Th Cell Phenotypes and Their Correlations with Disease Activity in Patients with Rheumatoid Arthritis

Abstract

Objectives: This study compared the frequencies of circulating CD4+ T helper (Th) cell subsets in rheumatoid arthritis (RA) patients and healthy controls (HCs) and investigated their relationship with RA disease activity. Methods: Peripheral blood samples and demographic/clinical data were collected from 75 RA patients and 28 HCs from the A3BC Biobank. Flow cytometry was utilized to identify cell subsets. Data were analyzed using FlowJo and GraphPad Prism. Results: RA patients displayed altered Th cell subset frequencies compared with HCs, including a higher overall proportion of Th cells (p = 0.02) but lower proportions of memory Th (p = 0.03), Th1 (p < 0.0001), and Th17.1 (p = 0.004) cells. In DMARD-naïve RA patients (n = 16), lower proportions of Th1 (p = 0.0005), Th9 (p = 0.04), and Th17.1 (p = 0.003) cells, alongside a higher Th17 cell proportion (p = 0.017), were observed compared with those in HCs. Further analysis of matched treatment-naïve, new-onset RA patients and HCs confirmed these findings. Within the RA cohort, lower proportions of Th1 (p = 0.002) and Th17.1 (p = 0.025) cells and a higher proportion of Th2 cells (p = 0.015) were correlated with increased disease activity. Inverse correlations were also found between the proportions of Th1 (p = 0.002), Th9 (p = 0.024), and Th17.1 (p = 0.00017) cells and CRP levels in RA patients. Conclusions: This study demonstrates an imbalance in circulating Th cell subset frequencies in RA patients compared with those in HCs, with notably lower Th1 and Th17.1 cell proportions in RA patients. Decreased frequencies of these cell subsets were linked to increased disease activity, indicating that restoring the balance of Th cell subsets could be a potential therapeutic strategy for RA.

Keywords: C-reactive protein; Th cells; anti-cyclic citrullinated peptide; disease activity; erythrocyte sedimentation rate; rheumatoid arthritis; rheumatoid factor.

Figure 1

Th cells and their subsets in patients with rheumatoid arthritis (RA) and healthy controls (HCs). Peripheral blood mononuclear cells from RA patients (n = 75) and HCs (n = 28) were analyzed by flow cytometry to determine the levels of Th cells and their subsets. (a) Relative levels of total Th cells, memory Th cells, Th1 cells, and Th17.1 cells in HCs and RA patients. (b) Relative levels of Th1, Th9, Th17, and Th17.1 cells in HCs (n = 28) and DMARD-naïve RA patients (n = 16). Data presented are means with 95% confidence intervals (CIs). Statistical significance was assessed using non-parametric Mann–Whitney U tests. (c) Spearman’s rank correlation coefficients showing significant correlations between Th cell subsets and demographical/clinical parameters (sex, age, disease duration, serological status, and DMARD use) in RA patients and HCs. * p < 0.05, ** p < 0.001, *** p < 0.0001. Anti-CCP: anti-cyclic citrullinated peptide antibodies; RF: rheumatoid factor; csDMARD: conventional synthetic disease-modifying antirheumatic drug; b/tsDMARD: biologic or targeted synthetic DMARD; Mem: memory.

Figure 2

tSNE visualization and automated clustering of Th cell subsets in patients with DMARD-naïve, new-onset rheumatoid arthritis (RA) and healthy controls (HCs). Peripheral blood mononuclear cells from 6 age- and sex-matched pairs of DMARD-naïve, new-onset RA patients and HCs were analyzed by flow cytometry using a T cell antibody panel. (a) tSNE plots displaying the expression of CD3, CD4, CD25, CD127, CD183, CD185, CD196, CD294, and CD45RA, with corresponding heatmaps and histograms. Marker expression levels are color-coded on the tSNE plots. (b) Cluster analysis identified 4 clusters enriched in RA patients compared with HCs. (c) tSNE visualization of Treg cells and non-Treg cells (memory Th and Tfh cells) identified by automated clustering of Th cells. tSNE: t-distributed stochastic neighbor embedding. Tfh cells: T follicular helper cells.

Figure 3

tSNE visualization and automated clustering of memory Th cell subsets in patients with DMARD-naïve, new-onset rheumatoid arthritis (RA) and healthy controls (HCs). Peripheral blood mononuclear cells from 6 age- and sex-matched pairs of DMARD-naïve, new-onset RA patients and HCs were analyzed by flow cytometry using a T cell antibody panel. (a) Cluster analysis of memory Th cells identified 2 clusters that were significantly less abundant in RA patients than in HCs. (b) tSNE visualization of Th1, Th2, Th9, Th17, and ThG cells identified by automated clustering of memory Th cells. Tfh cells: T follicular helper cells; tSNE: t-distributed stochastic neighbor embedding.

Figure 4

The correlations between Th cell subsets and disease activity in rheumatoid arthritis (RA). Linear regression analysis was used to assess correlations between Th cell subset frequencies and disease activity measures in RA patients. (a) The correlations of Th cell frequencies with DAS28-CRP and tender joint count (TJC) in all RA patients (n = 75). (b) The correlations of Th1 cell frequency with DAS28-CRP and TJC in female RA patients (n = 53). (c) The correlations of Th2 and memory Th cell frequencies with disease activity in male RA patients (n = 22). CRP: C-reactive protein.

Figure 5

The correlations of Th cell subsets with disease activity in seropositive and seronegative patients and DMARD-naïve patients with rheumatoid arthritis (RA). The correlations of Th cell subsets with disease activity in RA patients were analyzed using a linear regression test. (a) The correlations of Th1 and Th2 cell frequencies with disease activity in seropositive patients (n = 52). (b) The correlations of Th1, Th9, and fTh17 cell frequencies with disease activity in seronegative patients (n = 18). (c) The correlations of Th2 and Th17.1 cell frequencies with disease activity in DMARD-naïve RA patients (n = 16). CRP: C-reactive protein; DMARD: disease-modifying antirheumatic drug.

Figure 6

The correlations between Th cell subsets and C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in rheumatoid arthritis (RA). Nonparametric correlation matrix analysis was used to assess associations between Th cell subset frequencies and the levels of CRP and ESR in RA patients. (a) Overall RA patients (n = 75). (b) Female RA patients (n = 53). (c) Male RA patients (n = 22). (d) Seropositive RA patients (n = 52). (e) Seronegative RA patients (n = 18). * p < 0.05, ** p < 0.001, *** p < 0.0001.