Background and Aims: Renal transplant recipients with operational tolerance have changes in their lymphocyte subsets especially B cell subsets.  Our understanding of the role of B cells in organ transplantation remains limited due to majority of studies being directed towards the role of antibodies in pathogenesis of rejection responses. B cells play an important role in antigen presentation, cytokine production and co-stimulation and thereby can affect transplantation outcome. Through the expression of a set of markers, B cell can be segregated into various subsets, each with its unique function in immune responses.  Here, we studied changes in B cell subsets in peripheral blood of renal transplant patients (RT) on immunosuppression with kidney graft surviving over 10 years and compared to patients on dialysis and healthy volunteers (HV). 
Methods: Peripheral blood from HV and renal patients was collected. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll density gradient. Freshly isolated PBMC from healthy volunteers (n=13), patients on dialysis (n=12) and those with a kidney graft surviving for 1-2 years (n=12), 5-10 years (n=10) and >10 years (n=23) were stained with panel of eight monoclonal antibodies (CD19/CD21/CD24/CD27/CD38/CD45/IgD/IgM) to identify B cell subpopulations.  Data was acquired on BD FACSCanto II using BD FACSDiva software (v8.0) and analysed using FlowJo.
Results: Staining PBMC with a panel of B cell-related antibodies showed significant reduction in CD19⁺ cells in RT with >10 years surviving graft compared to RT with 1-2 year surviving graft or to patients on dialysis (p<0.05).  All other B cell subsets were examined as proportions of total CD19⁺ B cells. There were less CD21^hi and more CD21^lo cells in RT with >10 years surviving graft compared to HV and dialysis patients, with no difference compared to other groups.  Dialysis patients had lower proportion of marginal zone B cells (CD19⁺CD27⁺IgD⁺) (p<0.05). Naïve B cells (CD19⁺CD27^-IgM⁺IgD⁺) were lower in RT (>10 years (p<0.01) compared to those on dialysis. Conversely, total memory B cells (CD19⁺CD27⁺) and switched memory cells were higher in RT (>10 years) with about half of patients having >40% of B cells as memory B cells compared to dialysis patients (p<0.01) where <20% B cells were of memory phenotype and to HV with memory B cell proportions ranging from 20-60% of B cells (36 ± 13%), although the difference was not significant.  No difference was noted in transitional B cells (CD19⁺IgM⁺IgD⁺CD27^-CD38⁺CD24⁺) among groups however, majority of patients had lower proportions than HV or to those on dialysis.  Proportion of plasmablasts (CD19⁺IgM^-IgD^-CD27⁺CD38⁺) was not different either.
Conclusions: We observed changes in B cell subsets in patients with kidney graft over time. These changes may be a consequence of long-term non-specific immunosuppression, especially by mycophenolate and azathioprine.  Patients with operational tolerance have higher numbers of B cells, but the suppressor B cell phenotype is not identified in this study.