Mechanisms of lymphocyte-mediated cytotoxicity

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Dr. Ronald Smeltz

rbsmeltz@vcu.edu

Lecture objectivesLecture objectives Identify the immune cells that mediate cytotoxicity

Identify the receptor-ligand interactions that regulate cytotoxicity

Understand the molecular basis of activating/inhibitory receptors

Distinguish the granule exocytosis and receptor-mediated pathways of apoptosis

Understand the structure-function relationship of lytic granule components

Visualize the steps involved in exocytosis, apoptosis

Recognize the importance of extrinsic/intrinsic factors in development of cytotoxic T cells

Identify the immune cells that mediate cytotoxicity

Identify the receptor-ligand interactions that regulate cytotoxicity

Understand the molecular basis of activating/inhibitory receptors

Distinguish the granule exocytosis and receptor-mediated pathways of apoptosis

Understand the structure-function relationship of lytic granule components

Visualize the steps involved in exocytosis, apoptosis

Recognize the importance of extrinsic/intrinsic factors in development of cytotoxic T cells

Examples

• Pathogens

• Transformed cells

• Transplantation

• Homeostasis

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Cells that mediate cytotoxicity

• NK cells:– Innate – Receptors– Effector molecules– Circulation– No memory

• CTL:– Adaptive – Receptors– Effector molecules– Circulation– Memory

NK cell recognition of target cells

The “Missing Self” Hypothesis

• States that a lack of/down-regulation of MHC Class I on target cells leads to spontaneous NK-mediated destruction of the target cell

The “Altered Self” hypothesis

NK cell recognition: activating and inhibitory receptors

• Ly49 (mouse) H-2K, H-2D• KIR (human) HLA-A, HLA-B, HLA-C

• CD94/NKG2 Qa-1b

HLA-E

• NKG2D Rae-1MIC-A,MIC-B

• NKp ??

NK cell recognition molecules

NK cell receptors Target cell ligands

Genetic p

oly

morp

hism

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Ly49 family (mouse)

• Most Ly49 members are inhibitory receptors

• Bind to MHC Class I– Inhibitory receptors – Affinity

• Example: Ly49A

– Activating receptors– Affinity

• Examples: Ly49D, Ly49H

Journal of Molecular BiologyVolume 320, Issue 3, 12 July 2002, Pages 573-585

Basis of activation/inhibition

• ITAM– Increased tyrosine kinase

activity

• ITIM– Increased tyrosine

phosphatase activity

J Exp Med. 1997 February 17; 185(4): 673 ﾐ 684.

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Ly49A: Inhibition in action

KIR family (humans)

• KIR (Killer cell Ig-like receptors):– Immunoglobulin (Ig)-like domains

– Two types of KIR• Long: “L”, inhibitory

• Short: “S”, activating

• Similar to Ly49 family • inhibitory KIR molecules bind with high affinity

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Similarities between Ly49/KIR

• Expressed on NK cells, differentiated CD8+ T cells

• Bind to MHC Class I on target cell

• Inhibitory receptors have cytoplasmic ITIM

• Activating receptors possess ITAM

CD94/NKG2

• Inhibitory and activating receptors– CD94/NKG2A heterodimer:

• Inhibitory

– CD94/NKG2C heterodimer – CD94/NKG2E heterodimer

• Activating• Associates with DAP12

• Recognize HLA-E (Qa-1b in mouse) on target cell – Binds leader peptides derived from other

MHC class I alleles (HLA-A,B,C (humans), H-2 (mouse), HLA-G

!

NKG2D: activating receptor

Structurally linked but not encoded by the MHC locus

Pan NK cell activating receptors

Summary

Ly49/KIRLy49/KIR

polymorphic MHC encoded H-2/HLA-A,B,C-peptide

CD94/NKG2CD94/NKG2

polymorphic MHC encoded Qa-1/HLA-E+peptide

NKG2D non-poly Non-MHC encoded

Rae/MUC ligands

NKp30,40,46 non-poly ?? ??

Important points!

• NK receptors dictate the response of NK cells

• T cell receptor-MHC Class I-peptide dictate the response of CTL

• Once an activating response is initiated, the effector mechanisms are identical

Effector mechanisms of cytotoxicity

Effector molecules of cytotoxicity

• Used by NK cells and CTL

• Granule exocytosis pathway• Perforin, Granzymes• Trigger apoptosis

• Fas/Fas-L pathway• Receptor-mediated death• Trigger apoptosis

Mediators of cytotoxicity

• Granzymes, perforin• Calreticulin• Serglycin (-)• Cathepsins

– Cat C– Cat B

Lytic granules: secretory lysosomes

The immunological synapse

Hours

20-30 minutes

(movie file)

Movement of granules

Delivery of granules

(movie file)

1. Exocytosis

2. Granzymes can enter independent of perforin

3. Binding of perforin

a) Lipid attachment

b) Polymerization

4. Facilitated entry of granzymes

5. Osmotic damage

Entry of granules

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Cell Microbiol. 2008 September; 10(9): 1765 ﾐ1774.

Effects of Granzyme B

(movie file)

Granzyme B mode of action

Bcl-2

Granzyme A mode of action

Fas-Fas-L

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Granzymes, perforin, and Fas can synergistically promote cytotoxicity

against pathogens

Similarities between CTL and NK cells

• Recognition of MHC class I molecules

• Formation of immunological synapse

• Effector cytokines– IFN-

• Lytic granules

CD8+ T cell differentiation into cytotoxic T cells

MIGRATION

Steps in effector CTL differentiation

CTL gene transcription Eomesodermin (Eomes)

T-box 21 (T-bet)

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CD27L/OX40L

CD27L/4-1BBL

CD40-CD40L

IL-12

CD8

DC

Extrinsic factors

Intrinsic factors

CD4”Help”

Extrinsic factors: IL-12

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Intrinsic factors: T-bet and Eomes

Transition of cytotoxic T cells into memory cells

Annual Reviews

Marking memory

Linear progression

Asymmetric model

Reading

• Chapters 8, 10 of Janeway Book (7th edition)

• Quicktime movies from CD of Janeway book

• Supplemental readings posted on website

• Pub Med for additional information/help:

– http://www.ncbi.nlm.nih.gov/pubmed/