Dynamics of circulating follicular helper T cell subsets and follicular regulatory T cells in rheumatoid arthritis patients according to HLA-DRB1 locus

Abstract

B cells, follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are part of a circuit that may play a role in the development or progression of rheumatoid arthritis (RA). With the aim of providing further insight into this topic, here we evaluated the frequency of different subsets of Tfh and Tfr in untreated and long-term treated RA patients from a cohort of Argentina, and their potential association with particular human leukocyte antigen (HLA) class-II variants and disease activity. We observed that the frequency of total Tfh cells as well as of particular Tfh subsets and Tfr cells were increased in seropositive untreated RA patients. Interestingly, when analyzing paired samples, the frequency of Tfh cells was reduced in synovial fluid compared to peripheral blood, while Tfr cells levels were similar in both biological fluids. After treatment, a decrease in the CCR7^loPD1^hi Tfh subset and an increase in the frequency of Tfr cells was observed in blood. In comparison to healthy donors, seropositive patients with moderate and high disease activity exhibited higher frequency of Tfh cells while seropositive patients with low disease activity presented higher Tfr cell frequency. Finally, we observed that HLA-DRB1*09 presence correlated with higher frequency of Tfh and Tfr cells, while HLA-DRB1*04 was associated with increased Tfr cell frequency. Together, our results increase our knowledge about the dynamics of Tfh and Tfr cell subsets in RA, showing that this is altered after treatment.

Keywords: DAS28-ESR; DMARDs; HLA-DRB1; Tfh cells; Tfr cells; rheumatoid arthritis.

Figure 1

Frequency of total Tfh cells and different Tfh cell subsets in RA patients. (A) Frequency of CD25^loCD127^hi total (t) Tfh cells (left), CCR7^loPD1^hi Tfh cell subset (middle) and activated CCR7^loPD1^hiICOS⁺ Tfh cell subset (right) in RA patients (n=99, 40 and 40, respectively) and HD (n=46, 16 and 16, respectively). (B) Frequency of tTfh cells in seropositive (Seropos, n=72) and seronegative (Seroneg, n=27) RA patients and HD (n=46) (left), frequency of CCR7^loPD1^hi Tfh cell subset (middle) and activated CCR7^loPD1^hiICOS⁺ Tfh cell subset (right) in seropositive (Seropos, n=27) and seronegative (Seroneg, n=13) RA patients and HD (n=16). (C) Frequency of tTfh cells in untreated (n=52), patients treated with different drugs: csDMARDs (n=24), TNF inhibitors (TNF inh, n=14), tofacitinib (Tofa, n=9), and HD (n=46). (D) Frequency of tTfh cells in Seropos (n=41), Seroneg (n=11) untreated RA patients and HD (n=46). Symbols represent individual subjects. Mean ± DS is shown. P values were determined by Welch-test or T-Test in (A). One-way Analysis of Variance (ANOVA) followed by Bonferroni Multiple Comparisons Test were used in (B, D) and Kruskal-Wallis followed by Dunn’s Multiple Comparisons Test were used in (C). Two-tailed probability, *p < 0.05, **p < 0.01; ns, not significant.

Figure 2

Frequency of Tfr cells in RA patients. (A) Frequency of Tfr cells in blood from RA patients (n=99) and HD (n=46). (B) Frequency of Tfr cells in seropositive (Seropos, n=72) and seronegative (Seroneg, n=27) RA patients and HD (n=46). (C) Frequency of Tfr cells in untreated (n=52), RA patients treated with different drugs: csDMARDs (n=24), TNF inhibitor (TNF inh, n=14), tofacitinib (Tofa, n=9) and HD (n=46). (D) Frequency of Tfr cells in Seropos (n=41) and Seroneg (n=11) untreated RA patients and HD (n=46). Symbols represent individual subjects. Mean ± DS is shown in (B) and (D). Median ± interquartile range is shown in (A) and (C); P values were determined by T-Test in (A). Kruskal-Wallis Test followed by Dunn’s Multiple Comparisons Test were determined in (B–D). Two-tailed probability *p < 0.05, **p < 0.01, ns, not significant.

Figure 3

Correlation between Tfh and Tfr cells in RA patients. (A) Correlation between tTfh and Tfr cells frequency in all RA patients (n=99, left) or in seropositive RA patients (n=72, right). (B) Tfr/Tfh ratio in RA patients (n=99) vs HD (n=46) (left) or in seropositive (Seropos, n=72) and seronegative (Seroneg, n=27) RA patients and HD (right). Symbols represent individual subjects. ^#Pearson’s correlation coefficient r and P values are shown in (A); lines of best fit were drawn. Median ± interquartile range is shown in (B); T-Test (left) and Kruskal-Wallis Test followed by Dunn’s Multiple Comparisons Test (right) were determined in (B). ns, not significant.

Figure 4

Distribution of HLA-DRB1 and its relationship with tTfh cells in RA patients. (A) HLA-DRB1 variants distribution in RA patients (n=94) and HD (n=43). (B) HLA-DRB1 variants frequency in all RA patients (n=94) and HD (n=43, left), and HLA-DRB1*04 or HLA-DRB1*10 frequency in RA patients with anti-CCP antibodies (n=58) and HD (n=43, right). (C) Frequency of tTfh cells in RA patients (n=94) and in RA patients with anti-CCP antibodies (n=58) divided by HLA-DRB1*04 presence (n=34 out of 94 RA patients and n=30 out of 58 patients with anti-CCP) and HD (n=43). (D) Frequency of tTfh cells in RA patients (n=94) and in RA patients with anti-CCP antibodies (n=58) divided by HLA-DRB1*10 presence (n=10 out of 94 RA patients and n=9 out of 58 patients with anti-CCP) and HD (n=43). (E) Frequency of tTfh cells in RA patients (n=94) and in RA patients with anti-CCP antibodies (n=58) divided by HLA-DRB1*09 presence (n=10 out of 94 RA patients and n=8 out of 58 patients with anti-CCP) and HD (n=43). Symbols represent individual subjects and mean ± DS is shown in (C–E). Chi square test or Fisher Exact test were used in (B) as appropriate. P values were determined by Kruskal-Wallis test followed by Dunn’s Multiple Comparisons Test in (C, D); One-way Analysis of Variance (ANOVA) and Bonferroni Multiple Comparisons Test were used in (E). Two-tailed probability *p < 0.05, ns, not significant.

Figure 5

Frequency of Tfr cells according to HLA-DRB1 variants in RA patients. (A) Frequency of Tfr cells in RA patients (n=94) and in anti-CCP positive RA patients (n=58) divided by HLA-DRB1*04 presence (n=34 out of 94 RA patients and n=30 out of 58 patients with anti-CCP) and HD (n=43). (B) Frequency of Tfr cells in RA patients (n=94) and in anti-CCP positive RA patients (n=58) divided by HLA-DRB1*10 presence (n=10 out of 94 RA patients and n=9 out of 58 patients with anti-CCP) and HD (n=43). (C) Frequency of Tfr cells in RA patients (n=94) and in anti-CCP positive RA patients (n=58) divided by HLA-DRB1*09 presence (n=10 out of 94 RA patients and n=8 out of 58 patients with anti-CCP) and HD (n=43). Symbols represent individual subjects. Mean ± DS is shown. P values were determined by One-way Analysis of Variance (ANOVA) followed by Bonferroni Multiple Comparisons Test in (A), (B) all RA patients and (C). Kruskal-Wallis Test followed by Dunn’s Multiple Comparisons Test were used in (B) anti-CCP positive RA patients. Two-tailed probability *p < 0.05, **p < 0.01, ns, not significant.

Figure 6

Relationship between tTfh cells and the activity of the disease. (A) Correlation between DAS28-ESR and tTfh cell frequency in all RA patients (n=99, empty black circles and line) or in only seropositive patients (n=72, pink circles and line) (left), and in untreated RA patients (n=52) (right). (B) Frequency of tTfh cells in seropositive RA patients (Seropos, n=72) with low (n=17) or moderate-high (n=55) disease activity and HD (n=46). (C) Frequency of tTfh cells at baseline (0 months) and after 3 months of treatment in all RA patients (n=27) (left), in responder (n=20, middle) and non-responder (n=7, right) RA patients. (D) Frequency of CCR7^loPD1^hi Tfh cell subset (left) and activated CCR7^loPD1^hiICOS⁺ Tfh cell subset (right) at baseline (0 months) and after 3 months of treatment in responder RA patients (n=7). Symbols represent individual subjects. Mean ± DS is shown in (B). ^#Pearson’s correlation coefficient r and P values are shown in (A). Lines of best fit were drawn. One-way Analysis of Variance (ANOVA) followed by Bonferroni Multiple Comparisons Test were calculated in (B). P values were determined by Paired samples t-test in (C) and (D). Two-tailed probability *p < 0.05, **p < 0.01, ns, not significant.

Figure 7

Relationship between Tfr cells and the activity of the disease. (A) Correlation between DAS28-ESR and Tfr cell frequency in all RA patients (n=99, empty black circles and line) or in seropositive patients (n=72, pink circles and line left), and in untreated RA patients (n=52, right). (B) Frequency of Tfr cells in seropositive RA patients (Seropos, n=72) with low (n=17) or moderate-high (n=55) disease activity and HD (n=46). (C) Frequency of Tfr cells at baseline (0 months) and after 3 months of treatment in all RA patients (n=27) (left), in responder (n=20) (middle) and non-responder (n=7) (right) RA patients. Symbols represent individual subjects. ^#Pearson’s correlation coefficient r and P values are shown in (A). Lines of best fit were drawn. Mean ± DS is shown in (B). One-way Analysis of Variance (ANOVA) followed by Bonferroni Multiple Comparisons Test were calculated in (B). P values were determined by Paired samples T-Test in (C). Two-tailed probability *p < 0.05, **p < 0.01, ns, not significant.