Immunology 9Th Edition Male David Full Chapter
Immunology 9Th Edition Male David Full Chapter
Immunology 9Th Edition Male David Full Chapter
IMMUNOLOGY
David Male, BA, MA, PhD
Professor of Biology
Department of Life Sciences
The Open University
Milton Keynes, United Kingdom
The right of David Male, R. Stokes Peebles, Jr., and Victoria Male to be identified as authors of this work has been
asserted by them in accordance with the Copyright, Designs and Patents Act 1988.
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Notices
Practitioners and researchers must always rely on their own experience and knowledge in evaluating
and using any information, methods, compounds or experiments described herein. Because of rapid
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ISBN: 978-0-7020-7844-6
International ISBN: 978-0-7020-7845-3
Printed in Poland
This is the first edition of Immunology that has not included two elements that we consider essential for understanding basic
of our original editors, Ivan Roitt and Jonathan Brostoff. We and clinical immunology; the online version includes additional
would like to pay tribute to their foresight in developing this information at appropriate points (indicated by a symbol in
text, which was originally planned as a slide atlas of immuno- the margin), for readers who want to delve deeper. The critical
logy. They have steered the book through its eight previous edi- thinking sections that follow each chapter require an under-
tions, during which time the subject has advanced beyond all standing of the material presented and the implications in a lab-
recognition. In 1985 when the first edition was published, the oratory or clinical setting—they may be used as the basis of class
structure and function of antibodies were well known and discussion. Another important teaching tool is the summaries
MHC molecules had just been described, but how T cells which distil the key points of each chapter and are a solid basis
became activated was still a matter of conjecture and debate. for revision for exams.
Nowadays, antibodies have become key therapeutic agents, The contributors to this edition include many experts in dif-
not just for immunological conditions, but particularly for treat- ferent fields of immunology, with seven new contributors who
ment of cancer and the targeting of therapeutic agents. Cytokine- have brought their own expertise to individual chapters. We also
based treatments for many diseases are following closely behind. greatly appreciate the hard work of colleagues at Elsevier, par-
Hence, the subject of immunology impinges on diverse areas ticularly Trinity Hutton, Alex Mortimer and Karthikeyan
of clinical practice, as well as providing tools and important Murthy.
theoretical concepts for many of the biological sciences. Immunology bridges basic science and medicine and encom-
For this edition, and with two new editors, we have made passes genetics, cell biology and molecular biology. Advances in
a major reorganisation in the first half of the book, with innate biotechnology in the last 10 years have driven forward antibody-
immunity and cell-mediated immunity introduced first. based therapies. In the next 10 years we anticipate that under-
This rearrangement responds to our improved understanding standing of genetic diversity in the immune system will lead to
of these areas of immunology, and it also presents material advances in personalised medicine, while gene therapies are
in a more logical chronological order, since innate immune reac- becoming available to correct primary immunodeficiencies.
tions and lymphocyte activation precede antibody production. For the past century, immunology has fascinated and inspired
Despite these changes we have maintained the overall bal- some of the greatest scientific thinkers and Nobel prize winners.
ance of the text with the first two sections describing how the Most recently the prize for Medicine or Physiology was awarded
immune system works. Section three is concerned with immune to James Allison and Tasuko Honjo for advances in cancer
responses against pathogens—the primary function of the immunotherapy. We wish our readers well in their study of
immune system—and the final three sections deal with aspects immunology, a subject that continues to excite and surprise
of clinical immunology, including autoimmune disease, immu- us, and which underpins many areas of medicine and biomed-
nodeficiency, transplantation, tumour immunology and hyper- ical science.
sensitivity. All chapters have been fully updated with many new David Male
diagrams. R. Stokes Peebles, Jr.
We have followed the style of the 8th edition by including two Victoria Male
levels of detail in the text. The printed text includes those 2019
vii
CONTRIBUTORS
The editors would like to acknowledge and offer grateful thanks for the input of all previous editions’ contributors, without whom this
new edition would not have been possible.
Gregory J. Bancroft, BSc Hons, PhD David Isenberg, MD, FRCP, FAMS Luisa Martinez-Pomares, BSc, PhD
Professor Professor Associate Professor
Department of Infection Biology The Centre for Rheumatology Research, School of Life Sciences
Faculty of Infectious and Tropical Diseases Department of Medicine University of Nottingham
London School of Hygiene & Tropical University College London Nottingham, United Kingdom
Medicine London, United Kingdom
London, United Kingdom
Bryan Paul Morgan, BSc, MBBCh, PhD,
Roy Jefferis, BSc, PhD, FRSC, CChem, FRCPath, MRCP
David Bending, BA, MA, PhD
MRCP, FRCPath, DSc Professor of Immunology
Institute of Immunology and
Emeritus Professor School of Medicine
Immunotherapy
Institute of Immunology & Immunotherapy Cardiff University
University of Birmingham
University of Birmingham Cardiff, United Kingdom
Birmingham, United Kingdom
Birmingham, United Kingdom
Persephone Borrow, BA, MA, PhD
Professor of Viral Immunology Thomas Kamradt, Dr. med. Luigi D. Notarangelo, MD
Nuffield Department of Clinical Medicine Professor Chief
University of Oxford Department of Immunology Laboratory of Clinical Immunology
Oxford, United Kingdom University Hospital Jena and Microbiology
Jena, Germany National Institute of Allergy and
Colin Casimir, BSc, PhD Infectious Diseases, National
Department of Natural Sciences Institutes of Health
Middlesex University Yasmin Khan, MD Bethesda, Maryland, United States
London, United Kingdom Assistant Professor of Pediatrics
Department of Pediatric Allergy,
Daniel Cook, MD, PhD Immunology, and Pulmonary Medicine R. Stokes Peebles, Jr., MD
Resident Physician Vanderbilt University Medical Center Elizabeth and John Murray
Department of Internal Medicine Nashville, Tennessee, United States Professor of Medicine
Vanderbilt University Medical Center Division of Allergy, Pulmonary,
Nashville, Tennessee, United States and Critical Care Medicine
Peter Maldwyn Lydyard, BSc, MSc, PhD, Vanderbilt University School of Medicine
FRCPath Nashville, Tennessee, United States
David P. D’Cruz, MD, FRCP Emeritus Professor
Consultant Rheumatologist University College London
The Louise Coote Lupus Unit Visiting Professor
Guy’s and St Thomas’ Hospitals Thomas A.E. Platts-Mills, MD,
University of Westminster PhD, FRS
London, United Kingdom London, United Kingdom Head, Asthma and Allergic Disease Center
Daniel Dulek, MD Department of Medicine
Assistant Professor Arti Mahto, BSc, MBBCh, MRCP, PhD University of Virginia
Department of Pediatric Infectious Diseases Department of Rheumatology Charlottesville, Virginia, United States
Vanderbilt University Medical Center University College Hospital,
Nashville, Tennessee, United States London, United Kingdom
Richard John Pleass, BSc, MSc, PhD
Professor
Hakimeh Ebrahimi-Nik, Doctorate of
David Male, BA, MA, PhD Department of Parasitology
Veterinary Medicine, PhD
Professor of Biology Liverpool School of Tropical Medicine
Postdoctoral Fellow
Department of Life Sciences Liverpool, Merseyside, United Kingdom
Department of Immunology
The Open University
UConn Health
Milton Keynes, United Kingdom
Farmington, Connecticut, United States
Nina Porakishvili, BSc, MSc, PhD
School of Life Sciences
Andrew George, MBE, MA, PhD, DSc, Victoria Male, BA, MA, PhD
University of Westminster
FRCPath, FHEA, FRSA, FRSB Sir Henry Dale Fellow
London, United Kingdom
Emeritus Professor Department of Metabolism
Brunel University London Digestion and Reproduction
Uxbridge, United Kingdom Imperial College London
London, United Kingdom
ix
x CONTRIBUTORS
Theo Rispens, PhD Pramod K. Srivastava, PhD, MD Gestur Vidarsson, BSc, MSc, PhD
Department of Immunopathology Professor of Immunology and Medicine Head of Laboratory
Sanquin Research, Amsterdam Director, Carole and Ray Neag Department of Experimental
Amsterdam, Netherlands Comprehensive Cancer Center and Immunohematology/Immunoglobulin
Department of Immunology Research Laboratory
University of Connecticut School of Medicine Sanquin Research
Farmington, Connecticut, United States Amsterdam, Netherlands
SECTION 1 The Immune System and Innate Immunity
1
Introduction to the Immune System
SUMMARY
• The immune system has evolved to protect us from pathogens. Intra- • Antigens are molecules that are recognized by receptors on B cells
cellular pathogens infect individual cells (e.g. viruses), whereas extracellular and T cells. B cells usually recognize intact antigen molecules, whereas T
pathogens divide outside cells in blood, tissues or the body cavities (e.g. many cells recognize antigen fragments displayed on the surface of the body’s
bacteria and parasites). These two kinds of pathogen require fundamentally own cells.
different immune responses. • An immune response occurs in two phases – antigen recognition and
• Phagocytes and lymphocytes are key mediators of immunity. Phago- antigen eradication. In the first phase, clonal selection involves recognition
cytes internalize pathogens and degrade them. Lymphocytes (B and T cells) of antigen by particular clones of lymphocytes, leading to expansion of specific
have receptors that recognize specific molecular components of pathogens clones of T and B cells and differentiation to effector and memory cells. In the
and have specialized functions. B cells make antibodies (effective against effector phase, these lymphocytes coordinate an immune response, which
extracellular pathogens), cytotoxic T lymphocytes (CTLs) kill virally infected eliminates the source of the antigen.
cells and helper T cells coordinate the immune response by direct cell–cell • Vaccination depends on the specificity and memory of adaptive
interactions and the release of cytokines. immunity. Vaccination is based on the key elements of adaptive immunity,
• Inflammation is a response to tissue damage. It allows antibodies, com- namely specificity and memory. Memory cells allow the immune system to
plement system molecules and leukocytes to enter the tissue at the site of mount a much stronger and more rapid response on a second encounter with
infection, resulting in phagocytosis and destruction of the pathogens. Lympho- antigen.
cytes are also required to recognize and to destroy infected cells in the tissues. • The immune system may fail (immunopathology). This can be a result of
• Specificity and memory are two essential features of adaptive immunodeficiency, hypersensitivity or dysregulation leading to autoimmune
immune responses. As a result, the adaptive arm of the immune system diseases.
(B and T lymphocytes) mounts a more effective response on second and sub- • Normal immune reactions can be inconvenient in modern medicine,
sequent encounters with a particular antigen. Non-adaptive (innate) immune for example blood transfusion reactions and graft rejection.
responses (mediated, for example, by complement and phagocytes) do not
alter on repeated exposure to an infectious agent.
The immune system is fundamental to survival, as it protects • It introduces the basic elements of the immune system and
the body from pathogens: viruses, bacteria and parasites that of immune responses, which are mediated principally by
cause disease. To do so, it has evolved a powerful collection white blood cells or leukocytes (from the Greek for white
of defence mechanisms to recognize and protect against poten- cell) and are detailed in Chapters 2–13.
tial invaders that would otherwise take advantage of the rich Over many millions of years, different types of immune
source of nutrients provided by the vertebrate host. At the same defence, appropriate to the infecting pathogens, have evolved
time it must differentiate between the individual’s own cells and in different groups of organisms. In this book, we concentrate
those of harmful invading organisms while not attacking the on the immune systems of mammals, especially humans.
beneficial commensal flora that inhabit the gut, skin and other Because mammals are warm-blooded and long-lived, their
tissues. immune systems have evolved particularly sophisticated sys-
This chapter provides an overview of the complex network tems for recognizing and destroying pathogens.
of processes that form the immune system of higher Many of the immune defences that have evolved in other
vertebrates: vertebrates (e.g. reptiles, amphibians) and other phyla (e.g.
• It illustrates how the components of the immune system fit sponges, worms, insects) are also present in some form in
together to allow students to grasp the big picture before mammals. Consequently the mammalian immune system con-
delving into the material in more depth in subsequent sists of multi-layered, interlocking defence mechanisms that
chapters. incorporate both ancient and recently evolved elements.
1
2 SECTION 1 The Immune System and Innate Immunity
leukocytes
other
cell lymphocytes phagocytes auxiliary cells
B T ILC
Fig. 1.1 Components of the immune system The principal cells of the immune system and the mediators they
produce are shown. Neutrophils, eosinophils and basophils are collectively known as polymorphonuclear gran-
ulocytes (see Chapter 2). B cells and T cells have highly specific receptors for foreign material (antigens),
whereas innate lymphoid cells (ILCs) do not have the specific receptors. Cytotoxic describes the function of
different cells, including cytotoxic T lymphocytes (CTLs), natural killer (NK) cells (a type of ILC) and eosinophils.
Complement is made primarily by the liver, although there is some synthesis by mononuclear phagocytes. Note
that each cell produces and secretes only a particular set of cytokines or inflammatory mediators.
Treg
antigen antigen
presentation presentation
B
Fig. 1.5 Functions of different types of lymphocyte Macrophages present antigen to TH1 cells, which then
activate the macrophages to destroy phagocytosed pathogens. B cells present antigen to TH2 cells, which acti-
vate the B cells, causing them to divide and differentiate into antibody-secreting plasma cells. TH17 cells help to
protect mucosal surfaces by attracting and activating other leukocytes. Cytotoxic T lymphocytes (CTL) and nat-
ural killer cells (NK) recognize and destroy virally infected cells. Regulatory T cells (Treg) modulate activity of
other T-cell populations.
of immunity towards a site of infection. Inflammation is medi- Complement proteins mediate phagocytosis, control
ated by a variety of other cells, including basophils, mast cells inflammation and interact with antibodies in immune
and platelets. defence. The complement system, a key component of innate
Basophils and mast cells have granules that contain a variety immunity, is a group of about 20 serum proteins whose overall
of mediators, which induce inflammation in surrounding tissues function is to promote inflammation (Fig. 1.6) and clearance of
and are released when the cells are triggered. Basophils and mast microbes and damaged cells. The components interact with
cells can also synthesize and secrete a number of mediators that each other and with other elements of the immune system.
control the development of immune reactions. Mast cells lie For example, a number of microorganisms spontaneously acti-
close to blood vessels in most tissues and some of their media- vate the complement system, via the so-called ‘alternative path-
tors act on cells in the vessel walls. Basophils are functionally way’, which is an innate immune defence. This results in the
similar to mast cells, but are mobile, circulating cells. microorganism being opsonized (i.e. coated by complement
Platelets are small cellular fragments that are essential in molecules, leading to its uptake by phagocytes). The comple-
blood clotting, but they can also be activated during immune ment system can also be activated by antibodies bound to the
responses to release mediators of inflammation. pathogen via the ‘classical pathway’ or by mannose binding
lectin bound to the pathogen surface via the ‘lectin pathway’.
Soluble Mediators of Immunity Complement activation is a cascade reaction, where one
A wide variety of molecules are involved in the development of component acts enzymatically on the next component in the
immune responses, including antibodies, opsonins and comple- cascade to generate an enzyme, which mediates the following
ment system molecules. The serum concentration of a number step in the reaction sequence, and so on. (The blood clotting sys-
of these proteins increases rapidly during acute infection and tem also works as an enzyme cascade.)
they are therefore called acute phase proteins. Activation of the complement system generates protein mol-
One example of an acute phase protein is C-reactive protein ecules or peptide fragments, which have the following effects:
(CRP), so-called because of its ability to bind to the C protein • opsonization of microorganisms for uptake by phagocytes
of pneumococci; it promotes the uptake of pneumococci by pha- and eventual intracellular killing;
gocytes. Molecules such as CRP that promote phagocytosis are • attraction of phagocytes to sites of infection (chemotaxis);
said to act as opsonins. There are a number of these evolution- • increased blood flow to the site of activation and increased
arily ancient molecules in mammals and they recognize conserved permeability of capillaries to plasma molecules;
structures on the surface of pathogens called pathogen-associated • damage to plasma membranes on cells, Gram-negative bac-
molecular patterns (PAMPs) (see Chapter 3). Another important teria, enveloped viruses, or other organisms that have caused
group of molecules that can act as opsonins are components of complement activation;
the complement system (see Chapter 4). • release of inflammatory mediators from mast cells.
CHAPTER 1 Introduction to the Immune System 5
complement
IFN␣
IFN
virus
infected cell
virus-resistant
bacteria phagocyte bacteria T IFN␥ cell
antigen
1. lysis 2. chemotaxis 3. opsonization
Fig. 1.7 Interferons Host cells that have been infected by virus secrete
interferon-α (IFNα) and/or interferon-β (IFNβ). TH1 cells secrete interferon-
γ (IFNγ) after activation by antigens. IFNs act on other host cells to induce
resistance to viral infection. IFNγ has many other effects.
1 2 3
Fig. 1.8 Three phases in neutrophil migration across endothelium A neutrophil adheres to the endothelium
in a venule (1). It extends its pseudopodium between the endothelial cells and migrates towards the basement
membrane (2). After the neutrophil has crossed into the tissue, the endothelium reseals behind (3). The entire
process is referred to as diapedesis. (Courtesy Dr I Jovis.)
CHAPTER 1 Introduction to the Immune System 7
infected cell
MHC molecule presents
peptide
2
complement antigen peptide bound
C3b to MHC molecule
4
antibody and
complement
C3b
Fig. 1.15 T-cell recognition of antigen Major histocompatibility com-
plex (MHC) molecules transport peptides to the surface of an infected cell
Fig. 1.14 Opsonization Phagocytes have some intrinsic ability to bind to where they are presented to T cells, which may recognize the MHC–
bacteria and other microorganisms via their pattern recognition receptors peptide combination. If a cell is infected, MHC molecules present
(1). Binding is much enhanced if the bacteria have been opsonized by peptides derived from the pathogen and the cell’s own proteins.
complement C3b (2) or antibody (3), each of which cross-links the bacteria
to receptors on the phagocyte. Antibody can also activate complement,
and if antibody and C3b both opsonize the bacteria, binding is further
enhanced (4). ANTIGEN PRESENTATION
Virtually all cells of the body can present antigen to CTLs, but
there is a more limited group of specialized antigen-presenting
other circumstances, not just phagocytosis. For example, anti- cells (APCs) which process and present antigens to helper T
bodies bound to parasitic worms allow them to be recognized cells. Several different types of leukocyte can act as APCs,
by eosinophils. Another type of antibody binds to receptors including dendritic cells, macrophages and B cells. All of these
on mast cells and allows them to recognize soluble antigens cells internalize antigens from the extracellular space by
(see Chapter 10). phagocytosis or endocytosis. These APCs then display antigenic
peptide–MHC complexes on the cell surface and they express
co-stimulatory molecules that are essential for initiating
Peptides from intracellular pathogens are displayed on the immune responses. Activation of a TH cell requires both the
surface of infected cells. Antibodies are present only in extra- signal from antigenic peptide–MHC and co-stimulation.
cellular spaces, including blood, lymph and tissue fluids, and Co-stimulatory signals are upregulated by the presence of
they can usually only target extracellular pathogens. Intracellu- pathogens, which can be detected by the engagement of innate
lar pathogens (such as viruses) can escape antibody-mediated immune receptors that recognize PAMPs.
responses once they are safely located within a host cell. The Most immune responses to infectious organisms are made up
adaptive immune system has therefore evolved a specific of a variety of innate and adaptive components. In the earliest
method of displaying portions of virtually all cell proteins on stages of infection, innate responses predominate; later the lym-
the surface of each nucleated cell in the body so that they can phocytes start to generate adaptive immune responses. After
be recognized by T cells. recovery from infection, immunological memory remains
For example, a cell infected with a virus will present fragments within the population of lymphocytes, which can then mount
of viral proteins (peptides) on its surface that are recognizable by a more effective and rapid response if there is re-infection with
T cells. The antigenic peptides are transported to the cell surface the same pathogen at a later date.
and presented to the T cells by MHC molecules (a group of mol- The two major phases of any immune response are antigen
ecules encoded within the major histocompatibility complex, see recognition and a reaction to eliminate the antigen.
Chapter 6). T cells use their antigen-specific receptors (TCRs) to
recognize the antigenic peptide–MHC molecule complex Antigen activates specific clones of lymphocytes. In adap-
(Fig. 1.15). If an infected cell (target) is recognized by a cytotoxic tive immune responses, lymphocytes are responsible for
T cell (CTL), the T cell can signal to the target cell to induce immune recognition, which is achieved by clonal selection. Each
apoptosis (programmed cell death). The process by which lymphocyte is genetically programmed to produce one specific
MHC molecules facilitate recognition of antigenic peptides is antigen receptor (BCR or TCR) capable of recognizing just one
one component of a wider process called antigen presentation particular antigen. However, the immune system as a whole can
(see Chapter 7). specifically recognize many thousands of antigens and the
10 SECTION 1 The Immune System and Innate Immunity
lymphocytes that recognize any particular antigen are only a Lymphocytes that have been stimulated, by binding to their
tiny proportion of the total. specific antigen, take the first steps towards cell division. They
How then is an adequate immune response to an infectious express new receptors that allow them to respond to cytokines
agent generated? The answer is that, when an antigen binds to from other cells and will usually go through a number of cycles
the few lymphocytes that can recognize it, they are induced to of division before differentiating into mature cells, again under
proliferate rapidly. Within a few days there is a sufficient num- the influence of cytokines. They may also start to produce sets of
ber to mount an adequate immune response. In other words, the cytokines themselves.
antigen selects and activates the specific clones to which it binds Even when the infection has been overcome, some of
(Fig. 1.16), a process called clonal selection. This operates for the newly produced lymphocytes remain, available for re-
both B cells and T cells. stimulation if the antigen is ever encountered again. These cells
How can the immune system know which specific antibodies are called memory cells, because they are generated by past
will be needed during an individual’s lifetime? It does not know. encounters with particular antigens. Memory is partly the result
The immune system generates antibodies (and T-cell receptors) of the expansion of the responding population of lymphocytes
that can recognize an enormous range of antigens even before it in the first immune response and partly because these cells are
encounters them. Many of these specificities, which are gener- more easily activated on subsequent encounters with the anti-
ated more or less at random, will never be called upon to protect gen. Memory cells confer lasting immunity to a particular
the individual against infection. pathogen.
What is the advantage of generating billions of lymphocytes
that do not recognize any known infectious agent? Many path-
ogens mutate their surface antigens. Indeed the immune system ANTIGEN ELIMINATION
provides selective pressure for the evolution of new strains of
pathogen with altered antigens. If the immune system could There are numerous ways in which the immune system can
destroy pathogens, each being suited to a given type of infection
not recognize new variants of pathogens, it would not be able
at a particular stage of its life cycle. These defence mechanisms
to make an effective immune response. By having a wide range
are often referred to as effector systems.
of antigen receptors, at least some of the lymphocytes will be
able to recognize any pathogen that enters the body.
Antibodies can directly neutralize some pathogens. In one of
the simplest effector systems, antibodies can combat certain
antigen selection
pathogens just by binding to them. For example, antibody to
the outer coat proteins of some rhinoviruses (which cause colds)
can prevent the viral particles from binding to and infecting
host cells.
BCR
innocuous
primary secondary antigen
vaccination antibody natural antibody
infection hypersensitivity
response response
toxoid toxin acquired
immunity
time
memory cells formed
SUMMARY
• Most cells of the immune system derive from haematopoietic stem cells. • There are three major subpopulations of T cells that have helper,
The primary lymphoid organs in mammals are the thymus and bone marrow, cytotoxic and regulatory activities (TH, TC and Treg).
where lymphocyte differentiation occurs. • B cells can differentiate into antibody-secreting plasma cells and
• Phagocytic cells are found in the circulation as monocytes and granulocytes. memory cells.
Monocytes differentiate into macrophages that reside in tissues (e.g. Kupffer • T cells developing in the thymus are subject to positive and negative selec-
cells in the liver). Neutrophils are short-lived phagocytes present in high num- tion processes.
bers in the blood and at sites of acute inflammation. • Mammalian B cells develop mainly in the fetal liver and from birth
• Eosinophils, basophils, mast cells and platelets, together with cyto- onwards in the bone marrow. This process continues throughout life. B cells
kines and complement, take part in the inflammatory response. also undergo a negative selection process at the site of B-cell generation.
• Innate lymphoid cells are of two kinds: those that produce cytokines in • Lymphocytes migrate to, and function in, the secondary lymphoid organs and
response to micro-environmental signals and the cytotoxic NK cells that rec- tissues.
ognize and kill virus-infected cells and certain tumour cells by inducing • Secondary lymphoid organs and tissue protect different body sites:
apoptosis. the spleen responds to blood-borne organisms; the lymph nodes respond to
• Antigen-presenting cells link the innate and adaptive immune systems and lymph-borne antigens; and the mucosa-associated lymphoid tissue (MALT)
are required by T cells to enable them to respond to antigens. protects the mucosal surfaces.
• Lymphocytes are phenotypically, functionally and morphologically • Most lymphocytes recirculate around the body: there is continuous lym-
heterogeneous. phocyte traffic from the blood stream into lymphoid tissues and back again into
• B lymphocytes and T lymphocytes express specific antigen receptors the blood via the thoracic duct and right lymphatic duct.
called the B-cell receptor (BCR) and T-cell receptor (TCR), respectively.
14
CHAPTER 2 Cells, Tissues and Organs of the Immune System 15
thymus
T
T
tissues tissues
platelets mega-
karyocyte
B B
blood
lymphocytes
recirculating
haematopoietic lymphocytes
polymorphonuclear stem cell
granulocyte
bone marrow/
NK
fetal liver
monocytes
NK
mast cell blood circulation
tissue
macrophages
macrophages interdigitating dendritic
cell cell
APCs lymphocytes
Fig. 2.1 Origin of cells of the immune system All cells shown here arise from the haematopoietic stem cell.
Platelets (cellular fragments produced by megakaryocytes) are released into the circulation. Polymorphonuclear
granulocytes and monocytes pass from the circulation into the tissues. Mast cells are identifiable in all tissues.
B cells mature in the fetal liver and bone marrow in mammals, whereas T cells mature in the thymus. The origin
of the large granular lymphocytes with natural killer (NK) activity is probably the bone marrow. Lymphocytes
recirculate through secondary lymphoid tissues. Interdigitating cells and dendritic cells act as antigen-presenting
cells (APCs) in secondary lymphoid tissues.
microbe
antigens
antigen presentation
cytokines cytokines
phagocytes
cytokines
NK
B
Fig. 2.2 Antigen-presenting cells (APCs) Specialized APCs are involved in both innate and adaptive immunity
to bacteria and viruses by the production of cytokines and by presentation of processed antigens to T cells.
Antigen-presenting cells (APCs) link the innate so that they can be recognized by T cells and by producing cyto-
and adaptive immune systems. A specialized group of cells kines. APCs enhance innate immune cell function and they are
termed antigen-presenting cells (APCs) link the innate and essential for activation of T cells (Fig. 2.2).
adaptive immune systems by taking up and processing antigens
16 SECTION 1 The Immune System and Innate Immunity
Adaptive immune system cells are lymphocytes. and basic dyes, they are classified into neutrophils, basophils
Lymphocytes (T and B cells) recognize antigens through clon- and eosinophils, and have distinct effector functions:
ally expressed, highly specific antigen receptors (see Chapters 6 • The neutrophils, also called polymorphonuclear neutrophils
and 9). T cells are produced in the thymus (see Fig. 2.1) and (PMNs), are most numerous and constitute the majority of
require antigen to be processed and presented to them by leukocytes (white blood cells) in the blood stream (around
specialized APCs. 60%–70% in adults).
Whereas the cells of the innate immune system are found in • The primary actions of eosinophils and basophils, both of
the blood stream and in most organs of the body, lymphocytes which can function as phagocytes, involve granule release
are localized to specialized organs and tissues. (exocytosis).
The lymphoid organs where the lymphocytes differentiate The mononuclear phagocytes and polymorphonuclear gran-
and mature from stem cells are called the primary lymphoid ulocytes develop from a common precursor.
organs and include:
• the thymus: the site of T cell development; Mononuclear phagocytes are widely distributed throughout
• the fetal liver and postnatal bone marrow: the sites of B-cell the body. Cells of the mononuclear phagocytic system are found
development. in virtually all organs of the body where the local micro-
It is in the primary lymphoid organs that the lymphocytes environment determines their morphology and functional char-
undergo the antigen-independent part of their differentiation acteristics, e.g. in the lung as alveolar macrophages, in kidney as
program. Cells of the T- and B-cell lineages migrate from the glomerular mesangial cells and in the liver as Kupffer cells
primary lymphoid organs to function in the secondary lym- (Fig. 2.3 and see Fig. 1.4).
phoid organs. These can be subdivided into: The main role of the mononuclear phagocytes is to remove
• encapsulated organs: the spleen and lymph nodes; particulate matter of foreign origin (e.g. microbes) or self-origin
• non-encapsulated tissues, e.g. MALT. (e.g. aged erythrocytes).
In the secondary lymphoid organs and tissues, lymphocytes Myeloid progenitors in the bone marrow differentiate into
undergo their final differentiation steps, which occur in the pro-monocytes and then into circulating monocytes, which
presence of antigen. Effector B and T cells generated in the sec- migrate through the blood vessel walls into organs to become
ondary lymphoid tissues account for the two major cell types macrophages.
participating in adaptive immune responses of humoral and cel- The human blood monocyte:
lular immunity, respectively. • is large (10–18 μm in diameter) relative to the lymphocyte;
As the cells of the immune system develop, they acquire mol- • has a horseshoe-shaped nucleus;
ecules that are important for their function. These specific func- • contains primary azurophilic (blue-staining) granules; and
tional molecules are referred to as lineage markers because they • possesses ruffled membranes, a well-developed Golgi com-
identify the cell lineage. For example: plex, and many intracytoplasmic lysosomes (Fig. 2.4).
• myeloid cells: polymorphs and monocytes; and The lysosomes contain peroxidase and several acid hydro-
• lymphoid cells: T and B cells. lases, which are important for killing phagocytosed micro-
Other marker molecules include those involved in regulating organisms. Monocytes/macrophages actively phagocytose
cell differentiation (maturation, development), proliferation microorganisms (mostly bacteria and fungi) and the body’s
and function and those involved in regulating the number of own aged and dead cells, or even tumour cells.
cells participating in the immune response.
MYELOID CELLS
Mononuclear phagocytes and polymorphonuclear
granulocytes are the two major phagocyte lineages.
Phagocytes are found both in the circulation and in tissues; they
belong to two major lineages that differentiate from myeloid
precursors:
• mononuclear phagocytes: monocytes/macrophages; and
• polymorphonuclear granulocytes.
The mononuclear phagocytes consist of circulating cells (the
monocytes) and macrophages that differentiate from mono-
cytes and reside in a variety of organs (e.g. spleen, liver, lungs,
kidneys) where they display distinctive morphological features
and perform diverse functions.
Fig. 2.3 Kupffer cells Kupffer cells in the normal mouse liver stain
The other family of phagocytes, polymorphonuclear granu- strongly positive with antibody to F4/80 (arrow). Sinusoidal endothelial
locytes, have a lobed, irregularly shaped (polymorphic) nucleus. cells and hepatocytes are F4/80-negative. (Courtesy Professor S Gordon
On the basis of how their cytoplasmic granules stain with acidic and Dr DA Hume.)
CHAPTER 2 Cells, Tissues and Organs of the Immune System 17
PV
M
Go
Granulocytes and mononuclear phagocytes develop from a • derived mainly from stromal cells (connective tissue cells) in
common precursor. Studies in which colonies have been grown the bone marrow;
in vitro from individual stems cells have shown that the progen- • also produced by mature forms of differentiated myeloid and
itor of the myeloid lineage (CFU-GEMM) can give rise to gran- lymphoid cells.
ulocytes, monocytes and megakaryocytes (Fig. 2.6). Monocytes Bone marrow stromal cells, stromal cell matrix and cytokines
and neutrophils develop from a common precursor cell, the form the micro-environment to support stem cell differentiation
CFU-granulocyte macrophage cells (CFU-GMs). Myelopoiesis into individual cell lineages. Stromal cells produce an extracellular
(the development of myeloid cells) commences in the liver of the matrix, which is very important in establishing cell–cell interac-
human fetus at about 6 weeks of gestation. tions and enhancing stem cell differentiation. The major compo-
CFU-GEMMs mature under the influence of colony- nents of the matrix are proteoglycans, fibronectin, collagen,
stimulating factors (CSFs) and several interleukins. These laminin, haemonectin and thrombospondin.
factors, which are relevant for the positive regulation of haema- Other cytokines, such as transforming growth factor-β
topoiesis, are: (TGFβ) may downregulate haematopoiesis. CFU-GMs taking
the monocyte pathway give rise initially to proliferating mono-
blasts. Proliferating monoblasts differentiate into pro-
monocytes and finally into mature circulating monocytes,
pluripotent which serve as a replacement pool for the tissue-resident mac-
haematopoietic lymphoid rophages (e.g. lung macrophages).
stem cell stem cell
Neutrophils constitutively express FcγRIII and FcγRII, and • provokes bronchospasm, a symptom of allergic asthma.
FcγRI is induced on activation. Other proteins with similar effects are found in the granule
It is difficult to assess the functional activity of different matrix, for example:
developmental stages of granulocytes, but it seems likely that • eosinophil cationic protein (ECP); and
the full functional potential is realized only when the cells are • eosinophil-derived neurotoxin (EDN).
mature. Release of the granules on eosinophil activation is the only
To become active in the presence of opsonins, neutrophils way eosinophils can kill large pathogens (e.g. schistosomula),
must interact directly with microorganisms and/or with cyto- which cannot be phagocytosed. Eosinophils are therefore
kines generated by a response to antigen. This limitation could thought to play a specialized role in immunity to parasitic
reduce neutrophil activity in early life. worms using this mechanism (see Fig. 16.11).
Activation of neutrophils by cytokines and chemokines is
also a prerequisite for their migration into tissues (see
Basophils and mast cells play a role in immunity against
Chapter 3).
parasites. Basophils are found in very small numbers in the
circulation and account for less than 0.2% of leukocytes
EOSINOPHILS, BASOPHILS AND MAST CELLS (Fig. 2.8).
IN INFLAMMATION The mast cell (Fig. 2.9), which is present in tissue and not in
the circulation, is indistinguishable from the basophil in a num-
Eosinophils play a role in immunity to parasitic worms.
ber of its characteristics but displays some distinctive morpho-
Eosinophils comprise 2%–5% of blood leukocytes in healthy,
logical features (Table 2.w1). Their shared functions may
non-allergic individuals. Human blood eosinophils usually have
indicate a convergent differentiation pathway.
a bi-lobed nucleus and many cytoplasmic granules, which stain
The stimulus for mast cell or basophil degranulation is
with acidic dyes such as eosin (Fig. 2.7). Although not their pri-
often an allergen (i.e. an antigen causing an allergic reaction).
mary function, eosinophils appear to be capable of phagocytos-
To be effective, an allergen must cross-link IgE molecules
ing and killing ingested microorganisms.
bound to the surface of the mast cell or basophil via its
The granules in mature eosinophils are membrane-bound high-affinity Fc receptors for IgE (FcεRI). Degranulation of
organelles with crystalloid cores that differ in electron density a basophil or mast cell results in all contents of the granules
from the surrounding matrix. The crystalloid core contains being released very rapidly. This occurs by intracytoplasmic
the major basic protein (MBP), which: fusion of the granules, followed by discharge of their contents
• is a potent toxin for helminths;
(Fig. 2.10).
• induces histamine release from mast cells;
Mediators such as histamine, released by degranulation,
• activates neutrophils and platelets; and
cause the adverse symptoms of allergy but, on the positive side,
also play a role in immunity against parasites by enhancing
acute inflammation.
Nu
G
G
N
ER
Fig. 2.7 Morphology of the eosinophil The ultrastructure of a mature Fig. 2.8 Morphology of the basophil Morphology of the basophil: ultra-
eosinophil shows granules (G) with central crystalloids. 17 500. ER, structural analysis shows a segmented nucleus (N) and the large cyto-
Endoplasmic reticulum; Nu, nucleus; P, nuclear pores. Inset: A mature plasmic granules (G). Arrows indicate nuclear pores. 11 000. Inset:
eosinophil in a blood smear is shown with a bi-lobed nucleus and eosin- This blood smear shows a typical basophil with its deep violet-blue gran-
ophilic granules. 1000. (Reprinted from Shiland, BJ, Medical Assistant: ules. 1000. (Reprinted from Shiland, BJ, Medical Assistant: Urinary,
Urinary, Blood, Lymphatic and Immune Systems with Laboratory Blood, Lymphatic and Immune Systems with Laboratory Procedures—
Procedures—Module E, Second Edition, Copyright Elsevier Inc. 2015.) Module E, Second Edition, Copyright Elsevier Inc. 2015.)
19.e1
Granule Content
Histamine Yes Yes
Heparin ? Yes
Cytokine Production
IL-4 Yes Yes
IL-13 Yes Yes
19.e3
Platelets have a role in inflammation and clotting. Blood • other molecules important for their function, such as the
platelets (Fig. 2.w1) are not cells, but cell fragments derived from GpIIb/IIIa complex (CD41) responsible for binding to fibrin-
megakaryocytes in the bone marrow. They contain granules, ogen, fibronectin, vitronectin (tissue matrix), and von Wille-
microtubules and actin/myosin filaments, which are involved brand factor (another clotting factor).
in clot contraction. Platelets also participate in immune Both receptors and adhesion molecules are important in the
responses, especially in inflammation. activation of platelets.
The adult human produces 1011 platelets each day. About Following injury to endothelial cells, platelets adhere to and
30% of platelets are stored in the spleen but may be released aggregate at the damaged endothelial surface. Release of platelet
if required. granule contents, which include de novo synthesized serotonin
Platelets express MHC class I products and receptors for IgG and endocytosed fibrinogen, results in:
(CD32; FcγRII), which are important in platelet activation via • increased capillary permeability, a feature of inflammation;
IgG immune complexes. In addition, megakaryocytes and plate- • activation of complement (and hence attraction of
lets carry: leukocytes); and
• receptors for clotting factors (e.g. factor VIII); and • clotting.
Fig. 2.w1 Ultrastructure of resting platelets Designations: α, α-granules; m, mitochondria; mt, microtubules;
OCS, open canalicular system. Scale bar is 0.2 μm. (Reprinted from Andrianova IA, et al., In systemic lupus erythe-
matosus anti-dsDNA antibodies can promote thrombosis through direct platelet activation. J Autoimmun. 2019
Nov 12:102355.)
20 SECTION 1 The Immune System and Innate Immunity
1 2
Fig. 2.10 Electron micrograph of rat mast cells Rat peritoneal mast cells show electron-dense granules (1).
Vacuolation with exocytosis of the granule contents has occurred after incubation with anti-IgE (2). Transmission
electron micrographs. 2700. (Courtesy Dr D Lawson.)
CHAPTER 2 Cells, Tissues and Organs of the Immune System 21
skin
T
T T
Langerhans cell T
Mb I
migrating
veiled cell
T
afferent
lymph nodes, spleen, lymphatic
and MALT
macrophage
Fig. 2.13 Ultrastructure of an interdigitating dendritic cell In the T-cell
cortex FDC in germinal
(B-cell area) area of a lymph node, intimate contacts are made by antigen-presenting
centre
cells with the membranes of the surrounding T cells. The cytoplasm con-
tains a well-developed endosomal system and does not have Birbeck
IDC granules. 2000. I, IDC nucleus; Mb, IDC membrane; T, T-cell nucleus.
paracortex
(Courtesy Dr BH Balfour.)
(T-cell HEV
area)
medulla
thymus
thymocyte
epithelial
cortex
network
macrophage
IDC in medulla
medulla Fig. 2.14 Follicular dendritic cell An isolated follicular dendritic cell (FDC)
from the lymph node of an immunized mouse 24 hours after injection of
Fig. 2.12 Migration of antigen-presenting cells (APCs) into lymphoid antigen. The FDC is of intermediate maturity with smooth filiform dendrites
tissues Bone marrow-derived dendritic cells (DCs) are found especially in typical of young FDCs, and beaded dendrites, which bind immune
lymphoid tissues, in the skin and in mucosa. DCs in the form of Langer- complexes (iccosomes) in mature FDCs. The adjacent small white cells
hans cells are found in the epidermis and in mucosa and are characterized are lymphocytes. (Electron micrograph kindly provided by Dr Andras
by special (tennis racquet-shaped) Birbeck granules. Langerhans cells are Szakal, Isolated follicular dendritic cells: cytochemical antigen localization,
rich in MHC class II molecules and carry processed antigens. They Nomarski, SEM and TEM morphology. J.Immunology 1985;134:
migrate via the afferent lymphatics (where they appear as veiled cells) 1349–1359. Reproduced by permission of the Journal of Immunology.)
into the paracortex of the draining lymph nodes. Here they make contact
with T cells. These interdigitating dendritic cells (IDCs), localized in the
T-cell areas of the lymph node, present antigen to T helper cells. The anti- complement associated with immune complexes (iccosomes;
gen is exposed to B cells on the follicular dendritic cells (FDCs) in the Fig. 2.14). They also express Fc receptors. The FDCs produce
germinal centres of B-cell follicles. Some macrophages located in the
chemokines that are important in B cells homing to the follic-
outer cortex and marginal sinus may also act as APCs. In the thymus,
APCs occur as IDCs in the medulla. HEV, High endothelial venule; MALT, ular areas in lymphoid tissues. They are not bone-marrow
mucosa-associated lymphoid tissue. derived but are of mesenchymal origin.
LYMPHOCYTES
network by establishing strong intercellular connections via
desmosomes. Lymphocytes are phenotypically and functionally
FDCs lack class II MHC molecules, but bind antigen via heterogeneous. Large numbers of lymphocytes are produced
complement receptors (CD21 and CD35), which attach to daily in the primary or central lymphoid organs (i.e. thymus
CHAPTER 2 Cells, Tissues and Organs of the Immune System 23
and postnatal bone marrow). Some migrate via the circulation The LGL morphological pattern displayed in Figure 2.15(2)
into the secondary lymphoid tissues (i.e. spleen, lymph nodes is shown by less than 5% of TH cells and by about 30%–50% of
and MALT). TC cells. These cells display LGL morphology with primary lyso-
The average human adult has about 2 1012 lymphoid cells somes dispersed in the cytoplasm and a well-developed Golgi
and lymphoid tissue as a whole represents about 2% of total apparatus, as shown in Figure 2.15(3).
body weight. Lymphoid cells account for about 20% of the leu- Most B cells, when resting, have a morphology similar to that
kocytes in the adult circulation. seen in Figure 2.15(1).
Many mature lymphoid cells are long-lived and persist as
memory cells for many years. Lymphocytes express characteristic surface and
cytoplasmic markers. Lymphocytes (and other leukocytes)
Lymphocytes are morphologically heterogeneous. In a con- express a large number of different functionally important mol-
ventional blood smear, lymphocytes vary in size (from 6 to 10 μm ecules mostly on their surfaces but also in their cytoplasm,
in diameter) and morphology. which can be used to distinguish (mark) cell subsets. Many of
Differences are seen in: these cell markers can be identified by specific monoclonal anti-
• nuclear to cytoplasmic (N:C) ratio; bodies (mAb) and can be used to distinguish T cells from B cells
• nuclear shape; and (Table 2.2).
• the presence or absence of azurophilic granules. Lymphocytes express a variety of cell surface molecules that
Two distinct morphological types of lymphocyte are seen in belong to different families, which have probably evolved from a
the circulation as determined by light microscopy and a haema- few ancestral genes. These families of molecules are shared with
tological stain such as Giemsa (Fig. 2.15): other leukocytes and are distinguished by their structure. The
• The first type is relatively small, is typically agranular and has major families include:
a high nuclear to cytoplasmic (N:C) ratio (see Fig. 2.15(1)). • the immunoglobulin superfamily;
• The second type is larger, has a lower N:C ratio, contains • the integrin family;
cytoplasmic azurophilic granules and is known as the large • selectins; and
granular lymphocyte (LGL). • proteoglycans.
LGLs should not be confused with granulocytes, monocytes,
or their precursors, which also contain azurophilic granules. Marker molecules allow lymphocytes to communicate with
Most T cells express the αβ T-cell receptor (see later) and, when their environment. The major function of these families of
resting, can show either of the above morphological patterns. molecules is to allow lymphocytes to communicate with their
Most TH cells (approximately 95%) and a proportion environment. They are extremely important in cell trafficking,
(approximately 50%) of cytotoxic T cells (TC or CTL) have adhesion and activation. Markers expressed by lymphocytes
the morphology shown in Figure 2.15(1). can often be detected on cells of other lineages.
GA
PL
1 2 3
Fig. 2.15 Morphological heterogeneity of lymphocytes Lymphocyte morphology. (1) The small lymphocyte
has no granules, a round nucleus and a high N:C ratio. (2) The large granular lymphocyte (LGL) has a lower N:C
ratio, indented nucleus and azurophilic granules in the cytoplasm. Giemsa stain. (3) Ultrastructure of the LGL
shows characteristic electron-dense peroxidase-negative granules (primary lysosomes, PL), scattered through-
out the cytoplasm, with some close to the Golgi apparatus (GA) and many mitochondria (M). 10 000.
(Reprinted from Shiland, BJ, Medical Assistant: Urinary, Blood, Lymphatic and Immune Systems with Labora-
tory Procedures—Module E, Second Edition, Copyright Elsevier Inc. 2015.)
23.e1
The immunoglobulin superfamily consists of molecules with The selectins (CD62, E, L and P) are expressed on leukocytes
structural characteristics similar to those of the immunoglobu- (L) or activated endothelial cells and platelets (E and P). They
lins and includes CD2, CD3, CD4, CD8, CD28, MHC class I and have lectin-like specificity for a variety of sugars expressed on
II molecules and many more. heavily glycosylated membrane glycoproteins (e.g. CD43).
The integrin family consists of heterodimeric molecules with The proteoglycans, typically CD44, have a number of gly-
α and β chains. There are several integrin subfamilies and all cosaminoglycan (GAG) binding sites (e.g. for chondroitin
members of a particular subfamily share a common β chain, sulfate), and bind to extracellular matrix components (typically,
but each has a unique α chain: hyaluronic acid).
• One integrin subfamily (the β2-integrins) uses CD18 as the β
chain, which can be associated with CD11a, CD11b, CD11c Other families include:
or αd – these combinations make up the lymphocyte func- • the tumour necrosis factor (TNF) and nerve growth factor
tion antigens LFA-1, Mac-1 (CR3), p150, 95 and αdβ2 surface (NGF) receptor superfamily;
molecules, respectively – and are commonly found on • the C-type lectin superfamily;
leukocytes. • the family of receptors with seven transmembrane segments
• A second subfamily (the β1-integrins) has CD29 as the β (tm7); and
chain, which again is associated with various other peptides • the tetraspanins, a superfamily with four membrane-
and includes the VLA (very late activation) markers. spanning segments (tm4), for example CD20.
23.e2
Identification of lymphocyte subsets. Human lymphocytes measured as separated populations. Immunofluorescence tech-
can be identified in tissues and their function can be niques to identify leukocytes are shown in Method Box 2.1.
ELISPOT Assays
Individual B cells producing specific antibody or individual T cells secreting par-
ticular cytokines may be detected by enzyme-linked immuno SPOT (ELISPOT)
wash assay (Fig. 2.w4).
Continued
23.e3
sample
vibrating
sheath flow cell
fluid red
fluorescence
beam green
splitters fluorescence
laser
collection
tubes
waste
2.1 2.2
104 104
CD8 fluorescence
102 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
CD3 fluorescence CD3 fluorescence
lymphocytes lymphocytes
cytokine
T T T
B B B
Marker molecules allow lymphocytes to be isolated from their size and fluorescent staining (see Method Box 2.1). These
each other. The presence of characteristic surface molecules techniques, together with the expression of surface molecules
expressed by cell populations allows them to be identified using allowing the cell populations to be isolated from each other
fluorescent antibodies as probes. These can be applied to tissue using cell panning and immunomagnetic beads (Method
sections to identify cell populations or be used in flow cytometry Box 2.2) have allowed a detailed dissection of lymphoid cell
to enumerate and to separate cells in suspension on the basis of populations.
Isolating Cell Populations Using Their Characteristic Surface Cell separation by Immunomagnetic Beads
Molecules Direct Method (shown in Fig. 2.w7)
The presence of characteristic surface molecules expressed by cell populations The beads are coated with a monoclonal antibody to the cellular antigen of inter-
allows the cell populations to be isolated from each other using cell panning and est by either:
immunomagnetic beads. • direct binding to the bead; or
• binding the primary antibody to secondary antibody-coated beads.
Isolation of Lymphocyte Subpopulations – Panning The coated beads are then incubated with the cell suspension (or even whole
Cell populations can be separated on antibody-sensitized plates. The antibody blood) and the cells bound by the antibody on the beads (positively selected
binds non-covalently to the plastic plate (as for ELISPOT immunoassay) and the cells) are immobilized by applying a magnetic field to the tube (concentrating
cell mixture is applied to the plate. Antigen-positive cells (Ag+) bind to the antibody on the test-tube wall around the magnet). The non-immobilized cells (negatively
and the antigen-negative cells (Ag ) can be carefully washed off (Fig. 2.w6). selected) are removed from the tube and the positively selected cells are recov-
By changing the culture conditions or by enzyme digestion of the cells on the ered after washing and dissociation from the antibody-coated beads. This
plate, it is sometimes possible to recover the cells bound to the plate. method can be also used to remove an unwanted population of the cells from
Often the cells that have bound to the plate are altered by their binding (e.g. a mixture. In this case, the non-immobilized cells will be collected for further
binding to the plate cross-links the antigen, which can cause cell activation). The experiments.
method is therefore most satisfactory for removing a subpopulation from the pop-
ulation, rather than isolating it. Indirect Method
Examples of the application of this method include: The monoclonal antibody to the target cellular antigen is first added to the cell
• separating TH and TC cell populations using antibodies to CD4 or CD8; and suspension. Following incubation with the antibody, the cells are washed and
• separating T cells from B cells using anti-Ig (which binds to the surface anti- mixed with beads coated with the appropriate secondary anti-Ig antibody. Cells
body of the B cell). bound to the magnetic beads (positively selected) are then immobilized with a
In reverse, by sensitizing the plate with antigen, antigen-binding cells can be magnetic field and the non-immobilized cells (negatively selected) removed.
separated from non-binding cells. The positively selected cells are then washed and dissociated from the beads.
Continued
23.e5
antibody-coated The microbeads available these days allow us to abandon the dissociation pro-
magnetic beads cedure. They are so small that they can be left attached to the cell surface and
will not interfere with their function.
antigen-positive
cells Cell Sorting
Cells can be isolated by their surface markers using a flow cytometer with a sort-
ing function (cell sorter). The sorter will direct the cells fluorescing because of a
specific fluoresceinated antibody attached to them into a collection vessel. Using
cell the example discussed in Method Box 2.1, cells with attached fluorescein isothio-
suspension cyanate (FITC)-conjugated antibody would be directed to one collection vessel for
green cells and cells with attached PE-labelled (orange) antibody – to a separate
collection vessel for orange cells.
antibody-coated
magnetic beads
dissociating agent
antigen-positive
cells
and B Cells
T
CD number T cells B cells T
Fig. 2.16 Naive T-cell subsets In the thymus, naïve T cells develop as
T cells can be distinguished by their different antigen two distinct populations of cells, based on the composition of their T-cell
receptors. The definitive T-cell lineage marker is the TCR. receptor (TCR); 90%–95% have an α/β TCR and 5%–10% a γ/δ TCR. αβ T
The two defined types of TCR are: cells diverge into CD4+ or CD8+ cells through the ability of their TCR
• a heterodimer of two disulfide-linked polypeptides (α and to interact, respectively, with MHC class II (CD4+) or class I (CD8+)
molecules. CD4+ and CD8+ αβT cells have very distinct effector func-
β); and tions. About 5% of CD4+ cells are so-called natural regulatory cells
• a structurally similar heterodimer consisting of γ and δ (Tregs). CD4 subpopulations differentiate in response to the cytokines
polypeptides. indicated and the differentiated cells go on to produce distinct sets of
Both receptors are associated with a set of five polypeptides cytokines.
(the CD3 complex) and together form the TCR complex (TCR–
CD3 complex; see Chapter 6).
Approximately 90%–95% of blood T cells in humans are αβ • TH2 cells develop in response to IL-4 and IL-33 and produce
T cells and the remaining 5%–10% are γδ T cells. IL-4, IL-5, IL-6 and IL-10.
TH1 cells mediate several functions associated with cytotox-
There are three major subpopulations of αβ T cells. icity and local inflammatory reactions. They help cytotoxic
• TH cells that express the CD4 marker (CD4+ T cells) and T-cell precursors develop into effector cells to kill virally
mainly help or induce immune responses, divided into two infected target cells and to activate macrophages infected with
main subsets (TH1 and TH2). intracellular pathogens (e.g. Mycobacteria and Chlamydia),
• Tregs that express the CD4 marker (CD4+ T cells) and regu- enhancing intracellular killing of the pathogens by the produc-
late immune responses. tion of IFNγ. Consequently, they are important for combating
• TC cells that express the CD8 marker (CD8+ T cells) – also intracellular pathogens, including viruses, bacteria and para-
called CTLs. sites. Some TH1 cells also help B cells to produce different
classes of antibodies.
CD4+ T cells recognize their specific antigens in association
TH17 cells are similar to the TH1 subset but their development
with MHC class II molecules, whereas CD8+ T cells recognize
from TH0 cells is dependent on TGFβ and IL-21. Their induction
antigens in association with MHC class I molecules (see
by TGFβ suggests that they are related to the regulatory T-cell
Chapter 7). Thus, the presence of CD4 or CD8 limits (restricts)
subsets. They produce both IL-17 and IL-22 and play an impor-
the type of cell with which the T cell can interact.
A small proportion of αβ T cells express neither CD4 nor tant role in maintaining the integrity of mucosal epithelia and
thus in protection against microbial entry into the body.
CD8; these double negative T cells might have a regulatory func-
Another subset of T cells, TH9, can be produced from TH0
tion. Most circulating γδ cells are double negative.
cells by a combination of IL-4 and TGFβ. They, in turn, can pro-
duce IL-9 and IL-21 cytokines. The suggested function of TH9
TH subsets are distinguished by their cytokine profiles. cells is to carry out immune responses to intestinal worms. In
CD4+ TH cells can be further divided into functional subsets addition, they are implicated in autoimmune and allergic reac-
on the basis of the spectrum of the cytokines they produce. They tions and in immunosurveillance of melanoma.
initially develop from TH0 naive (antigen inexperienced) pre- TH2 cells are effective at stimulating B cells to proliferate and
cursor cells (Fig. 2.16): to produce antibodies of some IgG subclasses and especially IgE
• TH1 cells develop in response to IL-12, IL-18 and IFNγ and and therefore function primarily to protect against free-living,
secrete IL-2 and IFNγ. extracellular microorganisms (humoral immunity).
CHAPTER 2 Cells, Tissues and Organs of the Immune System 25
Several CD4+ regulatory T-cell populations have been • The third population is Vδ3 T cells, which make up about 0.2%
described as being capable of suppressing T-cell responses of circulating T cells. They express CD56, CD161, HLA-DR
(see later). and NKG2D but not NKG2A or NKG2C and although poorly
represented in the blood, they are rich in the liver and in patients
γδ T Cells and NKT Cells with leukaemias and some chronic viral infections.
γδ T cells maintain epithelial integrity, kill stressed cells γδ T cells show some long-term memory characteristics
and contribute to antimicrobial immunity. γδT cells display even though they are generally regarded, like NKT cells, as being
LGL characteristics (see Fig. 2.15) and some have a dendritic part of innate immunity.
morphology in lymphoid tissues (Fig. 2.17). They have a broad
specificity for recognition of unconventional antigens such as iNKT cells recognize glycolipid antigens. NKT cells have
heat shock proteins, phospholipids and phosphoproteins. T-cell markers and also some NK-cell markers (see later): they
Unlike αβ T cells, they do not generally recognize antigens in express CD3 and have a unique αβ TCR (expressing an invariant
association with classical MHC class I and II molecules. γδT Vα and Vβ11, hence called invariant NKT cells (iNKT cells).
cells express several of the Toll-like receptor families either Human iNKT cells are infrequent in the blood, being between
spontaneously or after activation and most express CD16. 0.01% and 1% of the peripheral mononuclear cells. iNKT cells
Human γδ T cells have been divided into three main popula- recognize glycolipid antigens presented by CD1d molecules (see
tions based on the expression of their δ chain. Fig. 7.11), but not conventional MHC molecules. iNKT cells are
• γδ T cells expressing Vδ1 chains are prominent in the intra- therefore thought to act as an interface between the innate and
epithelial layer of mucosal surfaces in the skin, lung and gut. adaptive systems by initiating T-cell responses to non-peptide
Here, as the majority of the intra-epithelial T cells, they are antigens.
involved in the maintenance of epithelial tissue integrity NKT cells are also thought to regulate immune responses
when facing damage, infection or responding to stressed or (especially dendritic cell function) through the production of
neoplastic epithelial cells. They produce IL-17 and some cytokines (e.g. IL-4, IL-10 and IFNγ) in response to antigens.
IL-10, but little or no IL-2, IL-4 or IFN γ. Vδ1 cells are also
present as a minor population in the peripheral blood. B cells recognize antigen using the B cell receptor complex.
• The second population using Vδ2 chains represent the About 5%–15% of the circulating lymphoid pool are B cells,
majority of circulating γδ T lymphocytes (50%–90%) in which are defined by the presence of surface immunoglobulin,
healthy human adults. Vδ2 chain pairing is mainly with transmembrane molecules, which are constitutively produced
Vγ9 (also termed Vγ2) and is thought to recognize confor- and inserted into the B-cell membrane, where they act as spe-
mational changes in the butyrophilin molecule (a widely cific antigen receptors.
expressed member of the Ig supergene family) and may rep- Most human B cells in peripheral blood express two immu-
resent a mechanism to identify infected or transformed cells. noglobulin isotypes on their surface: IgM and IgD (see
Interestingly, Vδ2 T cells have been reported to act as profes- Chapter 9).
sional APCs on activation acquiring co-stimulatory mole- On any B cell, the antigen-binding sites of these IgM and IgD
cules, MHC class II, CD80 and CD86. isotypes are identical.
Fewer than 10% of the B cells in the circulation express IgG,
IgA, or IgE, but B cells expressing IgG, IgA or IgE are present in
larger numbers in specific locations of the body (e.g. IgA-
bearing cells in the intestinal mucosa).
Immunoglobulin associated with other accessory molecules
on the B-cell surface forms the B cell antigen receptor complex
(BCR). These accessory molecules consist of disulfide-bonded
heterodimers of Igα (CD79a) and Igβ (CD79b).
The heterodimers interact with the transmembrane seg-
ments of the immunoglobulin receptor (see Fig. 10.1), and, like
the separate molecular components of the TCR/CD3 complex
(see Fig. 6.17), are involved in cellular activation. Intracellular
domains of CD79a/b have immunoreceptor tyrosine-based acti-
vation motifs (ITAMs). BCR interaction with specific antigen
triggers ITAM phosphorylation and this initiates a downstream
cascade of intracellular events, leading to the activation-related
Fig. 2.17 Dendritic morphology of γδ T cells in the tonsil The γδ T-cell changes in gene expression.
population is predominantly localized in the interfollicular T-cell-
dependent zones. Note the dendritic morphology of the cells. Anti-γδ Other B-cell markers include MHC class II antigens and
T-cell monoclonal antibody and immunoperoxidase. 900. (Courtesy
Dr A Favre, from Arancia G, Malorni W, Iosi F, et al. Morphological fea- complement and Fc receptors. Most B cells carry MHC class
tures of cloned lymphocytes expressing gamma/delta T cell receptors. II antigens, which are important for co-operative (cognate)
Eur J Immunol 1991;21:173, with permission.) interactions with T cells (see Fig. 7.3).
25.e1
Complement receptors for C3b (CD35) and C3d (CD21) are Marginal zone B cells are thought to protect against
commonly found on B cells and are associated with activation polysaccharide antigens. Much has been learned about mar-
and, together with chemokine receptors, possibly homing of ginal zone B cells over the past few years. These cells accumu-
the cells in the peripheral lymphoid organs and tissues. late slowly in the marginal zone of the spleen – a process that
CD19/CD21 interactions with complement, associated with takes between 1 and 2 years in humans.
antigen, play a role in antigen-induced B-cell activation via Like B-1 cells, marginal zone B cells respond to thymus-
the antigen-binding antibody receptor. independent antigens and they are thought to be our main pro-
Fc receptors for exogenous IgG (FcγRII, CD32) are also pre- tection against polysaccharide antigens. They also produce nat-
sent on B cells and play a role in negative signalling to the B cell ural antibodies and, together with B-1 cells, have recently been
(see Fig. 10.15). CD19 and CD20 are the main markers currently termed innate-like B cells.
used to identify human B cells. Other human B-cell markers are
CD22 and CD72–CD78. B cells can differentiate into antibody-secreting plasma
Murine B cells also express CD72 (Lyb-2) and B220, a high cells. Following B-cell activation, many B-cell blasts mature
molecular weight (220 kDa) isoform of CD45 (Lyb-5). into antibody-forming cells (AFCs), which progress in vivo
CD40 is an important molecule on B cells and is involved to terminally differentiated plasma cells, and a subset of B cells
in cognate interactions between T and B cells (see Fig. 9.10) with remains in the periphery as long-lived memory B cells.
T cells expressing CD40 ligand (CD40L). Activated B cells upre- Some B-cell blasts do not develop rough endoplasmic retic-
gulate expression of B7.1 (CD80) and B7.2 (CD86) molecules ulum cisternae. These cells are found in germinal centres and
that interact with their CD28 expressed by T cells. This provides are named follicle centre cells or centrocytes.
a co-stimulatory signal for T/B cognate interactions. Under light microscopy, the cytoplasm of the plasma cells is
basophilic because of the large amount of RNA being used for
antibody synthesis in the rough endoplasmic reticulum. At the
CD5+ B-1 cells and marginal zone B Cells produce natural ultra-structural level, the rough endoplasmic reticulum can
antibodies. Many of the first B cells that appear during ontogeny often be seen in parallel arrays (Fig. 2.18).
express CD5, a marker originally found on T cells. These cells Plasma cells are infrequent in the blood, comprising less than
(termed B-1 cells) are found predominantly in the peritoneal cav- 0.1% of circulating lymphocytes. They are normally restricted to
ity in mice and there is some evidence for a separate differentia- the secondary lymphoid organs and tissues, but are also abun-
tion pathway from conventional B cells (termed B-2 cells). dant in the bone marrow. Since their sole function is to produce
CD5+ B-1 cells express their immunoglobulins from unmu- immunoglobulins, plasma cells have few surface receptors and
tated germline genes (see Chapter 9) and produce mostly IgM, do not respond to antigens. Unlike resting B cells or memory
but also some IgG and IgA. These so-called natural antibodies B cells, plasma cells do not express surface BCR or MHC class II.
are of low avidity, but, unusually, they are polyreactive and are Antibodies produced by a single plasma cell are of one spec-
found at high concentration in the adult serum. CD5+ B-1 cells: ificity (idiotype) and immunoglobulin class (isotype and allo-
• respond well to TI (T-independent) antigens (i.e. antigens type; see Chapter 10).
that can directly stimulate B cells without T cell help);
• may be involved in antigen processing and antigen presenta-
tion to T cells; and
• probably play a role in both tolerance and antibody
responses.
Functions proposed for natural antibodies include:
• the first line of defence against microorganisms;
• clearance of damaged self components; and
• regulatory interactions within the immune system.
Characteristically, natural antibodies react against auto-
antigens including:
• DNA;
• Fc of IgG;
• phospholipids; and
• cytoskeletal components.
CD5 is expressed by B-2 cells when they are activated appropri-
ately and there is therefore some controversy about whether CD5
represents an activation antigen on B cells. Current theories there- Fig. 2.18 Plasma cell, transmission electron micrograph (TEM) This
fore support the notion for two different kinds of CD5+ B cells. section has revealed the cell’s large central nucleus. Surrounding this
A ligand for CD5 is CD72 and there is some suggestion that it is large amounts of rough endoplasmic reticulum thin lines. Plasma cells,
which are found in the blood and lymph, are mature B lymphocytes (white
self-associates and can bind directly to IL6. However, the exact blood cells) that produce and secrete antibodies during an immune
function of CD5 on human B cells is currently unclear but is response. Magnification x6000 when printed at 10 centimetres wide.
highly likely to be involved in the regulation of B-cell activation. (© Steve Gschmeissner/Science Photo Library.)
CHAPTER 2 Cells, Tissues and Organs of the Immune System 27
IL-25
virally infected IL-12 IL-33 IL-13 IL-7
& stressed cells IL-15 TSLP IL-23 CXCL13
Fig. 2.21 Innate lymphoid cells Innate lymphoid cells are subdivided
into five categories according to cytokines required to induce their
Fig. 2.19 Immunofluorescent staining of intracytoplasmic immuno- differentiation (top), the transcription factors that are required to
globulin in plasma cells Fixed human plasma cells, treated with fluores- induce their differentiation and the cytokines they produce. NK, Natural
ceinated anti-human-IgM (green) antibody and rhodaminated anti-human- killer cells.
IgG (red) antibody, show extensive intracytoplasmic staining. As the dis-
tinct staining of the two cells shows, plasma cells normally produce only
one class or subclass (isotype) of antibody. 1500.
role in defence against pathogens, wound healing and mainte-
nance of epithelial integrity. Unlike T and B cells, they do not
have rearranged antigen receptors and are therefore not able to
recognize specific antigens.
Fig. 2.20 Plasma cell death by apoptosis Plasma cells are short-lived NK cells kill virally infected and cancerous cells. NK cells
and die by apoptosis (cell suicide). Note the nuclear chromatin changes, are very important cytotoxic cells in innate immunity. They
which are characteristic of apoptosis. 5000. account for up to 15% of blood lymphocytes, are derived from
the bone marrow and morphologically have the appearance
of large granular lymphocytes (see Fig. 2.15). The function
of NK cells is to recognize and to kill virally infected cells
Immunoglobulins can be visualized in the plasma cell (Fig. 2.22) and certain tumour cells by mechanisms described
cytoplasm by staining with fluorochrome-labelled specific anti- in Chapter 8.
bodies (Fig. 2.19).
Many plasma cells have a short life span, surviving for a few
days and dying by apoptosis (Fig. 2.20). However, there is a sub- NK cells are identified by their expression of CD56
set of plasma cells with a long life span (months) in the bone and CD16. In humans, NK cells are most often identified by
marrow that might be important in giving rise to sustained anti- the absence of the T-cell receptor component CD3 and the
body responses. expression of CD56 (NCAM), a homophilic adhesion molecule
of the immunoglobulin superfamily. CD16 (FcγRIII) is another
marker that may be used to identify NK cells, although about
INNATE LYMPHOID CELLS (ILC) 10% of human NK cells do not express this molecule and are
ILCs are lymphocytes that do not express rearranged thought to represent immature NK cells.
antigen receptors. ILCs are a heterogeneous population of CD16 is involved in the activation pathways of NK cells by
non-T and non-B lymphoid cells that carry out many of the antibody-coated targets and is also expressed by neutrophils,
functions of conventional T cells and play an important first-line some macrophages and γδ T cells. However, on neutrophils,
28 SECTION 1 The Immune System and Innate Immunity
Fig. 2.22 NK cell attached to a target A natural killer (NK) cell attached to
a target cell (TC) 4500. (Courtesy Drs G Arancia and W Malorni, Rome.) LYMPHOCYTE DEVELOPMENT
Lymphocytes, the effector cells of the adaptive immune
response, are the major component of organs and tissues that
CD16 is linked to the surface membrane by a glycoinositol collectively form the lymphoid system.
phospholipid (GPI) linkage, whereas NK cells, some macro- Within the lymphoid organs, lymphocytes interact with
phages and γδ T cells express the transmembrane form of the other cell types of both haematopoietic and non-
molecule. haematopoietic origin that are important for lymphocyte
In mice, NK cells do not express CD56 and have historically maturation, selection, function and disposal of terminally dif-
been defined by their expression of the activating NK cell recep- ferentiated cells.
tors NK1.1 or NKp46. However, the recent discovery that some These other cell types are termed accessory cells and include:
helper ILCs also express these molecules means that this defini- • antigen-presenting cells;
tion is now treated with some caution. • macrophages;
Resting NK cells in both species express the β chain of the IL- • reticular cells; and
2 receptor and the signal transducing common γ chain of IL-2 • epithelial cells.
and other cytokine receptors (see Fig. 7.16). Therefore, direct The lymphoid system is arranged into either discrete encap-
stimulation with IL-2 activates NK cells. sulated organs or accumulations of diffuse lymphoid tissue,
which are classified into primary (central) and secondary
Helper ILCs polarize the immune response by producing (peripheral) organs or tissues (Fig. 2.23).
cytokines. ILC1, ILC2 and ILC3 are ubiquitous but are most In essence, lymphocytes:
often found at protective barriers in tissues associated with • are produced, mature and are selected in primary lymphoid
gastrointestinal, bronchial tracts and skin. Many of the ILCs organs; and
are found in higher numbers during neonatal development • exert their effector functions in the secondary lymphoid
than in adult life. There is some evidence that ILC2 and ILC3 organs and tissues.
subsets can change into ILC1 in the appropriate cytokine Tertiary lymphoid tissues are anatomical sites that under
micro-environment. normal conditions contain sparse lymphocytes, if any, but
The phenotypic definition of most helper ILC subsets has not may be selectively populated by these cells in pathological con-
yet been fully agreed upon. One complicating factor is that the ditions (e.g. skin, synovium, lungs).
phenotype of these cells varies between species and even
between organs in the same species. One definition that is cur- Lymphoid stem cells develop and mature within primary
rently widely used is expression of IL-7Rα (CD127) in the lymphoid organs. In the primary lymphoid organs, lympho-
absence of lineage markers that define other immune cell sub- cytes (B and T cells) differentiate from lymphoid stem cells, pro-
sets (for example, CD3 and CD19). liferate, are selected and mature into functional cells.
One key role of the helper ILCs is to sense changes in the In mammals, T cells mature in the thymus and B cells mature
environment through their intracellular and extracellular cyto- in the fetal liver and postnatal bone marrow. Birds have a spe-
kine receptors and to produce cytokines in response (see cialized site of B-cell generation, the bursa of Fabricius.
Chapter 7). For example, ILC1 are activated by IL-12 released In the primary lymphoid organs:
from macrophages following ingestion of pathogens; in turn, • lymphocytes acquire their repertoire of specific antigen
they produce IFNγ and TNFα, both polarizing the immune receptors to cope with the antigenic challenges that individ-
response to an anti-viral state. Expression of NKp44 and uals encounter during their lifetime;
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“He did it in a way that none would ever discover. He trusted Higgins, and
Sabine was an accident. Perhaps ... perhaps ... he did it to keep me here ... to
save the thing he believed in all his life.”
It was a horrible thought which she tried to kill, but it lingered, together
with the regret that she had never finished what she had begun to tell him as
they stood by the hedge talking of the letters—that one day Jean might take
the name of John Pentland. He had, after all, as much right to it as he had to
the name of de Cyon; it would be only a little change, but it would allow the
name of Pentland to go on and on. All the land, all the money, all the
tradition, would go down to Pentland children, and so make a reason for
their existence; and in the end the name would be something more then than
a thing embalmed in “The Pentland Family and the Massachusetts Bay
Colony.” The descendants would be, after all, of Pentland blood, or at least
of the blood of Savina Dalgedo and Toby Cane, which had come long ago
to be Pentland blood.
And she thought grimly, “He was right, after all. I am one of them at last
... in spite of everything. It’s I who am carrying on now.”
On the morning of the funeral, as she stood on the terrace expecting Jean
and Sybil, Higgins, dressed in his best black suit and looking horribly
awkward and ill at ease, came toward her to say, looking away from her,
“Mr. O’Hara is going away. They’re putting up a ‘For Sale’ sign on his
gate. He isn’t coming back.” And then looking at her boldly he added, “I
thought you might want to know, Mrs. Pentland.”
For a moment she had a sudden, fierce desire to cry out, “No, he mustn’t
go! You must tell him to stay. I can’t let him go away like that!” She wanted
suddenly to run across the fields to the bright, vulgar, new house, to tell him
herself. She thought, “He meant, then, what he said. He’s given up
everything here.”
But she knew, too, that he had gone away to fight, freed now and moved
only by his passion for success, for victory.
And before she could answer Higgins, who stood there wanting her to
send him to Michael, Miss Egan appeared, starched and rigid and wearing
the professional expression of solemnity which she adopted in the presence
of bereaved families. She said, “It’s about her, Mrs. Pentland. She seems
very bright this morning and quite in her right mind. She wants to know
why he hasn’t been to see her for two whole days. I thought....”
Olivia interrupted her quietly. “It’s all right,” she said. “I’ll go and tell
her. I’ll explain. It’s better for me to do it.”
She went away into the house, knowing bitterly that she left Miss Egan
and Higgins thinking of her with pity.
As she climbed the worn stair carpet to the north wing, she knew
suddenly a profound sense of peace such as she had not known for years. It
was over and done now, and life would go on the same as it had always
done, filled with trickiness and boredom and deceits, but pleasant, too, in
spite of everything, perhaps because, as John Pentland had said, “One had
sometimes to pretend.” And, after all, Sybil had escaped and was happy.
She knew now that she herself would never escape; she had been too
long a part of Pentlands, and she knew that what the old man had said was
the truth. She had acted thus not because of duty, or promises, or nobility, or
pride, or even out of virtue.... Perhaps it was even because she was not
strong enough to do otherwise. But she knew that she had acted thus
because, as he said, “There are things, Olivia, which people like us can’t
do.”
And as she moved along the narrow hall, she saw from one of the deep-
set windows the figure of Sabine moving along the lane in a faint cloud of
dust, and nearer at hand, at the entrance of the elm-bordered drive, Aunt
Cassie in deep black, coming along briskly in a cloud of crape. No, nothing
had changed. It would go on and on....
The door opened and the sickly odor of medicines flooded the hallway.
Out of the darkness came the sound of a feeble, reed-like voice, terrible in
its sanity, saying, “Oh, it’s you, Olivia. I knew you’d come. I’ve been
waiting for you....”
THE END
Cold Spring Harbor, Long Island
June 4, 1925
St. Jean-de-Luz, B. P., France
July 21, 1926
Typographical errors corrected by
the etext transcriber:
lay figure=> clay figure {pg 6}
sarcely giving=> scarcely giving
{pg 205}
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