Human CAR19 CD8+ iTreg exceed CAR19 CD8 CTLs both in suppressing GVHD lethality and eliminating CD19+ Nalm-6 lymphoma cells in vivo

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Abstract Simultaneously achieving a graft versus leukemia (GVL) effect while suppressing graft versus host disease (GVHD) would facilitate the development of immunotherapies for hematopoietic stem cell transplantation. Here, we show that human peripheral blood CD8+CD25− T-cells cultured with IL-2, TGF-β and rapamycin generates CD8 induced Tregs (iTregs). CD8 iTregs expressed FoxP3, CD25 and higher CD103 and CD39, but lower Helios, CTLA-4, and TIGIT than CD4 Tregs. While in vitro suppressor function was modestly reduced with CD8 iTreg, in vivo suppression of GVHD lethality was comparable to that of CD4 Treg. In vitro, cytolytic activity with an αCD3×αCD19 bispecific engager against CD19+ Nalm-6 lymphoma cells was comparable to CD8 CTLs. We subsequently generated CD8 iTreg and CTLs expressing αCD19scFv (CAR19). Following CAR19-EGFR sort purification, CAR19 CD8 iTreg and CAR19 CTL had similar expression of perforin, IFNγ and TNFα. In a 48h Incucyte killing assay against CD19+Nalm-6 targets, using CAR19 CD8 iTreg and CAR19 CTLs generated from the same donors, no significant differences were noted; CD8 iTreg killing was perforin-dependent and granzyme-independent. No killing was observed against CD19 koNalm-6 targets. Unexpectedly, CAR19 CD8 iTreg were found to be significantly more effective at limiting tumor burden in vivo and significantly delayed tumor-related mortality in a xenogeneic Nalm-6 tumor model compared to CAR19 CTLs (p = 0.0018). Mechanistic studies are ongoing. Together, these findings highlight distinct advantages of CD8 iTreg as a concurrent immunotherapy and provide a rationale for CD8 iTreg based CAR therapies to prevent GVHD and promote GVL. Supported by grants from NIH (R01 HL11879-07 and 2P01 CA065493-25) as well as funding from the Childrens’ Cancer Research Fund

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