Higher IL10 Production by Transitional B Cells in Tolerant Kidney Transplant Recipients

Abstract

Background: Induction of transplantation tolerance would reduce the risk of infection, malignancy and cardiovascular disease caused by immunosupresive drugs in renal transplant recipients. Indices of Tolerance Project, one of the largest studies identifying biomarkers of tolerance, revealed that tolerant kidney transplant recipients, had an expansion of B cells in peripheral blood. In parallel, a B cell subset with regulatory properties has been described in humans as a transitional B cell, characterised by high expression of CD38, CD24 and IL10 production. We set out to test the hypothesis that the expansion of this B cell subset was central in the process of spontaneous tolerance observed in these rare recipients and to test the ability of these cells to present alloantigens. Methods: Peripheral Blood Mononuclear Cells (PBMCs) samples from <tolerant: functionally stable recipients without immunosuppressive drugs, <stable: functionally stable recipients on standard immunosuppression, <chronic rejector: immunosuppressed recipients that presented signs of chronic rejection and <healthy controls were used in the study. B cell subsets from patients were identified by flow cytometry with anti-CD20, anti-CD19, anti-CD27, anti-IgM, anti-IgG, anti-CD24 and anti-CD38. PBMCs were cultured for 3 days alone, with CD40L and CpG in RPMI 10% FCS. After 3 days, activation markers (CD25, CD86 and CD40) were measured by surface staining and IL10, IFN-g and TNF-a were measured by intracellular staining and ELISA. Results: B cells were identified as CD20+CD19+ and memory B cells as CD20+CD19+CD27+. Within the CD27- non-memory B cells we selected the IgD+IgM+ population to study the non-class-switching B cells and then we identified Transitional B cells (regulatory B cells or Bregs) as CD24hiCD38hi. Within the C27- population, transitional B cells were more prevalent in tolerant patients (12,1%), in comparison with stable patients (4,5%), chronic rejectors (2,1%) and healthy controls (7,8%). B cells from all groups of patients increased activation markers and TLR-9 after CD40L and CpG activation, respectively. Tolerant patients displayed a higher percentage of IL10+Bcells in comparison with the rest of the groups in response to CD40L, however the response to CpG was similar in all patient groups. IFN-g-producing Bcells were not identified after any activation and levels of TNF-a were higher after activation with CD40L but not CpG in all of the tested patients. Discussion: CD40L interaction with B cells occurs after encounter with activated T cells. Only in this context B cells from tolerant recipients were producing higher amounts of IL-10. This is a first step towards a firm link between an expansion of transitional B cells and transplantation tolerance. Further studies are under way, to establish alloantigen specificity in this interaction.