*Purpose: B cells are known to play a significant role in allograft tolerance or rejection, largely determined by production of cytokines and antibodies that suppress or promote inflammation. In this study, we investigate whether human B cells mediate an indirect mode of allorecognition via alloantigen (alloAg) cross-presentation (CP) to drive CD8⁺ T cell responses.

*Methods: To study CP pathways in human B cells under allogeneic conditions, we analyzed the transcriptional profile of responder (host) B cells negatively selected from mixed lymphocyte reactions (MLRs). B cell subset phenotypes of interest and relevant functional molecules were validated in MLRs, human splenocytes and peripheral blood with flow cytometry. CD8⁺ T cell activation (HLA-DR/CD25), cytotoxicity (granzyme B/perforin) and B cell endocytosis capacity were assessed with flow cytometry-based functional assays.

*Results: In MLRs that demonstrate high pro-inflammatory CD8⁺ T cell responses, we identify the emergence of an activated B cell (aB) subpopulation. In contrast with other activated B cells, these cells show markedly higher expression of molecules, such as tapasin (p<0.0001) involved in processing antigens for presentation on HLA-I molecules, suggesting a role for these aB in antigen cross-presentation (aB-CP). Based on increased expression of genes associated with proteasomal processing and antigen transport pathways in transcription profiling of B cells in MLRs, aB-CP likely adopt an endosome-to-cytosol pathway for processing endocytosed antigens. Using proteasomal inhibition during MLR to examine this, we observed reduced aB-CP numbers and CP-molecule expression with a concurrent decrease in host CD8⁺ T cell responses to alloAgs. aB-CP also show distinctively higher endocytic activity, consistent with subset specialization for uptake and processing of exogenous alloAgs. As predicted by expression profiling, the marked elevation of the IgG receptor, FcRL5 (p<0.0001), in aB-CP supports this hypothesis. Furthermore, we observed that a small subpopulation of B cells with an aB-CP profile are readily identifiable in disaggregated splenocytes and peripheral blood of healthy individuals. These in vivo counterparts to our ex vivo observations support consideration of the relevance of aB-CP in transplant patients.

*Conclusions: We have characterized a distinct human B cell population which emerges and expands upon alloAg challenge in MLR and has substantial capacity for alloAg cross-presentation. These findings could lay the foundation for novel approaches to decreasing the cytotoxic activity of CD8⁺ T cells and minimizing post-transplant morbidity.

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