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Mechanisms of lymphocyte-mediated cytotoxicity
Dr. Ronald SmeltzMedical Sciences Building
Room [email protected]
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