Low Th2 and high PD1+ TFh cells in blood predict remission after CTLA-4Ig treatment for 48 weeks in early rheumatoid arthritis

Abstract

Objective: To determine whether baseline CD4+ T helper (Th) cell subset proportions in blood may serve as predictive biomarkers for achieving remission 48 weeks after initiating CTLA-4Ig, anti-tumor necrosis factor (TNF), or anti-interleukin 6 receptor (IL6R) treatment in patients with early rheumatoid arthritis (eRA).

Methods: This study included 60 untreated eRA patients from the larger randomized treatment trial NORD-STAR. They were treated with methotrexate (MTX) combined with either CTLA-4Ig (n = 17), anti-TNF (n = 22), or anti-IL6R (n = 21). Disease activity was assessed by clinical disease activity index (CDAI), C-reactive protein, and erythrocyte sedimentation rate. The primary outcome was remission (CDAI ≤ 2.8) at week 48, and the secondary outcomes were time to reach remission or sustained remission during the 48-week follow-up. CD4+ T cell subset proportions were analyzed fresh by flow cytometry at baseline and at 24 and 48 weeks.

Results: In CTLA-4Ig + MTX-treated patients, baseline Th2 together with PD1+ T follicular helper (TFh) cell proportions predicted CDAI remission at week 48 (AUC: 0.986, 95% CI 0.94-1.0). Survival analysis revealed that patients with Th2 proportions below 16.8% or PD1+ TFh proportions above 7.6% at baseline were more likely to achieve remission (log-rank p = 0.002 and p = 0.007, respectively), and sustained remission (log-rank p = 0.01 and p = 0.001, respectively), over the 48-week follow-up. CD4+ T cell subset proportions did not predict remission in patients treated with anti-TNF + MTX or anti-IL6R + MTX. Only CTLA-4Ig treatment reduced PD1+ TFh and PD1neg TFh fractions after 48 weeks.

Conclusion: Circulating Th2 and PD1+ TFh cell proportions at baseline may serve as predictive biomarkers for achieving CDAI remission after 48 weeks of CTLA-4Ig treatment in eRA.

Fig 1. Gating strategy for CD4⁺ T cell subsets.

(A) Memory Th cell subsets, i.e., Th1, Th1Th17, CXCR3⁺Th17, Th17, CXCR3⁺Th2 and Th2, were defined based on the expression of CCR4, CCR6, and CXCR3. Th cell subset fractions are presented as proportions of memory CD45RA^neg cells. (B) CXCR5 and PD1 positive gates were based on their expression on CD4^neg and CD4⁺ T cells among lymphocytes. (C) Treg cells were defined as CD25^highCD127^low/neg, and the remaining conventional CD4⁺ T cells were defined as Non-Treg. PD1⁺ cells were gated among Tregs and Non-Tregs (excluding TFh cells), respectively. TFh was defined as CXCR5⁺ Non-Tregs and further divided into PD-1^neg TFh or PD1⁺ TFh. (D) Positivity for CTLA-4 was determined by comparing the control that contained the SA-PE fluorophore but not the CTLA-4-biotin antibody to the stained sample. (E) CTLA-4-positive cells were gated among Tregs and Non-Tregs, respectively. All Treg and Non-Treg cell subset fractions are presented as proportions of CD4⁺ cells. Parts of this gating strategy have previously been described [10]. TFh, T follicular helper; Th, T helper; Treg, T regulatory; SA, streptavidin.

Fig 2. Treatment reduces the clinical disease activity index after 48 weeks.

RA disease activity measured by the CDAI at baseline (0w) and 48w in patients treated with MTX in combination with (A) CTLA-4Ig (abatacept, n = 17), (B) anti-TNF (certolizumab-pegol, n = 22), or (C) anti-IL6 receptor (tocilizumab, n = 21). The dotted line indicates the cutoff value for remission (CDAI ≤ 2.8). Paired Wilcoxon matched pairs signed rank test, **** p ≤ 0.0001. Two patients with missing data in the anti-TNF + MTX group and four patients with missing data in the anti-IL6R + MTX group at 48w are shown in the graph but not included in the pairwise comparisons. 0w, 0 weeks/baseline; 48w, 48 weeks; anti‐IL6, anti–interleukin‐6 receptor; anti‐TNF, anti–tumor necrosis factor; CDAI, clinical disease activity index; MTX, methotrexate.

Fig 3. Baseline CD4⁺ T cell subset proportions predict remission in patients treated with CTLA-4Ig.

(A) Orthogonal projection to latent structures discriminant analysis of Rem (CDAI ≤ 2.8) at 48w (binary Y-variable) and baseline Th cell subset proportions (X-variables) in CTLA-4Ig + MTX-treated patients (n = 17). (B) Comparison of baseline Th2, Th1Th17, PD1⁺ TFh, and PD1⁺ Non-Treg proportions in patients who did or did not achieve Rem at 48w. (C) Comparison of baseline CDAI in patients who did or did not achieve Rem at 48w. Mann-Whitney U-test, *p ≤ 0.05 and ***p ≤ 0.001. Bars indicate median. ROC curves of baseline Th2, PD1⁺ TFh, and Th1Th17 proportions in (D) individual regression models, or (E) in combined multiple regression models. Probability cutoffs were determined by maximizing the Youden index for each model. 0w, 0 weeks/baseline; 48w, 48 weeks; anti‐IL6, anti–interleukin‐6 receptor; anti‐TNF, anti–tumor necrosis factor; AUC, area under the curve; CDAI, clinical disease activity index; MTX, methotrexate; NPV, negative predictive value; PPV, positive predictive value; PD1, programmed cell death‐1; Rem, remission; ROC, receiving operating curve; TFh, T follicular helper; Th, T helper.

Fig 4. Baseline CD4⁺ T cell subset proportions are associated with time to achieve remission in CTLA-4Ig-treated patients.

Kaplan-Meier cumulative incidence analysis showing the percentage of patients in remission (CDAI ≤ 2.8) and in sustained remission, i.e., CDAI remission for two or more consecutive visits, with (A) Th2 proportions ≤ 16.8% (dotted line) or > 16.8% (blue line), and (B) PD1⁺ TFh proportions ≤ 7.6% (dotted line) or > 7.6% (orange line), and (C) Th1Th17 proportions ≤ 5.8% (dotted line) or > 4.4% (pink line) at baseline. The groups for the Kaplan-Meier estimates were determined by the probability threshold that maximizes the Youden index. Significance was tested with the log-rank test, and unadjusted HR for achieving remission were estimated using Cox regression. Reference groups were patients with low Th2, PD1⁺ TFh, or Th1Th17 proportions. Each Cox regression model fulfilled the proportional hazards assumption. CDAI, clinical disease activity index; CI, confidence interval; HR, hazard ratio; PD1, programmed cell death‐1; Rem, remission; SR, sustained remission; TFh, T follicular helper; Th, T helper.

Fig 5. Association of baseline CD4⁺ T cell subset proportions and disease activity after 48 weeks of CTLA-4Ig treatment.

(A) Unsupervised PCA of disease activity measures at 48w, patient demographics, and baseline T cell subset proportions (X-variables) in CTLA-4Ig + MTX-treated patients (n = 17). (B) Orthogonal projection to latent structures analysis of CDAI at 48w (Y-variable) and baseline Th cell subset proportions (X-variables). (C) Unadjusted Spearman’s rank correlation test and simple linear regression analyses of CDAI at 48w and the Th2, PD1⁺ Non-Treg, and PD1⁺ TFh proportions at baseline. 48w, 48 weeks; MTX, methotrexate; PD1, programmed cell death‐1; PCA, principal component analysis; TFh, T follicular helper; Th, T helper; Treg, T regulatory.

Fig 6. Treatment for 48 weeks distinctly affects CD4⁺ T cell subset proportions.

Comparison of the proportions of circulating (A) PD1⁺ TFh and PD1^neg TFh, (B) PD1-expressing Non-Treg (excluding TFh) and Treg, and (C) CTLA-4-expressing Non-Treg and Treg at 0w and at weeks 24 and 48 in patients treated with MTX + CTLA-4Ig (abatacept, n = 17), MTX + anti-TNF (certolizumab-pegol, n = 22), or MTX + anti-IL6 receptor (tocilizumab, n = 21), respectively. Friedman’s test with Dunn’s test for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Patients with missing data for one or more time points were not included in the pairwise comparisons. 0w, 0 weeks/baseline; 24w, 24 weeks; 48w, 48 weeks; anti‐IL6, anti–interleukin‐6 receptor; anti‐TNF, anti–tumor necrosis factor; MTX, methotrexate; PD1, programmed cell death‐1; TFh, T follicular helper; Th, T helper; Treg, T regulatory.

Fig 7. Treatment for 48 weeks distinctly affects T-helper cell subset proportions.

Comparison of the proportions of (A) CXCR3⁺ Th2 and CXCR3⁺ Th17 and (B) Th2 and Th17 and (C) Th1 and Th1Th17 of CD45RAneg memory cells in blood at 0w and 24w and 48w in patients treated with MTX + CTLA-4Ig (abatacept, n = 17), MTX + anti-TNF (certolizumab-pegol, n = 22), or MTX + anti-IL6 receptor (tocilizumab, n = 21), respectively. Friedman’s test with Dunn’s test for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01 and ***p ≤ 0.001. Patients with missing data for one or more time points were not included in the pairwise comparisons. Data for 0w and 24w have previously been published [10]. 0w, 0 weeks/baseline; 24w, 24 weeks; 48w, 48 weeks; anti‐IL6, anti–interleukin‐6 receptor; anti‐TNF, anti–tumor necrosis factor; MTX, methotrexate; PD1, programmed cell death‐1; TFh, T follicular helper; Th, T helper; Treg, T regulatory.