Cytotoxicity

1. Cytotoxicity Mitzi Nagarkatti Professor, Dept. Microbiology and Immunology Massey Cancer Center 533 Medical Sciences Building Tel. # 827-1555

2. Ag Ab C’ Cytotoxicity = Cell killing or lysis Effectors • Complement (C’) • Macrophages (Mf) • Granulocyte • Natural Killer (NK) cells • Cytotoxic T lymphocytes (CTL)

3. Target Cells • Tumors • Virally infected cells • Cells infected with intracellular bacteria

4. Macrophage • Monocyte - blood • Alveolar Mfs–lung • Histiocytes – connective tissue • Kupffer cells – liver • Mesangial cells – kidney • Microglial cells – brain

5. Function of macrophages • Phagocytosis • Antigen Processing and Presentation • Cytotoxicity • Direct Cytotoxicity • Antibody dependent cell-mediated cytotoxicity (ADCC) Ag Target Mf Ab Fc receptor

6. Target Target Mf Mf • Normal Mfs are not lytic • Activated Mfs are lytic Activation mediated by Th1 2 signals required: Cytokine - Interferon-g (IFN-g) CD40L

7. Mechanisms of Macrophage-mediated cytotoxicity • Reactive oxygen intermediates (ROIs): O2-, OH-, H2O2 • Reactive nitrogen intermediates (RNIs): NO, NO2 • Tumor necrosis factor-a • Lysosomal enzymes

8. Activation of Cytotoxic T lymphocytes IL-2 IL-2R CTL Precursor Ag Activated CTL MHC Cl. I CD8 Target TCR Ag Proliferation, Differentiation Activation

9. CTL Activation • Ag + Class I MHC on infected cells/grafts CTL • Ag + Dendritic cells (Class I) – cross priming of naïve CD8+ cells • Ag + APC + CD4+ Th CTL

10. Characteristics of Effector CTL • Increased adhesion molecule expression (LFA-1, CD2, CD44, CD45RO) • Decreased expression of L-selectin (prevents homing to lymph nodes) • Expression of VLA-4 (Very Late Ag-4) which interacts with VCAM-1 (vascular cell adhesion molecule) on endothelial cells leading to inflammation • Production of effector lytic molecules

11. Mechanism of CTL-mediated killing of targets Pathways • FasL • Perforin/Granzyme • Cytokines: IFN-g, TNF-a and TNF-b Perforin TCR Ag CTL MHC Granzyme Fas FasL Perforin

12. Immunologic Synapse between CTL and target cell Supramolecular Activation Cluster (SMAC) cSMAC (central) MHCp:TCR CD28:CD80/CD86 Exocytic vesicles pSMAC (Peripheral) LFA-1:ICAM-1 Microtubules Outside of SMAC CD43/CD45

13. Natural Killer Cells • Innate immunity • Large granular lymphocytes • Kill virus infected cells and tumor cells • Lysis non-MHC restricted • CD3-, TCR-, Ig- • Present in SCID (severe combined immunodeficiency disease) mice • Asialo GM1+, NK1.1+ • Intermediate affinity IL-2R+ • FcR(CD16)+, Mediate ADCC

14. ITIM ITIM Natural Cytotoxicity Receptors Altered-self Hypothesis • Activating Receptor: Contain immunoreceptor tyrosine-based activation motif (ITAM) • Inhibitory Receptor: Contain Immunoreceptor tyrosine-based inhibitory motif (ITIM) ITAM

15. NK Cell Receptor • KLR (Killer cell lectin-like receptor): CD94, NKG2 • KIR (Killer cell Ig-like receptor): Ly49 Human: NKG2A and B are inhibitory NKG2C and D are activating Mouse: Ly49H is activating Other Ly49 is inhibitory Recognize: Nonclassical MHC: HLA-E – human; Qa-1 – mouse (Classical MHC : HLA-A,B,C,D –human H-2K,I,D – mouse

16. Beneficial and Deleterious Effects of Cytotoxicity • Protection against • Tumors • Virus-infected cells • Intracellular bacteria • Parasites • Fungal infections • Cause • Autoimmune disorders • Transplant rejection • Immunopathology

17. Apoptosis (Programmed cell death) Nuclear and cytoplasmic condensation Membrane blebs DNA fragmentation (early, 180bp multiples) Apoptotic bodies Phagocytosis Localizes infection Ex. Development 3H-thymidine-release assay Necrosis (Pathologic cell death) Cell Swelling and lysis Inflammation DNA fragmentation (late, varying size) Spreads infection Ex. Stress 51Cr-release assay Cell Death

18. Role of Apoptosis • Embryogenesis • Organogenesis • Cytotoxic Lymphocyte killing of targets • FasL • Perforin/Granzyme TCR Ag CTL MHC Granzyme Fas FasL Perforin

19. Activation induced cell death (AICD) • In the Periphery Ag + Ag IL-2 Memory Resting T Cell - Ag Activated T Cell Cycling T Cell

20. Mature T cells Thymus Bone Marrow T Cell Development Spleen, LN, Peripheral blood Thymus – Central Tolerance • Death by Neglect • Positive Selection: T cells with low affinity for self MHC • Negative Selection: Deletion of autoreactive T cells

21. Caspase 9 Procaspase 8 Caspase 8 Procaspase 3 Receptor mediated Pathway Mitochondrial Pathway Apoptosis Signaling Bcl-2 Apaf-1 FasL (CD95L) Cytochrome c Fas (CD95) Procaspase 9 FADD (Fas associated death domain) Caspase 3 Activates DNase, Inactivate DNA repair enzymes DNA fragmentation

22. Molecules involved in apoptosis • Tumor necrosis factor receptor (TNFR) superfamily: Fas (CD95) TNFR (I and II) Ligands: Induce apoptosis FasL (CD96L) TNF – a and b (LymphotoxinLT a+b) TRAIL (TNF-related apoptosis inducing ligand) • Bcl-2 family Bcl-2, Bcl-xL –Inhibit apoptosis Bax, Bad – Pro-apoptotic

23. Apoptosis and Disease Lack of apoptosis • Cancer • Autoimmunity: • Mouse • Lpr – CD95 deficient • Gld – CD95 Ligand defective • Lprcg – CD95 intracellular death domain defect • Human • Type Ia Autoimmune lymphoproliferative disease (ALPS) – CD95 defect • Type Ib ALPS – CD95L defect • Type II ALPS – CD95 signaling defect

24. Induction of apoptosis • AIDS and other viral infections • Neurodegenerative disorder

25. Suggested Reading • Immunobiology: The Immune System in Health and Disease by Janeway et al. 6th ed., 2005. Pg 89-95; 341-361;717.

26. Exam Format • Multiple Choice • Essays • Both