*Purpose: TIM-1+ identifies the majority (~75%) of IL-10+ B regulatory cells (Bregs). However, TIM-1+ B cells are also enriched for IL-4+ B effector 2 cells (Be2). We previously demonstrated that TIM-1+ B cells can prolong islet graft survival (GS) and that this effect is IL-10 dependent. We also established that anti-TIM-1 mAb (RMT1-10) can prolong islet GS and this is associated with a 3-fold increase in IL-10+ and a 2-fold increase in IL-4+ TIM-1+ B cells. The concomitant increase in Foxp3+Tregs and Th2 cells and decrease in Th1 cells after anti-TIM-1 are all dependent on IL-10-competent B cells. Interestingly, our work revealed that both IL-4KO and IL-4Rα-KO mice lacked IL-10+ Bregs. Moreover, anti-TIM-1 treatment could not induce Bregs when IL-4Rα-KO B cells were transferred into B cell-deficient JHD mice. This implies IL-4 signaling may be indispensable for normal Breg induction.

*Methods: To clearly establish the importance of IL-4R signaling for Breg induction, we now developed hCD20Cre.ERT2+/- X IL-4Rαfl/fl (B-IL-4RαKO) mice, in which tamoxifen (TAM) treatment specifically deletes IL-4Rα in B cells.

*Results: 14d after allo-immunization and anti-TIM-1, TAM-treated B-IL-4RαKO mice had 50% less TIM-1+ B cells than TAM-treated Cre-neg XIL-4Rαfl/fl littermate controls. In addition, B cells from these B-IL-4RαKO mice expressed 50% less IL-4 and 40% less IL-10 than littermate controls. Next, we examined islet GS. Untreated B6 B-IL-4RαKO and control mice rejected BALB/c islets with MST 28d and MST 25d, respectively. Surprisingly, anti-TIM-1 treatment accelerated rejection in B-IL-4RαKO mice (MST 20d; p=0.01), while 75% of control mice treated with anti-TIM-1 achieved long-term GS (>100d; p=0.03). To try to understand this marked difference in GS, we examined the cytokine profile of T cells from B-IL-4RαKO vs. control mice. Splenic CD4 cells from B-IL-4RαKO mice expressed 33% more IFNγ and 33% less IL-10, and splenic CD8 cells expressed 20% more IFNγ than littermate controls.

*Conclusions: Collectively, our results suggest that B cell IL-4 signaling helps induce IL-10 secreting TIM-1+ Bregs which then reduce inflammatory T cell cytokines. Whether IL-4 produced by B cells themselves has an autocrine role and/or directly inhibits inflammatory T cell cytokines is currently being examined. Regardless, targeting B cell IL-4 signaling can have a major impact on transplant outcomes.

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