The Imbalance of Circulating Follicular T Helper Cell Subsets in Primary Sjögren's Syndrome Associates With Serological Alterations and Abnormal B-Cell Distribution

Abstract

Since B-cell hyperactivity and pathologic antibody response are key features in the immunopathogenesis of primary Sjögren's syndrome (pSS), the role of follicular T helper (T_FH) cells as efficient helpers in the survival and differentiation of B cells has emerged. Our aim was to investigate whether a change in the balance of circulating (c)T_FH subsets and follicular regulatory T (T_FR) cells could affect the distribution of B cells in pSS. Peripheral blood of 38 pSS patients and 27 healthy controls was assessed for the frequencies of cT_FH cell subsets, T_FR cells, and certain B cell subpopulations by multicolor flow cytometry. Serological parameters, including anti-SSA, anti-SSB autoantibodies, immunoglobulin, and immune complex titers were determined as part of the routine diagnostic evaluation. Patients with pSS showed a significant increase in activated cT_FH cell proportions, which was associated with serological results. Frequencies of cT_FH subsets were unchanged in pSS patients compared to healthy controls. The percentages and number of cT_FR cells exhibited a significant increase in autoantibody positive patients compared to patients with seronegative pSS. The proportions of transitional and naïve B cells were significantly increased, whereas subsets of memory B cells were significantly decreased and correlated with autoantibody production. Functional analysis revealed that the simultaneous blockade of cT_FH and B cell interaction with anti-IL-21 and anti-CD40 antibodies decreased the production of IgM and IgG. Imbalance in T_FH subsets and T_FR cells indicates an ongoing over-activated humoral immune response, which contributes to the characteristic serological manifestations and the pathogenesis of pSS.

Keywords: B cell; chemokine receptors; follicular T helper cell; follicular regulatory T cell; primary Sjögren’s syndrome.

Figure 1

The distribution of cT_FH subsets and T_FR cells in patients with pSS and their association with laboratory serological markers. PBMCs were isolated from 38 pSS patients and 29 healthy controls (HC) then were stained with fluorochrome-labeled antibodies as described previously. Peripheral circulating (c)T_FH subsets were quantified as their percentage within CD4⁺CXCR5⁺ lymphocyte population. (A) Representative dot plots show the gating strategy and the division of T_FH cell subpopulations. Percentages of cT_FH1 (B), cT_FH1/17 (C), cT_FH2 (D), cT_FH17 (E) in pSS patients (n = 38), pSS without anti-SSA(−) (n = 14), pSS with anti-SSA(+) (n = 24) and HCs (n = 29). (F) Representative dot plots show the gating of ICOS⁺PD-1⁺ activated cT_FH cells in pSS and HC. Percentages of activated cT_FH cells. (G) Representative dot plots display the gating of T_FR cells in pSS and HC. Frequencies of T_FR cells. (H) Correlation analysis of the percentages of T_FH cell subsets and T_FR cells with different serological parameters in patients with pSS. One-way ANOVA with Tukey’s multiple comparisons test (B, D, E) or Kruskal–Wallis test with Dunn’s multiple comparisons test (C, F, G) was used. Correlation analysis was performed using Spearman’s test (H). Box plots represent the interquartile range (IQR) with a line in the middle as median and “+” sign as the mean value. Statistically significant differences are indicated by *p < 0.05; **p < 0.01.

Figure 2

Abnormal distribution of different B cell subsets reflects the pathophysiology of pSS. Peripheral blood B cell subsets were quantified as their percentage in CD19⁺ lymphocyte population. (A) Representative dot plots indicate the gating strategy and determination of different B cell subsets. Percentages of IgD⁺CD27^- naive B cells (B), IgD⁺CD27⁺ un-switched memory B cells (C), IgD^-CD27⁺ switched memory B cells (D), CD38^hiCD24^hiCD27⁻ transitional B cells (E), CD38⁺CD24⁺ mature-naive B cells (F), CD38^-CD24^hiCD27⁺ primarily memory B cells (G) in pSS patients (n =38), pSS without anti-SSA(−) (n = 14) and pSS with anti-SSA(+) (n = 24) and healthy controls (HC; n = 29). (H) Frequencies of certain B cell subsets of pSS patients were acquired, and correlation analysis with serological parameters as well as with the percentages of cT_FH subsets was carried out. Data analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test (D, E) or Kruskal–Wallis test with Dunn’s multiple comparisons test (B, C, F, G). Correlation analysis was carried out with Spearman’s test (H). Box plots represent the interquartile range (IQR) with a line in the middle as median and “+” sign as the mean value. Statistically significant differences are indicated by *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

The effects of anti-human CD40/TNFRSF5 and anti-IL-21 on B-cell differentiation and antibody production. (A) Graphical representation of B-T_H/cT_FH co-culture assay. Magnetically isolated CD19⁺ B cells were pre-activated with F(ab′)2 fragment in the presence of recombinant IL-2 and IL-10 for 24 h. Isolated CD4⁺CXCR5⁺ cT_FH or CD4⁺CXCR5⁻ T_H cells were co-cultured for 7 days with autologous B cells under stimulation by SEB and in the presence or absence of anti-human CD40/TNFRSF5 and anti-human IL-21 antibodies. (B) Representative dot plots show the neutralization effect of anti-human CD40 and IL-21 on plasmablast differentiation. Percentages of 7-AAD⁻CD4^-CD27⁺CD38^hi viable plasmablasts were measured by flow cytometry in healthy controls (HC; n = 6) and pSS patients (n = 3). Two-way ANOVA followed by Bonferroni’s posttest was used. Bars represent mean with SD. (C–F) Concentration and fold decrease of IgM and IgG were measured by ELISA in patients with pSS (n = 5) and HCs (n = 7). Bars show the mean with SD and each data point represents an individual subject. Data analysis performed with two-way ANOVA followed by Tukey’s multiple comparison tests. Statistically significant differences are defined as *p < 0.05.