Krogman, Erica

Abstract Introduction Immunotherapy has achieved remarkable success in hematologic malignancies, but its efficacy in solid tumors remains limited due to tumor heterogeneity, immune suppression within the tumor microenvironment, and inefficient T cell infiltration. I hypothesize that enhancing tumor cell sensitivity to cytokine-mediated killing can potentiate paracrine killing, in which T cell—derived cytokines induce tumor cell death even without direct cell—cell contact. This mechanism can overcome antigen heterogeneity and limited T cell penetration, broadening the therapeutic reach of immunotherapy. Methods I propose targeting ADAR1, an RNA-editing enzyme that suppresses innate immune activation, to enhance paracrine killing. Loss of ADAR1 leads to dsRNA accumulation and activation of PKR, MDA5, and RIG-I, sensitizing tumor cells to T cell—derived cytokines such as IFNγ and TNF. To test this, I performed in vitro paracrine killing co-culture assays using T cells from healthy donor PBMCs, antigen-positive tumor targets, and GFP-labeled antigen-negative ADAR1KO tumor targets. GFP signal loss in ADAR1KO cells will indicate enhanced paracrine killing independent of antigen recognition. Results My data show that ADAR1 inhibition sensitizes tumor cells to cytokine-mediated cytotoxicity, though how dsRNA sensor and cytokine receptor signaling intersect remains unclear. Supernatant from antigen-positive T cell—tumor co-cultures induced cell death in ADAR1KO but not WT tumor targets, indicating selective cytokine sensitivity. Consistently, Western blot analysis revealed increased cleaved Caspase-3 and PARP in ADAR1KO cells. The paracrine killing assay is ongoing. Conclusion This study aims to elucidate the interplay between dsRNA sensing and cytokine signaling in the absence of ADAR1 and to define how this crosstalk shapes the efficacy of paracrine killing in solid tumors. These insights could inform new therapeutic strategies to enhance the potency of immunotherapy against resistant and heterogeneous solid tumors. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Introduction It is critical for the immune system to destroy cells harboring intracellular infection to remove pathogen burden. This function is widely attributed to cytotoxic T cells, which can physically engage with their targets through the T cell receptor complex, releasing cytotoxic granules, resulting in a specific death of infected targets. However, effector cytokines tumor necrosis factor-α (TNF) and interferon-γ (IFNγ), secreted by T cells, are themselves cytotoxic in combination to a degree. While TNF has appreciated cell death function through the TNF receptor 1 (TNFR1) signaling pathway, no clear cytotoxic function is attributed to IFNγ. Because of the diffusible nature of these cytokines, it is possible for T cells to elicit cytotoxicity without requiring direct contact with their targets, termed paracrine killing. Whether paracrine killing occurs during viral infection and how it is regulated is unclear. Paracrine killing poses issues of both sensitivity to these cytokines in that high concentrations of these cytokines are needed to affect target cells, as well as specificity in discriminating infected from uninfected host cells. Accounting for both issues, we hypothesize that paracrine killing is elicited through pathogen restriction whereby only infected cells are susceptible to death. Methods Analysis was performed in vitro using the Incucyte live cell imaging system and Western blots with fibroblast targets and a VSV infection model and OT1 T cells were leveraged ex vivo. Results TNF and IFNγ protect cells against the cytopathic effect of the virus. TNF specifically synergizes with VSV resulting in death of the infected cells and the two cytokines have distinct roles that work in concert. We demonstrate that TNF and IFNγ released by activated T cells suppress viral infection of nearby cells in a paracrine manner. Conclusion Here we provide insight and context into how distinct cytokines function in a cooperative manner for immune protection during an infection. Funding Source Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Viral Immunology (VIR)