Fig 1.
Calcineurin inhibitors reduce the expression of CD24 and CD38 on CD19+ B lymphocytes.
The expression of CD24, CD38 and IL-10 after gating on CD19+ B-cells is shown in a representative healthy subject, indicating that only a minority of IL-10 producing B-cells highly express CD38 (A). The dot blots in B depict CD24hiCD38hi B-cells (red-framed boxes) in a representative healthy subject, a renal transplant recipient receiving a CsA or tacrolimus based immunosuppression. The respective isotype control staining is depicted in between. The scatter plot graphs in C and D summarize the results and show that treatment with tacrolimus (n = 35) or CsA (n = 11) not only reduce the percentage of peripherally circulating B-lymphocytes (C) but also affect the CD24hiCD38hi B-cell subset (D). In contrast to healthy subjects (n = 16), the CD24hi38hi expressing B-cell subset of renal transplant patients receiving a calcineurin inhibitor were significantly reduced or even blunted (B, D). No correlation was found between the amount of CD24hiCD38hi B-cells and time of sample assessment after transplantation (E, Spearman test, r = -0,01, p = 0,9450).
Table 1.
Patient data.
Fig 2.
The calcineurin inhibitors tacrolimus and CsA inhibit IL-10 expression of B-cells in vitro and in vivo.
Freshly isolated PBMCs from healthy subjects (n = 9) and renal transplant recipients (n = 9) were mitogen/toll-like-receptor 9 stimulated for 72 hours and subsequently stained for surface CD19 and intracellular IL-10 or with the respective isotype control antibodies (A). The representative dot plots on the upper left in A show the intracellular IL-10 expression of stimulated CD19+ B-cells from a healthy subject and representative renal transplant recipients receiving either tacrolimus or CsA (after gating on CD19+ B-cells). The scatter plot in B summarizes the results of multiple unrelated experiments. PBMCs of healthy subjects were stimulated like described before in presence or absence of different concentrations of tacrolimus (n = 4) (C) or CsA (n = 4) (D), as indicated. The bar graphs in E depict the decline of IL-10 production (in %) of PBMCs vs. positively isolated CD19+ B-cells after stimulation co-cultured with CsA (n = 9 healthy subjects). 7-AAD staining was performed to ensure viability of cell culture.
Fig 3.
CsA reduced both the percentage of CD19+CD24hiCD38hi B-cells and IL-10+ B-cells in healthy subjects.
Healthy subjects (n = 4) were treated with CsA for 14 days, two days receiving a high dosage followed by an additional 12 days with a low dosage treatment. The bargraphs in panels A-C show the decline of peripheral circulating CD19+CD24hiCD38hi B-cells and their intracellular production of IL-10+ compared to day 0 (in %) while the percentage of CD4+CD25+CD127- Tregs was unaltered during CsA treatment (D). The intracellular IFN-γ production of CD3+ T-cells was assessed and served as a positive control reaction (E). Corresponding representative dotplots or histograms are displayed on the right (A-E).
Fig 4.
The amount of peripheral circulating CD19+CD24hiCD38hi cells correlated with the clinical outcome after kidney transplantation A low amount of peripheral circulating CD19+CD24hiCD38hi cells was associated with a lower eGFR (A). The orange-framed box in B indicates a subgroup of 29 patients that exhibited <1% of CD24hi38hi expressing CD19+ peripherally circulating B-cells. 31% (n = 9) of these patients experienced a biopsy proven rejection event 24 months before or after the analyses (5 patients before and 4 patients within 24 months after sample assessment).