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Chapter 9

T cell mediated immunity

T and B lymphocytes are found in distinct locations in secondary lymphoid tissues

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Lymphocytes and peripheral lymphoid tissue

 B and T cells find their way
to their respective
destinations by responses to
chemokines

 B and T cells possess

receptors that bind to
ligands in these zones

Peripheral lymphoid tissue development

 TNF and TNFR (receptor) have roles in the development and maintenance
of the secondary lymphoid tissue structure

 Lymph node development depends on expression of a subset of TNF family

of proteins-
 Lymphotoxin (LT)
 Required for normal segregation of T and B cell zones

 Not all lymph nodes are dependent on signals from same family of TNF
proteins

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Peripheral lymphoid tissue development

 LTά3
 supports development of cervical, lumbar, sacral and mesenteric
lymph nodes
 LT ά3 is thought to bind to TNFR-I and HVEM

 LT ά2 and LTß1 bind only to LTß receptor

 Supports development of all the other lymph nodes

 Signaling through LTß is required for:

 lymph node and follicular dendritic cell
development
 Normal development of spleen

 Signaling through TNFR-I is required for

 Follicular dendritic cell development
 Normal development of spleen

 NOT peripheral lymph node development

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Homing of lymphocytes in peripheral lymphoid

tissue
 Newly formed lymphocytes enter lymph nodes through HEV
(high endothelial venules)

 HEV is in the T zone

 B cells then move to follicle

 If no antigen is encountered, they stay for a day

 Location of cells controlled by chemokines

 produced by stromal and bone marrow derived cells

Homing of lymphocytes in peripheral lymphoid

tissue
 CCL19, CCL18 and CCL21 account for T cells localization in T
zones
 Also account for B cells presence in developing lymph node
 Their ligand is CCR7 on T cells
 Low levels of CCR7 expressed on B cells

 Dendritic cells also express CCR7

 B cells are attracted to the follicles by CXCL13

 CXCR5 is the ligand on B cells

 B cells also are a source of LT

 required for development of follicular dendritic cells

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Peripheral lymphoid tissue development

 Activated B cells express low levels of CCR7
 Explains their migration pattern after activation
 First T zone, then to the follicle (germinal center)

 T cells also express low levels of CXCR5

 Explains how T cells enter B cell follicles

Lymphocytes and peripheral lymphoid tissue

T and B cells are partitioned into distinct regions of secondary lymphoid tissues
by the actions of chemokines

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Peripheral lymphoid tissue development

 Immature lymphocytes leaving the primary lymphoid tissues are non
reactive against self antigens
 They must now undergo peripheral tolerance
 Ex. thyroglobulin

 Lymphocytes in the periphery have three fates

 Deletion, anergy and survival
 T cells undergo activated-induced cell death

 There is no receptor editing in the periphery

 advanced stage of the B cell
 Can no longer rearrange light chain loci

Effector T cells

 To participate in adaptive immune response, naïve T cells

 must encounter its specific antigen (target cell)
 Be induced to proliferate and differentiate

 T cells differentiate to
 cytotoxic
 helper cells (TH1, TH2, TH17, T reg)

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Production of effector cells

 Activation, differentiation of naïve T cells require three signals:
 1- antigen binding
 2- co stimulatory signals
 3- cytokines (control differentiation)

Adaptive response begins in peripheral lymphoid tissues

 Pathogens infecting:
 mucosal surfaces accumulate in peyer’s patches (gut and tonsils)

 Blood are trapped in the spleen

 Other sites are trapped at nodes downstream of site of infection

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Naïve T cell sampling

 Naïve T cells enter lymphoid tissue through HEV-high endothelial

venules

 Circulate continuously making contact with thousands of APCs

 Reinforces positive selection
 High chance of encountering pathogens

 If antigen is not encountered, T cells enter blood and continue routine

 If antigen is encountered:
 migration stops
 Clonal expansion and differentiation
 effector T cells are generated several days later

Naive T cells migrate through secondary lymphoid tissues, sampling peptide:MHC

complexes on dendritic cells
T cells recognizing peptides are activated to proliferate and differentiate

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T cells exit the lymph node to affected site (s)

Effectiveness of sampling

 Recirculation and recognition by T cell is very effective

 All T cells specific for an antigen is trapped within 2 days
 Clones exit by day 5

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Lymphocyte and cell adhesion molecules

 Migration, activation and effector functions are dependent on
interaction with adhesion molecules

 Classes of adhesion molecules include:

 Selectins
 Integrins
 Members of the Ig superfamily
 Mucin-like molecules (addressins)

Entry of lymphocyte into lymph node

 Occurs in distinct stages

 Chemokine signaling increases affinity of T cells to adhesion molecules

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T cells Leaving lymphoid tissue

 Sphingosine1-phosphate (S1P):
 Lipid molecule
 Binds to S1PR1 on T cells
 Chemotactic and signaling properties
 Large G protein

 Concentration gradient of this molecule influences migration of T cells

from lymph node
 Naïve T cells expressing S1P are drawn away from lymphoid tissues to
blood/lymph

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The exit of T cells from lymph nodes is controlled by a chemotactic lipid

Different maturation stages of dendritic cells

T-cell responses are

initiated in secondary
lymphoid organs by
activated dendritic
cells

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Antigen presenting cells

Macrophages
 Found in many areas of lymph node
 Especially at the afferent and efferent sites
 prevent microbes and other antigens from getting into blood
 Stimulate immune response in T cell areas

B cells
 Bind, phagocytose, degrade soluble antigens
 present the peptides
 Can also activate T cells if B cells express co stimulatory
receptors
 Least efficient in initiating adaptive response

Antigen presenting cells

 Dendritic cells process and
present a wide variety of
pathogens

 Macrophages process and

present ingested pathogens

 B cells process and presents

soluble antigens

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Dendritic cell types

 Two types
 Conventional and plasmacytoid (pDc)
 pDC produce interferons (α, β) due to viral infection
 Not important in activating T cells
 Express fewer MCH II molecules and co-stimulatory molecules
 Less efficient antigen processing

Dendritic cells and maturity

 Mature cells express co-stimulatory molecules
 loose ability to ingest antigens
 secrete CCL18 attracting naïve T cells

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 Take up pathogens by:

 macropinocytosis
 large volumes of fluid engulfed
 Phagocytosis
 Dec 205 receptors used in taking up antigens
 Viral infection in cytosol

 Langerhan’s cells are immature dendritic cells found under the skin
 Transfer of antigens ensures antigen presentation after death of infected dendritic cell

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Maturation stages in dendritic cells

 TLR and chemokine signaling transform
immature cells to mature dendritic cells
 Change known as licensing

 Activated dendritic cell through TLR induces

CCR7 receptor
 Binds to CCL21

 Signaling through CCR7 lead to the following:

 Processing and presentation rate are increased
 Expression of co stimulatory molecules
 High levels of adhesion molecules
 DC-SIGN
 Secrete CCL18
 Attract naïve T cells

Macrophages and activation of T cells

 Macrophages become APCs when a pathogen is ingested but evades
destruction

 Resting macrophages do not express B7

 Expression is thought to differentiate pathogens from harmless proteins

 Few or no MHC class II molecules are expressed

 presence of antigen induces expression

 No toll like receptor (TLR-4) in some macrophages -spleen and

liver

Why? Any guesses?

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B cells as APCs
 Efficiently binds soluble antigens

 Internalized antigen is processed and presented by MHC II molecules

 If induced to express B7, they can activate naïve T cells

 Cannot activate T cells in the absence of an infection

 Renders T cell anergic

B cells are highly efficient at presenting antigens that bind to their surface
immunoglobulin

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Clonal expansion and differentiation of T

cells
 Require three signals
1. Binding of the complex by
TCR transmits one signal

2. Co-stimulatory signals (B7)

must also be transmitted by
the APC
 Found only on cell
surface that stimulate T
cell proliferation

 CD8 T cells require a

stronger signal compared to
CD4
3. Cytokine signals

Proliferation of T cells (C28. B7)

 On activation, T cells
 enter the G1 phase of cell cycle
 During G, the cell is metabolically active and continuously grows
 Produce the α chain of the IL-2 receptor
 drives its proliferation and promotes differentiation
 Without IL-2 T cells do not proliferate

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 T cells secrete IL-2 and bind it as

well (autocrine)

 IL2 signaling used by drug

companies

 Suppresses immune responses of

transplant rejection

 Cyclosporing A, tracrolimus inhibit

IL2 production (TCR)

 Rapamycin inhibits signaling through

IL2 receptor

Clonal expansion of T cells and signaling

 Activation leads to expression of more proteins on T cells
1. ICOS (inducible co-stimulator, B7h)
• Binds to LICOS
• Induces IL10 expression, not IL2
• inhibits differentiation of TH1 cells
• Drives T cell growth

2. TNF family
 CD40 ligand on T cells
 Binds to CD40 on APCs and results in
 Transmission of activating signals to T cell
 activates APC to express B7 molecules
 stimulate further T cell proliferation

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3. CD152 (CTLA-4) is also

expressed on activation
 another B7 ligand which closely
resembles CD28

 Binds B7 about 20x more than

CD28

 Delivers inhibitory signals to

TCR
 limiting proliferative response of
activated T cells

Co-stimulatory molecules and T cells

 In the absence of co-stimulatory molecules
 small amounts of IL-2 are produced
 cell does not proliferate
 antigen recognition renders the cell anergic
 Self tolerance induced

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 Two signals from one cell are required for activating T cells
 prevent autoimmune responses
 Cells are rendered anergic in the absence of a co-stimulation

T cell differentiation
 After 4-5 days of proliferation, T cells differentiate into effector T cells
 Synthesize all effector molecules required for function
 Effector cell’s encounter with specific antigen does not require co-
stimulation. Why?

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T cell differentiation
 Activated T cells change expression of surface molecules
 Presence of VLA-4
 necessary to bind to vascular endothelium to site of infection
 Lose of L-selection

CD4 T cell differentiation

 Activated CD4 cells differentiate into TH1, TH2, TH17, TFH or
regulatory T cells
 Differ in cytokines produced and function

 Two types of regulatory T cells

 Natural
 Committed in the thymus
 Adaptive
 From naïve T cell
 Influence of environmental conditions

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CD8 T cell activation/differentiation

 Require more co-stimulatory
activity to become cytotoxic T
cells

 Two ways of activation

1. dendritic cells, B7
molecules
 potent activators

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CD8 T cell differentiation

2. Some may require the help of
CD4 T cells

 Use of CD40 ligand

 Activates the APC to express high
levels of co-stimulatory molecules
 Produces IL2, promoting CD8
differentiation

 Both T cells must recognize

related antigens on the surface of
same APC

Signaling through cytokines- signal 3

 Differentiating into which CD4 T cell occurs during priming
 Regulated by signals provided by environment and by APC

 Differentiation is dependent on the types of cytokines produced

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Cytokines induce the differentiation of naive CD4 T cells down distinct effector
pathways

Different STAT transcription factors lead to specific CD4 T cells

CD4 T-cell subsets can cross-regulate each other’s differentiation through the
cytokines they produce

TGF-B secreted
by T reg cells
inhibit TH1 and
TH2 proliferation

IFN-y produced
by TH1 inhibits
TH2 proliferation

IL4 produced by
TH2 inhibits TH1

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T cell’s interaction with target cells

 After differentiation, T cells search
for targets displaying antigens they
recognize

 Some T cells go into circulation

looking for targets

 Others encounter targets in

lymphoid tissues

 CD4 cells have longer contact with

target cells compared to CD8 cells

T cell interaction with target cells

 TCR and co-receptor cluster at site of cell-cell contact
 forms supramolecular adhesion complex---SMAC or
immunological synapse

 Antigen specific TCR binding sends signals with three outcomes

 Induces stable binding of effector cells to target cells
 Narrow space for concentration of molecules

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T cell interaction with target cells

 Reorientation of the cytoskeleton
 Cortical actin fibers are reorganized
 Microtubules are also reorganized
 Results in the focusing of exocytosis of the lytic granules at site of contact

 Triggers toxic molecules release and synthesis

T cell interaction with target cells

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Effector T cells and secreted molecules

Effector T cells secrete
 Cytotoxins (CD8 cells)
 Non specific
 Stored in specialized granules
 Cytokines and related proteins

Cytokines
 Small soluble proteins

 Most cytokines produced by T cells are termed interleukins

 Main cytokine released by CD8 T cell is IFN-γ

 blocks viral replication
 Eliminate virus from infected cell

 TH2 cells predominantly secrete IL4, IL5, IL9, IL10 and IL13

 All except IL 10 activate B cells

 IL 10 inhibits macrophage activation

 TH1 cells secrete IFN-γ and lymphotoxin (LTά)

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Cytokines and their receptors

 Both hematopoietin and interferon receptors signal through

Jak-STAT pathway

Key molecules are members of the

 Janus family of tyrosine Kinases (JAKs)
 4 members

 STATs (signal transducing activators of transcription)

 7 types
 STAT4 TH1 cell dev’t
 STAT6 TH2

Effector function of CD8 T cells

 All viruses and some bacteria are intracellular pathogens

 Are not accessible to antibodies after entry

 Can only be eliminated by modification or destruction of the

infected cell

 That’s why CD8 cells are most needed!!

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Effector function of CD8 T cells

 They can induce apoptosis
 First, nuclear blebbing (budding)
 Alteration in cell morphology
 Fragmentation of DNA (200 base pairs)

 Can program death within five minutes

 Death is evident after some hours

 Infectious agent can also be eliminated

 Activated nucleases can degrade viral DNA
 prevents assembly of the particle, which prevents further
spread of the virus

Apoptosis via Fas controls lymphocyte population

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Effector function of CD8 T cells

 Mode of action of CD8
apoptosis is by calcium
dependent release of lytic
granules

 Granules are modified

lysosomes

 Contains 3 classes of proteins

 Produced in the active form, but

are inactive in granule
environment

 effective only after their release

Effector function of CD8 T cells

 Previouly thought Perforin makes pores through which granzymes can move
into target cell (channel)

 Now complex formation

 Perforin and granzyme complex with serglycin
 Perforin aids in translocation

 Granzyme B cleaves and activates caspase-3

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Apoptosis (programmed cell death)

 Apoptotic program is present in all cells

 Useful in Getting rid of:

 Unneeded cells after infection
 Non functional lymphocytes
 Lymphocytes reacting with self cells

 Can be triggered by
 an absence of appropriate survival signals
 Presence of specific signals

Apoptosis (programmed cell death)

 Generally involves the following steps:
 Plasma membrane blebbing
 Membrane lipid distribution changes
 DNA fragmentation
 In the nucleus, CAD cleaves DNA into 200 base pair
fragments, characteristic of apoptosis
 Two main pathways are involved in signaling cell death
 Extrinsic pathway- activation of death receptors
 Extracellular ligands
 Intrinsic or mitochondrial pathway
 Ex. Some Drugs, Uv irradiation

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Apoptosis (programmed cell death)

 Both pathways involve activation of proteases known as caspases

 Two classes exist:

 Initiator
 Cleaving and activating other caspases
 Intrinsic pathway-caspase 9
 Extrinsic pathway- caspase 8
 Effector
 Initiate cellular changes; degrading DNA
 Both pathways use caspases 3,6, and 7

Intrinsic pathway
 Occurs when cell is stressed due to
 Exposure to toxic substances
 Lack of survival signals

 Activation of caspases is due to release of cytochrome C from

mitochondria

 Cells also have proteins that inhibit cell death

 First member of family of proteins is Bcl-2

 Can be divided into:

 death inhibited genes Bcl2, Bcl-xl
 death promoting genes Bax, Bak, Bok

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Intrinsic pathway

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 After a series of activations,

 caspase- activated deoxyribonuclease
(CAD) is activated (ICAD cleaved)

 CAD then degrades DNA (200 bp)

 Macrophages recognize these

by a change in cell membrane and ingest them

 Phosphatidylserine replaces
phosphotidylcholine (phospholipid)

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Effector function of CD8 T cells

 Once the cell starts
dying, the T cell is
released from the target
cell and starts making
new granules to kill the
next infected cell

 See the reorientation of

lytic granules in red!!

Effector function of CD8 T cells

 CD8 and some CD4 T cells express Fas ligand

 CD4 cells do not contain lytic granules

 Therefore CD4 can also kill some cells

 Why is Fas ligand so important?

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Effector function of CD8 T cells

 Binding a specific pathogen
signals the cell to release the
lytic granules, which is
delivered directly to the cell

 T cell delivers its toxin at the

site of infection; thereby,
protecting healthy
surrounding cells

 Thus, a single cytotoxic T

cell has the potential of
killing many infected cells in
succession

Effector function of CD8 T cells

 CD8 cells also secrete cytokines such as interferon-γ

 inhibits replication of viruses in infected cells

 Antigen processing and presentation by MHC 1

molecules are also increased

 IFN-γ:
 Activates macrophages
 Recruits macrophages as effector cells and APCs

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