*Purpose: We have previously reported that signaling via the transmembrane receptor neuropilin 2 (NRP2) regulates PI3K/mTOR/Akt activity in several human cell lines. Furthermore, it is reported to be expressed at high levels on dysfunctional CD4⁺ T cells in vivo. We find high levels of NRP2 expression on subpopulations of human CD4⁺ T effector (Teff) and T regulatory (Treg) cells in vitro following mitogen activation and in vivo in a xenogeneic huSCID mouse model. In this study, we wish to evaluate its mechanism of function.

*Methods: We generated CD4- and Foxp3-specific NRP2 knockout (KO) mice by standard breeding.

*Results: CD4⁺ KO T cells respond to mitogen (anti-CD3: 0.1-1 μg/ml) with enhanced proliferation (by ³H-thymidine uptake; P<0.05) and increased cytokine secretion (by qPCR and Luminex: IFN-γ [up to 5-fold] and IL-17A [up to 8-fold]; all P<0.05) compared to WT cells. To test NRP2 function in vivo, we performed MHC class II mismatched B6.C-H2^bm12 heart transplantation into C57BL/6 WT or CD4-NRP2KO mice. CD4-NRP2KO recipients had an accelerated rejection response (Mean Survival Time [MST]=21 days, n=7; P<0.001) vs. WT recipient (MST >50). To investigate the role of NRP2 expression on Teff vs. Treg cells, we deleted NRP2 expression on Tregs using Foxp3-cre mice. Foxp3-NRP2KO recipients did not reject B6.C-H2^bm12 cardiac allografts (MST>50 days) suggesting that the dominant effect of NRP2 relates to its expression on CD4⁺ Teff cells. Because constitutive CD4-cre strains also delete floxed sites in CD8⁺ T cells during thymic development, we depleted CD8⁺ cells from WT and CD4-NRP2KO recipients of B6.C-H2^bm12 hearts using anti-CD8 (clone 53-6.7). Allograft survival was unchanged confirming that NRP2 functions via CD4⁺ T cell subsets (KO + anti-CD8: MST=17 days vs. WT + anti-CD8: MST>50 days; P<0.001). These collective findings indicate that NRP2 acts as a novel coinhibitory molecule on CD4⁺ Teff cells. To confirm this interpretation, we tested the regulatory function of NRP2 in a syngeneic C57BL/6 melanoma tumor model in vivo, where B16F10 cells are injected subcutaneously (10⁶ cells/injection) and growth is monitored over 15-30 days. We found that tumor growth was significantly retarded in CD4-NRP2KO mice (n=12, P<0.001), suggestive that enhanced immune responsiveness may be functional in anti-tumor immunity. Finally, we performed RNA-seq of mitogen-activated WT and KO CD4⁺ T cells to identify molecular targets of NRP2 expression. Using Gene Set Enrichment Analysis, we find that NRP2 signaling is potent to regulate mTOR/Akt and Myc activity in CD4⁺ Teffs.

*Conclusions: In summary, we identify NRP2 as a novel coinhibitory receptor that is expressed on CD4⁺ Teffs and we show that it is functional to regulate alloimmunity. We postulate that pharmacological NRP2 ligands or targeting biologics will serve as promising immunomodulatory or immunostimulatory therapeutics respectively.

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