Peripheral blood CD4posCD25posFoxP3pos cells and inflammatory cytokines as biomarkers of response in rheumatoid arthritis patients treated with CTLA4-Ig

Abstract

Background: Prognostic biomarkers of treatment response to distinct biologic disease-modifying anti-rheumatic drugs (b-DMARDs) are still lacking within the management of rheumatoid arthritis (RA).

Methods: Thirty-four b-DMARDs naive RA patients, divided by disease duration into early (cohort 1) and long standing (cohort 2), received CTLA4-Ig. At study entry, and every 3 months for 1 year, each patient underwent peripheral blood (PB)-derived CD4^pos cell subpopulation assessment by flow cytometry, STAT3 and STAT5 expression by RT-PCR and IL-6, IL-12p70, TGFβ, and IL-10 serum levels by ELISA. The DAS and CDAI remission was assessed at 6 and 12 months.

Results: DAS- and CDAI-defined remission within 12 months was achieved by 16 (47.1%) and 8 (23.5%) RA patients, respectively. Considering the whole RA cohort, CTLA4-Ig induced a significant decrease of IL-6 serum levels from baseline to 6 and 12 months, as well as of PB CD4^posCD25^posFoxP3^pos cells at 6 and 12 months, and of CD4^posIL17^pos cells after 12 months. PB CD4^pos cells of RA patients showed higher STAT3 and STAT5 expression than healthy controls, which remained unchanged within 12 months of treatment. At study entry, RA patients achieving DAS remission had significantly lower IL-6 serum levels than RA patients not achieving this outcome. In particular, having baseline IL-6 serum levels ≤ 8.4 pg/ml, significantly identified naïve to b-DMARDs RA patients more likely to achieve DAS-remission under CTLA4-Ig at 6 months (66.7%) compared to RA patients with baseline IL-6 serum levels > 8.4 pg/ml [15.4%, OR (95%Cis) 11.00 (1.75-55.82)]. Moreover, having CD4^posCD25^posFoxP3^pos cells rate ≥ 6.0% significantly identifies naïve to b-DMARDs early RA patients more likely to achieve DAS remission at 6 months (83.3%) compared to RA patients with baseline CD4^posCD25^posFoxP3^pos cells < 6.0% [16.7%, OR (95% Cis) 25.00 (1.00-336.81)].

Conclusions: Baseline IL-6 serum levels and peripheral blood-derived CD4^pos subpopulations are putative novel prognostic biomarkers of CTLA4-Ig response in RA patients.

Keywords: Biomarkers; CTLA4-Ig; Rheumatoid arthritis.

Fig. 1

A–C Clinical outcome of CTLA4-Ig treatment in RA patients. A DAS value across 12 months of follow-up of CTLA4-Ig treatment in RA patients (whole cohort, cohort 1, and cohort 2, respectively); p values were calculated using Wilcoxon matched-pairs signed rank test comparing DAS at pre-treatment and after 3, 6, and 12 months of CTLA4-Ig administration in RA patients (whole RA cohort, RA cohort 1, or RA cohort 2). B CDAI value across 12 months of follow-up of CTLA4-Ig treatment in RA patients (whole cohort, cohort 1, and cohort 2, respectively); p values were calculated using Wilcoxon matched-pairs signed rank test comparing CDAI at pre-treatment and after 3, 6, and 12 months of CTLA4-Ig administration in RA patients (whole RA cohort, RA cohort 1, or RA cohort 2). C Kaplan–Meier survival curve of treatment with CTLA4-Ig across 12 months of follow-up in RA patients (whole cohort, cohort 1, and cohort 2, respectively) for DAS (log-rank test: chi-square = 0.029 p = 0.863; Breslow test: chi-square = 0.346 p = 0.841) and CDAI (log-rank test: chi-square = 0.217 p = 0.641; Breslow test: chi-square = 0.823 p = 0.663). DAS, Disease Activity Score; CDAI, Composite Disease Activity Index; RA, rheumatoid arthritis

Fig. 2

A–C Modulation of peripheral blood cytokines and T-lymphocyte subpopulations by CTLA4-Ig treatment in RA. A IL-6, TGF-b, and IL-10 serum levels of RA patients at study entry and after 6 and 12 months of follow-up of CTLA4-Ig treatment in RA patients (whole cohort, cohort 1, and cohort 2, respectively) and healthy controls. B Peripheral blood CD4^posCD25^posFoxP3^pos, CD4^posCD25^posCD127^neg, and CD4^posIL17^pos cell percentage at study entry and after 6 and 12 months of follow-up of CTLA4-Ig treatment in RA patients (whole cohort, cohort 1, and cohort 2 respectively) and healthy controls. C STAT3 and STAT5 gene expression in CD4^pos cells from peripheral blood of RA patients at study entry and after 6 and 12 months of follow-up of CTLA4-Ig treatment (whole cohort, cohort 1, and cohort 2, respectively) and healthy controls. All comparisons between groups were done using the Mann–Whitney U test while all comparisons between different time points within the same cohort were done using the Wilcoxon matched-pairs signed rank test. IL, interleukin; TGFβ, transforming growth factor beta; CD, cluster designation; RA, rheumatoid arthritis

Fig. 3

A–D Baseline inflammatory and peripheral blood CD4^pos subpopulations associated with remission achievement under CTLA4-Ig treatment in RA. A Spearman correlation test between IL-6, IL-10, TGFβ serum concentrations, CD4^pos cell subpopulation rates, STAT3, and STAT5 expression in CD4^pos cells in the whole RA cohort with DAS and CDAI at baseline and at 6 months of follow-up of treatment with CTLA4-Ig. Each number indicates the coefficient of the Spearman correlation test. “*” indicates correlations with p ≤ 0.05 from the Spearman test. B IL-6, TGF-β, and IL-10 serum levels of RA patients at study entry based on the achievement of DAS- and CDAI-defined remission after 6 months of follow-up of CTLA4-Ig treatment in the whole RA cohort and healthy controls. C Peripheral blood CD4^posCD25^posFoxP3^pos and CD4^posCD25^posCD127^neg cell percentage at study entry based on the achievement of DAS- and CDAI-defined remission after 6 months of follow-up of CTLA4-Ig treatment in the whole RA cohort and healthy controls. D STAT3 and STAT5 expression in peripheral blood-derived CD4^pos cells of RA patients at study entry based on the achievement of DAS- and CDAI-defined remission after 6 months of follow-up of CTLA4-Ig treatment in the whole RA cohort and healthy controls. B–D All comparisons between groups were done using the Mann–Whitney U test. HC, healthy controls; IL, interleukin; TGFβ, transforming growth factor beta; CD, cluster designation; RA, rheumatoid arthritis

Fig. 4

A–E Baseline inflammatory and peripheral blood CD4^pos subpopulations associated with remission achievement under CTLA4-Ig treatment in b-DMARDs naive RA. A Rate of naive to b-DMARDs RA achieving DAS remission at 6 months of treatment with CTLA4-Ig based on pre-treatment IL-6 serum levels. B Kaplan–Meier survival curve of naive to b-DMARDs RA achieving DAS remission at 6 months of treatment with CTLA4-Ig based on pre-treatment IL-6 serum levels. C Spearman correlation test between composite disease activity indices (i.e., DAS and CDAI, respectively) and peripheral blood CD4^posCD25^posFoxP3^pos cell rate in early naive to b-DMARDs RA at 6 months of treatment with CTLA4-Ig. D Rate of early naive to b-DMARDs RA achieving DAS remission at 6 months of treatment with CTLA4-Ig based on pre-treatment CD4^posCD25^posFoxP3^pos cells rate; p value was calculated using the chi-square test. E Rate of naive to b-DMARDs RA achieving DAS remission at 6 months of treatment with CTLA4-Ig based on pre-treatment IL-6 serum levels and/or CD4^posCD25^posFoxP3^pos cell rate. p values were calculated using the chi-square test; RA, rheumatoid arthritis; DAS, Disease Activity Score; CDAI, Composite Disease Activity Index; CD, cluster designation; b-DMARDs, biologic disease-modifying anti-rheumatic drugs