Abstract 6860: Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment

Abstract Radiation therapy (RT) has long been a cornerstone in cancer treatment, yet the mechanisms underlying its impact on the immune system remain incompletely understood. RT is recognized for its ability to induce DNA damage within cancer cells, leading to cancer cell death. For those cancer cells that do not die as a result of radiation, tumor infiltration by immune cells is closely associated with treatment outcomes. T cell infiltration is a dynamic process, with subsets taking up residence in the tumor while the majority recirculate through lymph nodes into the peripheral blood. T regulatory cells (Tregs) have been recognized as potent suppressors of anti-tumor immune responses, dampening immune-mediated clearance of cancer cells. While RT can initially contribute to an improved anti-tumor immune response, this beneficial effect is often counterbalanced by the presence of suppressive Tregs. Tregs can infiltrate the tumor microenvironment, ultimately impeding and overwhelming the immune response generated by RT. Our objective was to gain insights into the migratory patterns of Tregs to and from tumor sites and to elucidate how RT influences the dynamics of Treg recirculation. Utilizing the established photoconvertible Kaede mouse model we can track photoconverted cells following RT. Following photoconversion, photoconverted T cells were observable within the tumor and subsequently moved to the TdLN and on to other NdLN at later time points. In the TdLN of mice bearing MC38, Moc1, and Moc2 tumors, Tregs represented a significantly higher proportion of photoconverted cells compared to non-Treg CD4 or CD8 T cells. To validate their active departure from the tumor site, mice underwent FTY720 treatment, resulting in a reduction in both photoconverted Treg and non-Treg migration to the TdLN. When RT was administered to the tumor, there was a decrease in photoconverted Treg and non-Treg CD4 T cells within the TdLN, consistent with the expected cytotoxic effects on the irradiated tumor upstream. To focus on recruitment back from the TdLN, mice were analyzed after photoconversion of the TdLN. This revealed that a majority of TdLN Tregs exhibited a preference for relocating to the tumor, with only a minority migrating to the NdLN. The newly entered Tregs were analyzed in the tumor, at D1 and D3 post RT, and we found that these Tregs exhibited increased expression of CCR8, CD69, KLRG1 and Ly6C as compared to those in non-irradiated tumors, suggesting that these are recent entrants with limited suppressive capacity. These findings illuminate the intricate dynamics between RT, Tregs, and the use of site-specific photoconversion to track immune cell movement and changes over time to understand treatment responses. Our ongoing studies focus on the interplay between the Treg and the tumor environment that dictates the success of CD8 T cell mediated control of residual cancer cells following RT. Citation Format: David J. Friedman, Tiffany Blair, Gwen Kramer, Aanchal Preet Kaur, Jason Baird, Alejandro Alice, Megan Potuznik, Marka R. Crittenden, Michael J. Gough. Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6860.