Mechanisms of lymphocyte-mediated cytotoxicity

Dr. Ronald SmeltzMedical Sciences Building

Room 325rbsmeltz@vcu.edu

Outline

• Identify the effector cells of cytotoxicity

• NK cell recognition of target cells

• CTL differentiation into cytotoxic T cells

• Mechanisms of cytotoxicity– Granules– Receptor-mediated– Signaling cytotoxicity

• Transition of cytotoxic T cells into memory cells

Examples

• Pathogens

• Tumors

• Transplantation

• Homeostasis– Tolerance– Elimination of antigen-bearing dendritic cells

Cells that mediate cytotoxicity

• Natural killer (NK) cells

• CD8+ cytotoxic T cells (CTL)

NK cell recognition of target cells

NK cell recognition

The “Missing Self” Hypothesis

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

• Down-regulation of MHC Class I OR over-expression of NK cell activating molecules leads to NK cell-mediated killing of target cell

The “Altered Self” hypothesis

Activation of NK cells is the net effect of inhibitory and activating signals

• 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

Ly49 Family (mouse)

• Most Ly49 members are inhibitory receptors, some are activating receptors

• Bind to Class I– Inhibitory receptors bind Class I with high

affinity• Example: Ly49A

– Activating receptors bind Class I with low affinity, but bind additional ligands with high affinity

• Examples: Ly49D, Ly49H

KIR family (humans)

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

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

– 1-2 ITIM motifs• Short: “S”, activating

– No ITIMS, no cytoplasmic domains

– Similar to Ly49 family, inhibitory KIR molecules bind Class I with high affinity

Similarities between Ly49/KIR

• Expressed on NK cells, activated CD8+ T cells

• Bind to determinants of MHC Class I expressed by target cell

• Inhibitory receptors have cytoplasmic ITIMs

• Activating receptors bind to ITAM-bearing DAP12 adaptor proteins– Asp- in transmembrane domain of DAP; Lys+ in transmembrane domain

of activating receptor

• Different structures, very similar functions!

CD94/NKG2

• Inhibitory and activating receptors– CD94/NKG2A heterodimer: Inhibitory

• NKG2A has a cytoplasmic ITIM

– CD94/NKG2C heterodimer: – CD94/NKG2E heterodimer

• Activating• NKG2C must associate 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

CD94/NKG2 interactions exhibit peptide specificity!

NKG2D: Activating receptor

Structurally linked but not encoded by the MHC locus

Pan NK cell activating receptors

Summary

Ly49/KIRLy49/KIR

polymorphic MHC encoded-peptide

H-2/HLA-A,B,C-peptide

CD94/NKG2CD94/NKG2

polymorphic MHC encoded+peptide

Qa-1/HLA-E+peptide

NKG2D non-poly Non-MHC encodedRae/MUC ligands

Rae/MUC ligands

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

CTL differentiation into cytotoxic T cells

Requirements for generating CD8+ cytotoxic T cells

• Extrinsic factors:– Antigen-bearing DC (Signal 1)– Co-stimulation (Signal 2)

• CD27L/OX40L and CD27L/4-1BBL• CD40/CD40L

– CD4 help

– Cytokines (Signal 3)• Inverse relationship b/w inflammation and help

• Intrinsic factors – Transcription factors

• T-bet, Eomes

See posted PDF for missing slide

See posted PDF for missing slide

See posted PDF for missing slide

See posted PDF for missing slide

Mechanisms of cytotoxicity

Effector molecules of cytotoxicity

• Used by NK cells and CTL!

• Granule exocytosis pathway• Perforin, Granzymes• Trigger apoptosis

– Caspase-dependent/independent

• Fas/Fas-L pathway• Receptor-mediated death, caspase-dependent

• Granzymes, perforin Cause apoptosis• Calreticulin Inhibitor of perforin• Serglycin Complex with granzymes• Cathepsins

– Cat C Processes granzymes– Cat B Protection

Lytic granules:Secretory lysosomes

Mediators of cytotoxicity

Delivery of granules

The immunological synapse

Hours

20-30 minutes

Movement of granules

1. Membrane reorganization

2. Cytoskeletal polarization

3. Docking

4. Exocytosis

5. Binding

6. Entry

Summary

C2 domain of perforin acts as a lipid-recognition domain; pH-dependent

Effects of Granzyme B

Granzyme B mode of action

Bcl-2

Granzyme A mode of action

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

The granzymes synergistically promote cytotoxicity in a perforin-dependent manner

Fas-Fas-L

Similarities between CTL and NK cells

• Importance of MHC class I molecules– Peptide requirements

• CD8, Ly49/KIR, CD94/NKG2

• Formation of immunological synapse

• Effector molecules, lytic granules

Differences between CTL and NK cells

• NK cells:– Innate– Pre-formed effector molecules– Surveillance

• CTL:– Adaptive – Must synthesize effector molecules de novo– Restricted circulation

• Synapse

Transition of cytotoxic T cells into memory cells

Annual Reviews

Marking memory

Linear progression

Asymmetric model

Reading

• Chapters 7, 10 of Janeway Book (7th edition)• Suggested readings posted on MICR505

website