1. Cytotoxic T Lymphocytes (CTLs) and NK Cells

2. After activation, naïve T cells differentiate into effector and memory T cells

3. After activation, T cells remain in lymph nodes for 5-6 days

4. Effector T cells

5. CTLs do not require costimulation to kill infected targets

6. Chromium Release Assay Experiments demonstrating CTL killing of target cells are usually done with a chromium ( 51 Cr) release assay, which measures target cell lysis in 4-8 hours. Infect mouse with virus to generate CTLs specific for virus infected targets. Wait until mouse has generated immune response. Use Spleen as source of CTLs. Incubate with syngeneic targets infected with virus that are radioactively labeled with 51 Cr that will be released when cell is lysed. Chromium attaches to proteins in the cytoplasm of target cells

7. Chromium Release CTL Assay Target cells mixed with effector cells at various ratios. Measure release of 51 Cr into media. Express as percentage lysis relative to non-specific effector cell.

9. Perforin/ Granzyme B Pathway Directional release of granules (in red). How do CTL kill target cells?

10. CTL Killing Perforin Contained in CTL granules Pore forming protein. Pokes holes in target cell membrane. Homologous to C9.

11. Perforin forms polymers that poke holes in membranes to allow enzymes inside cell. CTL Killing

12. CTL Killing Granzymes A family of proteases which are involved in induction of apoptosis in target cells after entry through perforin pores. Granzyme = granule enzyme Granzyme B is most active granzyme.

13. CTL Killing Perforin/Granzyme pathway Antigen specific killing of infected target cells allows for reduced bystander killing of uninfected cells.

14. CTL Killing Selective (antigen specific) killing happens in minutes. CTLs can be serial killers (repeatedly killing).

15. Why don't CTLS kill themselves? Cathepsin B-- a protease which cleaves perforin!!! control Anti-cathepsinB

16. WildtypePerforin KO Virus 51 Cr labeled targets Harvest splenocytes Harvest splenocytes Perforin deficient mice can still kill virally-infected target cells. How? Specific killing of virally infected targets

17. Fas/Fas Ligand Pathway CTLs express Fas ligand interact with Fas expressed on the target cell surface.

18. Fas Induced Apoptosis Cascade of Caspases  Proteases that cut at C-terminal side of an aspartate.  Pro-enzyme form becomes active through cleavage into subunits.  Proteolytic cascade must be activated for eventual DNA fragmentation.

19. Fas Induced Apoptosis

20. Bcl-2 Proteins Control Apoptosis Pro-apoptosis and anti-apoptosis proteins  Bcl-2 proteins can inhibit or activate apoptosis.  Proteolytic cascade must be activated and not inhibited by anti-apototic bcl-2 proteins. (Target cell has to want to commit suicide).

21. CTL Pathways of Cytotoxicity

22. Summary Naïve CD8 T cells are activated in secondary lymphoid organs and differentiate into CTL effector cells. CTLs can kill targets independently of costimulation. Once a CTL encounters a target cell it releases cytotoxic granules containing perforin and granzymes. Perforin forms pores in the membrane of the target cell allowing granzymes to enter the cell. Granzymes induce apoptosis in the target cell by cleaving caspases. CTL also express FasL and can kill targets via Fas expressed on target cells.

23. But CTLS are not enough….. Viruses are tricky! Class I MHC inhibition by viruses VirusProteinEffect on class I AdenovirusE3-k19Retain in ER HSV-1,2ICP47Blocks TAP EBVEBNA1Block peptides CMVUS2ER to cytosol CMVUS3Retain in ER CMVUS6Blocks TAP

24. NK Cells Detect "Missing Self" Protection Against Viruses Non-infected anti-class I CMV infected anti-class I cIg CMV infection down-regulated MHC class I on human fibroblasts Immune evasion mechanism of viruses to decrease Class I MHC. NK cells preferentially kill cells that have lost expression of MHC class I.

25. NK Cells Detect "Missing Self" Immune surveillance for Tumors Tumor cells often have decreased expression of Class I MHC to escape T cell recognition. NK cells kill tumor cells.

26. NK cells  Distinct lineage of lymphocytes.  Do not rearrange  or  TCR.  CD3-,CD56+ in humans.  CD3-,NKR-P1+ (NK1.1) in rodents  Effector functions include cell-mediated cytotoxicity & cytokine secretion.

27. CD3 + Stem Cell T / NK Progenitor Pre-TPre-T Mature T Thymus CD34 ++ CD34 + CD16 +, CD56 + Mature NK Pre-NK CD4 + or CD8 + CD3 + CD3 + CD3 + TCR Rearrangement CD3- CD4+, CD8+ NK and T cell Development Bone Marrow Stroma IL-2, IL-7, stem cell factor (SCF) CD3-, CD4-, CD8- Thymus not required for development. Normal NK cells in scid mice and mice without RAG1 or RAG2

28. NK Cells - Distribution  ~5-20% peripheral blood lymphocytes  ~5% lymphocytes in spleen  Rare in uninfected lymph nodes  >90% of lymphocytes in placenta

29. NK Cells - Effector Functions  Cell mediated-cytotoxicity –Perforin granzyme pathway –Secreted or membrane TNF-   Antibody-dependent cellular cytotoxicity (ADCC)  Cytokine secretion –Early  -interferon production –Secretion of TNF- , LT- , GM-CSF, IL-5, M- CSF, IL-3, IL-10, IL-13.

30. Antibody-dependent cellular cytotoxicity (ADCC) Cells that perform ADCC must have FC receptors to bind Ig molecules and trigger killing of target cell.

31. Cytokine secretion of NK cells Role for IFN-  Viruses Varicella zoster virus & CMV are life- threatening in humans lacking NK cells. Bacteria NK cells protect against intracellular bacteria which tend to infect macrophages. (e.g. Listeria Toxoplasma, Leishmania)

32. Natural Killer (NK) Cells Part of Early Immune Response

33. NK are lymphocytes without traditional antigen receptors How do they get activated? NK cells express both activating and inhibitory receptors Inhibitory receptors recognize MHC class I (self) on target cells Activating receptors recognize ligands upregulated on infected cells or tumor cells + –

34. NK cell Activating Receptors Have ITAMs Intracellular signal of lymphocyte receptors through ITAMs. Immunoreceptor Tyrosine based Activating motifs (ITAMs).

35. NK cell Activating Receptors Have ITAMs STIMULATORY RECEPTOR (FcR, NKR-P1, NKG2D) Y X X L Ligand P Phosphorylation of substrates Immunoreceptor tyrosine-based Activation motif (ITAM) YxxL x 2 e.g. SYK Protein Tyrosine Kinase Y X X L DAP12 + - -

36. Class I MHC Specific NK Inhibitory Receptors human mouse IgSF C-typelectin- related family = Immunoreceptor tyrosine-based inhibitory motif (ITIM) KIR CD94NKG2A/C/ELy49 KIR2DL KIR3DL Ly49 ~10 genes Extensive allelic polymorphism Deleted from the human genome. Killer Inhibitory Receptors(KIR) 10-12 genes Extensive allelic polymorphism KIR genes found in primates, but not rodents

37. ITIMs on Lymphocyte Receptors Inhibitory receptors have ITIMs to prevent activation. (Act in opposition to ITAMs)

38. ITIM Signaling through Inhibitory Receptors V X Y X X L P PROTEIIN TYROSIINE PHOSPHATASE Shp-1 E.g. Immunoreceptor tyrosine-based Inhibitory motif= ITIM V/IxYxxL/V De-phosphorylation of signaling molecules Inhibitory receptor MHC

39. NK cell activation is regulated by integrated positive and negative signals Target NK Cell + - Activate InhibitOutcome No No killing No YesNo killing Yes No Killing Yes No killing/killing NK Cells kill cells expressing activating ligands but need to have inhibitory receptors to protect MHC expressing cells.

40. Ligands for NK cell activating receptors Very little known about NK activating receptors and their ligands NKG2D (activating receptor) Recognizes “MHC-like” ligands (  2m-independent) MIC-A, MIC-B (humans) Rae-1 family (mice) These ligands are induced during viral infection and cellular stress Ligands for many of the activating receptors have not been identified yet…

41. NK Cell- Opposing Signal Model

42. + – MHC – + No killing Normal Cell Infected Cell Ligand Killing Ligand induced by stress or infection In these situations, activating receptor can overcome inhibitory signal NK cell Inducible Ligand Model

43. Summary NK cells activation is controlled by the balance between activating and inhibitory receptors. Inhibitory receptors bind MHC class I molecules and prevent inappropriate lysis of self cells. NK cells are activated by “missing self”, which can occur when viruses or tumor cells downregulate MHC class I to avoid recognition by CTL. Some ligands for activating receptors are constitutively expressed. Others are induced upon viral infection or stress.