Abstract 4858: NKG2D co-stimulation via anti-sMIC/MIC immunotherapy reprograms intratumoral CD8 T cells toward TCF-1+ stemness to enhance anti-tumor immunity

Effective co-stimulation of CD8 T cells is critical for maintenance of TCF-1+ stem-like CD8 T cells necessary for long-term tumor control. NKG2D co-stimulation of CD8 T cells via binding of stress ligands such as membrane-bound MIC enhances CD8 T cell effector functions and is important for promoting memory development. Cancer cells often shed MIC into a soluble form (sMIC) that inhibits CD8 T cell activation. TRAMP prostate tumor mice expressing human MICB (TRAMP/MICB) treated with anti-sMIC/MIC antibody elicited potent anti-tumor responses with re-invigorated CD8 T cell immunity. However, mechanisms of CD8 T cell reprogramming by NKG2D co-stimulation in the tumor microenvironment are poorly understood. In this study, we used scRNA-seq of CD8 TILs from TRAMP/MICB mice treated or untreated with the anti-sMIC/MIC antibody to investigate the hypothesis that anti-sMIC/MIC therapy reprograms CD8 T cells via NKG2D pathway co-stimulation, optimizing TCR-dependent effector and memory responses. Adult male TRAMP/MICB mice were treated with 200µg anti-sMIC/MIC antibody intraperitoneally twice per week for 4-6 weeks. Prostate tumors were collected, and CD45+ cells were flow sorted for scRNA-seq. scRNA-seq analyses were validated using flow cytometry. Tumors from mice treated with anti-sMIC/MIC therapy revealed enhanced functional heterogeneity of CD8 T cell subtypes. In contrast to tumors from untreated mice that were populated with primarily effector and/or exhausted CD8 T cells, tumors from treated mice were enriched in NKG2D+ TCF-1+ stem-like CD8 T cells. This effect of anti-sMIC/MIC therapy was then recapitulated in the syngeneic TRAMP-C2-MICB tumor model. TCF-1+ CD8 T cells expressed higher levels of NKG2D compared to TCF-1- CD8 T cells, and displayed differential utilization of PI3K/Akt signaling pathways as well as upregulation of epigenetic regulators of CD8 T cell function such as Kdm6b and Setd2. Antigen-specific CD8 T cells chronically stimulated ex vivo with anti-sMIC/MIC antibody in complex with sMICB maintained greater proportions of TCF-1+ stem-like CD8 T cells compared to antigen stimulation alone or in combination with sMICB. The results suggest that NKG2D co-stimulation via anti-MIC/sMIC therapy reprograms CD8 TILs to promote their development of TCF-1+ stem-like function necessary for persistence of the anti-tumor response, and provides proof-of-concept for the translational potential of NKG2D signaling modulation in CD8 T cells to treat cancer. Citation Format: Tyler A. Smith, Sizhe Liu, Ryan J. Brown, Weiguo Cui, Jennifer D. Wu. NKG2D co-stimulation via anti-sMIC/MIC immunotherapy reprograms intratumoral CD8 T cells toward TCF-1+ stemness to enhance anti-tumor immunity [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 4858.