Donor HLA-DQ reactive B cells clonally expand under chronic immunosuppression and include atypical CD21 low CD27 ⁻ B cells with high-avidity germline B-cell receptors

Long-term allograft survival is limited by humoral-associated chronic allograft rejection, suggesting inadequate constraint of humoral alloimmunity by contemporary immunosuppression. Heterogeneity in alloreactive B cells and the incomplete definition of which B cells participate in chronic rejection in immunosuppressed transplant recipients limits our ability to develop effective therapies. Using a double-fluorochrome single-HLA tetramer approach combined with single-cell in vitro culture, we investigated the B-cell receptor (BCR) repertoire characteristics, avidity, and phenotype of donor HLA-DQ reactive B cells in a transplant recipient with end-stage donor specific antibody (DSA)-associated cardiac allograft vasculopathy while receiving maintenance immunosuppression (tacrolimus, mycophenolate mofetil, prednisone). Donor DQB1*03:02/DQA1*03:01 (DQ8)-reactive IgG+ B cells were enriched for minimally mutated and germline encoded high avidity BCRs (median K D 4.26×10 -09 ) with an atypical, antigen-experienced and proliferative phenotype (CD27 - CD21 low CD71 + CD11c +/- ). These B cells coexisted with a smaller subset of more highly mutated, affinity matured IgG+CD27+ B cells. Circulating donor-reactive B cells and DSA remained detectable after rituximab, contrasting with the marked reduction in DSA after allograft explant and retransplant. Together, these findings define the persistence of germline high-avidity HLA-DQ alloreactive B cells and their co-existence with affinity matured clones that were both driven by the allograft despite conventional immunosuppression.