Whereas a B cell-transcriptional profile has been recorded for operationally tolerant kidney graft patients, the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine production by effector CD4 + CD25 2 T cells were measured after anti-CD3/anti-CD28 stimulation with or without autologous B cells. We report that B cells inhibit CD4 + CD25 2 effector T cell response in a dosedependent manner. This effect required B cells to interact with T-cell targets and was achieved through a granzyme B (GzmB)-dependent pathway. Tolerant recipients harbored a higher number of B cells expressing GzmB and displaying a plasma cell phenotype. Finally, GzmB + B-cell number was dependent on IL-21 production, and B cells from tolerant recipients but not from other patients positively regulated both the number of IL-21 + T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases excessive B cell activation and allows regulation to take place. These data provide insights into the characterization of B cell-mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on effector T cells in blood from patients with operationally tolerant kidney grafts.

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Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation.