Elevated Circulating IL-10 Producing Breg, but Not Regulatory B Cell Levels, Restrain Antibody-Mediated Rejection After Kidney Transplantation

Abstract

Background: Antibody-mediated rejection (AMR) occupies a major position for chronic rejection after kidney transplantation. Regulatory B cell (Breg) has been reported to have an inhibitory immune function, which contributes to the resistance for AMR.

Methods: A nested case-control study for nine healthy donors, 25 stable (ST) patients, and 18 AMR patients was performed to determine the type of Breg in maintaining immune tolerance and preventing AMR.

Results: Compared to the ST group, circulating interleukin (IL)-10⁺ Bregs, but not Bregs, significantly decreased. The receiver operating characteristic (ROC) curve analysis revealed that rather than the circulating Bregs, decreased circulating IL-10⁺ Breg levels were positively associated with AMR. However, kidney B cell and IL-10 infiltration was significantly increased in the AMR group with high expression of C-X-C motif chemokine 13 (CXCL13). In addition, circulating IL-10⁺ Bregs, rather than Bregs, remained higher than those at pre-operation, during the 90-day post-operation in immune homeostasis.

Conclusion: The circulating IL-10⁺ Breg levels are more appropriate measures for assessing the resistance of AMR after kidney transplantation.

Keywords: Breg phenotyping; antibody-mediated rejection; dynamic; homeostasis; kidney transplantation.

Figure 1

The percentages of four subpopulations of circulating Bregs among the healthy, stable (ST) and antibody-mediated rejection (AMR) groups. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 9) at pre-operation, the ST group (n = 25) at day 90 post-operation, and the AMR group (n = 18) at day 1 before anti-rejection therapy. (A) Representative dot plots depict the gating strategy to determine B cell subsets in PBMC. CD19⁺ B cells were identified based on high expression of CD19. Memory Bregs (mBregs) were identified from CD19⁺ B cells based on the high expression of CD24 and positivity of CD27. IL-10-producing memory Bregs (IL-10⁺ mBregs) were further gated based on the high expression of IL-10. Transitional Bregs (tBregs) were identified based on high expression of CD38 and CD24. IL-10-producing transitional Bregs (IL-10⁺ tBregs) were further gated based on the high expression of IL-10. (B–D) The statistical summary for the percentages of CD19⁺ B cells in PBMCs (B), CD19⁺CD24⁺CD27⁺ mBregs or CD19⁺CD24⁺CD38⁺ tBregs in CD19⁺ B cells (C), CD19⁺CD24⁺CD27⁺IL-10⁺ or CD19⁺CD24⁺CD38⁺IL-10⁺ Bregs (IL-10⁺ mBregs or IL-10⁺ tBregs) in CD19⁺ B cells (D). Differences were evaluated by Kruskal–Wallis one-way analysis of variance (k samples) and post-hoc tests with all pairwise. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

ROC curves for the ratios of four subpopulations of circulating Bregs. Peripheral blood mononuclear cells (PBMCs) were isolated from the ST group (n = 25) at day 90 post-operation, the healthy group (n = 9) before surgery, and the AMR group (n = 18) at day 1 before anti-rejection therapy. (A, B) The percentages of four subpopulations of circulating Bregs in B cells were analyzed by the ROC assay.

Figure 3

Analysis of IL-10⁺ Bregs in kidney tissue and cells from healthy, stable (ST) and antibody-mediated rejection (AMR) groups. (A) Immunohistochemistry was performed on kidney tissues from six ST patients and six AMR patients to detect CD19, IL-10, and CXCL13 (brown). DAPI staining (blue) was performed to visualize the nuclei. Scale bars, 100 μm. (B) The statistical summary for the scores according to the counts of CD19, IL-10, and CXCL13. (C) Kidney cells from three healthy donors, three ST patients, and three AMR patients were measured by flow cytometry. Representative dot plots depict the gating strategy to determine B cell subsets in kidney. CD45⁺ leukocytes were identified based on high expression of CD45. CD19⁺CD24⁺ cells were gated based on the high expression of CD24 and CD19. IL-10-producing memory Bregs (IL-10⁺ mBregs) were further gated based on the high expression of IL-10 and CD27 while IL-10-producing transitional Bregs (IL-10⁺ tBregs) were identified based on high expression of IL-10 and CD28. (D) The statistical summary for the percentages of CD19⁺CD24⁺CD27⁺IL-10⁺ or CD19⁺CD24⁺CD38⁺IL-10⁺ Bregs (IL-10⁺ mBregs or IL-10⁺ tBregs) in CD45⁺ leukocytes. The score differences were evaluated by Mann–Whitney U-test. The percentage differences were evaluated by one-way analysis of variance, and multiple comparisons were made with the least significant difference test. *P < 0.05; **P < 0.01.

Figure 4

Dynamic variation of four subpopulations of circulating Bregs in healthy donors or stable (ST) patients. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) isolated from nine healthy donors at 0, 1, 7, and 14 days post-operation and 25 ST patients at 0, 1, 7, 14, 30, and 90 days post-operation. (A, B) Dynamic variation of four subpopulations of circulating Breg ratios in Bcells in healthy donors (A) and ST patients (B). (C) The differences of four subpopulations of circulating Breg ratios in Bcells between the healthy group and the ST group at the same time point. Comparisons between the healthy group and the ST group at the same time point were analyzed using independent-samples T test. Comparisons at different time points in healthy donors or ST patients were analyzed using one-way repeated measures analysis of variance, and multiple comparisons were made with the least significant difference test. *P < 0.05; **P < 0.01; ***P < 0.001.