Date: Saturday, May 30, 2020
Session Time: 3:15pm-4:00pm
Presentation Time: 3:30pm-4:00pm
*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-H2bm12 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-H2bm12 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-H2bm12 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.
To cite this abstract in AMA style:Wedel J, Lee J, Kochupurakkal N, Chernov E, Poreddy M, Liu K, Kong S, Bielenberg D, Nakayama H, Briscoe DM. Neuropilin 2 is a Novel Coinhibitory Molecule That Suppresses Alloimmune Responses Following Transplantation [abstract]. Am J Transplant. 2020; 20 (suppl 3). https://atcmeetingabstracts.com/abstract/neuropilin-2-is-a-novel-coinhibitory-molecule-that-suppresses-alloimmune-responses-following-transplantation/. Accessed September 21, 2021.
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