CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis

Abstract

Introduction: Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status.

Methods: We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA(-) treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration.

Results: ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions. Interestingly, CCR6(+) Th cells were inversely correlated with disease duration in ACPA(-) patients (R(2) = -0.35; p < 0.01) but not in ACPA(+) (R(2) < 0.01; p = 0.94) patients.

Conclusions: These findings demonstrate that increased peripheral blood CCR6(+) Th cells proportions distinguish ACPA(+) RA from ACPA(-) RA. This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

Fig. 1

Proportions of chemokine receptor expressing memory Th cells differ between ACPA⁺ and ACPA⁻ RA patients. a Fraction of CD4⁺ T cell population within the total lymphocyte population of 27 ACPA⁺ and 27 ACPA⁻ patients with RA. b Fraction of memory CD4⁺ T cell population within the total CD4⁺ T cell population of 27 ACPA⁺ and 27 ACPA⁻ patients with RA. c Chemokine receptor and CD161 expression on peripheral blood memory (CD45RO⁺) CD4⁺CD25⁻ T cells from matched ACPA⁺ and ACPA⁻ patients with RA, measured by flow cytometry. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).

Fig. 2

Memory CCR6⁺CD4⁺ T cell subpopulations are increased in ACPA⁺ patients compared to matched ACPA⁻ patients. a Gating strategy for the identification of peripheral blood Th17, Th17.1, Th22 and CCR4/CXCR3 DP cell subpopulations. CCR6⁺ cells were gated on CD4⁺CD45RO⁺CD25⁻ T cells. b Real-time PCR expression analysis for IL-17, IFNγ, RORC and TBX21 in sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6⁺ Th cells obtained from patients with RA (8–10 patients per population). Prior to RNA isolation cells were stimulated with antiCD3/CD28 and cultured for 3 days. c-d Proportions of the indicated CD4⁺ T cell subpopulations within the total memory CD4⁺ T cell population (c) and memory CCR6⁺CD4⁺ T cell population (d) of 27 ACPA⁺ and 27 ACPA⁻ patients with RA. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).

Fig. 3

Differences in CCR6⁻CD4⁺ T cell subpopulations and Tregs between ACPA⁺ patients and matched ACPA⁻ patients. a Gating strategy for the identification of peripheral blood Th1, Th2 and CCR4/CXCR3 DP cell subpopulations. CCR6⁻ cells were gated on CD4⁺CD45RO⁺CD25⁻ T cells. b Gating strategy to identify Treg (CD25^hiFOXP3⁺) cells within the memory CD4⁺ T cell population. Cells were gated on the total lymphocyte population. c Proportions of the indicated CD4⁺ T cell subpopulations within the total memory CD4⁺ T cell population of 27 ACPA⁺ and 27 ACPA⁻ patients with RA. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).

Fig. 4

Inverse correlation between proportions of CCR6⁺CD4⁺ T cell subpopulations and disease duration in ACPA⁻ patients. Correlation of the percentage of CCR6⁺CD4⁺ cells (as percentage of total memory CD4⁺ T cells) with self-reported disease duration in ACPA⁺ and ACPA⁻ patients with RA. Pearson correlation test was used to calculate the correlation coefficients (R²) and p-values.