*Purpose: We have previously reported that Neuropilin (NRP) 2-associated signaling events modulate PI3K/mTOR/Akt activity in several human cell lines. While intrinsic NRP1 activity has been linked to CD4⁺ Treg stability and CD8⁺ T cell memory formation, the role of NRP2 expression in T cells during alloimmune responses is not known.

*Methods: Human PBMC were adoptively transferred into SCID beige mice. The phenotype of transferred human CD4⁺ T cells was analyzed using FACS and scRNAseq 7, 14 and 21 days post-transfer. CD4-cre NRP2^lox/lox (ΔNRP2-CD4) and Foxp3-cre NRP2^lox/lox (ΔNRP2-Foxp3) mice were used to investigate NRP2 function in T cell subsets following cardiac transplantation.

*Results: By FACS, we found only minimal NRP2 expression in freshly isolated human CD4⁺ T cells. In contrast, adoptive transfer of human PBMC into SCID beige mice for >7 days resulted in a striking expansion of NRP2⁺ CD4⁺ T cell subsets (~38% NRP2⁺ within CD4⁺, P<0.01). scRNAseq (CITE-seq) identified NRP2⁺ CD4⁺ cells to co-express multiple coinhibitory receptors including PD1⁺Tim3⁺ and that NRP2 is expressed by late effector and exhausted Teff populations. Since NRP2 modulates cell intrinsic signaling, we performed bulk RNAseq to define NRP2-associated signaling networks and find that it regulates transcripts of co-stimulation, chemokines and cytokines. Comparisons of NRP2⁺ to NRP2^– transcriptomes further indicate that NRP2⁺ T cells represent a distinct and novel subpopulation. To test NRP2 function, we transplanted MHC class II mismatched B6.C-H2bm12 (BM12) donor hearts into C57BL/6 WT and NRP2 transgenic mice. We initially found that ΔNRP2-CD4 recipients mount accelerated rejection responses (Median Survival Time [MST] 21 days, n=7; P<0.001) vs. WT recipients (MST >50). To eliminate the possibility that depletion of NRP2 expression on CD8⁺ T cells is functional in this model, we treated recipients with anti-CD8 pre- and post-transplantation and find no change in the accelerated rejection response (KO + anti-CD8: MST 17 days vs. WT + anti-CD8: MST >50 days; P<0.001). Since these findings confirm that NRP2 functions via its inducible expression on CD4⁺ T subsets, we next transplanted BM12 hearts into ΔNRP2-Foxp3 mice to investigate if increased alloimmunity is related to impaired Treg function. ΔNRP2-Foxp3 recipients did not reject allografts (MST>50 days) further confirming that accelerated rejection in ΔNRP2-CD4 recipients relates to NRP2 function in exhausted CD4⁺ Teff cells.

*Conclusions: We identify NRP2 expression in human late effector and exhausted CD4⁺ T cell subsets where it is associated with the regulation of costimulation and trafficking. Furthermore, functional studies using KO mice identify NRP2 as a potent coinhibitory receptor that inhibits alloimmunity via its expression in Teffs. Our findings lead to the intriguing possibility that NRP2 ligands will be promising as future immunomodulatory therapeutics.

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