Follicular helper and follicular regulatory T cell subset imbalance is associated with higher activated B cells and abnormal autoantibody production in primary anti-phospholipid syndrome patients

Abstract

Primary anti-phospholipid antibody syndrome (pAPS) is a multi-organ autoimmune disease, and autoantibodies are involved in its pathogenesis. Follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr) are critical for B cell maturation and antibody production, but their roles in pAPS remain unknown. We enrolled 32 pAPS patients and 23 healthy controls (HCs) and comprehensively analyzed circulating Tfh and Tfr, as well as their subsets, using flow cytometry. Clinical data including autoantibody levels were collected and their correlations with Tfh and Tfr subsets were analyzed. In addition, correlation analyses between B cell functional subsets and Tfh and Tfr were performed. Changes and potential effects of serum cytokines on Tfr and Tfh were further explored. We found the circulating Tfr was significantly decreased while Tfh and Tfh/Tfr ratios were increased in pAPS patients. Tfh2, inducible T cell co-stimulator (ICOS)⁺ programmed cell death 1 (PD-1)⁺ Tfh and Ki-67⁺ Tfh percentages were elevated, while CD45RA^- forkhead box protein 3 (FoxP3)^hi , Helios⁺ , T cell immunoglobulin and ITIM (TIGIT)⁺ and Ki-67⁺ Tfr percentages were decreased in pAPS patients. New memory B cells and plasmablasts were increased and altered B cell subsets and serum autoantibodies were positively correlated with Tfh, Tfh2, ICOS⁺ PD-1⁺ Tfh cells and negatively associated with Tfr, CD45RA^- FoxP3^hi Tfr and Helios⁺ Tfr cells. In addition, pAPS with LA/aCL/β2GPI autoantibodies showed lower functional Tfr subsets and higher activated Tfh subsets. Serum interleukin (IL)-4, IL-21, IL-12 and transforming growth factor (TGF)-β1 were up-regulated and associated with Tfh and Tfr subset changes. Our study demonstrates that imbalance of circulating Tfr and Tfh, as well as their functional subsets, is associated with abnormal autoantibody levels in pAPS, which may contribute to the pathogenesis of pAPS.

Keywords: B cell; anti-phospholipid antibody syndrome; autoantibody; follicular helper T cell; follicular regulatory T cell.

FIGURE 1

Frequencies and numbers of peripheral blood follicular helper T cells (Tfh), follicular regulatory T cells (Tfr) and Tfh/Tfr ratios in primary anti‐phospholipid antibody syndrome (pAPS) patients and healthy controls. Peripheral blood mononuclear cells from peripheral blood of pAPS patients (n = 32) and healthy controls (HCs) (n = 23) were collected and Tfh cells and Tfr cells were measured by flow cytometry through staining of CD4, C‐X‐C motif chemokine receptor (CXCR)5, forkhead box protein 3 (FoxP3), CXCR3 and chemokine receptor type 6 (CCR6), inducible T cell co‐stimulator (ICOS), programmed cell death 1 (PD)‐1 and Ki‐67. Tfh1, Tfh2 and Tfh17 were defined as Tfh1 (CXCR3⁺CCR6⁻), Tfh2 (CXCR3⁻CCR6⁻) and Tfh17 (CXCR3⁻CCR6⁺) in CD4⁺CXCR5⁺FoxP3⁻Tfh cells; Tfh cells were also divided into different functional subsets according to ICOS, PD‐1 and Ki‐67 expression; namely, ICOS⁺PD⁺ Tfh and Ki‐67⁺ Tfh. (a) Representative dot‐plots for flow cytometry used in this study. CD4⁺CXCR5⁺FoxP3⁺ Tfr cells and CD4⁺CXCR5⁺FoxP3⁻ Tfh cells were analyzed. Numbers indicate the percentage of Tfr cells and Tfh cells in CD4⁺ lymphocytes. Frequencies and absolute numbers (per liter) of Tfh cells and Tfr cells; the Tfh/Tfr ratios in pAPS patients and HCs were compared. (b) Representative flow cytometry strategy of CXCR3 and CCR6 analysis in CD4⁺CXCR5⁺FoxP3⁻ Tfh cells. Numbers indicate the percentage of cells in each quadrant. The comparisons of the frequencies and absolute numbers (per liter) of Tfh1, Tfh2 and Tfh17 subsets between pAPS patients and HCs are shown below. (c) Representative flow cytometry strategy of ICOS and PD‐1 expression in Tfh cells. Numbers indicate the percentage of ICOS⁺PD‐1⁻ Tfh in the corresponding quadrant. The comparison of levels of ICOS⁺PD‐1⁺ Tfh between pAPS patients and HCs is shown below. (d) Representative flow cytometry strategy of Ki‐67 expression in Tfh cells. Numbers indicate the percentage of Ki‐67⁺ Tfh. Percentages and absolute numbers (per liter) of Ki‐67⁺ Tfh were compared between pAPS patients and HCs. Data are shown as mean ± standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

FIGURE 2

Flow cytometry analysis of follicular regulatory T cell (Tfr) cell subsets in primary anti‐phospholipid antibody syndrome (pAPS) patients and healthy controls. Peripheral blood mononuclear cells from pAPS patients (n = 32) and HCs (n = 23) were isolated and Tfr functional subsets were analyzed by staining CD4, C‐X‐C motif chemokine receptor (CXCR)5, forkhead box protein 3 (FoxP3), CD45RA, Helios and Ki‐67. (a) Representative flow cytometry dot‐plots of Tfr subsets classified by CD45RA and FoxP3 expression in CD4⁺CXCR5⁺FoxP3⁺ Tfr cells. Numbers indicate the percentages in each quadrant. The comparisons of the frequencies and absolute numbers (per liter) of CD45RA⁻FoxP3^hi, CD45RA⁻FoxP3^int and CD45RA⁺FoxP3^int Tfr cell subsets between pAPS patients and HCs are shown. (b) Representative flow cytometry analysis strategy of Helios expression in CD4⁺CXCR5⁺FoxP3⁺ Tfr cells. Numbers indicate the percentages of Helios⁺ Tfr. Comparison of the frequencies and absolute numbers (per liter) of Helios⁺ Tfr cell subsets between pAPS patients and HCs are shown. (c) Representative flow cytometry dot‐plots of Ki‐67 expression in Tfr cells. Numbers indicate percentages of Ki‐67⁺ Tfr cells. Frequencies and absolute numbers (per liter) of Ki‐67⁺ Tfr cell subsets were compared between pAPS patients and HCs. Data are shown as mean ± standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

FIGURE 3

Circulating B cell subset analysis in primary anti‐phospholipid antibody syndrome (pAPS) patients and healthy controls and their associations with follicular regulatory T cells (Tfr) and follicular helper T cells (Tfh) and their subsets. Peripheral blood from pAPS patients (n = 32) and HCs (n = 23) were collected and B cell subsets were analyzed by staining for CD19, CD24 and CD38. (a) Representative dot‐plots showing functional B cell subsets for primary memory B cells (I, CD19⁺CD24^hiCD38⁻), transitional B cells (II, CD19⁺CD24^hiCD38^hi), new memory B cells (III, CD19⁺CD24⁻CD38⁻) and plasmablasts (IV, CD19⁺CD24⁻CD38^hi). (b) Comparisons of both frequencies and absolute numbers (per liter) of each functional B cell subsets between pAPS patients (n = 32) and HCs (n = 23). (c) Correlation analyses between new memory B cells (top), plasmablasts (bottom) and percentages of CD4⁺C‐X‐C motif chemokine receptor (CXCR)5⁺forkhead box protein 3 (FoxP3)⁻ Tfh cells in CD4 cells, Tfh2 percentages in Tfh cells and inducible T cell co‐stimulator (ICOS)⁺ PD‐1⁺ percentages in Tfh in pAPS patients (n = 32). (d) Correlation analyses between new memory B cells (top), plasmablasts (bottom) and percentages of CD4⁺CXCR5⁺FoxP3⁺ Tfr cells in CD4 cells and CD45RA⁻FoxP3^hi/Helios⁺ percentages in Tfr cells in pAPS patients (n = 32). The r‐values were Spearman’s correlation coefficients, and p < 0.05 was linearly regressed to show relevant trends. **p < 0.01; ns, not significant.

FIGURE 4

Correlations analyses between β₂ glycoprotein 1 autoantibodies (β2‐GPI), anti‐cardiolipin antibodies (aCL), lupus anti‐coagulant (LA) and follicular regulatory T cell (Tfr) and follicular helper T cell (Tfh) subsets. (a) LA, β2‐GPI and aCL levels of 32 primary anti‐phospholipid antibody syndrome (pAPS) patients were detected. Correlation analyses were conducted between β2‐GPI, aCL, LA and percentages of CD4⁺C‐X‐C motif chemokine receptor (CXCR)5⁺forkhead box protein 3 (FoxP3)⁻ Tfh cells in CD4 cells, Tfh2 percentages in Tfh cells and inducible T cell co‐stimulator (ICOS)⁺PD‐1⁺ percentages in Tfh. (b) Correlation analyses between β2‐GPI, aCL, LA and percentages of CD4⁺CXCR5⁺FoxP3⁺ Tfr cells in CD4 cells and CD45RA⁻FoxP3^hi/Helios⁺ percentages in Tfr cells in 32 pAPS patients. The r‐values are Spearman’s correlation coefficients and p < 0.05 was linearly regressed to show relevant trends

FIGURE 5

Follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr) and their functional subset comparison between different autoantibody‐positive primary anti‐phospholipid antibody syndrome (pAPS) patient groups. We divided pAPS patients into three subgroups according to their autoantibodies [lupus anti‐coagulant (LA), anti‐cardiolipin antibodies (aCL) or β₂ glycoprotein 1 autoantibodies (β2‐GPI autoantibodies)] detected; i.e. single‐positive group (patients with single autoantibody detected positive) (n = 13), double‐positive group (patients with two kinds of autoantibodies detected positive) (n = 11) and triple‐positive group (patients with LA, aCL and β2‐GPI all positive) (n = 8). (a) The comparison of CD4⁺C‐X‐C motif chemokine receptor (CXCR)5⁺forkhead box protein 3 (FoxP3)⁻ Tfh and CD4⁺CXCR5⁺FoxP3⁺ Tfr cell percentages and Tfh/Tfr ratios among three groups. (b) Comparison of CXCR3⁻CCR6⁻ Tfh2, inducible T cell co‐stimulator (ICOS)⁺PD‐1⁺ and Ki‐67⁺ Tfh cell percentages (in CD4⁺CXCR5⁺FoxP3⁻ Tfh cells) among three groups. (c) Comparison of CD226⁺, Ki‐67⁺, CD45RA^‐FoxP3^hi, Helios⁺ and T cell immunoglobulin and ITIM (TIGIT)⁺ Tfr cell percentages (in CD4⁺CXCR5⁺FoxP3⁺ Tfr cells) among three groups. Data are shown as mean + standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

FIGURE 6

Serum cytokine levels in primary anti‐phospholipid antibody syndrome (pAPS) patients and their correlations with follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr) and their subsets. (a) Serum interleukin (IL)‐4, IL‐17A and IL‐21 levels of 23 healthy controls (HCs) and 32 pAPS patients were analyzed by enzyme‐linked immunosorbent assay (ELISA). Their levels were compared between two groups. Symbols represent results from included subjects and bars show the mean ± standard deviation (SD). ***p < 0.001; ns, not significant. (b) Correlation analyses were performed between serum IL‐4 and IL‐21 concentrations and percentages of CD4⁺C‐X‐C motif chemokine receptor (CXCR)5⁺forkhead box protein 3 (FoxP3)⁻ Tfh cells in CD4 cells, Tfh2 percentages in Tfh cells and inducible T cell co‐stimulator (ICOS)⁺PD‐1⁺ percentages in Tfh in pAPS patients (n = 32). (c) The correlation analyses were conducted between IL‐4 and IL‐21 and percentages of CD4⁺CXCR5⁺FoxP3⁺ Tfr cells in CD4 cells and CD45RA⁻FoxP3^hi/Helios⁺ percentages in Tfr cells in pAPS patients (n = 32). (d) Serum transforming growth factor (TGF)‐β1, IL‐12 and IL‐10 levels of 23 HCs and 32 pAPS patients were measured by ELISA. Their concentrations in two groups were compared. Symbols represent each subject and bars show the mean ± standard deviation (SD). ***p < 0.001; ns, not significant. (e) Correlation analyses were performed between serum TGF‐β1 and IL‐12 levels and percentages of CXCR5⁺FoxP3⁻ Tfh cells in CD4 cells, Tfh2 percentages in Tfh cells and ICOS⁺PD‐1⁺ percentages in Tfh cells in pAPS patients (n = 32). (f) The correlation analyses were conducted between TGF‐β1 and IL‐12 and percentages of CD4⁺CXCR5⁺FoxP3⁺ Tfr cells in pAPS patients (n = 32). The marked r‐values were Spearman’s correlation coefficients and p < 0.05 was linearly regressed to show relevant trends