Studies On The Signal Transduction Mechanisms Mediating Biological Effects Of Human γδT Cells

y8T cells are able to target a broad spectrum of tumors because of their unique properties, including major histocompatibility complex (MHC)-independent recognition, potent cytotoxicity, and cytokine (like IFN-γ) secretion. Therefore, γδ T cells have recently become attractive candidate effector cells for tumor immunotherapy.Besides T cell antigen receptor y8(TCRyδ), y8T cells also highly express natural killer group2, member D (NKG2D). TCRyS and NKG2D are considered as two important receptors of y8T cells that play important roles in the recognition of tumor cells. Evidences of TCRγδ-dependent y8T cells activation have been well documented, whereas NKG2D is generally accepted to act as a costimulatory receptor for γδ T cell cytotoxicity. However, why TCRγδ alone can activate γδ T cell cytotoxicity, and how does NKG2D costimulatory effect take place? Only when these questions are answered, the molecular mechanisms, especially the signal transduction underlying the biological effects of γδ T cells (mainly cytotoxic effect) could be well clarified.In view of these facts, our current study focused on the following three scientific questions. First, what is the principal pathway related to γδ T cell mediated tumor killing? Second, what are the specific functions of TCRγδ and NKG2D in the activation of γδ T cell cytotoxicity? Third, what is the regulative mechanism of y8T cell cytotoxicity?The first part of this work aimed at further clarifying y8T cell killing mechanism. First of all, the individual contribution of perforin-granzyme pathway and Fas-FasL pathway to y8T cell mediated tumor killing was compared. Five tumor cell lines were selected as the target cells, including Daudi (Human Burkkit lymphoma cells), G401(Human renal cancer Wilms cells), NCI-H446(Human small cell lung cancer cells), HR8348(Human colorectal cancer cells), and MGC-803(Human gastric cancer cells). Flow cytometry (FCM) analysis showed that these five tumor cell lines have different levels of Fas expression:Daudi5.14%, G4017.24%, NCI-H55644.10%, HR834869.40%, and MGC-80382.30%. Cytotoxicity assay following perforin-granzyme pathway and Fas-FasL pathway blockade showed that Fas-FasL signaling blockade with supplemented soluble anti-FasL antibody did not impair the specific lysis of tumor target cells or the release of IFN-y by γδ T cells. In contrast, concanamycin A (CMA), a perforin inhibitor that accelerates perforin degradation within lytic granules, dramatically blocked γδ T cell cytotoxicity. Hence, we can conclude that the perforin-granzyme pathway makes the major contribution to γδ T cell cytotoxicity.Later, the function of TCRy8receptor and NKG2D receptor in the activation of γδ T cell cytotoxicity was explored through using TCRγδ antibody and/or NKG2D antibody redirected P815cells as target. The results of P815redirected cytotoxicity showed that anti-TCRy5but not anti-NKG2D activating antibodies initiated γδ T cell specific killing of P815target cells, accompanied by the significant release of IFN-y. Interestingly, NKG2D ligation augmented TCRγδ activation-mediated cytotoxicity and IFN-y production. To compare the contribution of TCRγδ and NKG2D on perforin-granzyme pathway, we first measured cellular degranulation based on cell surface expression of CD107a (LAMP-1). No significant difference was observed in CD107a expression in γδ T cells after TCRγδ or NKG2D activation. Intracellular staining of the perforin-containing granules showed that TCRγδ but not NKG2D engagement induced lytic granule polarization, and NKG2D in combination with TCRγδ activation merely enhanced this effect. These results, taken together, suggest that TCRγδ-induced T cell cytotoxicity mainly depends on lytic granule polarization.The second part of the work was to look for the signal transduction mechanisms responsible for the regulation of γδ T cell cytotoxicity. First, we studied the activation signal pathways related to γδ T cell cytotoxicity. Four important signaling pathways that are associated with αβ T cell and NK cell cytotoxicitywere studied. They are Vavl, PLC-y, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) and PI3K. Western blot analysis showed that stimulation of TCRγδ but not NKG2D induced strong phosphorylation of Vav1, PLC-γ1and Erk, which were enhanced when both TCRy8and NKG2D were engaged. In contrast, phosphorylation of Akt, a downstream target of PI3K activation, showed no enhancement following the coengagement of TCRyS and NKG2D compared to either TCRγδ or NKG2D stimulation alone. These results combined with the cytotoxicity results, suggest that Vav1, PLC-yl and Erk pathways play the major role in y8T cell cytotoxicity. Knockdown of Vavl in γδ T cells by small interference RNA (siRNA) could block γδ T cell-mediated killing, lytic granule polarization and IFN-y release. In the mean while, knockdown of Vavl completely abrogated the phosphorylation of PLC-yl and Erk. Using inhibitor of signaling pathways, we found that the PLC-y inhibitor U73122can fully abrogate y8T cell cytotoxicity, lytic granule polarization and IFN-y release. However, MEK inhibitor U0126, an upstream activator of Erk, did not abrogate γδ T cell cytotoxicity and lytic granule polarization, even under high concentrations. Intriguingly, IFN-y production was inhibited by U0126. In addition, Erk phosphorylation was inhibited by U73122, indicating that Erk might be a downstream molecule of the PLC-y pathway. These results suggest that, unlike IFN-y production, y8T cell cytotoxicity is an independent event downstream of Vavl-PLC-yl but not Erk.Subsequent research confirmed that Cbl-b played inhibitory role in γδ T cell cytotoxicity activation. Knockdown of Cbl-b in γδ T cells by small interference RNA (siRNA) enhanced γδ T cell cytotoxicity towards redirected P815cells following TCRγδ and/or NKG2D engagement. After Cbl-b knockdown, lytic granule polarization toward target cells, phosphorylated Vavl, PLC-yl and Erk were all upregulated following TCRγδ or NKG2D activation. Interestingly, knockdown of Cbl-b enabled unresponsive γδ T cells to respond to the engagement of NKG2D. NKG2D engagement induced the phosphorylation of Vav1and PLC-γ1, as well as lytic granule polarization when Cbl-b expression was knocked down. Taken together, our data suggest that Cbl-b imposes a requirement of TCRγδ-dependent activation, and the activation of γδ T cell cytotoxicity requires a strong signal to overcome the activation threshold set by the inhibitory effect of Cbl-b. Vavl overexpression experiments further confirmed this conclusion. At the same time, RNAi results showed that Cbl-b might regulate y8T cell cytotoxicity by targeting phosphorylated Vavl.In conclusion, our major findings in this study include (1) Perforin-granzyme pathway mediates γδ T cell cytotoxicity towards tumor cells.(2) γδ T cell cytotoxicity is TCRγδ dependent. NK.G2D ligation augments γδ T cell cytotoxicity mediated by TCRγδ engagement.(3) Difference in lytic granule polarization is the main reason why TCRγδ and NKG2D have different function in the activation of γδ T cell cytotoxicity.(4) Vav1-PLCγ1is required for γδ T cell cytotoxicity.(5) Cbl-b inhibits Vavl-dependent γδ T cell activation signals.(6) The activation of γδ T cell cytotoxicity requires a strong signal to overcome the activation threshold set by the inhibitory effect of Cbl-b. This study further reveals the molecular mechanisms underlying TCRγδ dependent γδT cell cytotoxicity, and verifies that the activation of Vavl-PLCγ1pathway is required to overcome the inhibition by E3ubiquitin ligase Cbl-b in γδT cell cytotoxicity, thereby providing important information regarding the biological effects of γδT cells.