1pp imm3031 cytotoxic t cells (2016)

8/18/2019 1pp IMM3031 Cytotoxic T Cells (2016)

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Cytotoxic CD8 T cells

IMM 3031

Thursday April 21st 2016, 1pm, theatre S3

A/Prof Robyn Slattery

[email protected] Janeway – Chapter 9

Abbas - Chpt 13

Nature Immunol (2010) Vol 11 pp189-190

Nature Immunol (2013) Vol 14(4) pp311-363


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Cell-Mediated Immunity (CMI)

•

Two types of adaptive immunity

– Humoral immunity

•

B cell/antibody-based

•

primarily against extracellular microbes

– Cellular immunity or cell-mediated immunity

•

T cell-based

– helper and killer T cells

•

primarily against intracellular microbes


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T (Thymus-derived) cells

• B cells - main roles are antibody production

and antigen presentation

• T cells - more diverse functions

- CD8 T cells - killers or CTL

- kill virus infected cells, tumours (and grafts)

- most CD8 T cell responses require CD4 help for activation

- CD4 T cells - produce cytokines

- T helper cells (Th1, Th2, Th17, Tfh)

- produce stimulatory cytokines (e.g. IFN ! , IL-2 etc)

- T suppressor/regulatory cells (foxP3 T reg, NK1.1+ T)

- produce inhibitory/regulatory cytokines (e.g. TGFß, IL-4, IL-10).

- CD4 T cells can influence most cells of immune system.


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Types of Cell-Mediated Immune

Responses•

Different CMI responses deal with different

microbes

–

Pathogens residing/replicating in phagocytic

compartments (eg bacteria in macrophages)

•

Th1-based IFN-! responses

– Pathogens replicating in cell cytoplasm (eg virus)

•

CTL-based killing response

–

Large parasitic organisms (eg helminthic parasite)

•

Th2-based IgE activation of eosinophils


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Types of Cell-Mediated Immune

Responses•

Different CMI responses deal with different

microbes

–

Pathogens residing/replicating in phagocytic

compartments (eg bacteria in macrophages)

•

Th1-based IFN-! responses

– Pathogens replicating in cell cytoplasm (eg virus)

•

CTL-based killing response

–

Large parasitic organisms (eg helminthic parasite)

•

Th2-based IgE activation of eosinophils


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Development of CD8 T cellsRecognition of IL7induces nuclear

factor Runx3


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tissue DCs

(eg Langerhans cells)

HEV’s

naïve T cells

efferent lymphatic

afferent

lymphatic effector T cell

T cell priming

• Occurs within secondary lymphoid tissues

– eg lymph nodes


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Antigen-presenting cells deliver three

kinds of signals for the clonal expansion

and differentiation of naive T cells


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Naïve T

TCR

ActivatedAPC

MHC/

peptide CD4/8

CD3

Adhesion molecules

CD2

LFA-1

ICAM-1

LFA-3

• Eg. ICAM-1 or LFA-3 on APC; LFA-1 or CD2 on T cells

•

Facilitate initial interaction b/w APC and T cell

• Allow T cells to sample MHC peptide complexes on APC


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Initial contact between DC and naïve T cell

Signal 1 - engagement of TCR by MHC + peptide

–

Signal via CD3 complex associated with TCR

Coreceptor molecules CD4/CD8

• bind non-polymorphic regions of MHC-II/MHC-I respectively

Leads to upregulation of CD69 and CD40L on T cells

Naïve T

TCR

MHC/ peptide 1

CD3

CD2

LFA-1

ICAM-1

LFA-3

CD4/8

ActivatedAPC

CD69

CD40L


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Naïve T

TCR

MHC/ peptide

CD4/8

1

CD3

Increased adhesion

• On TCR recognition -> conformational change in LFA-1

–

Increases affinity for ICAM-1

– increases stability of T cell/APC complex

CD2

LFA-1

ICAM-1

LFA-3

ActivatedAPC

CD69

CD40L


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T cell activation – signal 1

•

TCR signal initiates cytokine gene transcription

–

Eg. for naïve CD4 T cells IL-2 Receptor mRNA and IL-2

mRNA

– But ! IL-2 mRNA is unstable

Naïve T

TCR

MHC/ peptide

CD4/8

1

CD3

IL-2R mRNA

IL-2 mRNA

IL-2R

unstable

CD2

LFA-1

ICAM-1

LFA-3

ActivatedAPC

CD69

CD40L


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T cell activation – signal 1

• Signal 2 - Co-stimulatory signal provided by APC

•

E.g. CD80, CD86 - binds to CD28 on T cells

• Stabilises IL-2 mRNA ->100x increase in IL-2 protein

• Proliferative signal to T cell

Naïve T

TCR

MHC/ peptide

CD4/8

1

CD3

IL-2 mRNA

IL-2R

unstable CD28 CD80/86

2 stabilised

IL-2R mRNA

IL-2

prolif

CD2

LFA-1

ICAM-1

LFA-3

ActivatedAPC

CD69


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Proliferating T cells differentiate

into effector T cells that do notrequire co-stimulation to act


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Most CD8 T cell responses

require CD4 T cell help


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Three phases of CD8 T cell

activation•

Phase I

– T cells contact DCs, ligate TCR and upregulateCD69 (~2-8hrs after Ag exposure)

•

Phase II

– T cells arrest motility with DCs and initiatecytokine expression (~8-60hrs after Ag exposure)

•

Phase III – T cell motility increases and cells proliferate


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activation•

Phase I


•

Phase II

– T cells arrest motility with DCs and initiatecytokine expression (~8-60hrs after Ag

exposure) • Phase III

– T cell motility increases and cells proliferate


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Recent evidence suggests that Phase IImay be split into early and late stages

Early phase II (~8-24hrs after Ag exposure)

antigen dependent T cell/DC synapse

Late phase II (~24-60hrs after Ag exposure) antigen independent

Dependent on LFA1/ICAM1 interactions between T cells clustering around DCs

Called critical differentiation period (CDP) and controls memory responses


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activation•

Phase I


•

Phase II

– T cells arrest motility with DCs and initiatecytokine expression (~8-60hrs after Ag exposure)

•

Phase III – T cell motility increases and cells proliferate


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• Effector T cells need to act on cell types other than activated APC - CTL must be able to kill any virus infected cells

- T helper cells need to help MØ or B cells that have taken up Ag

Effector T cells - less dependent on costimulation


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Types of armed effector T cells


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• virus infected or tumour cells are not

necessarily APCs

•

How can a CD8 T cell become primed to

these antigens?

•

DCs have the ability to ingest infected/tumour

cells, process and present antigen via MHCclass I and “cross prime” CD8 T cells

DCs can cross-present

antigen via MHC class I to

CD8 T cells


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DCs can cross-prime CD8 T

cells


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Dendritic cells the best APC for

priming CD8 T cells•

DCs strategically located

• DCs are able to ingest infected or tumour

cells and‘cross present

’ antigen via MHC I

• DCs migrate to T cell zone LNs (where naïve

T circulate)

•

DCs express high levels of co-stimulator

molecules


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Memory T cells

Memory cells - long lived, may require persistant Ag or cytokines

Upon Ag rechallenge - more vigourous response

APC Naïve T

Effector

T

Memory

T

1

2

Short lived (days)

Long lived

(years)


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Effector mechanisms - CTL• CTL

–

Killing of virus infected cells, cancer cells andforeign transplant cells

– Two major forms of killing

• perforin/granzyme mechanism

•

Fas/FasL

CTL

target

apoptosis

“lethal hit”

target

MHC class I


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Mechanism of CTL killing

Abbas et al Fig 13-16


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The granules of CTL (& NKcells)

–

Granzymes (A, B & C)

•

B most important in CTL killing –

serine proteases

–

activate caspase-3 and Bid

»

results in DNA fragmentation (both cellular and viral)

• A & C also induce apoptosis - caspase-independent

– Perforin

•

forms pores in target cell membrane

• Main function to facilitate delivery of granzymes intocytosol

•

can also kill by osmotic swelling

– Serglycin

• Assembles complex of perforin/granzymes


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Fas/FasL•

FAS (death receptor)

–

On many cell types

– member of the tumor necrosis factor receptor

(TNFR) family

– contains cytoplasmic “death domains”

Fas FasL

apoptosis

CTL


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Apoptosis•

Otherwise known as “programmed cell death”

– does not stimulate inflammation - unlike necrosis (cell disintegration)

– cells destroy themselves from “within”

– ‘apoptotic bodies’ cleared by tissue macrophages without inducing immune

response

•

Two major pathways (both involve intracellular signaling cascade and

activation of caspases) – Death receptor pathway – Mitochondrial pathway

•

Results in:

– Chromatin condensation

– Nucleolar disruption

–

Cytoplasmic contraction – membrane “blebbing”

– DNA fragmentation into “ladder ”


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Caspases

•

Cysteine proteases that cleave proteins

immediately after aspartic acid residues –

ie have cysteine in active site

• Present in cytoplasm of most (all?) cells

–

inactive (zymogen) form

• ‘initiator ’ caspase activated by signals from

– Death receptor pathway

–

Mitochondrial pathway

•

‘initiator ’ then activates downstream‘executioner ’ caspases

– common death pathway


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CTL killing mechanisms

Fas

FasL

granzyme B

caspase 8

caspase 3

caspase activatable

DNAse (CAD)

death-domain

See Abbas et al Box 11-2

ICAD


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-death receptor pathway


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-perforin/granzyme targetsdownstream in the death

receptor pathway at pro-

caspase-3

-as well as targeting the

mitochondrial pathway


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The release of cytochrome c into the cytosol initiates themitochondrial pathway of death

CTL killi h i


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-mitochondrial pathway


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Sequential killing

1st kill

2nd kill

CTL


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Directional killing

•

Granules accumulate at the immunologicalsynapse

– secretion at CTL-target junction

– seen with other T cell products

•

eg cytokines

See Abbas et al Fig 13-15

immune

synapse


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Do CTL kill themselves?

•

Some suggestion they are protected

– consequence of directional killing

– CTL membrane surface has cathepsin B

(proteolytic enzyme that degrades perforin)


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We use genetic engineering tools to studythe role of CD8 T cells in T1D Why do CD8 T cells escape thymic selection in T1D?

Why are they not controlled by Tregs?

Why do they become inappropriately activated? Are CD8 T cells responsible for killing the insulin producing beta

cells in T1D?

-cell

CD8

CD4 CD8

APC

1pp imm3031 cytotoxic t cells (2016)

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