Mechanisms of lymphocyte-mediated cytotoxicity
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Dr. Ronald Smeltz
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/