In B cell-deficient mice, prolonged islet allograft survival (GS) mediated by anti-TIM1 depends on transfer of IL-10 competent B cells. However, such regulatory B cells (Bregs) might also require other cytokines or differentiation into plasma cells (PCs) to provide effective suppression. In this regard, in EAE models, B cell IL-35 and IL-10 were both shown to be essential for Bregs to ameliorate disease. Moreover, PCs express very high levels of both cytokines and PCs were also essential for “Breg” activity in EAE. To determine the role of IL-35 in anti-TIM1 mediated GS, WT vs. IL-35KO (Ebi3-/-p35-/-) B cells were transferred into B cell-deficient [micro]MT (B6) recipients of BALB/c islets with/without anti-TIM-1 treatment. While anti-TIM1 had no effect on GS in [micro]MT mice without B cells (MST 17.5 vs. 21 d; p= NS), transfer of either WT or IL-35-/- B cells, restored responsiveness to anti-TIM-1 (100% vs.66.7% GS>50d p=NS) and both were significantly different than no B cell transfer (p<0.05). To examine the role of PCs (B220^lowCD138^hiIg⁺Blimp1⁺) in GS we first compared their IL-10 expression to (CD19⁺CD138^–) B cells in alloimmunized mice. Freshly isolated splenic PCs had increased frequency of IL-10 expression (~47%) vs. B cells (<1%). Thus, despite being infrequent (0.3%~0.6% of splenic B cells), PCs comprise ~20- 35% of all B-lineage IL-10. However, after in vitro stimulation to promote cytokine expression, B cell IL-10 expression increases markedly (4-7%), and PCs only comprise ~4% of B-lineage IL-10. To examine their Breg activity in vivo, WT and BLIMP1-/- B cells were transferred into [micro]MT islet recipients. Both types of B cells were similarly effective at mediating prolonged GS by anti-TIM-1 (100% vs.75% GS>80d p=NS). Finally, we used unique mice with an inducible B cell-specific KO of IL-10 (IL-10fl/flXCD20-ERT2-Cre; B6) vs Cre-neg littermate controls as recipients of islet allografts after treatment with Tamoxifen. B cell IL-10 was essential for prolonged GS by anti-TIM-1 in these otherwise entirely normal mice. Thus, anti-TIM-1 mediated prolongation of GS is completely dependent on induction of Bregs expressing IL-10, and unlike other models, requires neither IL-35 nor PC differentiation.

CITATION INFORMATION: Zhou Y, Ding Q, Cherukuri A, Rothstein D. Prolongation of Graft Survival by Anti-TIM1 Is Specifically Mediated by IL-10+ Bregs and Is Not Dependent on IL-35 or Plasma Cell Differentiation. Am J Transplant. 2016;16 (suppl 3).

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