The observation that operationally tolerant patients (TOL) display higher numbers of blood B cells and a B cell gene signature compared to stable kidney allograft recipients suggests that B cells may be critical to tolerance maintenance. If this is correct, we hypothesized that a tolerance signature would be most prominent in alloreactive B cells. Thus we investigated the function and phenotype of alloreactive B cells in a mouse model of allograft tolerance. BALB/c hearts transplanted into C57BL6 recipients that received anti-CD154 (D0,7,14) and DST (D0) were accepted for >150 days without detectable alloAbs. Whether the lack of an alloAb response was due to B cell deletion, absence of T cell help, B cell anergy or regulation was examined: i. To test for deletion, we used H2Kd tetramers to identify alloreactive B cells and determined that the numbers of H2Kd tetramer-binding B cells in long-term tolerant mice was comparable to that of naive mice. Thus deletion was not a feature of B cell tolerance. ii. To test the role of absent T cell help, we used alloreactive TCR75 CD4+ T cells that recognize H2Kd peptide presented by IAb. Transfer of 3×106 TCR75 cells into tolerant mice precipitated rejection and the differentiation of the TCR75 cells into Tfh cells. However, no alloreactive or Kd-specific Abs were detected, implying that the absence of T cell help is not the basis for B cell tolerance. iii. To test for B cell hyporesponsiveness, we transferred purified B cells from tolerant or control naïve mice into HEL-specific B cell transgenic recipients (with endogenous alloreactive T cells but no endogenous alloareactive B cells) and challenged them with BALB/c splenocytes. Mice receiving B cells from naïve controls produced alloreactive antibodies whereas those with B cells from tolerant mice did not, suggesting that alloreactive B cellsfrom tolerant mice were anergic or that they harbored a regulatory population of B cells. iv. Flow cytometric characterization of H2Kd-binding B cells from tolerant mice revealed that they were not enriched for CD5+ CD1Dhi IL10 producing B cells, did not upregulate inhibitory (CD22, Tim-1, TIM-3, PD-1, CTLA-4, FR4, CD73) or costimulatory (CD80, CD86) molecules and were not enriched at the T2 or T3 stage. Taken together, B cell tolerance induced with anti-CD154/DST is mediated by anergic or regulatory B cells that exhibit non-canonical phenotypes. Gene-profiling is being used to identify the basis of B cell tolerance.

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