B cells can exhibit potent IL-10-dependent regulatory (Breg) activity. IL-10-enriched Bregs can transfer tolerance in auto and allo-immune models. We showed that TIM-1 is an inclusive marker for IL-10+ B cells; that TIM-1+ but not TIM-1- B cells transfer allograft tolerance; and that anti-TIM-1 induces IL-10+ TIM-1+ Bregs. However, little is known about where Bregs are induced or where they act in vivo.

We now show most IL-10+ Bregs reside in the spleen (>85%), compared to LN (<1%) or marrow (∼15%), while few if any are in islet allografts. Moreover, IL-10+ Bregs in spleen increase from ∼2% to 7% of B cells in response to alloantigen plus anti-TIM-1, but remain unchanged in LN and marrow (0.5% and 1%, respectively). In splenectomized (spl-X) mice, anti-TIM-1 treatment did not increase IL-10+ B cells in LN or marrow. Another approach to induce Bregs is to treat mice with apoptotic cells (ACs). ACs increased IL-10+ B cells from 2% to 4.5% in spleen and from 0.5% to 2% in LN. However, after spl-X, ACs no longer increased IL-10+ Bregs in LN. This suggests that Bregs are induced in the spleen, but then may circulate to other sites.

Since Bregs are induced in spleen, we asked whether the spleen was required for Breg-mediated allograft survival (GS). Anti-TIM-1 was unable to induce longterm GS in spl-X islet recipients vs. controls (MST 24d vs. 100d; p=0.002). To confirm that anti-TIM-1 actually acts through IL-10+ Bregs, we generated hCD20ERT2-CreXIL-10fl/fl mice where IL-10 is deleted in B cells by tamoxifen treatment. In such B-IL-10KO mice, anti-TIM-1 was unable to prolong GS compared to Cre- littermates (MST 20d vs. >50d). In addition, LTβR-KO mice (lacking LNs) that received alloreactive Teff cells, rejected islets faster after spl-X (MST 7d) vs. no spl-X controls (MST 18d; p=0.02), consistent with a reservoir of Bregs in spleen.

Currently, it is unknown whether once induced, migrating Bregs can act outside the spleen. In ongoing experiments we will determine whether the spleen and/or SLO are essential for Breg activity, by transferring pre-generated TIM-1+ Bregs from alloimmunized mice into either WT spl-X or LTβR-KO spl-X allograft recipients vs. respective controls and determine GS. Thus, our data elucidate a vital role for the spleen in Breg induction and show that Breg suppressive function occurs in the spleen. Current experiments will clearly delineate whether once induced, Bregs can function outside of the spleen or SLO.

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