cytotoxicity mitzi nagarkatti professor, dept. microbiology

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

2. Cytotoxicity = Cell killing or lysis

  • Effectors
  • Complement (C)
  • Macrophages (M )
  • Granulocyte
  • Natural Killer (NK) cells
  • Cytotoxic T lymphocytes (CTL)

C Ag Ab 3. Target Cells

  • Tumors
  • Virally infected cells
  • Cells infected with intracellular bacteria

4. Macrophage

  • Monocyte - blood
  • Alveolar M slung
  • 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)

Target Fc receptor M Ag Ab 6.

  • Normal M s are not lytic
  • Activated M s are lytic
  • Activation mediated by Th1
  • 2 signals required:Cytokine - Interferon- (IFN- )
  • CD40L

M Target M Target 7. Mechanisms of Macrophage-mediated cytotoxicity

  • Reactive oxygen intermediates (ROIs): O 2 - , OH - , H 2 O 2
  • Reactive nitrogen intermediates (RNIs): NO, NO 2
  • Tumor necrosis factor-
  • Lysosomal enzymes

8. Activation of Cytotoxic T lymphocytes CTLPrecursor AgActivated CTL IL-2 TCR CD8 IL-2R Activation Proliferation, Differentiation Target MHCCl. I Ag 9. CTL Activation

  • Ag + Class I MHC on infected cells/grafts CTL
  • Ag + Dendritic cells (Class I) cross priming of nave 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- , TNF- and TNF-

Fas FasL Ag MHC TCR Perforin Granzyme CTL Perforin 12. Supramolecular Activation Cluster (SMAC) CD43/CD45 MHCp:TCR LFA-1:ICAM-1 Exocyticvesicles cSMAC (central) pSMAC (Peripheral) Microtubules CD28:CD80/CD86 Outside of SMAC Immunologic Synapse between CTL and target cell 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. 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 ITIM ITIM 15. NK Cell Receptor

    • KLR ( K iller celll ectin-liker eceptor): CD94, NKG2
    • KIR ( K iller cellI g-liker eceptor): Ly49
    • Human:NKG2A and B are inhibitory
    • NKG2Cand 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. Cell Death

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

18. Role of Apoptosis

  • Embryogenesis
  • Organogenesis
  • Cytotoxic Lymphocyte killing of targets
    • FasL
    • Perforin/Granzyme

Fas FasL Ag MHC TCR Perforin Granzyme CTL 19.

  • In the Periphery

Activation induced cell death (AICD) Memory RestingT Cell Ag + - ActivatedT Cell Cycling T Cell Ag Ag IL-2 20.

  • T Cell Development
  • Thymus Central Tolerance
  • Death by Neglect
  • Positive Selection:T cells with low affinity for self MHC
  • Negative Selection:Deletion of autoreactive T cells

Spleen, LN,Peripheral blood Bone Marrow Thymus Mature T cells 21. Apoptosis Signaling FasL (CD95L) Fas (CD95) Procaspase 9 Caspase 3 Cytochrome c Receptor mediated Pathway Mitochondrial Pathway Bcl-2 Activates DNase, Inactivate DNA repair enzymes DNA fragmentationFADD (Fas associateddeath domain) Apaf-1 Caspase 9 Procaspase 3 Procaspase 8 Caspase 8 22. Molecules involved in apoptosis

  • Tumor necrosis factor receptor (TNFR) superfamily:
    • Fas (CD95)
    • TNFR (I and II)
  • Ligands : Induce apoptosis
    • FasL (CD96L)
    • TNF and Lymphotoxin LT
    • 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
      • Lpr cg 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. 6 thed., 2005.Pg 89-95; 341-361;717.

26. Exam Format

  • Multiple Choice
  • Essays
  • Both

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