Altered Circulating Follicular T Helper Cell Subsets and Follicular T Regulatory Cells Are Indicators of a Derailed B Cell Response in Lupus, Which Could Be Modified by Targeting IL-21R

Abstract

Systemic lupus erythematosus (SLE) is characterized by the breakdown of self-tolerance, the production of high-affinity pathogenic autoantibodies and derailed B cell responses, which indicates the importance of central players, such as follicular T helper (T_FH) subsets and follicular T regulatory (T_FR) cells, in the pathomechanism of the disease. In this study, we aimed to analyze the distribution of the circulating counterparts of these cells and their association with disease characteristics and B cell disproportions in SLE. We found that the increased percentage of activated circulating T_FH (cT_FH) and cT_FR cells was more pronounced in cutaneous lupus; however, among cT_FH subsets, the frequency of cT_FH17 cells was decreased in patients with lupus nephritis. Furthermore, the decreased proportion of cT_FH17 cells was associated with low complement C4 levels and high disease activity scores. We also investigated whether the blocking of the IL-21 receptor (IL-21R) with an anti-IL-21R monoclonal antibody inhibits the B cell response, since IL-21 primarily produced by T_FH cells potentially promotes humoral immunity. We observed that anti-IL-21R inhibited plasmablast generation and immunoglobulin production. Our study demonstrated that, besides cT_FR/cT_FH imbalance, cT_FH17 cells play a crucial role in SLE pathogenesis, and modulating cT_FH-B cell interaction through the IL-21/IL-21R pathway may be a promising therapeutic strategy to suppress the pathological B cell response.

Keywords: B cell; chemokine receptors; follicular T helper cell; follicular T regulatory cell; interleukin-21 receptor; systemic lupus erythematosus.

Figure 1

The distribution of activated cT_FH and T_FR cells in lupus patients and healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from 48 SLE patients and 36 healthy controls (HC) were then stained with fluorochrome-conjugated monoclonal antibodies, as described in Materials and Methods. The proportions of peripheral activated (act.) cT_FH cells and cT_FR cells were quantified as their percentage within CD4⁺ lymphocytes. (A) Representative dot plots from an active lupus patient and HC show the identification of activated cT_FH cells using ICOS and PD-1. Merged tSNE plots incorporating 30,000 CD4⁺ T cells from the corresponding lupus patient and healthy control show the act.cT_FH cluster identified by tSNE clustering analysis (red color represents act.cT_FH). Histograms show the expression of markers used to identify act.cT_FH cells. (B) Percentages of act.cT_FH cells. (C) Representative dot plots from an active SLE patient and HC show the identification of cT_FR cells by CD127 and CD25. Merged tSNE plots incorporating 30,000 CD4⁺ T cells from the corresponding lupus patient and healthy control show the cT_FR cluster identified by t-SNE clustering analysis (magenta color represents cT_FR). Histograms show the expression of markers used to identify cT_FR cells. (D) Percentages of cT_FR cells. (E) The proportions of act.cT_FH and cT_FR clusters among CD4⁺ T cells were indicated by the horizontal bars. Mann–Whitney nonparametric test was used (C,E). Box plots represent the interquartile range (IQR) with a line in the middle as the median. Statistically significant differences are indicated by *** p < 0.001; **** p < 0.0001.

Figure 2

The imbalance of cT_FH subsets in SLE patients and their association with disease characteristics. PBMCs were isolated from 48 SLE patients and 36 healthy controls (HC) were then stained with fluorochrome-conjugated monoclonal antibodies, as described in Materials and Methods. The percentages of blood cT_FH cell subsets were quantified as their percentage within CD4⁺ lymphocytes. (A) Representative dot plots from an active SLE patient and HC show the identification of cT_FH subsets: cT_FH1 (CXCR3⁺CCR6⁻), cT_FH1/17 (CXCR3⁺CCR6⁺), cT_FH2 (CXCR3⁻CCR6⁻) and cT_FH17 (CXCR3⁻CCR6⁺) cells. Merged t-SNE plots incorporating 30,000 CD4⁺ T cells from the corresponding lupus patient and healthy control show cT_FH clusters identified by t-SNE clustering analysis (each color represents a different cluster: cT_FH1, purple; cT_FH1/17, blue; cT_FH2, orange; cT_FH17, green). Histograms show the expression of markers used to identify each cT_FH subset. (B) The proportions of cT_FH clusters among CD4⁺ T cells were indicated by the horizontal bars. (C) Percentages of cT_FH subsets. (D) The receiver operating characteristic (ROC) curves were performed to evaluate the efficiency of cT_FH cell subsets to predict lupus when compared to controls. Colored lines indicate areas under the ROC curves (AUC) which are considered acceptable. (E) Percentages of different cT_FH subsets in patients with lupus nephritis (LN, n = 15), cutaneous lupus (CL, n = 13) and HC. (F) Correlation matrix represents the association between the percentages of cT_FH cell subsets, cT_FR cells and different serological parameters (anti [α]-dsDNA, immune complex [IC], complement [C] 3, 4 and SLEDAI) in patients with SLE. Positive correlations are displayed in red and negative correlations in blue. Color intensity and the size of the circle are proportional to the Spearman’s correlation coefficient. Two-way analysis of variance (ANOVA) with a Sidak post hoc test was used. Box plots represent the interquartile range (IQR) with a line in the middle as the median. Statistically significant differences are indicated by * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Abnormal B cell distribution and their association with SLE laboratory and clinical features. PBMCs were isolated from 48 SLE patients and 36 healthy controls (HC), and were then stained with fluorochrome-conjugated monoclonal antibodies, as described in Materials and Methods. The percentages of B cell subsets were quantified as their percentages within CD19⁺ lymphocytes. (A) Representative dot plots show the determination of IgD⁺CD27⁻ naive B (nB) cells, IgD⁺CD27⁺ un-switched memory B (US.mB) cells, IgD⁻CD27⁺ switched memory B (S.mB) cells and IgD⁻CD27⁻ double negative (DN) B cells. (B) The proportions of naive and memory B cell subsets. (C) Representative dot plots show the identification of CD38^hiCD24^hiCD27⁻ transitional B (trans.B) cells, CD38^intCD24^int mature-naive B (mn.B) cells and CD38⁻CD24^hiCD27⁺ primarily memory B (pm.B) cells. (D) The proportions of trans.B, mn.B and pm.B cell subpopulations. (E) The percentages of CD38⁺CD27^hi plasmablasts. (F) Correlation matrix represents the association between the percentages of B cell subsets and cT_FR cells, cT_FH subpopulations, as well as routine laboratory parameters (anti [α]-dsDNA, immune complex [IC], complement [C] 3, 4 and SLEDAI) in patients with SLE. Positive correlations are displayed in red and negative correlations in blue. Color intensity and the size of the circle are proportional to the Spearman’s correlation coefficient. (G) Percentages of different B cell subsets in patients with lupus nephritis (LN, n = 15), cutaneous lupus (CL, n = 13) and HC. Two-way analysis of variance (ANOVA) with a Sidak post hoc test was used. Box plots represent the interquartile range (IQR) with a line in the middle as the median. Statistically significant differences are indicated by * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Anti (α)-IL-21R inhibits T_FH cell-dependent B cell differentiation in SLE patients and healthy individuals. (A) Representative contour plots show the gating strategy and the blocking of plasmablast differentiation by αIL-21R in SLE. As an isotype control, the IgG1 protein was used. (B) Percentages of 7-AAD⁻CD4⁻CD27⁺CD38^hi viable plasmablasts were measured by flow cytometry in healthy controls (HC; n = 3) and in SLE patients (n = 3). Fold change of IgM (C) and IgG (D) was measured by ELISA in patients with lupus and HC. Bar charts represent mean ± SD. Data analysis was performed with two-way ANOVA followed by Tukey’s multiple comparison tests. Statistically significant differences are defined as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5

Summarizing the outcome of T_FR/T_FH cell imbalance and altered B cell differentiation in SLE. Incomplete induction of self-tolerance and B cell hyperactivity induced by disturbed immune regulation are the hallmark of lupus pathogenesis. The breakdown of early B cell tolerance leads to consequential enrichment of the ratio of transitional B cells and naive/mature–naive B cells in the circulation. The microenvironment of GCs gives place to peripheral tolerance, where T_FR cells and T_FH cells collaborate to regulate the multistage differentiation of B cells. T_FH cells produce IL-21, which facilitates the GC reaction by promoting T_FH and B cell differentiation, while inhibiting T_FR function. The imbalance of T_FR cell and T_FH cell homeostasis in lymph nodes culminates in an increased number of activated cT_FH and cT_FR cells at the periphery, along with characteristic changes in the distribution of B cell subpopulations. We hypothesize that cT_FH17 cells may traffic to the site of inflammation, where they collaborate with the accumulated antibody-secreting cells and memory B cells within kidney tissues, which contributes to the progression of the disease. DC, dendritic cell; DN B, double negative B cell; GC, germinal center; IL-21, interleukin-21; IL-21R, interleukin-21 receptor; LL-PC, long-lived plasma cell; mB, memory B cell; SL-PC, short-lived plasma cell; SLE, systemic lupus erythematosus; Treg, regulatory T cell; T_FR, follicular T regulatory cell; T_FH, follicular T helper cells. [Some icons were inspired by Biorender.com accessed on 16 August 2022.].