Mechanisms of lymphocyte-mediated
cytotoxicity
Dr. Ronald Smeltz
Medical Sciences Building
Room 325
rbsmeltz@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
NK cell recognition molecules
NK cell receptors Target cell ligands
Genetic polymorphism
• Ly49 (mouse)
• KIR (human)
• CD94/NKG2
H-2K, H-2D
HLA-A, HLA-B, HLA-C
Qa-1b
HLA-E
• NKG2D
Rae-1
MIC-A,MIC-B
• NKp
??
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/KIR
Ly49/KIR
polymorphic
MHC encoded
-peptide
H-2/
HLA-A,B,C
-peptide
CD94/NKG2
CD94/NKG2
polymorphic
MHC encoded
+peptide
Qa-1/
HLA-E
+peptide
NKG2D
non-poly
Non-MHC
encoded
Rae/
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
Lytic granules:
Secretory lysosomes
•
•
•
•
Granzymes, perforin Cause apoptosis
Calreticulin
Inhibitor of perforin
Serglycin
Complex with granzymes
Cathepsins
–
Cat C
Processes granzymes
–
Cat B
Protection
Mediators of cytotoxicity
Delivery of granules
The immunological synapse
Hours
20-30 minutes
Movement of granules
Summary
1. Membrane reorganization
2. Cytoskeletal polarization
3. Docking
4. Exocytosis
5. Binding
6. Entry
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 a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (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