The immortalization rate was comparable for all subsets, showing that no significant bias would be introduced through the immortalization process. GUID:?0A430588-BAB0-43C0-8AE3-61F7CB2CF5D3 Abstract Antibody rejection is often accompanied by non-donor HLA specific antibodies (NDSA) and self-reactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of Rabbit polyclonal to IFIH1 these antibodies, we immortalized 107 B cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B cell MLS0315771 clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients. strong class=”kwd-title” Keywords: Humoral rejection, polyreactive antibodies, human Introduction Antibodies reactive to kidney grafts have been implicated in transplant rejection for more than 40 years, yet much remains to be elucidated regarding their generation, fine specificities and function [1C3]. The main targets of these humoral responses are HLA class I and class II molecules expressed on donor cells that are mismatched with those of the recipients. These antigens are recognized as nonself MLS0315771 by the host immune system due to their polymorphic allelic determinants and lead to the generation of donor MLS0315771 specific antibodies (DSA). Serological responses to other targets have also been reported following kidney transplantation, including non-donor specific antibodies (NDSA) [4C7], i.e. antibodies reactive to HLA molecules not expressed by the donor cells. How NDSA develop is still uncertain. The most widely accepted explanation is that NDSA and DSA cross-react to public epitopes shared by multiple HLA [8C11]. In other instances, especially in the absence of DSA, the generation of NDSA is thought to result from previous exposure to non-donor HLA, for example via blood transfusion and pregnancies. An abundant literature also attests to the development of antibodies to self-antigens alongside anti-HLA antibodies in transplant recipients [12C22]. In a recent study, we used a combination of solid phase techniques including protein microarrays, to characterize serological profiles of kidney transplant recipients with chronic humoral rejection (CHR) [23]. Our experiments showed that patients sera reacted to numerous autoantigens, at the time of CHR. Moreover, serum reactivity patterns appeared remarkably unique for each individual with only minimal overlap between patients. These complex serological responses, combining allogeneic and autoimmune components are intriguing and difficult to explain. In the present study, we investigated the antibody response associated with graft rejection at the cellular level by isolating, cloning and characterizing B cells responsible for the production of graft reactive antibodies. We examined the nature of these antibodies and looked for a possible relation between allo- and autoreactivity. Materials and Methods Patient characteristics and biological samples The patient studied in this report is a 43 year old male who underwent explantation of his MLS0315771 second kidney transplant consecutive to rejection. His original disease was focal segmental glomerulosclerosis (FSGS). The first kidney failed 12 years after the initial transplant, consecutive to cyclosporine vasculopathy and focal acute cellular rejection. The second kidney graft was transplanted 5 years after rejection of the first one and functioned for about 1.5 years. It was removed because of suspected pyelonephritis. Sections from the nephrectomy specimen showed a dense.