A multicolour HLA-specific B-cell FluoroSpot assay to functionally track circulating HLA-specific memory B cells

Emerging evidence suggests that donor-reactive memory B cells (mBC) play a key role inducing antibody-mediated rejection (ABMR) after solid organ transplantation and show a broader antigen repertoire than plasma cells thus, being potentially present even in absence of donor-specific antibodies. Therefore, the development of novel immune assays capable of quantifying circulating donor-reactive mBC in organ transplantation is highly warranted. We developed a novel HLA-specific B-cell FluoroSpot assay capable of enumerating multiple HLA-specific Antibody Secreting Cells (ASC) originated from circulating mBC after in-vitro polyclonal activation. We performed a thorough characterization of distinct selective in-vitro mBC activation methods based on either the TLR7,8 agonist R848 plus Interleukin-2 or an anti-CD40 agonist monoclonal antibody, assessed optimal activation culture conditions, cell sources, activation time-frame as well as the advantage of measuring HLA-specific IgG-ASC as compared to HLA-IgG Ab detected in supernatants of in-vitro stimulated B-cell to characterize anti-HLA alloreactivity. Notably, using fluorescently-labeled multimerized HLA molecules as detection matrix, we show the ability of this assay to precisely quantify multiple anti-HLA mBC specificities. In conclusion, evaluating circulating donor-reactive mBC using new technology may provide novel insight of the pathogenesis of humoral rejection and may help identifying transplant recipients at high risk of allograft rejection.