CELL MICB715-Advances

MEDIATED in Immunology
Fa2020
IMMUNITY WEEK 9 LECTURE 8

PREPARED BY
MS. Beenish Sarfraz
Department of Biology
Lahore Garrison University
 Preamble (Past Lesson Brief)

In Previous lecture, we came to know about B cell and T-helper cell

response

In this lecture we will discuss cellular immune ressponse

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 Learning Outcomes
Upon completing this lecture students will be able to:

Understand the cytotoxic T cell response

Understand the NK cell response

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ARE YOU READY ?

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TYPES OF T CELLS

TH CELLS TC CELLS TREG

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Cell-mediated immunity is the type of host defense that is
mediated by T lymphocytes, and it serves as the defense
mechanism against microbes that survive and replicate
inside phagocytes and nonphagocytic cells.

T cells recognize protein antigens of microbes that are

displayed on the surfaces of infected cells as peptides bound
to self major histocompatibility complex (MHC) molecules.
CELL
MEDIATE
cell-mediated immunity is specific for cell associated
microbes.
D
IMMUNIT
Defects in cell-mediated immunity result in increased
susceptibility to infection by viruses and intracellular
Y
bacteria as well as some extracellular bacteria and fungi that
are ingested by phagocytes.

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 Effector T cells of the CD4+ lineage link specific
recognition of microbes with the recruitment and
activation of other leukocytes that destroy the
microbes
TYPES OF The nature of the leukocytes that are recruited and
CELL activated is determined by the subset of CD4+ effector
T cells that are induced in the immune response.
MEDIATED
REACTIONS  In general, TH1 cells activate macrophages
 TH17 reactions are dominated by neutrophils (and
variable numbers of macrophages)
and TH2 cells recruit and activate eosinophils.

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CMI Response
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MACROPHAGES AND TH CELLS
The adaptive immune response to microbes that are phagocytosed and live within the
phagosomes of macrophages is mediated by TH1 cells, which recognize microbial antigens and
activate the phagocytes to destroy the ingested microbes.
 Macrophages are major cells of host defense early after infection, that is, during innate
immune responses
Their function is to ingest and kill microbes.
Many microbes have developed mechanisms that enable them to survive and even to replicate
within phagocytes, so innate immunity is unable to eradicate infections by such microbes. In
these situations, TH1 cells function to enhance the microbicidal actions of macrophages and thus
to eliminate the infection.

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Types cont…
The response to extracellular microbes, including many fungi and
bacteria, is mediated by TH17 cells. These cells recruit neutrophils
(and some monocytes), which ingest and destroy the microbes.
 The response to helminthic parasites is mediated by TH2 cells,
which stimulate the production of immunoglobulin E (IgE) antibodies
and activate eosinophils and mast cells to eliminate the helminths.

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 CTLS ROLE
The adaptive immune response to microbes that infect and replicate in the cytoplasm of
various cell types, including nonphagocytic cells, is mediated by CD8+ cytotoxic T lymphocytes
(CTLs), which kill infected cells and eliminate the reservoirs of infection
If the infected cells lack intrinsic microbicidal ability, the infection can be eradicated only by
destroying these cells.
CTL-mediated killing is also a mechanism for elimination of microbes that are taken up by
phagocytes but escape from phagosomes into the cytosol, where they are not susceptible to the
microbicidal activities of phagocytes.

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T CELL- dependent inflammation
T cell–dependent inflammation may damage normal tissues.
 Inflammation, consisting of leukocyte recruitment and activation, accompanies many of the
reactions of CD4+ T lymphocytes and may be injurious under various conditions.
 This T cell–dependent injurious reaction is called delayed-type hypersensitivity (DTH)
 the term hypersensitivity referring to tissue damage caused by an immune response.
 DTH frequently occurs together with protective cell-mediated immunity against microbes and
may be the cause of much of the pathology associated with certain types of infection

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CMIR Reaction Chain
Cell-mediated immune responses consist of the development of effector T
cells from naive cells in peripheral lymphoid organs

Migration of these effector T cells and other leukocytes to sites of

infection,;

Cytokine mediated activation of leukocytes to destroy microbes or direct

killing of infected cells

The development of effector T cells involves the sequence of antigen

recognition, clonal expansion, and differentiation that is characteristic of all
adaptive immune responses

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 Recruitment of other leukocytes.

The recruitment of neutrophils, monocytes, and eosinophils to the site

The functions of the reaction is mediated by chemokines produced by the T cells
themselves and by other cells in response to cytokines secreted by the
of CD4+ T cells.

effector cells in Activation of the recruited leukocytes.

cell-mediated
immunity: The mechanisms by which CD4+ T cells activate other leukocytes
STEPS involve T cell expression of the surface protein CD40 ligand (CD40L) and
secretion of cytokines.

The CD40Lmediated pathway is best defined for TH1-mediated

activation of macrophages.

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 RESPONSE TYPE
Amplification of the response.
cytokines produced by T cells activate macrophages to produce other cytokines that in turn act on the T cells
and increase their responses
Downregulation of the response.
Because effector T cells are typically short-lived, they die after performing their function.
As the antigen is eliminated, the stimuli for propagating the response are lost, and the response declines over
time. Special control mechanisms may also operate to limit effector responses.
both TH1 cells and activated macrophages produce the cytokine IL-10, which functions mainly to inhibit
further TH1 differentiation and macrophage activation.
Additional inhibitory mechanisms, such as other anti-inflammatory cytokines and receptors that turn off T cell
activation, may also be involved in controlling T cell–mediated responses.

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Functions of TH1 Cells
The principal function of TH1 cells is to activate macrophages to
ingest and destroy microbes
TH1 effector cells develop in response to the pathogens that these
cells are designed to eradicate, an excellent example of the
specialization of adaptive immunity.

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CYTOKINES PRODUCED BY TH1
Interferon-γ
 IFN-γ is the principal macrophage-activating cytokine and serves
critical functions in immunity against intracellular microbes.
 IFN-γ is also called immune or type II interferon.
it has some antiviral activity, it is not a potent antiviral cytokine, and
it functions mainly as an activator of effector cells of the immune
system

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 Mechanism of IFN-ɣ
IFN-γ activates macrophages to kill phagocytosed microbes, the hallmark of
“classically activated” macrophages.
These transcription factors stimulate the expression of several enzymes in the
phagolysosomes of macrophages, including phagocyte oxidase, which induces
the production of reactive oxygen species (ROS); inducible nitric oxide synthase
(iNOS), which stimulates the production of nitric oxide (NO); and lysosomal
enzymes.
These substances destroy ingested microbes in the vesicles and are
responsible for the microbicidal function of activated macrophages.

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 Functions of TH1 cells.
CD4+ T cells that differentiate into TH1 cells secrete
IFN-γ, which acts on macrophages to increase
phagocytosis and killing of microbes in
phagolysosomes and on B lymphocytes to
stimulate production of IgG antibodies that
opsonize microbes for phagocytosis. The cells also
produce TNF, which activates neutrophils and
promotes inflammation

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 TH1-Mediated Classical Macrophage Activation
and Killing of Phagocytosed Microbes
In cell-mediated immune responses against phagocytosed microbes, T cells specifically
recognize microbial antigens but phagocytes actually destroy the pathogens.
At any site of infection, as part of the innate immune response, monocytes are recruited
from blood into tissues by chemokines produced by macrophages and other cells resident at
the site
These monocytes mature into tissue macrophages and first attempt to phagocytose and
destroy the pathogen.
If the microbe has evolved to resist elimination by the macrophages, it survives within the
phagosomes. In these infected cells, microbial peptides are processed and presented as
peptides associated with class II MHC molecules.

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TH1 effector cells generation
At the same time, TH1 effector cells are generated in an adaptive immune
response in secondary lymphoid tissues.
These T cells are recruited to the site of infection, where they recognize
antigenic peptides (the same as those that initiated the response) displayed by
the microbe-bearing macrophages.
The macrophages are exposed to signals from the TH1 effector cells, which
activate the macrophages to kill the ingested microbes.
Activation consists of increased expression of various proteins that endow
activated macrophages with the capacity to perform specialized functions, such
as microbial killing.

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 ACTIVATION OF MACROPHAGES
CD4+ TH1 cells activate macrophages by contact mediated signals delivered by CD40L-CD40
interactions and by IFN-γ
When the TH1 cells are stimulated by antigen, the cells express CD40L on their surface and
secrete IFN-γ. The actions of IFN-γ on macrophages, synergize with the actions of CD40 ligand,
and together they are potent stimuli for macrophage activation.
Activated macrophages kill phagocytosed microbes mainly by the actions of reactive oxygen
species, nitric oxide, and lysosomal enzymes. All these potent microbicidal agents are produced
within the lysosomes of macrophages and kill ingested microbes after phagosomes fuse with
lysosomes
These toxic substances may also be released into adjacent tissues, where they kill extracellular
microbes and may cause damage to normal tissues. This pathway of macrophage activation is
called classical to distinguish it from alternative activation

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 INFLAMMATION
Activated macrophages stimulate inflammation through the secretion of cytokines, mainly TNF, IL-1, and chemokines, and short-lived lipid
mediators such as prostaglandins, leukotrienes, and platelet activating factor.

The collective action of these macrophage-derived cytokines and lipid mediators is to recruit more leukocytes, which improves the ability to
destroy infectious organisms.

Activated macrophages amplify cell-mediated immune responses by becoming more efficient APCs because of increased levels of molecules
involved in antigen processing and increased surface expression of class II MHC molecules and costimulators and by producing cytokines (such
as IL-12) that stimulate T lymphocyte differentiation into effector cells.

Some tissue injury may normally accompany TH1 cell–mediated immune reactions to microbes because the microbicidal products released by
activated macrophages and neutrophils are capable of injuring normal tissue and do not discriminate between microbes and host tissue.
However, this tissue injury is usually limited in extent and duration, and it resolves as the infection is cleared

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Functions of TH2
TH2 cells stimulate IgE- and eosinophil-mediated reactions that serve to
eradicate helminthic infections
 Helminths are too large to be phagocytosed by neutrophils and macrophages
and may be more resistant to the microbicidal activities of these phagocytes
than are most bacteria and viruses.
TH2 cells secrete IL-4, IL-5, and IL-13, which work cooperatively to eradicate
these infections.

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 The functions of TH2 cells are
mediated by

IL-4, which induces IgE antibody

Cytokines responses
Produced by
TH2 Cells
IL-5, which activates eosinophils

IL-13, which has diverse actions

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 FIGURE 10–8
Classical and alternative macrophage activation.
Subsets of activated macrophages.
Different stimuli activate monocytes-macrophages
to develop into functionally distinct populations.
• Classically activated macrophages are induced by
microbial products and cytokines, particularly
IFN-γ, and are microbicidal and involved in
potentially harmful inflammation.
• Alternatively activated macrophages are induced
by IL-4 and IL-13 produced by TH2 cells and other
leukocytes and are important in tissue repair and
fibrosis

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 IL-4
IL-4 is the major stimulus for the production of IgE antibodies and for the development of TH2 cells from
naive CD4+ helper T cells.
IL-4 stimulates B cell Ig heavy chain class switching to the IgE isotype
IL-4 stimulates the development of TH2 cells and functions as an autocrine growth factor for differentiated
TH2 cells
IL-4, together with IL-13, contributes to an alternative form of macrophage activation that is distinct from
the macrophage response to IFN-γ.
IL-4 (and IL-13) stimulate peristalsis in the gastrointestinal tract
IL-13 increases mucus secretion from airway and gut epithelial cells. Both these actions contribute to
elimination of microbes at epithelial surfaces.

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IL-13
 IL-13 is structurally and functionally similar to IL-4 and also plays a
key role in defense against helminths) and in allergic diseases
 IL-13 is produced mainly by the TH2 subset, but basophils,
eosinophils, and NKT cells may also produce the cytokine.

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IL-5
IL-5 is an activator of eosinophils and serves as the principal link between T cell
activation and eosinophilic inflammation.
It is produced by TH2 cells and by activated mast cells.
The principal actions of IL-5 are to activate mature eosinophils and to stimulate
the growth and differentiation of eosinophils.
Activated eosinophils are able to kill helminths. Eosinophils express Fc receptors
specific for IgE and some IgG antibodies and are thereby able to bind to microbes,
such as helminths, that are opsonized by these antibodies.
 IL-5 also stimulates the production of IgA antibodies.

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Roles of TH2 Cells in Host Defense
IgE- and eosinophil-mediated reactions.
 IL-4 (and IL-13) stimulates the production of helminth-specific IgE antibodies, which opsonize the
helminths and promote the binding of eosinophils.
IL-5 activates the eosinophils and these cells release their granule contents, including major basic
protein and major cationic protein, which are capable of destroying even the tough integuments of
helminths
Activation of mast cells.
Mast cells express high affinity Fcε receptors and may be activated by IgE coated helminths and other
antigens that bind IgE, resulting in degranulation.
The granule contents of mast cells include vasoactive amines, and mast cells secrete cytokines such as
TNF and chemokines, and lipid mediators, all of which induce local inflammation that helps to
destroy the parasites.

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 Cytokines produced by TH2 cells are
involved in blocking entry and promoting
expulsion of microbes from mucosal
organs.
Barrier For instance, IL-13 stimulates mucus
immunity. production, and IL-4 and IL-13 may
stimulate peristalsis in the gastrointestinal
system.
TH2 cells play an important role in host
defense at the barriers with the external
environment, sometimes called barrier
immunity.
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IL-4 and IL-13 activate macrophages to express enzymes that promote collagen synthesis and fibrosis.

The macrophage response to TH2 cytokines has been called alternative macrophage to distinguish it
from the activation induced by IFN-γ, which was characterized first (and hence the designation
“classical”) and which results in potent microbicidal functions.
Macrophages that are activated by TH2 cytokines contribute to tissue remodeling and fibrosis in the
setting of chronic parasitic infections and allergic disease.
Alternatively activated macrophages may also serve to initiate repair after diverse types of tissue
injury that may not involve infectious agents or immune responses; in these situations, the activating
cytokines, such as IL-4, may be produced by eosinophils and other cell types in tissues.

Alternative macrophage activation.

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 Cells TH17 cells secrete cytokines that
recruit leukocytes, mainly
neutrophils, to sites of infection
Functions of
TH17 Because neutrophils are a major
defense mechanism against
extracellular bacteria and fungi,
TH17 cells play an especially
important role in defense against
these infections.
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EFFECTOR FUNCTIONS OF CD8+
CYTOTOXIC T LYMPHOCYTES
CD8+ CTLs eliminate intracellular microbes mainly by killing infected
cells
The development of a CD8+ CTL response to infection proceeds through
similar steps as those described for CD4+ T cell responses, including
antigen-mediated stimulation of naive CD8+ T cells in lymphoid organs,
clonal expansion, differentiation, and migration of differentiated CTLs into
tissues.
In addition to direct cell killing, CD8+ T cells secrete IFN-γ and thus
contribute to macrophage activation in host defense and in
hypersensitivity reactions.

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Recognition of Antigen and Activation of
CTLs
The CTL binds and reacts to the target cell by using its antigen receptor, coreceptor (CD8), and adhesion
molecules.
To be efficiently recognized by CTLs, target cells must express class I MHC molecules complexed to a peptide
(the complex serving as the ligand for the T cell receptor (TCR) and the CD8 coreceptor) and intercellular
adhesion molecule 1 (ICAM-1, the principal ligand for the LFA-1 integrin). The CTLs and their target cells form
tight conjugates.
 This immunologic synapse formed between the two cells is characterized by a ring of close apposition
between the CTL and target cell membranes, mediated by LFA-1–ICAM-1 binding, and an enclosed gap or
space inside the ring.
This interaction results in the initiation of biochemical signals that activate the CTL, which are essentially the
same as the signals involved in the activation of helper T cells
once CD8+ T cells specific for an antigen have differentiated into fully functional CTLs, they can kill any
nucleated cell that displays that antigen.

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Killing of Target Cells by CTLs
Within a few minutes of a CTL’s antigen receptor recognizing its antigen
on a target cell, the target cell undergoes changes that induce it to die by
apoptosis.
 Target cell death occurs during the next 2 to 6 hours and proceeds even
if the CTL detaches. Thus, the CTL is said to deliver a lethal hit to the
target cell.
The principal mechanism of CTL-mediated target cell killing is the
delivery of cytotoxic proteins stored within cytoplasmic granules (also
called secretory lysosomes) to the target cell, thereby triggering
apoptosis of the target cell

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 CTL ACTION
1. CTL recognition of the target leads to activation of the CTL, one
consequence of which is cytoskeleton reorganization such that the
microtubule organizing center of the CTL moves to the area of the
cytoplasm near the contact with the target cell.
2. The cytoplasmic granules of the CTL are transported along microtubules
and become concentrated in the region of the synapse, and the granule
membrane fuses with the plasma membrane at the secretory domain.
3. Membrane fusion results in exocytosis of the CTL’s granule contents
into the confined space within the synaptic ring, between the plasma
membranes of the CTL and target cell.

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 The cytotoxic proteins in the granules of CTLs (and NK cells) include
granzymes and perforin.

Granzymes A, B, and C are serine proteases that cleave proteins

Perforin is a membrane-perturbing molecule homologous to the C9

STEPS CONT.. complement protein.

The granules also contain and a sulfated proteoglycan, serglycin, which

serves to assemble a complex containing granzymes and perforin. The
main function of perforin is to facilitate delivery of the granzymes into
the cytosol of the target cell.
Perforin may polymerize and form aqueous pores in the target cell
membrane through which granzymes enter

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Steps Cont…
According to another current model, complexes of granzyme B, perforin, and
serglycin are discharged from the CTL onto the target cell and are internalized
into endosomes by receptor-mediated endocytosis.
Perforin may act on the endosomal membrane to facilitate the release of the
granzymes into the target cell cytoplasm. Once in the cytoplasm, the granzymes
cleave various substrates, including caspases, and initiate apoptotic death of the
cell.
Another protein found in human CTL (and NK cell) granules, called granulysin,
can alter the permeability of target cell and microbial membranes, but its
importance in cell killing by CTLs is not established.

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Video demonstration
https://www.khanacademy.org/science/biology/human-biology/immunology/v/review-of-b-cell
s-cd4-t-cells-and-cd8-t-cells

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Q&A

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Solved examples

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Challenging exercise
Give response of virus infected cells?

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What did you learn?

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Home Assignment

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 Next Lesson Preview

In Next lecture, we will discuss the immune reactions

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 References

Kuby, Judith A. Owen, Jenni Punt, Sharon A. Stranford, Patricia P. Jones. 2013. Immunology. W.
H. Freeman and Company. New York.

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