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

Abstract

Regulatory T cells (Tregs) play an integral role in the adaptive immune system through suppression of self-reactive immune responses in order to prevent autoimmunity and maintain homeostasis. However, they are deleterious in cancer through suppression of the anti-tumor 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 Foxp3Cre-YFP heterozygous mice are cellular heterozygotes. We generated Nrp1L/LFoxp3Cre-YFP/+ heterozygous mice comprised of 50% WT Tregs and 50% Nrp1-deficient Tregs. Surprisingly, when given B16 melanoma, heterozygous mice phenocopyNrp1L/LFoxp3Cre-YFP homozygous mice (Figure ​(Figure1A).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 ​(Figure1B).1B). Neither WT nor Nrp1-deficient Tregs in the tumor from Nrp1L/LFoxp3Cre-YFP/+ mice can suppress in a standard microsuppression assay ex vivo, unlike WT Tregs from Foxp3Cre-YFP mice. Through various co-culture experiments, we revealed that destabilization of WT Tregs is possibly due to a soluble factor derived from Nrp1-deficient Tregs. Our data revealed that Nrp1-deficient Tregs produce large amounts of IFNγ in the tumor microenvironment. Indeed, when treated with IFNγ, WT Tregs lose suppressive capacity. In order to uncover potential novel pathways leading to this phenotype, we are performing global transcript studies using RNASeq. Overall, we have shown that Nrp1 is required for intratumoral Treg stability, and in its absence, there is an alteration in the tumor microenvironment, leading to an enhanced anti-tumor immune response. These studies uncover a novel potential target for future cancer immunotherapies that preserves peripheral immune health. Figure 1