Clinical data suggest that donor-specific antibody (DSA)-mediated rejection is currently the leading cause of kidney allograft failure, with anti-donor Class II Abs tending to be more pathogenic compared to anti-donor Class I Abs. We have previously reported on the use of MHC Class I tetramers to study the cellular dynamics driving anti-Class I antibody production in mice; here we focus on tracing the fate of endogenous B cells recognizing donor Class II using I-E^d tetramers. At 10 day after BALB/c splenocyte immunization of or heart transplant into C57BL/6 mice, a 7.9-9.8 fold increase in the total number of I-E^d-binding B cells was observed, with 54-58% of these cells expressing a germinal center (Fas⁺GL7⁺) phenotype. Following subcutaneous immunization, the I-E^d-specific B cell response was localized in draining lymph nodes whereas following heart transplantation into the abdominal cavity, the response was observed in the mediastinal lymph node and spleen. In sensitized recipients, transplantation of BALB/c hearts resulted in a recall I-E^d-binding B cell response that resulted in a modest acquisition of the germinal center phenotype (<5%). In contrast the recall anti-I-E^d response was associated with strong differentiation into IgG secreting cells, which was detected with an I-E^d-specific ELISPOT assay. By combining this visualization approach with the lineage tracking of memory B cells in AID-cre xRosa26-StopfloxEGFP recipients, we observed that I-E^d -specific memory B cells were predominantly generated between days 7-14 post-sensitization, consistent with their germinal center origin, and that CTLA-4Ig treatment starting as as late as day 7-14 post-immunization was able to significantly reduce memory B cell generation. In summary, we validated the use of MHC Class II tetramers to specifically track the in vivo fate of endogenous Class II-specific B cells, and provide insights into the timeframe whereby memory B cell generation can be prevented.

CITATION INFORMATION: Yang J, Chen J, Akl A, Sciammas R, Chong A. Tracking Endogenous B Cells Recognizing Allogeneic MHC Class II in Mice. Am J Transplant. 2016;16 (suppl 3).

« Back to 2016 American Transplant Congress