Date Published: April 5, 2016
Publisher: Public Library of Science
Author(s): Bastian Tebbe, Benjamin Wilde, Zeng Ye, Junyu Wang, Xinning Wang, Fu Jian, Sebastian Dolff, Manfred Schedlowski, Peter F. Hoyer, Andreas Kribben, Oliver Witzke, André Hoerning, Stanislaw Stepkowski.
CD19+CD24hiCD38hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.
PBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.
The amount of CD19+ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.
Calcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.
B-cells shape the humoral immunity and are classically considered to amplify the immune response because of their capability to produce antibodies (including autoantibodies) as well as acting as antigen-presenting cells to modulate T-cell-mediated immune responses [1, 2]. After solid organ transplantation, the production of donor-specific allo-antibodies (DSA) is involved in both acute and chronic allograft rejection [3, 4]. By contrast, immature subsets of B-cells termed regulatory B-cells have recently been shown in mice and humans to mediate protective immune responses by producing regulatory cytokines such as IL-10, TGF-b, IL-35 and directly interacting with pathogenic T-cells via cell-to-cell contact [1, 2, 5–9]. The Breg cell population appears to be heterogenous as different murine B-cell subsets such as CD1dhiCD5+  and Tim-1+IL-10+ B-cells [11, 12] have been described to exert immunoregulatory function. In humans, the identification of B-cells with regulatory properties has first been demonstrated in several studies in allergic  and autoimmune diseases such as systemic lupus erythematosus  or gianT-cell arteritis . An exact definition of human Breg cells by lineage-specific surface markers is lacking [9, 16]. Studies in patients with relapsing-remitting multiple sclerosis or type 1 diabetes demonstrated that ex vivo-activated B-cells produced less IL-10 than B-cells from healthy subjects, suggesting that insufficient IL-10 secretion by B-cells might facilitate autoimmune pathogenesis in humans [17, 18]. A widely accepted phenotypic characterization of Bregs in humans was suggested by Blair et al. . In their studies, the immature transitional CD19+CD24hiCD38hi B-cell subset was shown to enrich for IL-10+ Bregs and these cells suppressed CD3-mediated activation and differentation of Th1-cells via both secretion of IL-10 and cell-cell interaction . In addition, Tedder and colleagues  identified a CD19+CD24hiCD27+ memory B-cell subset harboring a similar number of IL-10 producing cells that have been identfied to inhibit TNFα production of CD4+ Th-cells and monocytes.
The presence and function of B regulatory cells are of increasing interest in solid organ transplantation. In particular, the immature transitional CD19+ B-cell phenotype characterized by the surface expression of CD24hiCD38hi was previously suggested to play an important role not only in maintaining long-term allograft function but also in promoting allograft tolerance [21–23].