Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

Abstract

Background: 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.

Methods: 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.

Results: 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%.

Conclusion: 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.

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 CD24^hiCD38^hi 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 CD24^hiCD38^hi B-cell subset (D). In contrast to healthy subjects (n = 16), the CD24^hi38^hi 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 CD24^hiCD38^hi B-cells and time of sample assessment after transplantation (E, Spearman test, r = -0,01, p = 0,9450).

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⁺CD24^hiCD38^hi 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⁺CD24^hiCD38^hi 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⁺CD24^hiCD38^hi cells correlated with the clinical outcome after kidney transplantation A low amount of peripheral circulating CD19⁺CD24^hiCD38^hi cells was associated with a lower eGFR (A). The orange-framed box in B indicates a subgroup of 29 patients that exhibited <1% of CD24^hi38^hi 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).