Novel mechanism of induction of tumor pyroptosis by IL-9-secreting Tc9 cells - PROJECT SUMMARY Blood CD8+ T cells can be subdivided into different subsets based on their cytokine section profiles and functions. We have shown that IL-9-secreting CD8+ Tc9 cells mediate a stronger antitumor effect in vivo compared to the classical IFN--secreting Tc1 or CTLs. However, the underlying mechanisms remain unclear. Recently, we discovered that Tc9 cells could eradicate large-established tumors by inducing an enhanced tumor pyroptosis, a form of programmed cell death dependent on caspase-1 activation. Our preliminary studies showed that Tc9 cell-treated tumors had increased expressions of IL-1 and IL-18 and activated caspase-1, and more tumor cell death compared to Tc1-treated tumors. Neutralizing IL-1 and IL-18 in vivo completely abrogated the therapeutic advantage of Tc9 cells over Tc1 cells in tumor controls. We further showed that tumor-specific Tc9 cells killed tumor cells by inducing both caspase-1-depedent pyroptosis and caspase-3- dependent apoptosis while Tc1 cells mainly induced apoptosis in tumor cells. Similarly, human tumor-specific Tc9 cells also displayed stronger antitumor effects in vivo compared to Tc1 cells, which was IL-1- and IL-18- dependent. Interestingly, we observed that IL-1 plus IL-18 induced apoptosis in cultured Tc1 but not Tc9 cells, Tc9 cell-derived IL-9 is critical for Tc9 cell activation of STAT3 and pro-growth and survival signaling and function, and IL-1 plus IL-18 can re-activate STAT3 and pro-growth and survival signaling in aged Tc9 cells with reduced IL-9 production. Thus, these findings reveal novel mechanisms underlying how Tc9 cells overcome the suppressive tumor microenvironment after transfer, survive and persist longer, and exert a greater antitumor activity as compared to the traditional Tc1 cells. We hypothesize that tumor-specific Tc9 subset may be superb effector T cells for cancer immunotherapy due to their capacity to induce both pyroptosis and apoptosis in tumor cells and utilize Tc9 cell-secreted IL-9 and tumor-produced IL-1 and IL-18 for their fitness, longevity, and function in vivo to effectively eradicate large established tumors. To test our hypothesis, Aim 1 will determine the importance and mechanisms of Tc9 cell-induced tumor cell pyroptosis in tumor clearance via IL-9- and GrzB-induced NFB-NLRP3-caspase-1-gasdermin activation, and Aim 2 will determine the role and mechanisms underlying Tc9 cell persistence and function mediated by Tc9 cell- secreted IL-9 and tumor-produced IL-1 and IL-18. Completing this project will reveal the importance and mechanism in induction of tumor pyroptosis by tumor-specific Tc9 cells and elucidate the mechanisms underlying the longevity and function of Tc9 cells in tumor microenvironment.
