Abstract 4865: Inhibition of PD-1 signaling during TCR stimulation promotes increased T cell activation, but upregulates compensatory inhibitory molecules and induces activation induced cell death

Abstract The PD1/PDL1 axis acts as a potent immune regulator for T cells. Inhibition of the PD1/PDL1 pathway has demonstrated success in the context of checkpoint immunotherapy, yet the biological consequences of the long-term effects of PD1 inhibition on T cells remain unclear. In murine models we have observed that inhibition of the PD1 signaling either through genetic ablation or PD1 checkpoint blockade results in increased activation and function. This has been demonstrated through in vitro stimulation of PD-1-/- T cells in which increased percentages of activation markers including CD25, CD69, and IFNγ were observed. Similar effects of increased activation markers MFI and percent were illustrated in vivo both through adoptive transfer of PD-1 -/- cells into allogeneic NSG recipients and in NSG recipients receiving allogenic wild type cells in combination with anti-PD-1. However, within both in vitro and in vivo models we observed decreases in T cells overtime. We hypothesized that due to increased early activation T cells with inhibited PD-1 signaling underwent increased apoptosis via activation induced cell death (AICD). We observed that apoptosis was increased in PD-1-/- T cells after culture indicated by elevated levels of Annexin V. Further, in vivo following anti-PD-1 treatment we observed increases in other inhibitory markers such as TIM3 and LAG3 via flow cytometry, and confirmed via PCR, suggesting an upregulation of other inhibitory markers following blocking of PD-1. The role PD-1 plays in CAR T cell therapy still requires additional study. Thus, to assess the role of PD-1 and its role in AICD on human cells we next implemented a CAR T xenograft model in which NSG mice were challenged with Raji cells transduced with PD-L1 and then treated with CAR T cells with and without Pembrolizumab. We observed that human CAR T cells transplanted into tumor bearing NSG mice had increased percentages of activation markers(HLA-DR and CD69) when treated with Pembrolizumab, but we also observed elevated flow percentages of inhibitory markers including TIGT, TIM3, and LAG3. These cells in addition to increased activation and upregulation of inhibitory molecules also had decreased numbers overtime and increased apoptosis identified by Annexin V staining. These data suggest a complex interplay in which PD-1 regulates T cell activation as a means of protection, but when this balance is manipulated, T cells can experience increased activation and effector function, followed by an upregulation of inhibitory molecules such as TIM3, LAG3 and TIGIT to compensate, but ultimately still resulting in AICD. Thus, targeting PD-1 unlocks a “short term gain” of activation and function of T cells at the cost of a “long term loss” in T cell numbers due to apoptosis in which cells undergo AICD. Citation Format: Logan V. Vick, Michael K. Sheng, Wahed Firoz, Craig Collins, Lam T. Khuat, Bruce Blazar, Sean Judge, William J. Murphy. Inhibition of PD-1 signaling during TCR stimulation promotes increased T cell activation, but upregulates compensatory inhibitory molecules and induces activation induced cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4865.