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

Regulatory T cells (T_regs) 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 T_regs results in normal tumor growth. Therefore, it is advantageous to understand the role of T_regs in the tumor microenvironment in order to create targeted cancer therapies. Our lab has shown that the Neuropilin-1 (Nrp1) pathway is required for T_reg 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 T_regs 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^Cre-YFP heterozygous mice are cellular heterozygotes. We generated Nrp1^L/LFoxp3^Cre-YFP/+ heterozygous mice comprised of 50% WT T_regs and 50% Nrp1-deficient T_regs. Surprisingly, when given B16 melanoma, heterozygous mice phenocopy Nrp1^L/LFoxp3^Cre-YFP homozygous mice (Figure 1A). This suggests that Nrp1-deficient T _regs are playing an active role in shifting the anti-tumor immune response by destabilizing surrounding WT T _regs as determined by DNA methylation status (Figure 1B). Neither WT nor Nrp1-deficient T_regs in the tumor from Nrp1^L/LFoxp3^Cre-YFP/+ mice can suppress in a standard microsuppression assay ex vivo, unlike WT T_regs from Foxp3^Cre-YFP mice. Through various co-culture experiments, we revealed that destabilization of WT T_regs is possibly due to a soluble factor derived from Nrp1-deficient T_regs. Our data revealed that Nrp1-deficient T_regs produce large amounts of IFNγ in the tumor microenvironment. Indeed, when treated with IFNγ, WT T _regs 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 T_reg 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