Friday, May 16, 2025
5/16/2025
Death receptor signaling as an immune checkpoint in tumor-specific iPSC-T cell function - PROJECT SUMMARY Realizing the potential of cell therapies for cancer requires advances in manufacturing, cell potency, and in vivo durability of anti-tumor responses. In vitro generation of T cells from induced pluripotent stem cells (iPSCs) can advance these goals by enabling complex gene editing in clonal, self-renewing, genomically validated “master” iPSC lines from which therapeutic T cells can be produced. This promise is tempered by the largely unknown objective function of these cells, particularly their in vivo persistence. Development of PSC-T cells as viable alternatives to autologous T cells therefore requires a mechanistic understanding of their responses to tumor encounter and, from this, clear strategies to achieve functional parity with PB T cells. In this proposal, we examine the hypothesis that autocrine/paracrine Fas/FasL signaling is a critical checkpoint limiting the persistence of antigen-specific PSC-T cells during serial antigen encounter. We base this hypothesis on our observations that: a) PSC-T cells exhibit a de novo effector/memory-like gene expression program, including expression of Fas and its ligand, FasL, despite expression of naïve surface markers; b) that Fas and FasL expression increases on PSC-T cells following serial antigen encounter; and c) that, even in the absence of tumor-expressed FasL, antibody blockade of FasL on PSC-T cells markedly enhanced serial tumor killing. To test this hypothesis, we will use the artificial thymic organoid (ATO) method, developed by our group, to generate mature, CD8+ TCR-T and CAR-T cells from human iPSCs. We will leverage the ability to generate clonal, isogenic iPSC lines with deletion of FAS, FASLG, or both, to interrogate the role each gene plays in PSC- T cell response to serial antigen exposure in a CD19 CAR-T model. This will include testing the effect of FAS/FASLG gene deletion on depth and duration of in vivo tumor responses and competitive fitness in a xenograft mouse model; and testing in vitro efficacy of FAS/FASL-deleted PSC-T cells against patient-derived B-ALL. As a potential strategy for therapeutic PSC-T cells development, we will use both immunocompetent syngeneic mouse models and long-term PSC-T cell engraftment in NSG mice to demonstrate the safety of Fas/FasL disruption with respect to autoimmune disease, uncontrolled proliferation, and transformation. Our goal in characterizing this regulatory pathway in PSC-T cells is to significantly advance our understanding of PSC-T cell anti-tumor function to further their potential as a promising T cell source for cancer therapy. Furthermore, mechanistic understanding of the role of autocrine/paracrine Fas/FasL in the antigen-specific responses of PSC-T cells may more broadly inform our understanding of this potentially important pathway in T cells from other sources.
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