Lymphocyte Effector Functions: Killing

1. Lymphocyte Effector Functions: Killing

2. Major Lymphocyte Subpopulations in Peripheral Blood and Selected Effector Functions B cells T cells B “Other” CD8 CD4 Ab production Cytotoxicity Help to B cells Ag presentation Help to CD8 T cells Cytokine secretion

3. Peptide Loading on MHC Class I in the ER

4. What Cells Do When the Blitz is On

5. Antigen Presentation Pathways; Two Old: and Two New: MHC Class I presentation of peptides MHC Class II presentation of peptides Cross-priming of exogenous peptides (MHC Class I) CD1-mediated presentation of glycolipids

6. Question: How do viruses that don’t infect “professional APCs” such as dendritic cells elicit a primary immune response? After all, virally-infected cells don’t normally traffic to 2˚ lymphoid organs

7. “Classic” view of CTL response against virus-infected cells PVR expressed on non-hematopoietic cells. Endocytosis of virus, nuclear entry, synthesis of viral proteins in cytosol. Infection with Poliovirus Presentation of viral peptides on MHC Class I to CD8+ cytotoxic T-cells Proliferation of cytotoxic T-cells (CTLs) Perforin/granzyme- mediated cell death Cross-priming of exogenous antigens by dendritic cells • Phagosome-to-cytosol protein export; ubiquitin-mediated proteolysis of viral proteins; Presentation of peptide via MHC Class I Perforin/granzyme- mediated cell death of DC; proliferation of CD8+ CTL; Killing of virus-infected epithelial cells by CTL Cytopathic changes; recognition and phagocytosis by dendritic cell PVR expressed on non-hematopoietic cells. Infection with Poliovirus

8. Cross-priming: A Dendritic Cell Engulfs aViral-infected Macrophages Influenza-infected MF Dendritic Cell From: Albert et al., J. Exp. Med. 188:1359, 1998

9. On the Job Training for CTLs

10. CD8 T Cells Need Help With Their Memory CD4 Help 1º effectors Memory 2º effectors No help

12. Cooperation in Killing:Granzyme and Perforin Tubulin X X Barry & Bleackely. Nature Rev. Immunol. 2:401, 2002 Lieberman. Nature Rev. Immunol. 3:361, 2003

13. Structure of Perforin

14. Cellular Events at the Synapse Between CTL and Target Cells Transfer of membrane proteins at the synapse. (a–e) A series of still frames from a movie showing CTLs killing a target cell, the membrane of which has been labeled with a fluorescent protein marker, and electron micrographs showing roughly equivalent stages of synapse formation. Granules polarize towards the immunological synapse as the CTL engages its target (a–c); as the CTL disengages, the granules withdraw from the synapse (d), where membrane from the target has accumulated. As the CTL detaches, target membrane is ripped off the dying target (e), and the CTL can engage a new target (color panel of e). Trambas & Griffiths. Nature Immunol. 4:399, 2003

15. Human Diseases Involving Defective Granule Killing Disease Gene Clinical Manifestations Chediak-Higashi CHS1 Syndrome Griscelli Syndrome Rab27a Hermansky-Pudlak HPS1 Syndrome Familial Hemophagocytic Perforin Lymphohistiocytosis (30% of cases) Lysosomal inclusions in all leukocytes Recurrent bacterial infections Decreased NK cell function Oculocutaneous albinism (melanosome defect) Bleeding (platelet storage granule defect) Partial albinism Hepatosplenomegaly (lymphohistiocytic infiltration) Decreased NK cell function Oculocutaneous albinism (melanosome defect) Bleeding (Platelet storage granule defect) Pulmonary fibrosis (Type II cell surfactant body inclusions) Hepatosplenomegaly (accumulation of activated T-cell and macrophages) Decreased NK cell function Pancytopenia

16. Life (and Death) in the Fas Lane Barry & Bleackely. Nature Rev. Immunol. 2; 401-409 (2002)

17. Viral Evasion of Immunity Barry & Bleackely. Nature Rev. Immunol. 2; 401-409 (2002)

18. Cross-priming: DCs Fight Back Infected DC Infected tissue cell Cross-presenting DC

19. B cells T cells Innate-like Lymphocytes B1 NK, NKT, gd

20. Time Course of Innate and Adaptive Immunity Time Course of thePrimary Immune Response Innate immunity Acquired immunity

21. Distinctions Between Innate and Adaptive Immunity Innate immune system Adaptive immune system Receptors Germline-encoded Somatically engineered Distribution Non-clonal Clonal Kinetics Rapid Slow (requires clonal expansion) Specificity Recognizes non-self Recognizes “altered self” “pattern recognition” Primary structure (TCR) Higher order structure (Immunoglobulin; BCR) Effector Cells All Primarily lymphocytes, DCs, Mf

22. Innate-like B Lymphocytes NK cells not MHC associated

23. B-1 Cells: B Prepared

24. Thymus-independent Antigens are Presented to Specialized B-cells Marginal zone B cells, like B-1 cells, respond to carbohydrate antigen and secrete mainly IgM Like students who cram, after the initial encounter of Ag, they demonstrate little memory MZ MZ Central arteriole GC MZ

25. Innate-like T Lymphocytes: NK Cells NK cells not MHC associated

26. Natural Killer Cell Immunology Course-General Principles

27. How do NK Cells Recognize Their Targets?

28. Major Genes in the MHC Class I Region Gene Structure of the Human MHC The human MHC covers ~4 Mbp of DNA on chromosome 6p21.3 and contains over 220 identified loci. It has been divided into three regions: class II (centromeric), class III, and class I (telomeric) with extended class I and class II regions on either side. This is one of the most gene-dense regions of the human genome. It encodes the most polymorphic human proteins known to date. Of the expressed loci in the MHC, roughly 40% are associated with the immune system. They include the classical class I, HLA-A, -B, and -C, nonclassical HLA-E, -F, and -G, as well as ''postmodern'' MICA and MICB genes (MHC class I chain-related genes). The products of classical polymorphic class I genes, HLA-A, B, and C, interact with T cell receptor (TCR) molecules as well as with the products of the killer immunoglobulin-like receptor (KIR) genes expressed on natural killer cells and some T cells. Trowsdale., Immunity. 15:363, 2001

29. One Mechanism of Triggering NK Cell Cytotoxicity The NKG2D-DAP10 receptor complex and its ligands. A representation of NKG2D-DAP10 receptor expression and the interaction of NKG2D-DAP10 with its ligands. Cytotoxicity requires the expression of ligands of NKG2D (e.g., MICA, MICB) in the target cells. Lanier, Nature Immunol. 2:23, 2001

30. Why do NK Cells Fail to Recognize Healthy Cells?

31. The Balance of Activating and Inhibitory Signals Determines the Outcome of the NK Effector Response KIR2DS KIR2DL NKG2D DAP12 DAP10 Y YP Y YP X X M Y YP x x L YP Y Y Y Syk SHP-2 PI 3-kinase + + - Syk: a protein tyrosine kinase PI 3-kinase: a phosphoinositide kinase SHP-2: A protein tyrosine phosphatase Cytotoxicity Cytokine Secretion

32. X-linked Lymphoproliferative Disease Rare (prevalence of 1/1,000,000) X-linked Defect in cytotoxicity Uncontrolled T-cell activation from excessive cytokine secretion, especially in response to EBV infection Defect apparent in NK cells and CTL Patients are treated with bone marrow transplantations of hematopoietic stem cells

33. Molecular Defect of X-linked Lymphoproliferative Desease Infected B cell NK cell

34. Innate-like T Lymphocytes NK cells not MHC associated

35. Structure of the CD1b Molecule--Look Familiar?

36. The NKT Cell Recognizes Glycolipid AntigenPresented by CD1 on the APC DCs that are infected with intracellular bacteria present foreign bacterial lipid antigens on the cell surfac bound to CD1 molecules. CD1-restricted T cells that are specific for the foreign microbial lipids are stimulated to carry out effector functions, including the secretion of cytolytic granules containing perforin and granulysin, which lyse the infected cells and have direct antimicrobial effects, respectively, and the production of IFN-g and TNF-a, which activate the microbicidal functions of macrophages.

37. Where Do NKT Cells Fit In? NKT CD1d-restricted High IL-4 production Rapid cytokine Production Non-MHC restricted CD3/abTCR+ Thymus-dependent TCR-dependent Cytotoxic IFN-g production MHC-restricted Thymus-independent T NK

38. Summary 1. For cytotoxic CD8 T-cells, ligation of the TCR by MHC I/peptide + co-stimulation results in release of granzymes and perforin and/or FasL, leading to apoptosis of the target cells. 2. Viruses evade host defense, in part, by down-regulating MHC Class I. Uninfected dendritic cells circumvent this by “cross-priming”: phagocytosis of virus-infected cell and presentation of “exogenous” viral antigens on MHC Class I. 3. The innate immune system has a rapid onset and recognizes molecular patterns in a non-clonal fashion. 4. NK cells lack TCRs, but instead express both activating and inhibitory (e.g., KIRs) receptors at their surfaces. The relative expression and ligation of these receptors determines the outcome (i.e., killing or not) of the NK effector response. 5. Innate immune B-cells (e.g., B-1 cells and marginal zone B cells) recognize carbohydrate antigens, secrete IgM, and are not long-lived. 6. Innate immune T-cells (gd T-cells, and NK T cells) recognize non-peptide antigens in non-classical MHC-like molecules. They mediate cytotoxicity & rapid cytokine secretion.