title: Defective proximal TCR signaling inhibits CD8+ tumor-infiltrating lymphocyte lytic function.
abstract: CD8+ tumor-infiltrating lymphocytes (TIL) are severely deficient in cytolysis, a defect that may permit tumor escape from immune-mediated destruction. Because lytic function is dependent upon TCR signaling, we have tested the hypothesis that primary TIL have defective signaling by analysis of the localization and activation status of TIL proteins important in TCR-mediated signaling. Upon conjugate formation with cognate target cells in vitro, TIL do not recruit granzyme B+ granules, the microtubule-organizing center, F-actin, Wiskott-Aldrich syndrome protein, nor proline rich tyrosine kinase-2 to the target cell contact site. In addition, TIL do not flux calcium nor demonstrate proximal tyrosine kinase activity, deficiencies likely to underlie failure to fully activate the lytic machinery. Confocal microscopy and fluorescence resonance energy transfer analyses demonstrate that TIL are triggered by conjugate formation in that the TCR, p56lck, CD3zeta, LFA-1, lipid rafts, ZAP70, and linker for activation of T cells localize at the TIL:tumor cell contact site, and CD43 and CD45 are excluded. However, proximal TCR signaling is blocked upon conjugate formation because the inhibitory motif of p56lck is rapidly phosphorylated (Y505) and COOH-terminal Src kinase is recruited to the contact site, while Src homology 2 domain-containing protein phosphatase 2 is cytoplasmic. Our data support a novel mechanism explaining how tumor-induced inactivation of proximal TCR signaling regulates lytic function of antitumor T cells.
authors.affiliation: Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.
authors.forename: Mythili
authors.initials: M
authors.lastname: Koneru
authors.forename: David
authors.initials: D
authors.lastname: Schaer
authors.forename: Ngozi
authors.initials: N
authors.lastname: Monu
authors.forename: Aidee
authors.initials: A
authors.lastname: Ayala
authors.forename: Alan B
authors.initials: AB
authors.lastname: Frey
mesh_terms: D000199:Actins / Q000172:deficiency / Q000378:metabolism; D000818:Animals; D018801:CD2 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D016827:CD8 Antigens / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D003594:Cytoplasmic Granules / Q000276:immunology / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000037:antagonists & inhibitors / Q000378:metabolism; D016246:Lymphocytes, Tumor-Infiltrating / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D021381:Protein Transport / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000378:metabolism; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000172:deficiency / Q000502:physiology*; D015398:Signal Transduction / Q000276:immunology*; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D000199:Actins; D018801:CD2 Antigens; D017252:CD3 Complex; D016827:CD8 Antigens; C485468:Cd3e protein, mouse; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse; D002118:Calcium