Elucidating the role of Neuropilin-1 in intra-tumoral regulatory T cell stability

Regulatory T cells (Tregs) play an integral role in the adaptive immune system through suppression of selfreactive immune responses in order to prevent autoimmunity and maintain homeostasis. However, they are deleterious in cancer through suppression of the antitumor immune response. In fact, we show that deletion of 50% of Tregs results in normal tumor growth. Therefore, it is advantageous to understand the role of Tregs in the tumor microenvironment in order to create targeted cancer therapies. Our lab has shown that the Neuropilin-1 (Nrp1) pathway is required for Treg stability in the tumor microenvironment, but is disposable for maintaining immune homeostasis in the periphery, identifying it as a prime therapeutic target. In order to further understand the role of Nrp1-deficient Tregs intratumorally, we constructed a competitive environment by utilizing Foxp3, which is located on the X chromosome, and as a result of X-inactivation, female Foxp3 heterozygous mice are cellular heterozygotes. We generated Nrp1Foxp3 heterozygous mice comprised of 50% WT Tregs and 50% Nrp1-deficient Tregs. Surprisingly, when given B16 melanoma, heterozygous mice phenocopyNrp1Foxp3 homozygous mice (Figure 1A). This suggests that Nrp1-deficient Tregs are playing an active role in shifting the anti-tumor immune response by destabilizing surrounding WT Tregs as determined by DNA methylation status (Figure 1B). Neither WT nor Nrp1-deficient Tregs in the tumor from Nrp1Foxp3 mice can suppress in a standard microsuppression assay ex vivo, unlike WT Tregs from Foxp3 mice. Through various co-culture experiments, we revealed that destabilization of WT Tregs