and ICOS
3 Cytokine signaling
indicate that these positive costimulatory molecules play
FIGURE 11-3 Three signals are required for activation of distinct roles in T-cell activation. Unlike CD28, ICOS is not
a naïve T cell. The TCR/MHC-peptide interaction, along with CD4 expressed on naïve T cells; rather, it is expressed on memory
and CD8 coreceptors and adhesion molecules, provide Signal 1. Co- and effector T cells. Investigations suggest that CD28 plays a
stimulation by a separate set of molecules, including CD28 (or ICOS, key costimulatory role during the initiation of activation and
not shown) provide Signal 2. Together, Signal 1 and Signal 2 initiate a ICOS plays a key role in maintaining the activity of already
signal transduction cascade that results in activation of transcription differentiated effector and memory T cells.
factors and cytokines (Signal 3) that direct T-cell proliferation (IL-2)
and differentiation (polarizing cytokines). Cytokines can act in an au- Negative Costimulatory Receptors: CTLA-4
tocrine manner, by stimulating the same cells that produce them, or
The discovery of CTLA-4 (CD152), the second member of
in a paracrine manner, by stimulating neighboring cells.
the CD28 family to be identified, caused a stir. Although
closely related in structure to CD28 and also capable of bind-
ing both CD80 and CD86, CTLA-4 did not act as a positive
Although most T cells express CD28, most cells in the costimulator. Instead, it antagonized T-cell activating signals
body do not express its ligands. In fact, only professional and is now referred to as a negative costimulatory receptor.
APCs have the capacity to express CD80/86. Mature den- CTLA-4 is not expressed constitutively on resting T cells.
dritic cells, the best activator of naïve T cells, appear to con- Rather, it is induced within 24 hours after activation of a
TABLE 11-1 T-cell costimulatory molecules and their ligands

Costimulatory receptor on T cell Costimulatory ligand Activity
Positive costimulation
CD28 CD80 (B7-1) or CD86 (B7-2) Activation of naïve T cells

Expressed by professional APCs, (and medullary
thymic epithelium)
ICOS ICOS-L Maintenance of activity of differentiated T cells;
Expressed by B cells, some APCs, and T cells a feature of T-/B-cell interactions
Negative costimulation
CTLA-4 CD80 (B7-1) or CD86 (B7-2) Negative regulation of the immune response
Expressed by professional APCs (and medullary (e.g., maintaining peripheral T-cell tolerance;
thymic epithelium) reducing inflammation; contracting T-cell pool
after infection is cleared)
PD-1 PD-L1 or PD-L2 Negative regulation of the immune response,
Expressed by professional APCs, some regulation of TREG differentiation
T and B cells, and tumor cells
BTLA HVEM Negative regulation of the immune response,
Expressed by some APCs, T and B cells regulation of TREG differentiation (?)
CLASSIC EXPERIMENT
Discovery of the First Costimulatory Receptor: CD28

In 1989, Navy immunologist Carl June the mitogen phorbol myristyl acetate remained quiescent, exhibiting no nucle-
took the first step toward filling (and (PMA), a protein kinase C (PKC) activator. otide uptake. Cell groups treated with
revealing) the considerable gaps in our To engage the CD28 molecule, they used stimuli that were known to cause T-cell
understanding of T-cell proliferation and an anti-CD28 monoclonal antibody. They proliferation—anti-CD3 and PMA—
activation by introducing a new actor: included two negative controls: one pop- showed evidence of activation, with CD3
CD28. CD28 had been recently identified ulation that was cultured in growth engagement producing relatively more of
as a dimeric glycoprotein expressed on all medium with no additives and another a response than PMA at the time points
human CD4 T cells and half of human population that was exposed to phyto- examined.
CD8 T cells, and preliminary data sug- haemagglutinin (PHA), which was known The cells treated with anti-CD28 only
gested that it enhanced T-cell activation. to activate T cells only in the presence of were just as quiescent as the negative
June and his colleagues specifically won- APCs. This last control was clever and control samples, indicating that engage-
dered if CD28 might be related to the would be used to demonstrate that the ment of CD28, alone, could not induce
Signal 2 that was known to be provided researchers’ isolated populations were not activation. However, when CD28 was
by APCs (sometimes referred to as “acces- contaminated with APCs, which would engaged at the same time cells were
sory cells” in older literature). express their own sets of ligands and con- exposed to PMA, incorporation of [3H]Uri-
June and his colleagues isolated T cells found interpretation. dine increased markedly. T cells cotreated
from human blood by density gradient June and colleagues measured the with anti-CD28 and anti-CD3 also took up
centrifugation and by depleting a T-cell- proliferation of each of these populations more [3H]Uridine than those treated with
enriched population of cells that did not by measuring incorporation of a radioac- anti-CD3 alone.
express CD28 (negative selection). They tive (tritiated) nucleotide, [3H]Uridine, What new RNA were these cells pro-
then measured the response of these which is incorporated by cells that are ducing? Using Northern blot analysis and
CD28 T cells to TCR stimulation in the synthesizing new RNA (and, hence, are functional assays (bioassays) to character-
presence or absence of CD28 engage- showing signs of activation). Their results ize the cytokines in culture supernatant of
ment (Figure 1a). were striking, particularly when responses the stimulated cells, June and colleagues
To mimic the TCR-MHC interaction, to PMA were examined (Figure 1b). As went on to show that CD28 stimulation
June and colleagues used either mono- expected, T cells grown without stimula- induced anti-CD3 stimulated T cells to
clonal antibodies to the CD3 complex or tion or with incomplete stimulation (PHA) produce higher levels of cytokines
naïve T cell and peaks in expression within 2 to 3 days post- PD-L1 (B7-H1) and PD-L2 (B7-DC), which are also mem-
stimulation. Peak surface levels of CTLA-4 are lower than bers of the CD80/86 family. PD-L2 is expressed predomi-
peak CD28 levels, but because it binds CD80 and CD86 with nantly on APCs; however, PD-L1 is expressed more broadly
markedly higher affinity, CTLA-4 competes very favorably and may help to mediate T-cell tolerance in nonlymphoid
with CD28. Interestingly, CTLA-4 expression levels increase tissues. Recent data suggest that PD-L1/PD-L1/2 interactions
in proportion to the amount of CD28 costimulation, sug- regulate the differentiation of regulatory T cells.
gesting that CTLA-4 acts to “put the brakes on” the pro- BTLA is more broadly expressed: not only has it been
proliferative influence of TCR-CD28 engagement. The found on conventional TH cells, as well as T cells and
importance of this inhibitory function is underscored by the regulatory T cells, but it is also expressed on NK cells, some
phenotype of CTLA-4 knockout mice, whose T cells prolif- macrophages and dendritic cells, and most highly on B cells.
erate without control, leading to lymphadenopathy (greatly Interestingly, BTLA’s primary ligand appears not to be a B7
enlarged lymph nodes), splenomegaly (enlarged spleen), family member, but a TNF receptor family member known
autoimmunity, and death within 3 to 4 weeks after birth. as herpesvirus-entry mediator (HVEM), which is also
expressed on many cell types. Studies on the role of this
Negative Costimulatory Receptors: PD-1 and BTLA interesting costimulatory receptor-ligand pair are ongoing,
PD-1 (CD279) and B and T lymphocyte attenuator (BTLA but there are indications that BTLA-HVEM interactions also
(CD272)) are relatively new additions to our list of negative play a role in down-regulating inflammatory and autoim-
costimulatory receptors. Although more distantly related to mune responses.
CD28 family members than CTLA-4, they also inhibit TCR- As the genome continues to be explored, additional
mediated T-cell activation. program death-1 (PD-1) is costimulatory molecules—both negative and positive in
expressed by both B and T cells and binds to two ligands, influence—are likely to be identified. Understanding their
BOX 11-1
PMA or anti-CD3 10
incorporation (CPMx10-3)
1 8
Gene
expression 7
2
6
Anti-CD28 5
3H-Uridine
FIGURE 1 3
(a) June’s experimental setup used anti-CD3 monoclonal antibodies or a
mitogen, PMA, to provide Signal 1, and anti-CD28 antibodies to provide 2
Signal 2. (b) June measured [3H]Uridine incorporation, an indicator of RNA
1
synthesis, in response to various treatments. Addition of stimulating anti-
CD28 antibody increased RNA synthesis in response to activation by PMA 0
or anti-CD3. [Part (b) adapted from C. Thompson et al., 1989, CD28 activation pathway MED PHA αCD28 PMA PMA αCD3 αCD3
regulates the production of multiple T-cell-derived lymphokines/cytokines. Proceedings + +
of the National Academy of Sciences of the United States of America 86:1333.] αCD28 αCD28
involved in antiviral, anti-tumor, and prolif- the critical Signal 2 during naïve T-cell even more subtly perceptive than we
erative activity, generating an increase in activation, a signal required for optimal once appreciated.
T-cell immune response. up-regulation of IL-2 and the IL-2 recep- Based on a contribution by Harper Hubbeling,
When this paper was published, June tor. Finding this second switch capable of Haverford College, 2011.
did not know the identity of the natural modulating T-cell activation was only the Thompson, C., et al. 1989. CD28 activation
ligand for CD28, or even if the homodi- beginning of a landslide of discoveries of pathway regulates the production of mul-
tiple T-cell-derived lymphokines/cytokines.
mer could be activated in a natural additional costimulatory signals—posi- Proceedings of the National Academy of Sci-
immune context. We now know that tive and negative—involved in T-cell acti- ences of the United States of America
CD28 binds to CD80/86 (B7), providing vation, and the recognition that T cells are 86(4):1333–1337.
regulation and function will continue to occupy the atten- appropriate MHC-peptide combination and CD80/86. How-
tion of the immunological community and has already pro- ever, glutaraldehyde-fixed APCs, which express MHC class
vided the clinical community with new tools for manipulating II-peptide complexes, but cannot be induced to express
the immune response during transplantation and disease CD80/86, render T cells unresponsive (Figure 11-4a). These
(see Clinical Focus Box 11-2). anergic T cells are no longer able to secrete cytokines or
proliferate in response to subsequent stimulation (Figure
Clonal Anergy Results if a Costimulatory 11-4b). T-cell anergy can also be induced in vitro by incubat-
ing T cells with normal, CD80/86-expressing APCs in the
Signal Is Absent presence of the Fab portion of anti-CD28, which blocks the
Experiments with cultured cells show that if a resting T cell’s interaction of CD28 with CD80/86 (Figure 11-4c).
TCR is engaged (Signal 1) in the absence of a suitable In vivo, the requirement for costimulatory ligands to
costimulatory signal (Signal 2), that T cell will become unre- fully activate a T cell decreases the probability that circulat-
sponsive to subsequent stimulation, a state referred to as ing autoreactive T cells will be activated and become dan-
anergy. There is good evidence that both CD4 and CD8 T gerous. For instance, a naïve T cell expressing a T-cell
cells can be anergized, but most studies of anergy have been receptor specific for an MHC class I insulin-peptide com-
conducted with CD4 TH cells. plex would be rendered nonresponsive if it encountered a
Anergy can be demonstrated in vitro with systems -islet cell expressing this MHC class I-peptide complex.
designed to engage the TCR in the absence of costimulatory Why? -islet cells cannot be induced to express costimula-
molecule engagement. For instance, T cells specific for a tory ligands, and the encounter would result in T-cell
MHC-peptide complex can be induced to proliferate in vitro anergy, preventing an immune attack on these insulin-
by incubation with activated APCs that express both the producing cells.
CLINICAL FOCUS BOX 11-2

Costimulatory Blockade
Taking an idea from the research and basic researchers was not always
setting to a therapeutic reality—from smooth.
CTLA4
“bench to bedside”—is a dream for many, In late 2005, the FDA approved the use
but a reality for few. Immunologists recog- of CTLA-4 Ig (abatacept) for rheumatoid
nized the therapeutic promise of costimu- arthritis (RA). As of 2012 it is marketed as
latory receptors as soon as they were Orencia by Bristol Meyers Squibb, which
discovered. Reagents that would block shares patent royalties with Repligen Cor-
these interactions could block the activa- IgG1 poration. Abatacept shows promise in
domain
tion of destructive T cells, which were delaying the joint damage seen with RA,
known to be responsible for many auto- and clinical trials are also underway to
immune disorders and for transplantation evaluate its potential to ameliorate psoria-
rejection. sis, lupus, Type 1 diabetes, and more.
Several investigators specifically rec- FIGURE 1 This true bench-to-bedside story is still
ognized that soluble versions of CD28 and Structure of CTLA-4 Ig The extracellular not finished and continues to be informed
domain of human CTLA-4 is fused to a modified
CTLA-4 could be very valuable. By block- by basic researchers’ growing knowledge
Fc region of human IgG1. [PDB IDs 1IGY and 3OSK.]
ing the interaction between costimula- of the complexities of costimulation.
tory receptors and their CD80/86 ligands, Investigators have already developed a
soluble CD28 and CTLA-4 could inhibit modified version of CTLA-4 Ig that differs
destructive T-cell responses (e.g., those bind to Fc receptors and cause unintended in two amino acids in the CD80/86 bind-
involved in autoimmune disease or trans- cytotoxicity. Using this new protein, Peter ing region and exhibits a higher affinity for
plantation rejection). Couple this idea Linsley et al. found that their soluble CTLA- CD86, which may play a more important
with technological advances in protein 4-Ig bound CD80/86 with higher affinity role in transplantation rejection. This drug
design and you had a novel reagent—and than CD28-Ig, and could therefore more is also showing promise in clinical trials
a potential drug. potently block costimulation. and may result in fewer side effects than
In the early 1990s, at least two groups The development of CTLA-4 Ig as a standard immunosuppressant therapies
converted human CTLA-4 into a soluble drug did not proceed without difficulty. (e.g., cyclosporine), which are not specific
protein by fusing the extracellular domain Originally designed and tested as a treat- for T cells. It is also important to recognize
of human CTLA-4 with the Fc portion of an ment for T-cell-mediated transplantation that CTLA-4 Ig not only blocks positive
IgG1 antibody (Figure 1). The Fc portion rejection, it did not originally perform as costimulatory reactions, but also has the
enhances (1) the ability to manipulate a well as expected. However, hope in its potential to inhibit negative ones and in
protein during production by taking utility was revived when it showed signifi- some circumstances could lead to
advantage of Fc binding as well as (2) the cant promise as a treatment for rheuma- enhancement of T-cell activity. Research-
distribution of the reagent in an organism toid arthritis. The marketing of CTLA-4 Ig ers will continue to draw from basic and
(the Fc portion confers some of the anti- was not without controversy, either. At clinical knowledge to determine how best
bodies’ tissue distribution behaviors). The least two groups claimed patent rights, to use the drug and to improve its design
Fc portion is modified so that it does not and communication between companies for enhanced safety and efficacy.
Interactions between negative costimulatory receptors Although anergy is a well-established phenomenon, the
and ligands can also induce anergy. This phenomenon, precise biochemical pathways that regulate this state of nonre-
which would typically only apply to activated T cells that sponsiveness are still not fully understood. During the past few
have up-regulated negative costimulatory receptors, could years, microarray analyses (see Chapter 20) have identified
help curb T-cell proliferation when antigen is cleared. several key enzymes expressed by anergic T cells, including
Unfortunately, negative costimulation may also contribute to ubiquitin ligases that appear to target key components of the
the T cell “exhaustion” during chronic infection, such as that TCR signaling pathway for degradation by the proteasome.
caused by mycobacteria, HIV, hepatitis virus, and more. T
cells specific for these pathogens express high levels of PD-1
Cytokines Provide Signal 3
and BTLA, and are functionally anergic. Some recent thera-
pies, in fact, are designed to block this interaction and allow We have now seen that naïve T cells will initiate activation
T cells to reactivate. when they are costimulated by engagement with both
(a) As we will see below, Signal 3 is also supplied by other

cytokines (produced by APCs, T cells, NK cells, and others),
1 known as polarizing cytokines, that play central roles not just
Anergic Fixed in enhancing proliferation, but also in determining what
genes APC
(no CD80/86) types of effector cells naïve T cells will become.
Antigen-Presenting Cells Have Characteristic

(b) Costimulatory Properties
Which cells are capable of providing both Signal 1 and Signal
Anergic 2 to a naïve T cell? Although almost all cells in the body
T cell express MHC class I, only professional APCs—dendritic
Normal
APC cells, activated macrophages, and activated B cells—express
the high levels of class II MHC molecules that are required
No response for T-cell activation. Importantly, these same professional
APCs are among the only two cell types capable of express-
ing costimulatory ligands. (The only other cell type known
(c) to have this capacity is the thymic epithelial cell; see Chapter
9.) Professional APCs are more diverse in function and ori-
1 gin than originally imagined, and each subpopulation differs
Anergic
Normal both in the ability to display antigen and in the expression of
genes
APC costimulatory ligands (Figure 11-5).
In the early stages of an immune response in secondary
Fab CD80/86 lymphoid organs, T cells encounter two main types of
anti-CD28 professional APCs: the dendritic cell and the activated B
FIGURE 11-4 Experimental demonstration of clonal an- cell. Mature dendritic cells that have been activated by
ergy versus clonal expansion. (a) Only Signal 1 is generated microbial components via their pattern recognition
when resting T cells are incubated with glutaraldehyde-fixed APCs, receptors (PRRs) are present throughout the T-cell zones.
which cannot be stimulated to up-regulate the costimulatory ligand They express antigenic peptides in complex with high
CD80/86. (b) Anergic cells cannot respond to subsequent challenge, levels of MHC class I and II molecules. They also express
even when APCs can engage costimulatory receptors. (c) Anergy can costimulatory ligands and are the most potent activators
also be induced when naïve T cells are incubated with normal APCs of naïve CD4 and CD8 T cells.
in the presence of the Fab portion of anti-CD28, which blocks interac- Resting B cells residing in the follicles gain the capacity to
tion with CD80/86. activate T cells after they bind antigen through their B-cell
receptor (BCR). This engagement stimulates the up-regulation
of MHC class II and costimulatory CD80/86, allowing the
MHC-peptide complexes and costimulatory ligands on B cell to present antigen to CD4 T cells they encounter at
dendritic cells. However, the consequences and extent of the border between the follicle and T-cell zone. Because of
T-cell activation are also critically shaped by the activity of their unique ability to internalize antigen (e.g., pathogens) via
soluble cytokines produced by both APCs and T cells. These specific BCRs and present them in MHC class II, B cells are
assisting cytokines are referred to, by some, as Signal 3 (see most efficient at activating CD4 T cells that are specific for
Figure 11-3). the same pathogen for which they are specific. This situation
Cytokines bind surface cytokine receptors, stimulating a serves the immune response very well, focusing the attention
cascade of intracellular signals that can enhance both prolif- of antigen-specific CD4 T cells activated in the T-cell zone on
eration and/or survival (see Chapter 4). IL-2 is one of the B cells activated by the same antigen in the neighboring follicle.
best-known cytokines involved in T-cell activation and plays The pairing of B cells with their helper T cells occurs at the
a key role in inducing optimal T-cell proliferation, particu- junction between the B- and T-cell zones (see Chapter 14) and
larly when antigen and/or costimulatory ligands are limiting. allows T cells to deliver the help required for B-cell prolifera-
Costimulatory signals induce transcription of genes for both tion, differentiation, and memory generation (see Chapter 12).
IL-2 and the chain (CD25) of the high-affinity IL-2 recep- Macrophages are also found in secondary lymphoid organs,
tor. Signals also enhance the stability of the IL-2 mRNA. The but can activate T cells in a wide range of other peripheral
combined increase in IL-2 transcription and improved IL-2 tissues. They also must be activated to reveal their full antigen-
mRNA stability results in a 100-fold increase in IL-2 produc- presenting capacity. They up-regulate MHC molecules and
tion by the activated T cell. Secretion of IL-2 and its subse- costimulatory ligands in response to interactions with patho-
quent binding to the high-affinity IL-2 receptor induces gens, as well as in response to cytokines produced by other
activated naïve T cells to proliferate vigorously. cells, including IFN-.
Dendritic cell Macrophage B lymphocyte

Resting Activated Resting Activated Resting Activated
Class I Class I
PAMPs and MHC
cytokines MHC
PAMPs Ag
T cell
help
Class I (IFN-γ)
MHC CD80/86 Class II Class I Class II
Class I Class II Class II
MHC MHC CD80/86
MHC MHC CD80/86 MHC MHC
Antigen-presenting
cell Dendritic cell Macrophage B cell
Antigen uptake Endocytosis Phagocytosis Receptor-mediated endocytosis

Phagocytosis
Activation Mediated by pattern recognition Mediated by pattern recognition Mediated by antigen recognition
receptors receptors and enhanced by T-cell
help
MHC Class II Increases with activation (may Increases with activation Increases with activation (expresses
expression express low levels constitutively) low levels of constitutively)
Costimulatory Up-regulation of CD80/86 with Up-regulation of CD80/86 with Up-regulation of CD80/86 with
activity activation activation activation
T-cell activation Naïve T cells Effector T cells Effector T cells

Effector T cells Memory T cells Memory T cells
Memory T cells
Location Resting: Resting: Resting:

Circulation Circulation Circulation
peripheral tissues peripheral tissues SLOs (follicles)
Activated: Activated: Activated:
SLOs (T-cell zones) SLOs (subcapsular cortex of lymph SLOs (B cell/T-cell zone interface,
Tertiary tissues node, marginal zones of spleen) germinal centers, and marginal
Peripheral tissues zones)
FIGURE 11-5 Differences in the properties of professional ary lymphoid tissues (SLOs) as well as interacting with effector cells in
antigen-presenting cells that induce T-cell activation. This fig- the periphery. It is important to recognize that the distinctions shown
ure describes general features of three major classes of professional are rules of thumb only. Functions among the APC classes overlap,
APCs. Dendritic cells are the best activators of naïve T cells. This may and the field now recognizes different subsets within each major
be due, in part, to relatively high levels of expression of MHC and co- group of APC, each of which may act independently on different
stimulatory molecules when they are mature and activated. Activat- T-cell subsets. This diversity may be a consequence of activation by
ed B cells interact most efficiently with differentiated TH cells that are different innate immune receptors or may reflect the existence of
specific for the same antigen that activated them. Macrophages play independent cell lineages. Note that activation of effector and memory
several different roles, processing and distributing antigen in second- T cells is not as dependent on costimulatory interactions.
It turns out that there are several different dendritic cell Superantigens Are a Special Class
and macrophage subtypes; however, their functions are of T-Cell Activators
still being clarified. Some are likely to activate or induce
differentiation of specific effector T cells that travel to the Superantigens are viral or bacterial proteins that bind
site of infection, and some may be involved in reactivating simultaneously to specific V regions of T-cell receptors
memory cells that reside in tissues. Others may help to and to the chain of class II MHC molecules. V regions
quell, rather than to activate, responses. Full understand- are encoded by over 20 different V genes in mice and 65
ing awaits more research. different genes in humans. Each superantigen displays a
Both endogenous superantigens and exogenous superanti-

gens have been identified. Exogenous superantigens are solu-
TH cell
ble proteins secreted by bacteria. Among them are a variety of
exotoxins secreted by Gram-positive bacteria, such as staphy-
β α TCR lococcal enterotoxins, toxic shock syndrome toxin, and exfo-
Vβ
Peptide for which
liative dermatitis toxin. Each of these exogenous superantigens
Superantigen TCR is not specific binds particular V sequences in T-cell receptors (Table 11-2)
and cross-links the TCR to a class II MHC molecule.
Endogenous Class II MHC
superantigen is α β Endogenous superantigens are cell-membrane proteins
membrane bound generated by specific viral genes that have integrated into
mammalian genomes. One group, encoded by mouse mam-
APC mary tumor virus (MTV), a retrovirus that is integrated into
the DNA of certain inbred mouse strains, produces proteins
called minor lymphocyte-stimulating (Mls) determinants,
FIGURE 11-6 Superantigen-mediated cross-linkage of T- which bind particular V sequences in T-cell receptors and
cell receptor and class II MHC molecules. A superantigen binds cross-link the TCR to class II MHC molecules. Four Mls
to all TCRs bearing a particular V sequence regardless of their superantigens, originating from distinct MTV strains, have
antigen specificity. Exogenous superantigens are soluble secreted been identified.
bacterial proteins, including various exotoxins. Endogenous super- Because superantigens bind outside the TCR antigen-
antigens are membrane-embedded proteins produced by certain binding cleft, any T cell expressing that particular V
viruses; they include Mls antigens encoded by mouse mammary sequence will be activated by a corresponding superantigen.
tumor virus. Hence, the activation is polyclonal and can result in massive
T-cell activation, resulting in overproduction of TH-cell
“specificity” for one of these V versions, which can be cytokines and systemic toxicity. Food poisoning induced by
expressed by up to 5% of T cells, regardless of their antigen staphylococcal enterotoxins and toxic shock induced by
specificity. This clamp-like connection mimics a strong toxic shock syndrome toxin are two examples of disorders
TCR-MHC interaction and induces activation, bypassing caused by superantigen-induced cytokine overproduction.
the need for TCR antigen specificity (Figure 11-6). Superan- Given that superantigens result in T-cell activation and
tigen binding, however, does not bypass the need for proliferation, what adaptive value could they possibly have
costimulation; professional APCs are still required for full for the pathogens that make them? The answer is not clear,
T-cell activation by these microbial proteins. but there is evidence that such antigen-nonspecific T-cell
TABLE 11-2 Exogenous superantigens and their V␤ specificity

V␤ SPECIFICITY
*
Superantigen Disease Mouse Human
Staphylococcal enterotoxins
SEA Food poisoning 1, 3, 10, 11, 12, 17 nd
SEB Food poisoning 3, 8.1, 8.2, 8.3 3, 12, 14, 15, 17, 20
SEC1 Food poisoning 7, 8.2, 8.3, 11 12
SEC2 Food poisoning 8.2, 10 12, 13, 14, 15, 17, 20
SEC3 Food poisoning 7, 8.2 5, 12
SED Food poisoning 3, 7, 8.3, 11, 17 5, 12
SEE Food poisoning 11, 15, 17 5.1, 6.1–6.3, 8, 18
Toxic shock syndrome toxin (TSST1) Toxic shock syndrome 15, 16 2
Exfoliative dermatitis toxin (ExFT) Scalded skin syndrome 10, 11, 15 2
Mycoplasma arthritidis supernatant (MAS) Arthritis, shock 6, 8.1–8.3 nd
Streptococcal pyrogenic exotoxins
(SPE-A, B, C, D) Rheumatic fever, shock nd nd
*
Disease results from infection by bacteria that produce the indicated superantigens.
ADVANCES
How Many TCR Complexes Must Be Engaged

to Trigger T-Cell Activation?
Until single-cell imaging techniques that can enter cells and fluoresces when cence.) The fluorescence intensity
were developed, indirect methods were intracellular calcium is released. By add- calculated from this particular image indi-
used to calculate how many ligands a T ing fura-2 “loaded” T cells to the APCs cated that only a single MHC-peptide
cell must recognize in order to be acti- bound to varying numbers of red fluo- combination was at the T cell-APC syn-
vated. Mark Davis and colleagues at Stan- rescent peptide (see Figure 1a), research- apse. The plot below these images shows
ford School of Medicine approached this ers could determine (1) if a T cell was the intensity of fura-2 fluorescence (i.e.,
question using an acutely sensitive, two- activated and (2) how many molecules of the increase in intracellular Ca2) over
part microscopic visualization technique peptide were present at the point of time after T cell-APC engagement. The
(Figure 1). APCs were cultured (pulsed) contact between the T cell and the initial spike is an indicator that this single
briefly with peptide bound to a biotin APC—in other words, how many TCR/ MHC-peptide could inspire robust Ca2
molecule. When APCs are exposed to MHC-biotinylated peptide complexes signals.
soluble peptides a small number will were required to stimulate the release of The investigators quantified many
exchange it with a peptide bound to an intracellular calcium. interactions in this way and definitively
MHC molecule on the cell surface. The One set of data from these experi- concluded that a single MHC-peptide
number of peptides that actually bound ments is illustrated in Figure 1b. An acti- combination could stimulate significant
to MHC could be determined in this sys- vated, fura-2-loaded T cell (blue) is shown Ca2 release. Maximal Ca2 release was
tem by adding a fluorescent (phycoery- interacting with an APC in the upper left. achieved in CD4 T cells when as few as
thrin) streptavidin conjugate, which The fluorescent micrograph of the pep- 10 TCR complexes were engaged. Simi-
binds the biotin of the biotinylated peptide at the junction of the T cell and APC is lar results were obtained with CD8 T
tide and can be detected and quantified shown in the top right image. The inten- cells.
by fluorescent microscopy. sity of the red fluorescence varies with the
Irvine, D. J., M. A. Purbhoo, M. Krogsgaard, and
The response of T cells specific for the number of peptides bound. (The image is M. M. Davis. 2002. Direct observation of
complex could also be quantified using “stretched” artificially because of the com- ligand recognition by T cells. Nature
another fluorescent tool: fura-2, a dye puter program used to quantify fluores- 419:845–849.
activation and inflammation hampers the development of a in combination with costimulation and signals received by
coordinated antigen-specific response that would most soluble cytokines, culminate in the activation of “effector”
effectively thwart infection. Some speculate that the large- molecules that regulate (1) cell survival, (2) cell cycle entry,
scale proliferation and cytokine production that occur and, as we shall see below, (3) cell differentiation.
harm the cells and microenvironments that are required to In 1 to 2 days after successful engagement with a den-
start a normal response; others argue that these events dritic cell in the T-cell zone of a secondary lymphoid organ,
induce T-cell tolerance to the pathogen. a naïve T cell will enlarge into a blast cell and begin under-
going repeated rounds of cell division. Signals 1 plus 2
induce up-regulation of expression and activity of pro-
T-Cell Differentiation survival genes (e.g., bcl-2), as well as the transcription of
genes for both IL-2 and the chain (CD25) of the high-
How does an interaction between a naïve T cell and a den- affinity IL-2 receptor (Figure 11-7). The combined effect on
dritic cell result in the generation of cells with effector func- a naïve T cell is activation and robust proliferation. Activated
tions? An activating, costimulatory interaction between a T cells divide 2 to 3 times per day for 4 to 5 days, generating
naïve T cell and an APC typically lasts 6 to 8 hours, a period a clone of progeny cells that differentiate into memory and
that permits the development of a cascade of signaling effector T-cell populations.
events (see Chapter 3) that alter gene programs and induce Activated T cells and their progeny gain unique func-
differentiation into a variety of distinct effector and memory tional abilities, becoming effector helper or cytotoxic T cells
cell subtypes. Just a few TCR-MHC interactions (as described that indirectly and directly act to clear pathogen. CD8 cyto-
in Advances Box 11-3) stimulates a signaling cascade that, toxic T cells leave the secondary lymphoid tissues and circulate
BOX 11-3
(a) (b)
T cell
T
Ca2+
APC
PE snapshot
APC 3
Ca2+ signal
2
0
-5 0 5 10 15 20
Time (minutes)
FIGURE 1
Engagement of a single T-cell receptor can induce Ca2ⴙ and APC is captured by bright field microscopy (top left) and by high-
signals in a T cell. (a) The experimental approach taken by the resolution fluorescence microscopy (top right), where the arrow points to the
investigators to determine the ability of a single TCR-MHC interaction to single TCR/MHC-peptide interaction. The Ca2 signal generated within the
generate Ca2 signals. See text for details. (b) One example of original data on T cell over a 20-minute period is depicted in the graph and measured using
Ca2 signaling generated from a single T cell that has engaged a single MHC- software that quantifies the intensity of fura-2 fluorescence, which increases
peptide ligand on the surface of an APC. The interaction between the T cell with a rise in cytosolic Ca2 levels. [Irvine, D.J. et al. 2002 Nature 419:845–849.]
to sites of infection where they bind and kill infected cells. the death of target cells, becoming “killer” or “cytotoxic” T
CD4 helper T cells secrete cytokines that orchestrate the lymphocytes (CTL, or TC). Because cytotoxic CD8 T cells
activity of several other cell types, including B cells, macro- recognize peptide bound to MHC class I, which is expressed
phages, and other T cells. Some CD4+ T cells, particularly by almost all cells in the body, they are perfectly poised to
those that “help” B cells and those that generate lymphocyte clear the body of cells that have been internally infected by
memory, stay within secondary lymphoid tissue to continue the pathogen that resulted in their activation. On the other
to regulate the generation of the response. Others return to hand, activated CD4 T cells (helper T cells or TH) acquire
the sites of infection and enhance the activity of macro- the ability to secrete factors that enhance the activation
phages and cytotoxic cells. and proliferation of other cells. Specifically, they regulate
Effector cells tend to be short-lived and have life spans activation and antibody production of B cells, enhance the
that range from a few days to a few weeks. However, the phagocytic, anti-microbial, cytolytic, and antigen-presenting
progeny of an activated T cell can also become long-lived capacity of macrophages, and are required for the develop-
memory T cells that reside in secondary and tertiary tissues ment of B-cell and CD8 T-cell memory.
for significant periods of time, providing protection against As immunologists developed and adopted more tools to
a secondary infection. distinguish proteins expressed and secreted by helper T cells,
Effector T cells come in more varieties than was originally it became clear that CD4 helper T cells were particularly
anticipated, and each subset plays a specific and important diverse, differentiating into several different subtypes, each
role in the immune response. The first effector cell distinc- of which secretes a signature set of cytokines. The cytokines
tion to be recognized was between CD8 T cells and CD4 secreted by CD4 TH cells can either act directly on the same
T cells. Activated CD8 T cells acquire the ability to induce cell that produced them (acting in an autocrine fashion) or
IL-2 Normal array of cytokines, hinted at this possibility. However, Mos-

APC mann and Coffman definitively identified two distinct func-
tional subgroups, TH1 and TH2, each of which produced a
1
different set of cytokines. Furthermore, they showed that
IL-2 gene
IL-2R gene these differences were properties of distinct T-cell clones:
2
each activated T cell expanded into a population of effector T
cells that secreted a distinct array of cytokines.
CD28 CD80/86
Specifically, these investigators developed over 50 indi-
Activation
vidual T-cell clones from a mixture of T cells with different
receptor specificities (i.e., a polyclonal T-cell population) that
had been isolated from the spleen of an immunized mouse.
IL-2 At a time when the community did not have the tools to
IL-2 identify most cytokines directly, these researchers developed
receptor G1 clever (and elaborate) strategies to define each clone’s cyto-
M S kine secretion pattern. They showed that the cytokines
G2
secreted by each of the 50 clones fell into one of two broad
categories, which they named TH1 and TH2.
Because TH1 and TH2 subsets were originally identified
from in vitro studies of cloned T-cell lines, some researchers
Several doubted that they represented true in vivo subpopulations.
divisions They suggested instead that these subsets might represent
different maturational stages of a single lineage. In addition,
the initial failure to locate either subset in humans led some
to believe that TH1, TH2, and other subsets of T helper cells
were not present in this species. Further research corrected
M M E E E E these views. In many in vivo systems, the full commitment
of populations of T cells to either a TH1 or TH2 phenotype
Population of memory and effector cells occurs late in an immune response. Hence, it was difficult to
find clear TH1 or TH2 subsets in studies employing healthy
FIGURE 11-7 Activation of a naïve T cell up-regulates
human subjects, where these cells would not have developed.
expression of IL-2 and the high-affinity IL-2 receptor. Signal 1
Indeed, TH1 and TH2 populations in T cells were ultimately
and Signal 2 cooperate to enhance transcription and stability of
isolated from humans during chronic infectious disease or
mRNA for IL-2 and IL-2R. Secreted IL-2 will bind the IL-2R, which gener-
chronic episodes of allergy, and studies in humans and mice
ates signals that enhance the entry of the T cell into the cell cycle and
definitively confirmed their lineage independence.
initiates several rounds of proliferation. Most of the cells differentiate
With the benefit of new tools and technology, we now
into effector helper or cytotoxic cells, but some will differentiate into
have a more detailed understanding of the panels of cyto-
effector or central memory cells.
kines that each group produces. The TH1 subset secretes
IL-2, IFN-, and Lymphotoxin- (TNF-), and is responsi-
can bind to receptors and act on cells in the vicinity (acting ble for many classic cell-mediated functions, including acti-
in a paracrine fashion). Below we describe the major features vation of cytotoxic T lymphocytes and macrophages. The
and functions of the best-characterized CD4 helper T-cell TH2 subset secretes IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13,
subsets. Although CD8 cytotoxic T cells also secrete cyto- and regulates B-cell activity and differentiation.
kines, and there are indications that CD8 T cells may also These experiments set the stage for the discovery over the
differentiate into more than one killer subtype, the diversity last decade that CD4 T cells can adopt not just two but at
of CD8 T-cell effector functions are clearly more restricted least five distinct effector fates after activation. The TH1 and
than those of CD4 TH cells. The generation and activity of TH2 subpopulations have been joined definitively by the
CTL cells are described in more detail in Chapter 13. TH17 and TREG subpopulations, each of which produces a
distinct cytokine profile and regulates distinct activities
Helper T Cells Can Be Divided within the body. Yet another subpopulation, T follicular
helper cells (TFH), has recently been characterized, and has
into Distinct Subsets achieved membership among the major helper subsets.
Tim Mosmann, Robert Coffman, and colleagues can be cred- More are bound to reveal themselves, although it will be
ited with one of the earliest experiments definitively demon- important to determine whether each represents distinct
strating that helper CD4 T cells were more heterogeneous in subgroups or variants within the major subgroups.
phenotype and function than originally supposed. Earlier In retrospect, we probably should have anticipated the
investigations, showing that helper T cells produced a diverse heterogeneity of helper responses, which allows an organism
to “tailor” a response to a particular type of pathogen. The PAMPs from pathogens

(or adjuvants) PRR
type of effector TH cell that a naïve T cell (also called a TH0
cell) becomes depends largely on the kind of infection that Other cellular
sources of
occurs. For example, extracellular bacterial infections result PRR cytokines
in the differentiation of activated CD4 T cells into TH2
cells, which help to activate B cells to secrete antibodies that
can opsonize bacteria and neutralize the toxins they pro- APC
Polarizing
duce. On the other hand, infection by an intracellular virus cytokines
or bacterium induces differentiation of CD4 T cells into
TH1 helpers that enhance the cytolytic activity of macro-
phages and CD8 T cells, which can then kill infected cells.
Responses to fungi stimulate the differentiation of different
helper responses than responses to worms, and so on. The STATs
reality is, of course, more complex. Infections evoke the dif-
TCR
ferentiation of more than one helper subtype, some of which
Effector
have overlapping roles. What regulates the differentiation of cytokines
each effector subset and what function each subset plays are Master gene
still being actively investigated. We describe the fundamen- regulators
tals of our current understanding below.
The Differentiation of T Helper Cell Subsets Is FIGURE 11-8 General events and factors that drive TH
Regulated by Polarizing Cytokines subset polarization. Interaction of pathogen with pattern rec-
ognition receptors (PRRs) on dendritic cells and other neighboring
As you know, T-cell activation requires TCR and costimula- immune cells determines which polarizing cytokines are produced
tory receptor engagement, both of which are supplied by an and, hence, into which T helper subset a naïve cell will differenti-
activated APC. It is now clear that the functional fate of acti- ate. In general, polarizing cytokines that arise from dendritic cells
vated T cells is determined by signals they receive from or other neighboring cells interact with cytokine receptors and
additional cytokines generated during the response. These generate signals that induce transcription of unique master gene
cytokines (Signal 3) are referred to as polarizing cytokines regulators. These master regulators, in turn, regulate expression of
because they are responsible for guiding a helper T cell various genes, including effector cytokines, which define the func-
toward one of several different effector fates. For example, T tion of each subset.
cells that are activated in the presence of IL-12 and IFN-
tend to differentiate, or polarize, to the TH1 lineage, whereas
T cells that are activated in the presence of IL-4 and IL-6 Hence, PRR signals regulate the fate a T cell will adopt fol-
polarize to the TH2 lineage. lowing activation (Figure 11-8).
Polarizing cytokines can be generated by the stimulating For example, double-stranded RNA, a product of many
APC itself, or by neighboring immune cells that have also viruses, binds TLR3 receptors on dendritic cells, initiating a
been activated by antigen. Which cytokines are produced signaling cascade that results in production of IL-12, which
during an immune response depends on (1) the cell of origin directly promotes TH1 differentiation. On the other hand,
(DC, macrophage, B cell, NK cell, etc.), (2) its maturation worms stimulate PRRs on innate immune cells, including
and activation status, (3) which pathogens and other inflam- mast cells, which generate IL-4. IL-4 directly promotes
matory mediators it encounters, and (4) in what tissue envi- polarization of activated T cells to the TH2 subset, which
ronment it encounters that pathogen. Innate interactions coordinates the IgE response to helminths (see Figure 11-8).
therefore have a critical role in shaping adaptive responses In this case, the main polarizing cytokine is not made by the
(see Figure 5-18). Specifically, by influencing APC secretions activating dendritic cell, but is generated by a neighboring
and the surface and the microenvironmental landscape that immune cell. The pathogen interactions that give rise to the
a T cell encounters, innate immune responses directly influ- polarizing cytokines that drive T helper cell differentiation
ence the functional fate of helper T cells. are often complex and a very active area of research.
Recall from Chapter 5 that APCs and other innate Adjuvants, which have been used for decades to enhance
immune cells are activated by interaction with pathogens the efficacy of vaccines, are now understood to exert their
bearing pathogen-associated molecular patterns (PAMPs). influence on the innate immune system by regulating the
These PAMPs bind PRRs, including, but not limited to Toll- expression of costimulatory ligands and cytokines by APCs,
like receptors (TLRs). PRR interactions activate dendritic events that ultimately shape the consequences of T-cell acti-
cells by stimulating the up-regulation of MHC and costimu- vation. PAMPs and cytokines such as IL-12, produced by
latory proteins. They also determine the type of cytokine(s) APCs themselves, are considered natural adjuvants. Dead
that dendritic cells and other immune cells will secrete. mycobacteria, which clearly activate many PRRs, have long
TABLE 11-3 Regulation and function of T helper subtypes

Polarizing cytokines Master gene regulators Effector cytokines Functions
TH1 IL-12 T-Bet IFN- Enhances APC activity

IFN- TNF Enhances TC activation
IL-18 Protects against intracellular pathogens
Involved in delayed type hypersensitivity,
autoimmunity
TH2 IL-4 GATA-3 IL-4 Protects against extracellular pathogens
IL-5 (particularly IgE responses)
IL-13 Involved in allergy
TH17 TGF- ROR IL-17A Protects against some fungal and
IL-6 IL-17F bacterial infections
(IL-23) IL-22 Contributes to inflammation,
autoimmunity
TREG TGF- FoxP3 IL-10 Inhibits inflammation
IL-2 TGF-
TFH IL-6 Bcl-6 IL-4 B cell help in follicles and germinal
IL-21 IL-21 centers
been used as a very potent adjuvant for immune responses nature of the pathogen the APC encountered at the site
in mice. Very few adjuvants are approved for human vacci- of infection.
nation, but given our new and evolving understanding of the
• Broadly speaking, TH1 and TH17 cells regulate cell-
molecules that stimulate PRRs and the consequences of that
mediated immunity (CD8 T cells and macrophages)
stimulation, investigators expect that we will one day be able
and TH2 and TFH cells regulate humoral immunity (B
to shape the effector response to vaccine antigens by varying
cells). However, it is important to recognize that all
the adjuvants—natural and/or synthetic—included in vac-
CD4 effector T-cell subsets may have the potential to
cine preparations (see Chapter 17).
provide help to B cells. TH1 and TH17 subsets generally
encourage B cells to produce antibodies that contribute
Effector T Helper Cell Subsets Are to cell-mediated immunity (e.g., isotypes like IgG2a that
can “arm” NK cells for cytotoxicity; see Chapter 13). TH2
Distinguished by Three Properties cells encourage B cells to produce antibodies that
Each helper T-cell subset is defined by an array of features, mediate the clearance of extracellular pathogens (e.g.,
the details of which can rapidly overwhelm a new (or old) isotypes like IgE that induce the release of molecules
student of immunology. Understanding these specifics is an that harm extracellular parasites).
important first step to clarifying the role each subset plays in
• Helper T-cell subsets often “cross-regulate” each other.
resolving infection and causing disease. Having a reference
The cytokines they secrete typically enhance their own
to them is also helpful when deciphering primary literature
differentiation and expansion and inhibit commitment to
describing advances. However, some generalizations provide
other helper T-cell lineages. This is particularly true of
a useful conceptual framework for organizing some of these
the TH1 and TH2 pair, as well as the TH17 and TREG pair.
details. Consider the following:
• Helper cell lineages may not be fixed; some subsets can
• Each of the major T helper cell subsets is characterized
assume the cytokine secretion profile of other subsets if
by (1) a distinct set of polarizing cytokines that induce
exposed to a different set of cytokines, particularly early
the expression of (2) a master gene regulator that
in the differentiation process.
regulates expression of (3) a signature set of effector
cytokines the T-cell population produces once it is fully • The precise biological function and sites of differentiation
differentiated (see Figure 11-8 and Table 11-3). and activity of each subset continue to be actively
investigated. Much remains unknown.
• Which effector subset an activated helper cell becomes
depends on the quality and quantity of signals its naïve We start our discussion of helper cell subset characteris-
cell precursor receives from APCs in a secondary tics with the first two subsets to be identified: TH1 and TH2
lymphoid organ; that activity, in turn, depends on the cells. They provide an illustrative example of the features that
c11T-CellActivation,Differentiation,andMemory.indd Page 373 12/21/12 4:39 PM user-fw429 /208/WHF00165/work/indd
Pathogens inducing
cell-mediated immunity TH1-polarizing
(most viruses, some factors Naïve TH cell
bacteria and fungi) TH1
IL-12 IFN-γ
TH1-polarizing
PRRs TCR Signal 3
Dendritic Signal 1 T-Bet
cell MHC Class II-
peptide Signal 2 GATA-3
CD80/86 CD28 TH2-polarizing
Signal 3
IL-4
TH2 IL-5
IL-13
Pathogens inducing TH2-polarizing Cellular

humoral immunity, factors IL-4 sources
particularly extracellular of IL-4
parasites (e.g., worms)
FIGURE 11-9 Regulation of TH1 and TH2 subset differen- On the other hand, extracellular pathogens activate signal cascades
tiation. This figure depicts some of the cellular events that drive TH1 that can polarize naïve T cells to the TH2 lineage. Parasitic worms inter-
and TH2 lineage commitment in more detail. Intracellular pathogens act with PRRs on neighboring immune cells (such as mast cells, baso-
activate a cascade of signals that polarize cells to the TH1 lineage. For phils, or germinal center B cells), triggering the release of the signature
example, viruses interact with PRRs (e.g., TLR-3) that induce dendritic TH2 polarizing cytokine IL-4. This interacts with receptors on T cells that
cells to generate IL-12. This binds to receptors on naïve T cells, acti- activate STAT6, up-regulating expression of the transcriptional regula-
vating a signal transduction pathway mediated by STAT4 that induces tor GATA-3. GATA-3, in turn, induces expression of the TH2 effector cy-
expression of the transcription factor T-Bet. T-Bet, in turn, activates tokines, including IL-4, IL-5, and IL-13. [Adapted from M. L. Kapsenberg, 2003,
expression of effector cytokines, including IFN-␥, which define the TH1 Dendritic-cell control of pathogen-driven T-cell polarization, Nature Reviews
subset’s functional capacities (and can also enhance TH1 polarization). Immunology 3:984.]
distinguish T helper cells as well as the relationship between the differentiation of fully cytotoxic TC cells from CD8⫹ pre-
subsets. We follow this section with summaries of what is cursors by activating the dendritic cells that engage naïve TC
currently understood about the more recently characterized cells. These combined effects make the TH1 subset particu-
helper subsets: TH17, TFH, and TREG. larly suited to respond to viral infections and other intracel-
lular pathogens. They also contribute to the pathological
The Differentiation and Function of TH1 and TH2 Cells effects of TH1 cells, which are also involved in the delayed
The key polarizing cytokines that induce differentiation of type hypersensitivity response to poison ivy (see Chapter 15).
naïve T cells into TH1 cells are IL-12, IL-18, and IFN-␥ Just as differentiation to the TH1 subset is promoted by
(Figure 11-9). IL-12 is produced by dendritic cells after an IL-12 and IFN-␥, differentiation to the TH2 subset is pro-
encounter with pathogens via PRRs (e.g., TLR4, TLR3). It is moted by a defining polarizing cytokine, IL-4 (see Figure
also up-regulated in response to IFN-␥, which is generated 11-9). Exposing naïve helper T cells to IL-4 at the beginning
by activated T cells and activated NK cells. IL-18, which is of an immune response causes them to differentiate into TH2
also produced by dendritic cells, promotes proliferation of cells. Interestingly, TH2 development is greatly favored over
developing TH1 cells and enhances their own production of TH1 development. Even in the presence of IFN-␥ and IL-12,
IFN-␥. These polarizing cytokines trigger signaling path- naïve T cells will differentiate into TH2 effectors if IL-4 is
ways that up-regulate the expression of the master gene present. IL-4 triggers a signaling pathway within the T cell
regulator T-Bet. This master transcription factor induces that up-regulates the master gene regulator GATA3, which,
commitment to the TH1 lineage, inducing expression of the in turn, regulates expression of TH2-specific cytokines,
signature TH1 effector cytokines, including IFN-␥ and TNF. including IL-4, IL-5, and IL-13.
IFN-␥ is a particularly potent effector cytokine. It acti- Dendritic cells do not make IL-4, so from where does it
vates macrophages, stimulating these cells to increase micro- come? Mast cells, basophils, and NKT cells can be induced
bicidal activity, up-regulate the level of class II MHC, and, as to make IL-4 after exposure to pathogens and could influ-
mentioned above, secrete cytokines such as IL-12, which ence helper T cell fate in the periphery. Germinal-center B
further enhance TH1 differentiation. IFN-␥ secretion also cells and TFH cells can also produce IL-4, which could influ-
induces antibody class switching in B cells to IgG classes ence helper T-cell polarization in the lymph nodes and
(such as IgG2a in the mouse) that support phagocytosis and spleen. And TH2 cells themselves are an excellent source
fixation of complement. Finally, IFN-␥ secretion promotes of additional IL-4 that can enhance polarization events.
Investigators, however, are still working to definitively iden-

tify the source of the IL-4 that initiates TH2 polarization in
APC
secondary lymphoid tissues.
The effector cytokines produced by TH2 cells help to clear
extracellular parasitic infections, including those caused by IL-12
TCR
IL-4
helminths. IL-4, the defining TH2 effector cytokine, acts on
both B cells and eosinophils. It induces eosinophil differen-
tiation, activation, and migration and promotes B-cell activa-
tion and class switching to IgE. These effects act synergistically Stat4 Stat6
because eosinophils express IgE receptors (FcR) which,
when cross-linked, release inflammatory mediators that are T-Bet − − GATA-3
particularly good at attacking roundworms. Thus, IgE anti-
body can form a bridge between the worm and the eosino-
+ IFN-γ −
phil, binding to worm antigens via its variable regions and
− IL-4 +
binding to FcR via its constant region. IgE antibodies also − IL-5 +
mediate allergic reactions, and the role of TH2 activity in
these pathological responses is described in Chapter 15.
Promotes Promotes
IL-5 can also induce B-cell class switching to IgG sub- TH1 TH2
classes that do not activate the complement pathway (e.g.,
IgG1 in mice), and IL-13 functions largely overlap with IL-4. FIGURE 11-10 Cross-regulation of T helper cell subsets by
Finally, IL-4 itself can suppress the expansion of TH1-cell transcriptional regulators. GATA-3 and T-Bet reciprocally regulate
populations. differentiation of TH1 and TH2 lineages. IL-12 promotes the expression
of the TH1-defining transcription factor, T-Bet, which induces expres-
TH1 and TH2 Cross-regulation sion of TH1 effector cytokines, including IFN-. At the same time, T-Bet
The major effector cytokines produced by TH1 and TH2 sub- represses the expression of the TH2 defining master transcriptional
sets (IFN- and IL-4, respectively) not only influence the regulator, GATA-3, as well as expression of the effector cytokines IL-4
overall immune response, but also influence the helper T cell and IL-5. Reciprocally, IL-4 promotes expression of GATA-3, which
subsets. First, they promote the growth (and in some cases up-regulates the synthesis of IL-4 and IL-5, and at the same time
even the polarization) of the subset that produces them; sec- represses the expression of T-Bet and the TH1 effector cytokine IFN-
ond, they inhibit the development and activity of the opposite . [Adapted from J. Rengarajan, S. Szabo, and L. Glimcher, 2000, Transcriptional
subset, an effect known as cross-regulation. For instance, regulation of Th1/Th2 polarization, Immunology Today 21:479.]
IFN- (secreted by the TH1 subset) inhibits proliferation of
the TH2 subset, and IL-4 (secreted by the TH2 subset) down- specifically by (1) inhibiting expression of class II MHC mol-
regulates the secretion of IL-12 by APCs, thereby inhibiting ecules, (2) suppressing production of bactericidal metabo-
TH1 differentiation. Furthermore, IL-4 enhances TH2 cell lites (e.g., nitric oxide) and various inflammatory mediators
development by making TH cells less susceptible to the TH1 (e.g., IL-1, IL-6, IL-8, GM-CSF, G-CSF, and TNF-), and
promoting cytokine signals (and vice versa). even by (3) inducing apoptosis.
Similarly, these cytokines have opposing effects on target The master regulators T-Bet and GATA-3 also play an
cells other than TH subsets. In mice, where the TH1 and TH2 important role in cross-regulation. (Figure 11-10) Specifi-
subsets have been studied most extensively, the cytokines cally, the expression of T-Bet drives cells to differentiate into
have distinct effects on the type of antibody made by B cells. TH1 cells and suppresses their differentiation along the TH2
Recall from Chapter 3 (and see Chapter 13) that the antibody pathway. Expression of GATA-3 does the opposite, promot-
isotype IgG2a enhances cell-mediated immunity by arming ing the development of naïve T cells into TH2 cells while sup-
NKT cells, whereas the isotypes IgG1 and IgE enhance pressing their differentiation into TH1 cells. Consequently,
humoral immunity by their activities on extracellular patho- cytokine signals that induce one of these transcription factors
gens. IFN- secreted by the TH1 subset promotes IgG2a sets in motion a chain of events that represses the other.
production by B cells but inhibits IgG1 and IgE production.
On the other hand, IL-4 secreted by the TH2 subset promotes TH17 Cells
production of IgG1 and IgE and suppresses production of The discovery of the TH17 subset of T cells, which, like the
IgG2a. Thus, these effects on antibody production are consis- TH1 subset, is involved in cell-mediated immunity, arose in
tent with TH1 and TH2 subsets’ overall tendencies to promote part from a recognition that IL-12, one of the polarizing cyto-
cell-mediated versus humoral immunity, respectively. kines that induces TH1 development, was a member of a fam-
IL-10 secreted by TH2 cells also inhibits (cross-regulates) ily of cytokines that shared a subunit (p40) with IL-23. The
TH1 cell development, but not directly. Instead, IL-10 acts on p40 knockout mouse was a favorite model for studying the
monocytes and macrophages, interfering with their ability to importance of TH1 cells because, in the absence of IL-12, it
activate the TH1 subset by abrogating their activation, failed to generate TH1 cells. However, once it was understood
T Helper Subset Differentiation
Naïve
CD4+
T cell
IL-1,
IL-6, IL-12,
Polarizing IL-2, IL-23, IL-6, IFN-γ,
cytokines TGF-β TGF-β IL-4 IL-21 IL-18
Master FOXP3 RORγt GATA3 Bcl-6 T-Bet

transcriptional
regulator
Induced TReg cell TH17 cell TH2 cell TFH cell TH1 cell
Effector IL-10, TGF-β IL-17A, IL-17F, IL-22 IL-4, IL-5, IL-13 IL-4, IL-21 IFN-γ, TNF
cytokines
Regulation, Inflammation Allergic and B cell help Cell-mediated

Effector suppression of anti-helminth in germinal immunity,
functions
immune and responses centers macrophage
inflammatory activation,
responses inflammation
This figure synthesizes current information about the distinguish- plasticity in differentiation among subsets is depicted, but both are
ing features of T helper subset differentiation and activity. Polarizing described in the text. [Adapted from S. L. Swain, K. K. McKinstry, and T. M.
cytokines, master transcriptional regulators, effector cytokines, and Strutt, Expanding roles for CD41 T cells in immunity to viruses, Nature Reviews.
broad functions in health and disease are depicted for each of the Immunology 12:136–148.]
major helper subsets. Neither cross-regulation nor the potential
that p40 was also required for IL-23 production, investigators which also plays a role in T-cell development. RORt knock-
quickly realized that the results from these mice were no out mice have reduced severity of experimental autoimmune
longer unambiguous. In fact, it became clear that these mice encephalitis (EAE, a mouse model of multiple sclerosis)
also failed to produce a T-cell subset that required IL-23. apparently because of the reduction in TH17 cells.
Some of the functions originally attributed to IL-12-induced TH17 cells are so named because they produce IL-17A, a
TH1 cells were actually carried out by an IL-23-induced T cytokine associated with chronic inflammatory and autoim-
helper cell subpopulation now referred to as TH17 cells. mune responses, including those that result in inflammatory
TH17 cells are generated when naïve T cells are activated bowel disease, arthritis, and multiple sclerosis. TH17 cells are
in the presence of IL-6 and TGF-, the key polarizing cyto- the dominant inflammatory cell type associated with chronic
kine for iTREG differentiation (Overview Figure 11-11). IL-23 autoimmune disorders. They also produce IL-17F and IL-22,
also plays a role in finalizing the commitment to the TH17 cytokines associated with tissue inflammation. We have only
fate and is induced in APCs by interactions with PAMPs begun to understand the true physiological function of TH17
including fungal wall components, with TLR2 and the CLR cells, which in healthy humans have been found at epithelial
Dectin-1. Like TH1 and TH2 differentiation, TH17 cell differen- surfaces (e.g., lung and gut) and may play a role in warding
tiation is also controlled by a master transcriptional regulator off fungal and extracellular bacterial infections (see Clinical
whose expression is induced by polarizing cytokines. In this Focus Box 11-4). However, a full appreciation of their bio-
case the master regulator is the orphan steroid receptor RORt, logical role awaits further investigations.
CLINICAL FOCUS
What a Disease Reveals about the Physiological

Role of TH17 Cells
Experiments that reveal the inner caused by an imbalance between TH1 and Signal 3) (Figure 1a). They stained the cells
workings of normal healthy immune cells TH2 responses. Initial work did not reveal a for intracellular expression of TH17’s signa-
have offered invaluable insights into what difference in these activities, but as the ture cytokine, IL-17, and performed flow
can and does go wrong. However, at diversity of T helper subsets was revealed, cytometry (Figure 1b; also see Chapter 20).
times we need “experiments of nature”— investigators pursued the possibility of a Although CD4 T cells from HIES patients
the diseases themselves—to clarify how helper imbalance more vigorously. A were able to develop into other helper lin-
the immune system works in healthy indi- number of groups from Japan and Amer- eages (and, as you can see from the flow
viduals. Job syndrome, a rare disease in ica independently discovered that lym- cytometry contour plots, were also able to
which patients suffer from defects in phocytes from HIES patients were unable make IFN-, indicating they could become
bone, teeth, and immune function, is to respond to select cytokines, including TH1 cells), they could not be induced to
helping us solve the mystery of the physi- IL-6, IL-10, and IL-23. An analysis of genes secrete IL-17. Specifically, whereas 18.3% of
ological function of TH17 cells. that could explain this signaling failure T cells from healthy patients that were
People with Job syndrome suffer from revealed a dominant negative mutation in subject to these conditions stained with
recurrent infections, typically of the lung STAT3, a key cytokine signaling molecule antibodies against IL-17, 0.05% (essentially
and skin. The painful abscesses that are (see Chapter 4). none) of the T cells from HIES patients
often a feature of the disease and the trials These investigators knew from the lit- stained with the same antibodies.
endured by its patients explain its name, erature that the absence of signaling These striking observations suggest
which comes from a biblical story of a man through these cytokines suggested a spe- that the recurrent infections HIES patients
(Job) who is subject to horrific hardship as cific problem with polarization to the TH17 experience are caused at least in part by
a test of his piety. Another cardinal feature helper subset. Indeed, circulating TH17 cells the absence of TH17 cells. Reciprocally,
of the disease, elevated IgE levels in serum, were absent in HIES patients with STAT3 they indicate that TH17 cells play an
is the basis for its more formal name, Hyper mutations. The investigators then directly important role in controlling the type of
IgE Syndrome (HIES). HIES comes in two tested their hypothesis that this absence infection that afflicts these patients,
major forms: Job syndrome, the most was due to a failure of CD4 T cells to polar- including Staphylococcus aureus skin
common, is also referred to as Type 1 HIES. ize normally. Specifically, they removed infections and pneumonia. The critical
Patients with Type 2 HIES, which we will T cells from blood, and exposed them to role of TH17 in controlling bacterial and
not discuss here, do not have trouble with conditions that would ordinarily polarize fungal infections at epithelial surfaces has
bone or dental development. them to the TH17 lineage: T-cell receptor been supported by studies in mice, too.
The abundance of IgE originally sug- stimulation (Signal 1) in the presence of J. D. Milner et al. 2008. Impaired TH17 cell
gested to some investigators who were costimulatory signals (CD28, Signal 2) and differentiation in subjects with autosomal
savvy about the roles of helper T-cell sub- cytokines known to drive human TH17 dif- dominant Hyper-IgE syndrome. Nature
452:773–776.
sets that Type 1 HIES symptoms may be ferentiation (IL-1, IL-6, TGF-, and IL-23,
(Induced) TREG Cells tion by interacting directly with T cells. The depletion of
Another major CD4 T-cell subset negatively regulates iTREG cells in otherwise healthy animals leads to multiple
T-cell responses and plays a critically important role in autoimmune outbreaks, revealing that even healthy organ-
peripheral tolerance by limiting autoimmune T-cell activity. isms are continually warding off autoimmune responses.
This subset of T cells, designated induced TREG (iTREG) Recent data also indicate that iTREG cells are critically impor-
cells, is similar in function to the natural TREG cells tant for maintaining a mother’s tolerance to her fetus.
(nTREGs) that originate from the thymus (see Chapter 9).
Induced TREG cells, however, do not arise in the thymus, but TH17 and TREG Cross-Regulation
from naïve T cells that are activated in secondary lymphoid Just as TH1 and TH2 cells reciprocally regulate each other,
tissue in the presence of TGF- (see Overview Figure TREG and TH17 cells also cross-regulate each other. TGF-
11-11). TGF- induces expression of FoxP3, the master induces TREG differentiation; however, when accompanied
transcriptional regulator responsible for iTREG commitment. by IL-6, TGF- induces TH17 differentiation. Specifically
The iTREG cells secrete the effector cytokines IL-10 and TGF- TGF- appears to up-regulate both FoxP3 and ROR
, which down-regulate inflammation via their inhibitory (which control TREG and TH17 differentiation, respec-
effects on APCs, and can also exert their suppressive func- tively). In combination with signals generated by IL-6,
BOX 11-4
IL-17?
TCR and CD28 engagement

+/–
polarizing cytokines (IL-1/IL-23)
12 days
Naïve CD4+ T cells TH17 cells?

(a)
(b)
FIGURE 1
CD4ⴙ T cells from Hyper-IgE patients do not differentiate into TH17 cells. (a) Condi-
tions used to polarize CD4 T cells to the TH17 lineage in vitro. The ability of the cells to make IL-17 (a
feature of TH17 cells) or IFN- (a property of TH1, not TH17 cells) after they were polarized was assessed
by staining for intracellular cytokines. (b) Flow cytometric analysis of intracellular staining. The boxes
outlined in red indicate the quadrant where IL-17 (TH17) cells would appear. [Irvine, D.J. et al. 2002.
Nature 419: 845–849. Reprinted by permission from Macmillan Publishers]
signals generated by TGF- inhibit FoxP3 expression, let- independent lineage or a developmental option for other
ting ROR dominate and induce TH17 development. The helper lineages remains controversial. Like TH2 cells, TFH
TH17 versus iTREG relationship may be very adaptive. At cells play a central role in mediating B-cell help and are
rest, a healthy organism may favor the development of an found in B-cell follicles and germinal centers. However, the
anti-inflammatory iTREG population, which would be rein- effector cytokines secreted by follicular helper T cells differ
forced by the iTREG cell’s own production of TGF-. partially from those secreted by TH2 cells.
Inflammation, however, would induce the generation of Cytokines that polarize activated T cells toward the TFH
acute response proteins, including IL-6. In the presence of lineage include IL-6 and IL-21. These polarizing cytokines
IL-6, TGF- activity would shift development of T cells induce the expression of Bcl-6, a transcriptional repressor
away from iTREGs toward the pro-inflammatory TH17, so a that is thought to be TFH’s master transcriptional regulator
proper defense could be mounted. (see Overview Figure 11-11). Cross-regulation is also a
feature of TFH function: Bcl-6 expression inhibits T-bet,
TFH Cells GATA3, and RORt expression, thus inhibiting TH1, TH2,
Follicular helper T (TFH) cells are a very recent addition to and TH17 differentiation, respectively, while inducing TFH
the helper T-cell subset family. Whether they represent an polarization.
Although both TFH and TH2 cells secrete IL-4, TFH cells leprae, an intracellular pathogen that can survive within the
are best characterized by their secretion of IL-21, which phagosomes of macrophages. Leprosy is not a single clinical
induces B-cell differentiation. Interestingly, they can also entity; rather, the disease presents as a spectrum of clinical
produce IFN- (the defining TH1 cytokine). How TFH and responses, with two major forms of disease, tuberculoid and
TH2 cells divide responsibilities for inducing B-cell antibody lepromatous, at each end of the spectrum. In tuberculoid
production is still an open question. leprosy, cell-mediated immune responses destroy most of
the mycobacteria. Although skin and peripheral nerves are
Other Potential Helper T-Cell Subsets damaged in tuberculoid leprosy, it progresses slowly and
Other T-cell subsets with distinct polarizing requirements and patients usually survive. In contrast, lepromatous leprosy
unique cytokine secretion profiles have been identified (e.g., is characterized by a humoral response; cell-mediated
TH9 cells, which secrete IL-9 and IL-10). However, because immunity is depressed. The humoral response sometimes
these subpopulations secrete cytokines that are also produced results in markedly high levels of immunoglobulin (hyper-
by TH1, TH2, TH17, or iTREG cells, some speculate that these gammaglobulinemia). This response is not as effective in
cell types do not represent distinct subclasses but rather are inhibiting disease, and mycobacteria are widely dissemi-
developmental or functional variants of one of the major sub- nated in macrophages, often reaching numbers as high as
populations. This perspective has, indeed, been applied to the 1010 per gram of tissue. Lepromatous leprosy progresses
follicular helper T-cell (TFH) subset, which also expresses cyto- into disseminated infection of the bone and cartilage with
kines shared by several other subtypes. However, this subset extensive nerve and tissue damage.
has a distinct gene signature and a distinct master regulator The development of lepromatous or tuberculoid leprosy
(bcl-6), so most now consider it a bona fide independent lin- depends in part on the balance between TH1 and TH2
eage. Clearly, our understanding of the developmental rela- responses (Figure 11-12). In tuberculoid leprosy, the immune
tionship among effector subtypes will continue to evolve. response is characterized by a TH1-type response and high
circulating levels of IL-2, IFN-, and Lymphotoxin- (LT-).
In lepromatous leprosy, there is a TH2-type immune
Helper T Cells May Not Be Irrevocably
response, with high circulating levels of IL-4 and IL-5 (and
Committed to a Lineage IL-10, which can also be made by TH2 cells). This cytokine
Investigations now suggest that the relationship among TH profile explains the diminished cell-mediated immunity and
cell subpopulations may be more plastic than previously increased production of serum antibody in lepromatous
suspected. At early stages in differentiation, at least, helper leprosy.
cells may be able to shift their commitment and produce Presumably each of these patients was exposed to the
another subset’s signature cytokine(s). For example, when same pathogen. Why did some develop an effective TH1
exposed to IL-12, young TH2 cells can be induced to express response while others did not? Studies suggest that genetic
the signature TH1 cell cytokine, IFN-. Young TH1 cells can differences among human hosts play a role. For example, dif-
also be induced to express the signature TH2 cytokine, IL-4, ferences in susceptibility may correlate with individual differ-
under TH2 polarizing conditions. ences in the expression of PRRs (TLR1 and TLR2) expressed
Interestingly, TH1 and TH2 cells do not seem able to adopt
TH17 or iTREG characteristics. On the other hand, TH17 and
iTREG cells are more flexible and can adopt the cytokine TH1 activity TH2 activity
expression profiles of other subsets, including TH1 and TH2. Tuberculoid Lepromatous Tuberculoid Lepromatous
The TH17 subset may be the most unstable or “plastic” lineage
and can be induced to express IFN- and IL-4, depending on
environmental input. Some iTREG cells can also be induced to IL-2 IL-4
express IFN-, and some can be redirected toward a TH17
phenotype if exposed to IL-6 and TGF-. This fluidity among
IFN-␥ IL-5
subsets makes it difficult to definitively establish the indepen-
dence of helper cell lineages. In fact, some of the emerging
subgroups may be variants of TH1, TH2, TH17, and iTREG sub- LT-␣ IL-10
sets that have been exposed to other polarizing environments.
FIGURE 11-12 Correlation between type of leprosy and
Helper T-Cell Subsets Play Critical Roles relative TH1 or TH2 activity. Messenger RNA isolated from lesions
from tuberculoid and lepromatous leprosy patients was analyzed by
in Immune Health and Disease
Southern blotting using the cytokine probes indicated. Cytokines
Studies in both mice and humans show that the balance of characteristic of TH1 cells (IFN- and TNF-, for instance) predominate
activity among T-cell subsets can significantly influence the in the tuberculoid patients, whereas cytokines characteristic of TH2
outcome of the immune response. A now classic illustration cells (IL-4) predominate in the lepromatous patients. [From P. A. Sieling
of the influence of T-cell subset balance on disease outcome and R. L. Modlin, 1994, Cytokine patterns at the site of mycobacterial infection,
is provided by leprosy, which is caused by Mycobacterium Immunobiology 191:378.]
by innate cells. This makes sense given that interactions for activation than naïve T cells. For example, naïve T cells
between pathogen and innate immune cells determine the are activated almost exclusively by dendritic cells, whereas
cytokine environment that influences the outcome of T-cell memory T cells can be activated by macrophages, dendritic
polarization. Differences in TLR expression or activity could cells, or B cells. Memory cells express different patterns of
alter the quality or quantity of cytokines produced. surface adhesion molecules and costimulatory receptors that
Progression of HIV infection to AIDS may also be influ- allow them to interact effectively with a broader spectrum of
enced by T-cell subset balance. Early in the disease, TH1 APCs. They also appear to be more sensitive to stimulation
activity is high, but as AIDS progresses, some have suggested and respond more quickly. This may, in part, be due to their
that a shift may occur from a TH1-like to a TH2-like response, ability to regulate gene expression more readily because of
which is less effective at controlling viral infection. In addi- differences in epigenetic organization that occurred during
tion, some pathogens may “purposely” influence the activity their formation. Finally, memory cells display recirculation
of the TH subsets. The Epstein-Barr virus, for example, pro- patterns that differ from those of naïve or effector T cells.
duces a homolog (mimic) of human IL-10 called viral IL-10 Some stay for long periods of time in the lymph node and
(vIL-10). Like cellular IL-10, vIL-10 tends to suppress TH1 other secondary lymphoid organs, some travel to and
activity by inhibiting the polarizing activation of macro- remain in tertiary immune tissues where the original infec-
phages. Some researchers have speculated that vIL-10 may tion occurred, anticipating the possibility of another infec-
reduce the cell-mediated response to the Epstein-Barr virus, tion with the antigen to which they are specific.
thus conferring a survival advantage on the virus. As our ability to identify different cell surface proteins
TH17 cells first received attention because of their asso- improved, so has our ability to identify and distinguish mem-
ciation with chronic autoimmune disease. Mice that were ory cell subpopulations. We now broadly distinguish two
unable to make IL-23, a cytokine that contributes to TH17 subsets of memory T cells, central memory T cells (TCM) and
polarization, were remarkably resistant to autoimmunity. effector memory T cells (TEM), on the basis of their location,
TH17 cells and their defining effector cytokine, IL-17, are their patterns of expression of surface markers, and, to some
often found in inflamed tissue associated with rheumatoid extent, their function. Recent work also reveals a great deal of
arthritis, inflammatory bowel disease, multiple sclerosis, diversity within these subsets, whose relationship is still being
psoriasis, and asthma. However, the role TH17 cells played in clarified. We describe some useful generalizations below, and
protecting organisms from infection was not immediately close with the many questions that remain.
obvious. Studies of individuals with an autosomal dominant
form of a disease known as Hyper-IgE syndrome or Job syn- Naïve, Effector, and Memory T Cells Display
drome confirmed indications from mice that TH17 cells were
important in controlling infections by extracellular bacteria
Broad Differences in Surface Protein Expression
and fungi (see Clinical Focus Box 11-4). Three surfaces markers have been used to broadly distin-
These disorders and those described in Chapters 15 and 16 guish naïve, effector, and memory T cells: CD44, which
are just some examples of the influence of helper T-cell subsets increases in response to activation signals; CD62L, an adhe-
on disease development. It is important to recognize that our sion protein; and CCR7, a chemokine receptor. Both CD62L
current perspectives of the roles of helper subsets in disease and CCR7 are involved in homing to secondary lymphoid
and health are still simplistic. Our developing appreciation of organs (Table 11-4). Naïve T cells express low levels of CD44,
the complexity of the interplay among subsets will improve reflecting their unactivated state, and high levels of the adhe-
and add more subtlety to our explanations in the future. sion molecule CD62L, directing them to the lymph node or
spleen. In contrast, effector helper and cytotoxic T cells have
the reciprocal phenotype. They express high levels of CD44,
T-Cell Memory indicating that they have received TCR signals, and low lev-
els of CD62L, which prevents them from recirculating to
T-cell activation results in a proliferative burst, effector cell secondary lymphoid tissue, allowing them to thoroughly
generation, and then a dramatic contraction of cell number. probe sites of infection in the periphery.
At least 90% of effector cells die by apoptosis after pathogen Both types of memory T cells also tend to express CD44,
is cleared, leaving behind an all-important population of indicating that they are antigen experienced (i.e., have
antigen-specific memory T cells. Memory T cells are gener- received signals through their TCR). Like naïve T cells, cen-
ally long-lived and quiescent, but respond with heightened tral memory cells (TCM) express CD62L and the chemokine
reactivity to a subsequent challenge with the same antigen. receptor, CCR7, consistent with their residence in secondary
This secondary immune response is both faster and more lymphoid organs. Effector memory cells (TEM), which are
robust, and hence more effective than a primary response. found in a variety of tissues, can express varying levels of
Until recently, memory cells were difficult to distinguish CD62L depending on their locale; however, they do not
from effector T cells and naïve T cells by phenotype, and for express CCR7, reflecting their travels through and residence
some time they were defined best by function. Like naïve T in nonlymphoid tissues. Other markers have been used to
cells, most memory T cells are resting in the G0 stage of the distinguish subtypes of memory cells, but these still provide
cell cycle, but they appear to have less stringent requirements a useful starting point for gauging the status of a T cell.
investigators contest the definitive distinction between TCM

Surface proteins that are used
and TEM cells and emphasize the diversity and continuum of
TABLE 11-4 to distinguish naïve, effector,
variations among these subtypes. Hence, memory cell biol-
and memory T cells
ogy is one of the most active fields of investigation, one that
Cell type CD44 CD62L CCR7 is critical to our ability to develop the best vaccines.
Naïve T cell low

How and When Do Memory Cells Arise?
Effector T cell low
Current work suggests that memory cells arise very early in the
Effector memory T cell variable
course of an immune response (e.g., within 3 days), but their
Central memory T cell cell of origin remains controversial. Some investigations sug-
gest that memory cells arise as soon as naïve T cells are acti-
vated. Others suggest that memory cells arise from more fully
TCM and TEM Are Distinguished by Their Locale differentiated naïve T cells. Still others raise the intriguing pos-
sibility that naïve T-cell activation generates a “memory stem
and Commitment to Effector Function cell” that is self-renewing and gives rise to memory effector cell
A small proportion ( 10%) of the progeny of a naïve cell populations. These models are not mutually exclusive, and it is
that has proliferated robustly in response to antigen differen- possible that memory cells can arise at several different stages
tiates into TCM and TEM cells. In general, these two subsets of T-cell activation throughout a primary response.
are distinguished by where they reside as well as their level The relationship between TCM and TEM cells is also
of commitment to a specific effector cell fate. debated. They may originate independently from naïve and
In general, TCM cells reside in and travel between second- effector cells, respectively, or may give rise to each other.
ary lymphoid tissues. They live longer and have the capacity Studies suggest, in fact, that TCM cells arise from TEM cells,
to undergo more divisions than their TEM counterparts. and one possible model of relationships is shown in Figure
When they reencounter their cognate pathogen in second- 11-13. Here, investigators speculate that TCM cells arise prior
ary lymphoid tissue, they are rapidly activated and have the to TEM cells, from cells at an earlier stage of differentiation
capacity to differentiate into a variety of effector T-cell sub- into effector (helper or cytotoxic) T cells. TEM cells arise late,
types, depending on the cytokine environment. and also may develop from fully differentiated effector cells.
On the other hand, TEM cells travel to and between ter- The model also suggests that effector cells can replenish cen-
tiary tissues (including skin, lung, liver, and intestine). They tral memory cells.
are arguably better situated to contribute to the first line of It should be stressed, however, that several other models
defense against reinfection because they have already com- have also been advanced. For instance, recent work suggests
mitted to an effector lineage during the primary response interactions experienced by effector cells determines their TCM
and exhibit their effector functions quite rapidly after reacti- versus TEM fate. Effector cells that interact with B cells may
vation by their cognate pathogen. preferentially develop into central and not effector memory T
It is important to note that some of these generalizations cells. New models may also need to incorporate intriguing
may not hold up to scientific scrutiny. For instance, some recent observations, including the possibilities that (1) memory
Proliferation and differentiation
Effector T cell
Naïve T cell
Central memory Effector memory

T cell ? T cell
FIGURE 11-13 One possible model for the development effector cell features. The model also includes the possibilities
of central and effector memory T cells. This model, only that (1) some effector memory T cells arise from fully differenti-
one of several that have been advanced, suggests that central ated effector T cells and (2) effector memory T cells can develop
memory cells arise early after naïve T-cell activation, perhaps from into central memory T cells. [Adapted from D. Gray, 2002, A role for
the first divisions. Effector memory T cells may arise later, after antigen in the maintenance of immunological memory, Nature Reviews
the progeny have divided more and have assumed at least some Immunology 2:60.]
cells arise from the asymmetric cell division of activated T cells, of cytokines produced. However, the cellular origin of this
where one daughter cell becomes an effector cell, and another diversity is still under investigation. Specifically, does this
contributes to the memory pool, and (2) that T-cell activation diverse memory response strictly reflect the functional effec-
generates a self-renewing memory stem cell population that tor diversity generated during the primary response? Or
provides a long-term source of memory T cells. does it develop anew from central memory T cells respond-
ing to different environmental cues during rechallenge? The
What Signals Induce Memory Cell answer is likely to be “Both,” but investigations continue.
Commitment?
Are There Differences between CD4
Most investigators agree that T-cell help is critical to generat-
ing long-lasting memory. For instance, CD8 T cells can be and CD8 Memory T Cells?
activated in the absence of CD4 T-cell help, but this “help- The simple answer is “Maybe.” Memory CD8 T cells are
less” activation event does not yield long-lived memory clearly more prevalent than memory CD4 T cells. This is
CD8 T cells. The relative importance of other variables in partly because CD8 T cells proliferate more robustly and
driving memory development is still under investigation. therefore generate proportionately more memory T cells. It
Although intensity of T-cell receptor engagement was may also be due to differences in the life span of memory T
thought to be a factor in memory cell commitment, recent cells: CD4 memory T cells may not be as long-lived as
data suggest that even low-affinity interactions can generate CD8 memory T cells.
memory T cells. All studies, however, appear consistent with
the recognition that the more proliferation a response How Are Memory Cells Maintained
inspires, the better the memory pool.
over Many Years?
Do Memory Cells Reflect the Heterogeneity of Whether memory cells can persist for years in the absence of
Effector Cells Generated during a Primary antigen remains controversial, although evidence seems to
favor the possibility that they do. Regardless, it does seem
Response? that memory persistence depends on the input of cytokines
We have seen that naïve T cells differentiate into a wide vari- that induce occasional divisions, a process known as homeo-
ety of effector T-cell subpopulations, largely determined by static proliferation, which maintains the pool size by balanc-
the cytokine signals they receive during activation. Studies ing apoptotic events with cell division. Both IL-7 and IL-15
indicate that the memory cell response is also very diverse, appear important in enhancing homeostatic proliferation,
in terms of both the T-cell receptor specificities and the array but CD4 and CD8 memory T-cell requirements may differ.
S U M M A R Y
■ T-cell activation is the central event in the initiation of the ■ CD8 T cells, which recognize MHC class I-peptide com-
adaptive immune responses. It results from the interaction plexes that are expressed by virtually all cells in the body,
in a secondary lymphoid tissue between a naïve T cell and become cytotoxic (TC) cells with the capacity to kill
an APC, specifically a dendritic cell. infected cells.
■ Activation of naïve T cells leads to the differentiation of effec- ■ CD4 T cells, which recognize MHC class II-peptide
tor cells, which regulate the response to pathogen, and of complexes that are expressed by professional APCs,
long-lived memory T cells, which coordinate the stronger and become helper (TH) cells, secreting cytokines that regulate
quicker response to future infections by the same pathogen. (positively and negatively) cells that clear infection,
■ Three distinct signals are required to induce naïve T-cell acti- including B cells, macrophages, and other T cells.
vation, proliferation, and differentiation. Signal 1 is generated ■ CD4 T cells differentiate into at least five main subpopula-
by the interaction of the TCR-CD3 complex with an MHC- tions of effector cells: TH1, TH2, TH17, iTREG, and TFH. Each
peptide complex on a dendritic cell. Signal 2 is a costimula- subpopulation is characterized by (1) a unique set of polar-
tory signal provided by the interaction between molecules of izing cytokines that initiate differentiation, (2) a unique mas-
the B7 family expressed by APC with the positive costimula- ter transcriptional regulator that regulates the production of
tory molecules CD28 or ICOS expressed by T cells. Signal 3 helper-cell-specific genes, and (3) a distinct set of effector
is provided by soluble cytokines and plays a key role in deter- cytokines that they secrete to regulate the immune response.
mining the type of effector cell that a T cell becomes. ■ TH1 and TH17 cells generally enhance cell-mediated
■ In the absence of a costimulatory signal (Signal 2), T-cell immunity and inflammatory responses. TH2 and TFH cells
receptor engagement results in T-cell inactivity or clonal enhance humoral immunity and antibody production,
anergy. and induced TREG cells inhibit T-cell responses.
■ Helper T-cell subsets have also been associated with dis- ■ Two types of memory T cells have been described. Central
ease and play a role in the development of autoimmunity memory (TCM) cells are longer lived, reside in secondary
and allergy. lymphoid tissues, and can differentiate into several differ-
■ Memory T cells, which are more easily activated than ent effector T cells. Effector memory (TEM) cells populate
naïve cells, are responsible for secondary responses. the sites of infection (tertiary tissues) and immediately
reexpress their original effector function after reexposure
■ The generation of memory B cells as well as CD4 and
to antigen.
CD8 memory T cells requires T-cell help.
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http://wenliang.my web.uga.edu/mystudy/
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Zhu, J., and W. Paul. 2010. Heterogeneity and plasticity of T Dependency_of_Mycobacterium_leprae You will
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microbiology resource” originating from Kenyon College.
Useful Web Sites http://users.rcn.com/jk imball.ma.ultranet/
The following are examples of well-organized Web sites B i o l o g y Pa g e s / T / T H 1 _ T H 2 . h t m l # Ty p e s _ o f _
developed by undergraduate students, graduate students, Helper_T_Cells This selection is from immunologist and
and teachers of immunology who have done an excellent job teacher John Kimball’s online version of his textbook.
S T U D Y Q U E S T I O N S
CLINICAL AND EXPERIMENTAL FOCUS QUESTION Multiple that the neuronal cell precursors secrete a cytokine called
sclerosis is an autoimmune disease in which TH cells partici- Leukemia Inhibiting Factor (LIF). In fact, the administration
pate in the destruction of the protective myelin sheath around of this factor, alone, ameliorated symptoms.
neurons in the central nervous system. Each person with These investigators were curious to know if this cytokine had
this disease has different symptoms, depending on which an effect on T-cell activity. They added LIF to cultures of (nor-
neurons are affected, but the disease can be very disabling. mal) T cells that were being stimulated under different polariz-
Recent work in a mouse model of this disease suggests that ing conditions (i.e., TCR and CD28 engagement in the presence
transplantation of cell precursors of neurons may be a good of cytokines that drive differentiation to distinct T helper sub-
therapy. Although these immature cells may work because sets). They stained T cells for cytokine production and analyzed
they can develop into neuronal cells that replace the lost my- the results by flow cytometry. The data below show their results
elin sheath, some investigators realized that they play another, from normal mouse T cells polarized to the lineage indicated in
perhaps even more important role. These scientists showed the absence (top, control) or presence (bottom) of LIF.
TH17 TH1 TREG
22.1 0.6 2.9 1.2 36.9

Control
3.4 27.8
11.2 0.7 2.7 0.6 37.7

LIF
FoxP3
IL-17
5.2 29.1
IFN-γ CD4
Which T helper lineage(s) is(are) most affected by the addi- 5. The following sentences are all false. Identify the error(s)
tion of LIF? Explain your answer. Why might these results and correct.
explain the beneficial effect of LIF on the disease? Knowing
a. Macrophages activate naïve T cells better than dendritic
what you know now about the molecular events that influence
cells.
T-cell differentiation, speculate on the molecular basis for the
b. ICOS enhances T-cell activation and is called a negative
activity of LIF.
coreceptor.
c. Virtually all cells in the body express costimulatory
1. Which of the following conditions would lead to T-cell
ligands.
anergy?
d. CD28 is the only costimulatory receptor that binds to
a. A naïve T-cell interaction with a dendritic cell in the B7 family members.
presence of CTLA-4 Ig. e. Signal 3 is provided by negative costimulatory recep-
b. A naïve T cell stimulated with antibodies that bind both tors.
the TCR and CD28. f. Toxic shock syndrome is an example of an autoimmune
c. A naïve T cell stimulated with antibodies that bind only disease.
the TCR. g. Superantigens mimic TCR-MHC class I interactions.

d. A naïve T cell stimulated with antibodies that bind only h. CD4 T cells interact with MHC class I on CD8 T
CD28. cells.
i. Naïve T cells produce IFN-.
2. A virus enters a cut in the skin of a mouse and infects den-
j. T-bet and GATA-3 are effector cytokines.
dritic cells, stimulating a variety of PRRs both on and
k. Polarizing cytokines are only produced by APCs.
within dendritic cells that induce it to produce IL-12. The
l. Bcl-6 is involved in the delivery of costimulatory
mouse subsequently mounts an immune response that suc-
signals.
cessfully clears the infection. Which of the following state-
m. TH17 and TFH cell subsets are the major sources of
ments is(are) likely to be true about the immune response
B-cell help.
that occurred? Correct any that are false.
n. iTREG cells enhance inflammatory disease.
a. The infected dendritic cells up-regulated CD80/CD86 o. Effector cytokines act exclusively on T cells.
and MHC class II. p. Central memory T cells tend to reside in the site of
b. The dendritic cells encountered and activated naïve T infection.
cells in the skin of the mouse. q. Like naïve T cells, effector memory cells express CCR7.
c. Naïve T cells activated by these dendritic cells generated
6. Like TH1 and TH2 cells, TH17 and TREG cells cross-regulate
signals that released internal Ca2 stores.
each other. Which of these two statements about this cross-
d. Naïve T cells activated by these dendritic cells were
regulation is (are) true? Correct either, if false.
polarized to the TH2 lineage.
e. Only effector memory T cells were made in this mouse. a. TGF- is a polarizing cytokine that stimulates up-
regulation of each of the master transcriptional regula-
3. Your lab acquires mice that do not have the GATA-3 gene
tors that polarize T cells to the TH17 and TREG lineages.
(GATA-3 knockout mice). You discover that this mouse
b. IL-6 inhibits polarization to the TREG lineage by inhibit-
has a difficult time clearing helminth (worm) infections.
ing expression of ROR.
Why might this be?
7. A new effector T-cell subset, TH9, has been recently identi-
4. You isolate naïve T cells from your own blood and want to
fied. It secretes IL-9 and IL-10 and appears to play a role in
polarize them to the TH1 lineage in vitro. You can use any of
the protection against intestinal worm infection. What
the following reagents to do this. Which would you choose?
other information about this subset would help you to
Anti-TCR antibody CTLA-4 Ig IL-12 determine if it should be considered an independent helper
IL-4 anti-CD80 antibody IL-17 T-cell lineage?
IFN anti-CD28 antibody

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c10B-CellDevelopment.

indd Page 329 12/20/12 3:23 PM user-t044 /Volumes/203/MHR00209/siL52070/disk1of1/0071052070

330 PA R T I V | Adaptive Immunity: Development

OVERVIEW FIGURE 10-1

B-Cell Development Begins in the Bone Marrow and Is

B-Cell Development | CHAPTER 10 331

Specification Emergence Maturation Expansion Quiescence/

Mesoderm Pre-HSC HSC HSC

7.5 10.5 12.5 15.5 days Birth

AGM Bone marrow

Primitive Yolk sac

332 PA R T I V | Adaptive Immunity: Development

B-Cell Development | CHAPTER 10 333

CLINICAL FOCUS Box 10-1

334 PA R T I V | Adaptive Immunity: Development

Pro-B cell IL-7

Pre-pro- Pre-B cell

B-Cell Development | CHAPTER 10 335

Characterization of progenitors bearing Knocking out particular transcription factors

Knocking out TF1 leaves only this population. Therefore

Determining sequence of marker expression by culturing cells

D-JH VHDJH recombination VH and VL VHDJH VH and VL

336 PA R T I V | Adaptive Immunity: Development

The Role of miRNAs in the Control of B-Cell Development

Geneticists have long known that

B-Cell Development | CHAPTER 10 337

338 PA R T I V | Adaptive Immunity: Development

c-Kit sca-1 flt-3 CD34 IL-7R Rag1/2

Common low low + – + +

B-Cell Development | CHAPTER 10 339

MPPs in the B-cell differentiation pathway. Signaling through

340 PA R T I V | Adaptive Immunity: Development

Pre-pro GL2 None – lo – – +

Early pro DJH None lo lo/+ – + +

Late pro Some None lo + +/– + +

Large Pre VHDJH Pre-BCR – + + + + FIGURE 10-6 Immunoglobulin gene

Classic Experiment Box 10-1.

B-Cell Development | CHAPTER 10 341

CLASSIC EXPERIMENT BOX 10-3

The Stages of B-Cell Development: Characterization

342 PA R T I V | Adaptive Immunity: Development

CLASSIC EXPERIMENT (continued)

rearrangements occur in fraction C

Pro-B Cells Chapter 3, as one of the components of the B-cell co-receptor.

B-Cell Development | CHAPTER 10 343

the VH to the D Ig gene segment, indicating that PAX5 is Pre-B Cells

344 PA R T I V | Adaptive Immunity: Development

B-Cell Development | CHAPTER 10 345

346 PA R T I V | Adaptive Immunity: Development

(a) H-2d transgenics

Mature B cells express

(b) H-2d/k transgenics

No mature B cells express

(c) H-2d/k transgenics

A few mature B cells with new

Experimental animal Number of animals tested As membrance Ab As secreted Ab (␮g/ml)

Nontransgenics 13 (⫺) ⬍0.3

B-Cell Development | CHAPTER 10 347

Bone marrow Central