Immunophenotypic Alterations in Adult Patients with Steroid-Dependent and Frequently Relapsing Nephrotic Syndrome

Abstract

Immune dysregulation plays a key role in the pathogenesis of steroid-dependent/frequently relapsing nephrotic syndrome (SDNS/FRNS). However, in contrast with evidence from the pediatric series, no major B- or T-cell alterations have been described for adults. In these patients, treatment with rituximab allows safe discontinuation of steroids, but long-term efficacy is variable, and some patients experience NS relapses after B cell reconstitution. In this study, we aimed to determine disease-associated changes in the B and T cell phenotype of adult patients with SDND/FRNS after steroid-induced remission. We also investigated whether any of these changes in immune cell subsets could discriminate between patients who developed NS relapses after steroid-sparing treatment with rituximab from those who did not. Lymphocyte subsets in SDNS/FRNS patients (n = 18) were compared to those from patients with steroid-resistant NS (SRNS, n = 7) and healthy volunteers (HV, n = 15). Before rituximab, SDND/FRNS patients showed increased frequencies of total and memory B cells, mainly with a CD38-negative phenotype. Within the T-cell compartment, significantly lower levels of FOXP3⁺ regulatory T cells (Tregs) were found, mostly due to a reduction in CD45RO⁺ memory Tregs compared to both SRNS and HV. The levels of CD45RO⁺ Tregs were significantly lower at baseline in patients who relapsed after rituximab (n = 9) compared to patients who did not (n = 9). In conclusion, patients with SDND/FRNS displayed expansion of memory B cells and reduced memory Tregs. Treg levels at baseline may help identify patients who will achieve sustained remission following rituximab infusion from those who will experience NS relapses.

Keywords: B cells; nephrotic syndrome; regulatory T cells; rituximab; steroid-dependent nephrotic syndrome; steroid-resistant nephrotic syndrome.

Figure 1

Subject groups and scheme of the experimental study design.

Figure 2

FACS analysis of circulating B cell subsets in SSNS, SRNS patients and HV. (A) Percentages of CD19⁺CD20⁺ B cells among total lymphocytes in the three groups of subjects; (B) Representative contour plots of memory, mature and transitional B cell gating strategy in SSNS, SRNS patients and HV; (C) Percentages of memory, transitional and mature B cells among total lymphocytes in patients with SSNS or SRNS and in HV; (D) Representative contour plots of IgM memory and Switched memory on gated CD27⁺ memory B cells and (E) the relative percentages among total lymphocytes in the three groups of subjects. Plots display the median, 25th and 75th percentiles of distribution (boxes) and whiskers extend to the minimum and maximum values of the series. * p < 0.05 between the indicated groups; ns: not significant.

Figure 3

FACS analysis of memory B cell subsets and correlation with prednisone dose. Percentages of (A) memory, transitional and mature B cells and (B) total, IgM and switched memory B cells on CD19⁺CD20⁺ B cells in SSNS and SRNS patients and HV. Plots display the median, 25th and 75th percentiles of distribution (boxes) and whiskers extend to the minimum and maximum values of the series; * p < 0.05 between the indicated groups; ns: not significant. (C) Correlation between percentages of memory, transitional and mature B cells with prednisone dose in NS patients. Correlations were analyzed using Pearson’s correlation coefficient.

Figure 4

Regulatory T cells are reduced in SDNS/FRNS patients. (A) Percentages of CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells on circulating CD45⁺ cells in SSNS, SRNS patients and HV; (B) gating strategy for the identification of memory (CD45RO⁺) and naïve (CD45RA⁺) Tregs on FOXP3⁺CD127- Tregs gated on CD4⁺CD25hi cells; (C) percentages of total, memory and naïve Tregs on CD4⁺ T cells in the three groups of subjects. Plots display the median, 25th and 75th percentiles of distribution (boxes) and whiskers extend to the minimum and maximum values of the series; * p < 0.05 between the indicated groups, ns: not significant.

Figure 5

Treg levels in relapsers and non-relapsers SDNS patients. (A) Percentages of total, memory and naïve Tregs on CD4⁺ T cells in SDNS/FRNS patients who relapsed or did not after rituxi-mab therapy and (B) their correlation with prednisone dose. Plots display the median, 25th and 75th percentiles of distribution (boxes) and whiskers extend to the minimum and maximum values of the series, correlations were analyzed using Pearson’s correlation coefficient. * p < 0.05 between the indicated groups, ns: not significant.