Relationship of transitional regulatory B and regulatory T cells and immunosuppressive drug doses in stable renal transplant recipients

Abstract

Objectives: Regulatory B cells (Bregs) and T cells (Tregs) are thought to be involved in the regulation of graft acceptance in renal transplant recipients. However, mechanisms that affect Breg differentiation and interaction with Tregs are rather unclear.

Methods: Using eight-color-fluorescence flow cytometry, Tregs and CD19+ CD24hiCD38hi Bregs were analyzed in whole blood samples of 80 stable kidney transplant recipients, 20 end-stage renal disease (ESRD) patients and 32 healthy controls (HC). In addition, differentiation of Bregs and Tregs was studied in different micromilieus using cocultures with strongly enriched B-lymphocytes and autologous peripheral blood mononuclear cells stimulated with CpG and phytohemagglutinin.

Results: Bregs were higher in HC than in ESRD patients and lowest in transplant recipients. Bregs were higher early as compared to late posttransplant. Posttransplant, high Bregs were associated with higher glomerular filtration rate (GFR) and lower C-reactive protein (CRP). Higher doses and blood levels of ciclosporine, tacrolimus, and mycophenolate mofetil as well as higher doses of steroids were not associated with low Bregs. In contrast, most Treg subsets were lower when blood levels of ciclosporine, tacrolimus, and mycophenolate mofetil were higher. Tregs were not associated with Bregs, GFR, CRP plasma levels, and occurrence of rejection or infection. In vitro, differentiation of Bregs was strongly dependent on T cell support and was blocked by excessive or lacking T-cell help. Tregs were not associated with Breg numbers in vitro.

Conclusion: Bregs appear to be insensitive to high doses of posttransplant immunosuppressive drugs. The protracted Breg decrease posttransplant might be caused by impaired T cell support attributable to immunosuppressive drugs.

Keywords: Foxp3; IL10; Tregs; blood; cell culture; immunoregulation; immunosuppressive drug doses; renal transplant recipients; transitional Bregs.

Figure 1

Gating strategy of total and IL10+CD19+CD24hiCD38hi Breg subsets, Tregs, and T cell proliferation. (A) Gating strategy for Bregs. Graph 1 shows all analyzed events; doublets are excluded (Graph 2). FSC versus SSC are dot‐plotted and lymphocytes are gated according to size (Graph 3). CD45 versus SSC dot plot permits focusing on lymphocytes and elimination of debris (Graph 4); B cells were identified using a CD19 versus SSC dot plot (Graph 5) and the CD24hiCD38hi Breg cluster was gated out of CD19+ lymphocytes (Graph 6). Graph 7 shows IL10+ Bregs gated from total Bregs. (B) Gating strategy for Tregs and their subsets. Graph 1 includes all the events analyzed in the tube. Graph 2 exhibits the exclusion of doublets. Graph 3 shows FSC versus SSC dot plot and gating of lymphocytes according to size, CD45 versus SSC dot plot permits elimination of debris and focuses on lymphocytes (Graph 4), CD25 versus CD4 dot plot allows the identification of CD4+CD25+ T lymphocytes (Graph 5), the Foxp3+CD127− cluster was gated out of CD4+CD25+ lymphocytes and identifies Tregs (Graph 6). In addition, Graphs 7–12 show Treg subsets gated out of total Tregs. (C) Gating strategy for T cell proliferation measured as proportion of blasts with low CFSE fluorescence (% CFSE low blasts). Graph 1 shows a dot plot of unstained cells in a cell culture incubated with cell culture medium for 3 days. Cells in the blast area R1 are depicted in red color. Graph 2 shows a cell culture incubated for 3 days with cell culture medium and CFSE. Lymphocytes are depicted in green color. Using a CFSE histogram, CFSE background staining of all cells in graph 2 was adjusted to less than 5% (M2 in graph 3). This CFSE gate was used for all further flow cytometric analyses when CFSE low blasts were measured in the blast area R2 and showed 1% CFSE low blasts for the 3‐day cell culture with cell culture medium and CFSE in the graphs 4 and 5. When cells were incubated with PHA and CFSE for 3 days, proportion of CFSE low blasts (proliferating T cells) increased to 50% (R2 in graph 6 and M2 in graph 7). Breg, regulatory B cell; CFSE, carboxyfluorescein succinimidyl ester; IL, interleukin; Treg, regulatory T cell

Figure 1

Gating strategy of total and IL10+CD19+CD24hiCD38hi Breg subsets, Tregs, and T cell proliferation. (A) Gating strategy for Bregs. Graph 1 shows all analyzed events; doublets are excluded (Graph 2). FSC versus SSC are dot‐plotted and lymphocytes are gated according to size (Graph 3). CD45 versus SSC dot plot permits focusing on lymphocytes and elimination of debris (Graph 4); B cells were identified using a CD19 versus SSC dot plot (Graph 5) and the CD24hiCD38hi Breg cluster was gated out of CD19+ lymphocytes (Graph 6). Graph 7 shows IL10+ Bregs gated from total Bregs. (B) Gating strategy for Tregs and their subsets. Graph 1 includes all the events analyzed in the tube. Graph 2 exhibits the exclusion of doublets. Graph 3 shows FSC versus SSC dot plot and gating of lymphocytes according to size, CD45 versus SSC dot plot permits elimination of debris and focuses on lymphocytes (Graph 4), CD25 versus CD4 dot plot allows the identification of CD4+CD25+ T lymphocytes (Graph 5), the Foxp3+CD127− cluster was gated out of CD4+CD25+ lymphocytes and identifies Tregs (Graph 6). In addition, Graphs 7–12 show Treg subsets gated out of total Tregs. (C) Gating strategy for T cell proliferation measured as proportion of blasts with low CFSE fluorescence (% CFSE low blasts). Graph 1 shows a dot plot of unstained cells in a cell culture incubated with cell culture medium for 3 days. Cells in the blast area R1 are depicted in red color. Graph 2 shows a cell culture incubated for 3 days with cell culture medium and CFSE. Lymphocytes are depicted in green color. Using a CFSE histogram, CFSE background staining of all cells in graph 2 was adjusted to less than 5% (M2 in graph 3). This CFSE gate was used for all further flow cytometric analyses when CFSE low blasts were measured in the blast area R2 and showed 1% CFSE low blasts for the 3‐day cell culture with cell culture medium and CFSE in the graphs 4 and 5. When cells were incubated with PHA and CFSE for 3 days, proportion of CFSE low blasts (proliferating T cells) increased to 50% (R2 in graph 6 and M2 in graph 7). Breg, regulatory B cell; CFSE, carboxyfluorescein succinimidyl ester; IL, interleukin; Treg, regulatory T cell

Figure 2

Total and IL10+CD19+CD24hiCD38hi regulatory B cell (Breg) subsets in stable transplant recipients, patients with end‐stage renal disease (ESRD) and healthy controls. Transplant recipients (Tx), patients with ESRD and healthy controls (HC) show different (A) relative (p < .001) and (B) absolute (p < .001) numbers of total CD19+CD24hiCD38hi, and (C) relative (p < .001) and (D) absolute (p < .001) numbers of IL10+CD19+CD24hiCD38hi Bregs (Kruskal–Wallis test). In addition, pairwise comparisons using Mann–Whitney U test are depicted. Horizontal bars represent medians. Relative cell numbers are related to CD45+ total lymphocytes

Figure 3

Total and IL10+CD19+CD24hiCD38hi regulatory B cells (Bregs), days posttransplant, glomerular filtration rate, and C‐reactive protein. Relative and absolute numbers of total and IL10+CD19+CD24hiCD38hi Breg subsets are associated with (B–E) days posttransplant and (J–M) C‐reactive protein (CRP) (Spearman's rank correlation test). There is no association of glomerular filtration rate (GFR) with (A) days posttransplant and only a weak trend of higher Bregs in patients with higher GFR (F–I)

Figure 4

Total and IL10+CD19+CD24hiCD38hi regulatory B cells (Bregs), doses and blood levels of immunosuppressive drugs. (A–D) Higher relative and absolute counts of total Bregs are associated with higher daily doses of MMF and steroids (Spearman's rank correlation test) (E, F) and higher MMF doses with higher relative and absolute numbers of IL10+ Bregs, (G, H) whereas higher steroids doses show only a weak trend with higher IL10+ Bregs, (I, J)

Figure 5

Proportions of CD19+CD24hiCD38hi regulatory B (Breg) cells in unstimulated and PHA‐stimulated cell cultures and cocultures. (A) Enriched CD19+ B lymphocytes separated from heparinized whole blood samples of 10 healthy controls consisted of 3% (median) CD19+CD24hiCD38hi Bregs. Proportion of Bregs decreased during 72‐h incubation in cell culture medium with or without CpG. When precultivated CD19+ B lymphocytes were added to autologous PBMCs and cocultured for another 72 h, Bregs recovered and Breg numbers were higher in cocultures with CpG precultivated B cells than in cocultures precultivated without CpG. In contrast, Bregs decreased strongly in all PHA‐stimulated cell cultures and cell cocultures

Figure A1

Regulatory T cells (Tregs) and days posttransplant. Absolute and relative numbers of CD4+CD25+Foxp3+CD127− Treg cells in the blood of 80 renal transplant recipients are higher late compared to early posttransplant (Spearman's rank correlation test)

Figure A2

Relationship of transitional regulatory B (Breg) and regulatory T (Treg) cells and immunosuppressive drug doses in stable renal transplant recipients. Bregs might be insensitive to high doses of immunosuppressive drugs posttransplant. The delayed Breg decrease posttransplant might be caused by impaired T cell support mediated by immunosuppressive drugs. Circulating Tregs show an inverse relationship to transitional Bregs in the present study suggesting that they do not induce each other