Transitional B cells represent a cortical link between the initial B cell compartment in bone marrow and the mature peripheral B cell repertoire. The prior characterisation of human transitional B-cells has suggested that this population could be subdivised into two major subsets: the immature T1-Bcells CD19+ CD10high CD24high CD38high IgMhigh IgDlow CD21low and tne intermediate cells mentionned as T2-B cells CD19+ CD10 + CD24high CD38high IgMhigh IgDhigh CD21high. Recently B-cell regulation have been shed in light and a lot of evidences suggested that regulatory B cells (Bregs) are contained within the CD24high CD38high transitional B-cell subsets. In this study, we wanted to fully characterised the composition of the CD24high CD38high population using phenotypic and functionnal approaches. First, we used a non manual and non subjective gating technique with 10 color flow cytometry to identify the different subsets contained in the CD24high CD38high population through FLOCK (Flow cytometry without K) algorythm program. We showed that non less than 5 different populations are present in the parent gate. Three population are included in transitional B-cell subset confirmed by in vitro functional analysis. The two other populations belong to memory B-cells. In the second part of the work, the different populations were sorted and their supressive capacities in a coculture model of B cells and activated T-cell were analysed. We demonstrated that B-cell subets have different regulatory potency on the inhibition of T-cell proliferation and Th1 cytokine production. These data bring new clarifications on the precise nature of regulatory B cells in human and their supressive functions.
Regulatory B-cell supress T-cell proliferation through a TGFβ/IDO axis and are deficient in chronic humoral rejection
In kidney transplantation, B cell is increasingly identified as an important determinant for graft outcome. Chronic antibody mediated rejection (cABMR) represent today the major complication in clinic. Patients with cABMR display a unique B cell phenotype characterised by a disturbance in B cell distribution. Here, the aim of our study was to research if cABMR B cells present also impaired regulatories functions.
Experiments are realised in 12 stable patients (ST) (delay from graft >12 months, no rejection, proteinuria <0.5g/24h, PRA <10%, no DSA and a one year biopsy without allograft glomerulopathy or rejection), 14 patients with cABMR (positive DSA, allograft glomerulopathy and/or C4d staining) and 17 controls (blood donors). B-cell functions were analysed using coculture with B and T cells. Proliferation and Th1 cytokine secretion of isolated CFSE labelled T cells in presence of autologous B cells was compared between the 3 groups. T cells were stimulated with anti CD3 and anti CD28, and co-cultured with CpG-activated B cells. Suppressive cytokines like TGFβ and IL-10 and the regulatory enzyme indoleamine 2,3-dioxygenase (IDO) were evaluated during the course of the culture.
First, we have demonstrated that compared with situation of stable graft function and healthy volunteers, B cells from cABMR patients display an impaired regulatory function on T cell proliferation and Th1 production. Furthermore, B cells induce more T regulatory cells (Tregs) in coculture after 4 days in HV and ST groups compared to cABMR group (p<10-3). We also evidenced that TGFβ and IDO expressed by B cells are involved in inhibition of T cell proliferation and Treg cells generation in HV and ST patients. We additionally shown that B cells from cABMR patients present a failure of TGFβ and IL-10 secretion in co-culture associated with a significantly decrease of IDO production leading to a defect in the capacity of activated-B cells to induce Treg cells and control T cell responses.
Finally, B cells from cABMR patients display functional abnormalities as evidenced by the loss of B cell suppressive activity. This work represents interesting perspectives with the discovered of new cellular targets who can be might guide therapy in transplantation in the future.
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