Neuropilin-2 functions as a coinhibitory receptor to regulate antigen-induced inflammation and allograft rejection
Johannes Wedel, … , Diane R. Bielenberg, David M. Briscoe
Johannes Wedel, … , Diane R. Bielenberg, David M. Briscoe
Published July 1, 2025
Citation Information: J Clin Invest. 2025;135(13):e172218. https://doi.org/10.1172/JCI172218.
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Research Article Immunology

Neuropilin-2 functions as a coinhibitory receptor to regulate antigen-induced inflammation and allograft rejection

Abstract

Coinhibitory receptors function as central modulators of the immune response to resolve T effector activation and/or to sustain immune homeostasis. Here, using humanized SCID mice, we found that neuropilin–2 (NRP2) is inducible on late effector and exhausted subsets of human CD4+ T cells and that it is coexpressed with established coinhibitory molecules including PD-1, CTLA4, TIGIT, LAG3, and TIM3. In murine models, we also found that NRP2 is expressed on effector memory CD4+ T cells with an exhausted phenotype and that it functions as a key coinhibitory molecule. Knockout (KO) of NRP2 resulted in hyperactive CD4+ T cell responses and enhanced inflammation in delayed-type hypersensitivity and transplantation models. After cardiac transplantation, allograft rejection and graft failure were accelerated in global as well as CD4+ T cell–specific KO recipients, and enhanced alloimmunity was dependent on NRP2 expression on CD4+ T effectors but not on CD4+Foxp3+ Tregs. Also, KO Tregs were found to be as efficient as WT cells in the suppression of effector responses in vitro and in vivo. These collective findings identify NRP2 as a potentially novel coinhibitory receptor and demonstrate that its expression on CD4+ T effector cells is of great functional importance in immunity.

Authors

Johannes Wedel, Nora Kochupurakkal, Sek Won Kong, Sayantan Bose, Ji-Won Lee, Madeline Maslyar, Bayan Alsairafi, Kayla MacLeod, Kaifeng Liu, Hengcheng Zhang, Masaki Komatsu, Hironao Nakayama, Diane R. Bielenberg, David M. Briscoe

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Figure 4

Patterns of NRP2 expression on murine CD4+ T cells after activation.

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Patterns of NRP2 expression on murine CD4+ T cells after activation. (A)...
(A) Representative dot plot of NRP2-GFP expression within CD4+ splenocytes of NRP2lox/lox mice (n = 4 independent experiments; Supplemental Figure 5, A–D). (B and C) Representative dot plots (left) and summary of n = 4 independent experiments comparing phenotype of NRP2-GFPpos with NRP2-GFPneg CD4+ splenocytes. (D) NRP2 mRNA expression by PrimeFlow cytometry on isolated murine CD4+ T cells stimulated with 1 μg/mL anti-CD3 for up to 48 hours in vitro. Dot plots are gated on CD4+ cells. Graph illustrates changes in the expression of NRP2 mRNA in CD4+ T cells over 48 hours of in vitro stimulation (n = 6 independent experiments; mean ± SD; Kruskal-Wallis test with Dunn’s multiple-comparison test, ***P < 0.001; ****P < 0.0001 vs. unstimulated). (E and F) Fully MHC-mismatched Balb/c donor hearts were transplanted into C57BL/6 recipients. Splenocytes were isolated 4–7 days after transplant; frequency of NRP2 mRNA-expressing CD4+ T cells was evaluated by PrimeFlow cytometry. Nontransplanted C57BL/6 mice were included to illustrate NRP2 mRNA expression before transplant (day 0). (E) Dot plots are gated on CD4+ cells. Graph illustrates changes in expression of NRP2 mRNA in CD4+ T cells before and up to 7 days after transplant (n = 4 mice per time point; mean ± SD; Kruskal-Wallis test with Dunn’s multiple-comparison test, **P < 0.01 vs. day 0). (F) CD44 expression on NRP2neg and NRP2pos CD4+ T cells isolated 7 days after transplant. Graph summarizes n = 4 experiments (paired t test). (G–J) 2.5 × 106 CD4+ T cells from CD45.2pos OT-II mice were adoptively transferred into congenic CD45.1+ hosts by tail vein injection. Host mice were immunized s.c. with ovalbumin (50 μg) in complete Freund’s adjuvant (CFA), and the phenotype of antigen-specific OT-II and host CD4+ T cells were assessed after 7 days by flow cytometry. (G) Representative dot plots of CD45.2pos OT-II (top) and CD45.1pos host (bottom) CD4+ T cells are shown. (H) Mean NRP2 positivity within CD4+ T cells ± SD of n = 5/condition (2-way ANOVA with Fisher’s least significant difference test). (I) Representative dot plots illustrate PD1 and Tim3 expression of NRP2pos (right) and NRP2neg (left) OT-II CD4+ T cells. (J)Graph summarizes mean ± SD of PD1+Tim3+ cells of n = 5/condition (paired t test).