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, CD3ζ, 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.

T Cells and Stromal Fibroblasts in Human Tumor Microenvironments Represent Potential Therapeutic Targets

“…In a murine adenocarcinoma model, Frey and colleagues [56] identified two populations of tumor-derived CD8 + T cells that differed in their ability to lyse target cells. The non-lytic TIL had deficiencies in the ability to flux calcium and tyrosine phosphorylation of the proximal TCR signaling molecules Zap-70 and LAT was significantly reduced when compared to the lytic TIL.…”

Cancer-Induced Signaling Defects in Antitumor T Cells

“…Since cognate MCA38 tumor cells cause induction of proximal TCR signaling defects in TIL Koneru et al, 2005;Radoja et al, 2001) but it is not certain that the mechanism for signaling inhibition which we describe will be utilized for all tumor types, in the following sections we present (to the best of our ability) a synopsis of proximal TCR signaling in T cells in the hope that other models of dysfunctional antitumor T-cell function may be understood in this context. It should be apparent that the complexity of regulation of proximal TCR-mediated signaling provides robust opportunities for interference by tumor-induced factors.…”

“…Since TCR signaling is required for cytolysis, the presence in tumor tissue of T cells whose antigenspecific functions can be demonstrated in vitro upon purification but whose lytic function in situ is not readily detected (Bronte et al, 2005) implies tumor-induced inhibition of TCR signaling in situ. Freshly isolated CD8 + TIL are, like TIL in situ, lytic-defective and when analyzed in in vitro signaling assays using cognate tumor cells as stimulus have been shown to have a blockade in the most proximal portion of the TCR signaling pathway (Koneru et al, 2005;Radoja et al, 2001). TIL receive initial antigen signals upon contact with cognate tumor cells but are unable to perpetuate the signal downstream past p56 lck activation.…”

“…Calcium flux was reduced (~30 %) in T cells from tumorbearing mice after stimulation with anti-CD3ε Ab implying defective TCR-mediated signaling. (Stimulation of T cells with cognate tumor cells was not performed, a technical necessity later shown to dramatically impact upon signaling in antitumor T cells (Koneru et al, 2005).) Stated (but not shown) was that activation of cells with calcium ionophore resulted in normal levels of calcium flux implying that the signaling block was in the proximal TCR pathway.…”

Homeostatic Proliferation as an Isolated Variable Reverses CD8+ T Cell Anergy and Promotes Tumor Rejection

“…In addition, CD8 ϩ T cells often lack expression of perforin and granzyme B, and thus poorly lyse target cells (10,11). Even TIL that express cytotoxic granules have been shown to exhibit defective TCR signaling that is associated with a failure to orient granules toward the T cell:target cell interface (12)(13)(14). Circulating melanoma Ag-specific T cells binding specific tetramers also have been observed to be dysfunctional in some cases (15).…”