T-bet is essential for natural regulatory T cells (nTreg) to regulate Th1 inflammation, but whether T-bet controls other Treg functions after entering the inflammatory site is unknown. In an islet allograft model, T-bet^−/− nTreg, but not induced Treg, failed to prolong graft survival as effectively as wild-type Treg. T-bet^−/− nTreg had no functional deficiency in vitro but failed to home from the graft to draining lymph nodes (dLN) as efficiently as wild type. T-bet regulated expression of adhesion- and migration-related molecules, influencing nTreg distribution in tissues, so that T-bet^−/− nTreg remained in the grafts rather than migrating to lymphatics and dLN. In contrast, both wild-type and T-bet^−/− CD4⁺ conventional T cells and induced Treg migrated normally toward afferent lymphatics. T-bet^−/− nTreg displayed instability in the graft, failing to suppress Ag-specific CD4⁺ T cells and prevent their infiltration into the graft and dLN. Thus, T-bet regulates nTreg migration into afferent lymphatics and dLN and consequently their suppressive stability in vivo.

Background Circulation of leukocytes via blood, tissue and lymph is integral to adaptive immunity. Afferent lymphatics form CCL21 gradients to guide dendritic cells and T cells to lymphatics and then to draining lymph nodes (dLN). Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 (VEGFR-3) are the major lymphatic growth factor and receptor. We hypothesized these molecules also regulate chemokine gradients and lymphatic migration. Methods CD4⁺ T cells were injected into the foot pad or ear pinnae, and migration to afferent lymphatics and dLN quantified by flow cytometry or whole mount immunohistochemistry. Vascular endothelial growth factor receptor 3 or its signaling or downstream actions were modified with blocking monoclonal antibodies (mAbs) or other reagents. Results Anti–VEGFR-3 prevented migration of CD4⁺ T cells into lymphatic lumen and significantly decreased the number that migrated to dLN. Anti–VEGFR-3 abolished CCL21 gradients around lymphatics, although CCL21 production was not inhibited. Heparan sulfate (HS), critical to establish CCL21 gradients, was down-regulated around lymphatics by anti–VEGFR-3 and this was dependent on heparanase-mediated degradation. Moreover, a Phosphoinositide 3-kinase (PI3K)α inhibitor disrupted HS and CCL21 gradients, whereas a PI3K activator prevented the effects of anti–VEGFR-3. During contact hypersensitivity, VEGFR-3, CCL21, and HS expression were all attenuated, and anti-heparanase or PI3K activator reversed these effects. Conclusions Vascular endothelial growth factor C/VEGFR-3 signaling through PI3Kα regulates the activity of heparanase, which modifies HS and CCL21 gradients around lymphatics. The functional and physical linkages of these molecules regulate lymphatic migration from tissues to dLN. These represent new therapeutic targets to influence immunity and inflammation.

Anemia and vitamin D deficiency are associated with allograft failure, and hence, are potential therapeutic targets among kidney transplant recipients (KTRs). We conducted a multicenter, two‐by‐two factorial, open‐label, randomized clinical trial to examine the effects of anemia correction and vitamin D supplementation on 2‐year change in eGFR among KTRs (CANDLE‐KIT). We enrolled 153 patients with anemia and >1‐year history of transplantation across 23 facilities in Japan, and randomly assigned them to either a high or low hemoglobin target (>12.5 vs. <10.5 g/dL) and to either cholecalciferol 1000 IU/day or control. This trial was terminated early based on the planned interim intention‐to‐treat analyses (α=0.034). Among 125 patients who completed the study, 2‐year decline in eGFR was smaller in the high vs. low hemoglobin group (i.e., ‐1.6±4.5 vs. ‐4.0±6.9 mL/min/1.73 m²; P=0.021), but did not differ between the cholecalciferol and control groups. These findings were supported by the fully‐adjusted mixed effects model evaluating the rate of eGFR decline among all 153 participants. There were no significant between‐group differences in all‐cause death or the renal composite outcome in either arm. In conclusion, aggressive anemia correction showed a potential to preserve allograft kidney function.