Increased frequency of PD-1hiCXCR5- T cells and B cells in patients with newly diagnosed IgA nephropathy

Abstract

Recent research has identified a population of PD-1^hiCXCR5^- 'peripheral helper' T (Tph) cells that simulate plasma cell differentiation by interactions between IL-21 and SLAMF5. However, the alteration of circulating Tph and CD138⁺ B in IgA nephropathy (IgAN) remains poorly understood. Flow cytometry analysis was used to measure the frequency of circulating PD-1^hiCXCR5^- T cells and CD138⁺ B cells in 37 patients with IgAN and 23 healthy controls (HCs). Estimated glomerular filtration rate (eGFR), 24 h urinary protein and serum cytokine concentrations were measured. The percentage of different subsets of circulating PD-1^hiCXCR5^- T cells and CD138⁺ B cells were significantly higher in patients with IgAN compared to HCs. Pretreatment, the percentage of different subsets of circulating PD-1^hiCXCR5^- T cells and CD138⁺ B cells were negatively correlated with eGFR, the percentage of circulating CD138⁺ B cells was positively correlated with 24-h urinary protein concentration, and the percentage of circulating PD-1^hiCXCR5^-, CD28⁺ and ICOS⁺ T cells. Posttreatment, the percentage of different subsets of circulating PD-1^hiCXCR5^- T cells and CD138⁺ B cells and serum IL-21 concentration were significantly reduced. Different subsets of circulating PD-1^hiCXCR5^- T cells contribute to the progression and pathogenesis of IgAN by regulating the differentiation of CD138⁺ B cells through a combination of surface molecules.

Figure 1

Flow cytometry analysis of the frequency of various subsets of PD1^hiCXCR5⁻ T cells. PBMCs from patients with IgAN and HCs were stained with anti-CD4, anti-CD3, anti-CXCR5, anti-PD-1, anti-CD28, anti-CD154, anti-ICOS, and anti-IL-21. Cells were gated on living lymphocytes and CD3⁺CD4⁺ T cells. The frequency of PD-1^hiCXCR5⁻, PD-1^hiCXCR5⁻ CD28⁺, PD-1^hiCXCR5⁻CD154⁺, PD-1^hiCXCR5⁻ICOS⁺, and PD-1^hiCXCR5⁻IL21⁺ T cells was analyzed by flow cytometry. (A) Flow cytometry analysis. (B–G) Quantitative analyses: data are representative dot plots or are expressed as the mean % of different subsets of Tph and Tfh cells in the total CD3⁺CD4⁺ T cells from individual subjects from three separate experiments. Horizontal lines represent the medians of all subjects.

Figure 2

Flow cytometry analysis of plasma cells. PBMCs from patients with IgAN and HCs were stained with anti-CD19, anti-CD138 antibody. Cells were gated on living lymphocytes and CD19⁺ B cells. (A) Flow cytometry analysis. (B) Quantitative analyses showing CD138⁺CD19⁺ B cells in individual subjects. Horizontal lines represent the medians of all subjects.

Figure 3

Correlations between the frequency of various subsets of circulating PD1^hiCXCR5⁻ T cells, circulating CD138⁺CD19⁺ plasma cells, and clinical parameters in patients with IgAN. (A–F) eGFR was negatively correlated with the percentage of the different subsets of circulating PD1^hiCXCR5⁻ T cells and CD138⁺ B cells; (G–J) The percentage of circulating CD138⁺ B cells was positively correlated with 24-h urinary protein concentration and the percentage of circulating PD-1^hiCXCR5⁻, PD-1^hiCXCR5⁻CD28⁺, PD-1^hiCXCR5⁻ICOS⁺ T cells; (K,L) The percentage of PD-1^hiCXCR5⁻ and PD-1^hiCXCR5⁻IL-21⁺ T cells was positively correlated with serum IL-21 concentration; (M) The percentage of PD-1^hiCXCR5⁻ T cells was positively correlated with the percentage of PD-1^hiCXCR5⁺ T cells.

Figure 4

Serum cytokine concentrations. (A–E) Serum Il-4, IL-10, IL-17A, IFN-γ and IL-21 concentrations in patients with IgAN and HCs were detected by CBA and ELISA. Data are expressed as the mean values of individual samples from three separate experiments. Horizontal lines represent the median values.

Figure 5

Altered frequency of TPH cells, B cells and levels of serum cytokines in IgAN patients after treatment. The percentages of different subsets of TPH cells and the levels of serum cytokines were compared in IgAN patients before and after the treatment. Data are expressed as the mean % or concentrations of individual subjects from two separate experiments. The numbers of circulating PD-1⁺, CD28⁺, CD154⁺, ICOS⁺, IL-21⁺ TFH cells, CD138⁺CD19⁺ B cells and the level of serum IL-21 of individual patients in the pre- and post-treatment stages, (A–G) respectively. The levels of serum IL-4 and IL-10 of individual patients in the pre- and post-treatment stages, (H–I) respectively.