Signal Transducer and Activator of Transcription 3 Hyperactivation Associates With Follicular Helper T Cell Differentiation and Disease Activity in Rheumatoid Arthritis

Abstract

Follicular helper T (Tfh) cells are the specialized CD4⁺ T cell subset that supports B cells to produce high-affinity antibodies and generate humoral memory. Not only is the function of Tfh cells instrumental to mount protect antibodies but also to support autoantibody production and promote systemic inflammation in autoimmune diseases. However, it remains unclear how the activation of Tfh cells is driven in autoimmune diseases. Here, we report that in patients with rheumatoid arthritis (RA), excessive generation of CXCR5⁺PD-1⁺ memory Tfh cells was observed and the frequency of memory Tfh cells correlated with disease activity score calculator for RA (DAS28). The differentiation of Tfh cells is dependent on signal transducer and activator of transcription 3 (STAT3), the key transcription factor downstream of cytokine signal pathways. A drastic increase of phosphorylated STAT3 (pSTAT3) in CD4⁺ T cells were detected in RA patients who also produced larger amounts of STAT3-stimulating cytokines, including IL-6, IL-21, IL-10, and leptin than those of healthy controls. Importantly, the phosphorylation status of STAT3 in CD4⁺ T cells positively correlated with the plasma concentration of IL-6 and the frequency of memory Tfh cells. This study reveals an IL-6-pSTAT3-Tfh immunoregulatory axis in the pathogenesis of RA and reinforces its candidature as biomarkers and targets for diagnosis and therapy.

Keywords: IL-6; follicular helper T cells; patient; phosphorylation; rheumatoid arthritis; signal transducer and activator of transcription 3.

Figure 1

Increased follicular helper T (Tfh) cell differentiation in patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from RA patients and healthy control individuals (HC) were analyzed by flow cytometry. (A) FACS plots showing the gating of ZA⁻ TCRab⁺ CD4⁺ viable CD4⁺ T cells for Treg (CD25^high) and conventional CD4⁺ T cell subsets: naïve (CD25⁻CD45RA⁺CD62L⁺), Th1 (CD25⁻CD45RA⁻CXCR3⁺CCR6⁻CCR4⁻), Th2 (CD25⁻CD45RA⁻CXCR3⁻CCR6⁻CCR4⁺), Th17 (CD25⁻CD45RA⁻CXCR3⁻CCR6⁺CCR4⁺), and Tfh (CD25⁻CD45RA⁻CXCR5⁺PD-1⁺). (B) Statistics showing the percentages of CD4⁺ T cell subsets in total CD4⁺ T cells. Each dot represents the value of an individual subject with columns showing the mean values of each group. The p-values were obtained using Student’s t-tests.

Figure 2

Increased follicular helper T (Tfh) cell differentiation correlates with rheumatoid arthritis (RA) disease activity. The percentages of CD4⁺ T cell subsets in the peripheral blood mononuclear cells from patients with RA were analyzed as Figure 1. The correlation between the frequencies of these subsets and the disease activities measured by DAS28 were determined using Spearman’s correlation coefficient.

Figure 3

Constitutive phosphorylation of signal transducer and activator of transcription 3 (STAT3) in CD4⁺ T cells from patients with rheumatoid arthritis (RA). The expression of intracellular phosphorylated STAT3 (pSTAT3) was analyzed using Phosflow assays in CD4⁺ T cells and B cells from RA patients. (A) FACS plots showing representative staining patterns for pSTAT3 (empty histograms) and an isotype control antibody (filled histograms) of indicated immune cell types; Numbers indicating the percentages of the pSTAT3 positive population. (B) Statistics showing the percentages of pSTAT3 positive population in total ZA⁻CD3⁻CD19⁺ viable B cells and ZA⁻TCRab⁺CD4⁺ viable CD4⁺ T cells from RA or HC groups. (C) FACS plots showing representative staining patterns for pSTAT3 and an isotype control antibody of indicated CD4⁺ T cell subsets; numbers indicating the percentages of the pSTAT3 positive population. (D) Statistics showing the percentages of pSTAT3 positive population in indicated CD4⁺ T cell subsets from RA or HC groups. Each dot represents the value of an individual subject with columns showing the mean value of each group. The p-values were obtained using Student’s t-tests.

Figure 4

Signal transducer and activator of transcription 3 (STAT3) hyperactivation correlates with aberrant follicular helper T (Tfh) differentiation in patients with rheumatoid arthritis (RA). Statistics showing the relationship between the frequencies of indicated CD4⁺ T cell subsets with the phosphorylated STAT3 (pSTAT3) expression in total CD4⁺ T cells (A) or in each individual subsets (B) in the peripheral blood mononuclear cells from patients with RA. The correlation was determined using Spearman’s correlation coefficient.

Figure 5

Signal transducer and activator of transcription 3 (STAT3) hyperactivation correlates with rheumatoid arthritis (RA) disease activity. Statistics showing the relationship between the disease activities measured by DAS28 with the phosphorylated STAT3 (pSTAT3) expression in total CD4⁺ T cells (A) or in each individual subsets (B) in the peripheral blood mononuclear cells from patients with RA. The correlation was determined using Spearman’s correlation coefficient.

Figure 6

Increased signal transducer and activator of transcription 3-stimulating cytokines in the plasma of rheumatoid arthritis (RA) patients. Plasma was isolated from blood from RA patients and healthy controls. The amount of IL-6 (A), IL-10 (B), IL-21 (C), and leptin (D) was measured by ELISA. Each dot represents the value of an individual subject with columns showing the mean values of each group. The p-values were obtained using Student’ t-tests.

Figure 7

Association between signal transducer and activator of transcription 3 (STAT3)-stimulating cytokines with STAT3 phosphorylation, disease activity or follicular helper T (Tfh) differentiation. Statistics showing the relationship between the expression of phosphorylated STAT3 (pSTAT3) in total CD4⁺ T cells, the disease activities measured by DAS28 or the frequency of Tfh cells with the amount of plasma IL-6 (A), IL-10 (B), IL-21 (C), or leptin (D). The correlation was determined using Spearman’s correlation coefficient.