The conceptual underpinning of our studies is that robust tolerance requires multiple mechanisms to enforce functional quiescence of alloreactive T and B cells. While much research has focused on the mechanisms that control alloreactive T cells, much less is known about the fate of alloreactive B cells. Clinical data suggest that donor-specific antibodies are a major cause of graft rejection despite immunosuppression, leading us to hypothesize that stable tolerance requires the donor-specific B cell responses to be profoundly inhibited, and by mechanisms that are not solely dependent on the absence of T cell help. To investigate the mechanism of B cell tolerance, endogenous donor-MHC Class I and Class II reactive B cells from C57BL/6 recipients of BALB/c hearts were identified using H-2K^d and I-E^d tetramers. Tolerance was induced with anti-CD154 plus BALB/c splenocytes, and the majority of allografts were accepted for >200 days without donor-specific antibodies. In tolerant recipients, alloreactive B cells were not deleted, and were present in numbers and phenotypes similar to naïve controls. Also there was no enrichment for CD93⁺transitional or marginal zone B cells, or for the expression of CD5, CD1d or TIM-1. Importantly there was no increased frequencies of IL-10-production within the alloreactive B cell subset in tolerant compared to naïve controls. Despite the lack of these phenotypic differences that have been described to mark regulatory B cells, B cells from tolerant mice were functionally unresponsive. Upon adoptive transfer of purified B cells into naïve MD4 recipients (~95% of their B cells express B cell receptors specific for HEL), B cells from tolerant mice (B-Tol) did not make DSA-IgG upon challenge with BALB/c splenocytes, even when additional alloreactive CD4⁺ T cells (TCR75) and polyclonal T cells from naïve donors were included in the adoptive transfer. In contrast, B cells from naïve mice (B-naïve) responded with strong DSA responses. Even more notable was the ability of B-Tol to inhibit B-naïve responses, when the cells were adoptively transferred at a 1:1 ratio. To our knowledge, this is the first demonstration that B cells from tolerant mice have the ability to mediate infectious tolerance to naïve B cells, even in naïve hosts harboring TCR-tg alloreactive T cells capable of providing T cell help.

CITATION INFORMATION: Chen J, Yang J, Akl A, Wang Q, Yin D, Sciammas R, Chong A. Fate of Endogenous Alloreactive B Cells in Transplant Tolerance. Am J Transplant. 2016;16 (suppl 3).

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