Donor-specific transplantation tolerance has long been an aspiration of clinical transplantation. Recently, we have reported that multiple mechanisms are required to reinforce profound hyporesponsiveness of alloreactive T cells and to mediate robust tolerance. In contrast, little is known about the fate of alloreactive B cells. Clinical observations suggest that donor-specific antibodies (DSA) are a major cause of graft rejection despite ongoing immunosuppression, leading us to hypothesize that stable tolerance will require the donor-specific B cell response to also be profoundly suppressed. We used a mouse model of tolerance, where C57BL/6 recipients treated with anti-CD154 (D0, 7,14) and DST (D0) accepted BALB/c hearts for >60 days and where minimal DSA is produced. Firstly, we showed that the lack of an alloantibody response was not due to B cell deletion. Donor-MHC class I and class II reactive B cells were identified using H-2K^d and I-E^d tetramers, and their total numbers in tolerant recipient was comparable to that of naïve controls. Second, there was no enrichment for donor-specific B cells with Breg phenotype or function; with comparable numbers of CD93⁺ T1, T2 and T3 B cells, folicullar zone B cells, and in percentage of donor-specific B cells expressing expression of CD5, CD1d, TIM-1 and IL-10. Third, B cell unresponsiveness is not due to the absence of T cell help, and is B cell intrinsic. Adoptive transfer of B cells from tolerant recipients into naive MD4 hosts did not produce DSA upon challenge with BALB/c splenocytes, even when additional alloreactive T cells were included in the transfer. In contrast, naïve B cells recipients counterparts showed strong DSA responses. Finally, we show that the donor-specific B cells intrinsic hyporesponsive signal is not reversed even when challenged with DST in the presence of CpG and agonistic anti-CD40. Taken together, we demonstrate the acquisition of a robust cell-intrinsic state of B cell hyporesponsiveness during the maintenance phase of transplant tolerance, that is maintained even in the absence of T cell regulation, and in the presence of T cell help and CpG plus anti-CD40. The mechanistic basis for this profound B cell hyporesponsive state is under investigation, but preliminary studies indicate defective proliferation and differentiation into germinal center B cells.

CITATION INFORMATION: Khiew S., Jain D., Young J., Chen J., Yang J., Wang Q., Yin D., Sciammas R., Chong A. Alloreactive B Cells Acquire Cell-Intrinsic Hyporesponsiveness in Tolerant Recipients Am J Transplant. 2017;17 (suppl 3).

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