Axonal guidance molecules are expressed within the immune system and elicit anti-inflammatory effects with potential to target auto- and alloimmunity. The neuropilin (NRP) family receptors bind class three semaphorins and elicit signals that induce cytoskeletal collapse within neuronal and tumor cells. NRP1 is well established to be expressed by regulatory T cells. We recently identified another member, NRP2, on CD4+ effector T cell and Foxp3+ CD4+ regulatory T cell subsets. NRP2 is expressed at baseline, is induced following mitogen activation and selectively binds semaphorin 3F (SEMA3F), resulting in immunomodulation following transplantation. In this study, we evaluated the function of NRP2 using CD4+ T cells derived from global NRP2-KO and CD4^creNRP2^lox/lox mice. We initially found that CD4+ T cells derived from these mice were hyperproliferative in response to mitogen (anti-CD3 0.1-1 μg/ml) and produced markedly increased amounts of IL-2 (∼3-fold), IFNγ (∼5-fold) and IL-17 (∼8-fold) vs. WT cells. Furthermore, both NRP2-KO and CD4^creNRP2^lox/lox recipients of minor MHC mismatched B6.C-H2^bm12 donor cardiac transplants reject grafts at an accelerated rate (NRP2-KO; mean graft survival (MST)=32 days, n=5, P<0.005, CD4^creNRP2^lox/lox; MST=19 days, n=4, P<0.0001) vs. WT recipients (MST>54 days, n=11). To evaluate immunoregulatory signaling elicited by NRP2, we analyzed the effect of SEMA3F on two human NRP2-expressing cell lines, U87MG and Jurkat cells. PI3K activity, measured by standard kinase ELISA, was reduced by SEMA3F in a time dependent manner. Using phosphokinase arrays and Western blot analysis, we also found that the activity of MAPK, pAkt (T308 and S473), pmTOR and pS6K was markedly inhibited by SEMA3F (n=4, P<0.05). When the cells were treated with rapamycin (10 nM) for 30 min to inhibit mTORC1 activity, SEMA3F inhibited pAkt (S473). By immunoprecipitation, SEMA3F disrupted the association of rictor with mTOR, further documenting that its effect is to inactivate mTORC2. Using a siRNA knockdown approach, we confirmed that these effects were elicited via NRP2, indicating that SEMA3F-NRP2 interactions modulate cellular activation via the targeting of mTORC2. Our findings for the first time define NRP2 as a novel immunomodulator, and our data suggest that its primary function is to suppress Akt/mTOR signaling. These observations have broad implications in alloimmunity.

« Back to 2015 American Transplant Congress