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Elias.haddad@umontreal

Elias.haddad@umontreal.ca. Questions for the Final: Slide presentation Immunity paper (survivin) Immunity paper (Gr. A induced apoptosis). Cell Mediated Cytotoxicity and Apoptosis. Cytotoxic T cells and granule exocytosis Trafficking of granules to the plasma membrane in CTL

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Elias.haddad@umontreal

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  1. Elias.haddad@umontreal.ca Questions for the Final: Slide presentation Immunity paper (survivin) Immunity paper (Gr. A induced apoptosis)

  2. Cell Mediated Cytotoxicity and Apoptosis • Cytotoxic T cells and granule exocytosis • Trafficking of granules to the plasma membrane in CTL • Entry of cytolytic molecules into target cells • Granule mediated apoptosis (Granzyme B and Granzyme A) • Discuss the Gr. A paper • 2. Discuss paper by Croft et al., (Survivin in memory T cell generation and maintenance)

  3. Why study CTL cytotoxicity? • In vitro Evidence: • MLR, Cr Release assay, killing virally infected cells and tumor cells. • In vivo Evidence: • Histological analysis: presence of CTL and its granules in pathological sites. • Adoptive transfer: Generating CTL specific for certain antigens and transferring them to naïve animals. • Knockout mice:Perforin is needed for the clearance of certain viral and bacterial infections. • Cancer: perforin deficient mice have shown increased occurrence of certain tumors.

  4. CTLs kill target by 2 different mechanisms: • Granule Exocytosis • Fas/FasL

  5. Mice with deficiencies or mutations in CTL death pathways

  6. Cytotoxic Lymphocyte Granule Components • Perforin/Cytolysin • Granzymes • Granzyme A, Granzyme B, Granzymes C-H • Serglycin/Chondroitin sulfate proteoglycan • Granulysin • Lysosomal enzymes • Proteases, Cathepsins BHLC(W) • Cathepsins DE • Glycosidases, Other degradative enzymes • Chemokines • MIP1-a, MIP1-b, RANTES • Calreticulin • Granule membrane proteins • CTLA-4 • LAMPs • Man-6-PR

  7. Granzymes: Subfamily of serine proteases found in the secretory granules of hematopoietic cells Cytotoxic T lymphocyte granules contain: 1) Granzyme A, with “tryptase” specificity cleaving after arg or lys residues. Measured by the sensitive BLT esterase assay. 2) Granzyme B, with an unusual “aspase” specificity cleaving after asp residues. Can process and activate caspases 3,7,8,9&10. 3) Granzymes C-G, expressed at lower levels, with poorly defined “chymase” specificity. Not detectable in CTL in vivo.

  8. Variations on a theme of lysosomal secretion. Summary of the protein machinery identified in lysosomal secretion in spermatid (acrosomal granule, orange) , platelets, fibroblasts, CTL, melanocytes, and mast cells. Secretion of lysosomes (white) is required for secretion in some cell types and can involve movement along microtubules (black) in a plus or minus direction.

  9. Trafficking of cytolytic granules to the plasma membrane in CTL • Natural mutations (Human & Mice) • Chediak-Higashi syndrome (lyst-function unknown)-beige mice • Griscelli syndrome (Rab27a- vesicle docking)-ashen mice • Familial haemophagocytic lymphohistiocytosis (FHL) • (pfp & Munc13-4 mutations • 4. Hermansky-Pudlak syndrome (Adaptor protein-3 (AP3).

  10. Protein secretion can utilize two export pathways (Disrupted by Brefeldin A) From: Molecular Biology of the Cell 00020

  11. Regulation of intracellular membrane vesicle fusion Munc-18 Pfeffer, Nature Cell Biology 1:E17 (1999)

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