Recent studies have indicated that the VEGF receptor NRP-1 is expressed on T regulatory cells, and that VEGFR1 and VEGFR2 are expressed on human T cells. Moreover, blockade of VEGF-VEGFR interactions has been found to inhibit T cell recruitment and graft arteriosclerosis in different in vivo animal models of rejection. However, the differential effects of VEGF on T effs and Tregs is unknown. Here, we evaluated the expression of each receptor on murine T cells derived from wild type and FoxP3^eGFP transgenic mice. Receptor profiling was evaluated on naive T cells as well as on T cells driven towards Th1 (anti-CD3⁺ IFNΓ+anti-IL-4), Th2 (anti-CD3⁺IL-4+anti-IFNΓ) and iTreg (anti-CD3/IL-2/TGFΒ) subsets. Consistently, we find that VEGFR expression is minimal on CD4⁺CD25^neg naive T cells, whereas Flt-1 (VEGFR1) is expressed at high levels on murine T effectors but not by FoxP3⁺CD25^hi T reg subsets. In contrast, NRP-1 expression is minimal on T effectors but is expressed at high levels on all iTreg populations examined. To assess whether VEGF functions to alter activation of Teff, Tregs or both subsets, we performed in vitro suppression assays, in which purified/sorted populations of CD4⁺ Flt-1⁺ T effectors were cultured with increasing numbers of sorted CD4⁺ CD25^hi Tregs in the absence or presence of VEGF (20ng/ml). We found that Tregs were potent to suppress proliferation of Flt-1⁺ T effectors, and the addition of VEGF failed to inhibit this suppressive effect (n=>5, P=NS). In contrast, VEGF augmented mitogen-induced (anti-CD3, 1Μg/ml) proliferation, IL-4 (p<0.05) and IL-10 (p=0.06) production, but not IFNΓ production (as determined by ELISPOT) of sorted populations of Flt-1⁺ splenic CD4⁺ T cells. Since NRP-1 also binds known semaphorin 3 family molecules, we also evaluated the effect of increasing concentrations of Sema 3A (25-1000ng/ml) in suppression assays and found that it failed to alter the suppressive effects of Tregs, but trended to increase cytokine production (IFNΓ) by T effectors. Collectively, these findings indicate that VEGF receptors are differentially expressed on T cells and that VEGFR1 is potent to elicit T eff activation. We suggest that VEGF-VEGFR interactions are tightly regulated on different T cell subsets. Our data also provide for the intriguing possibility that Sema3A-NRP-1 interactions elicit inhibitory signals for T reg function and/or inhibit immunoregulation.

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