Immune skew of follicular helper T cells and effector T cells in patients with Sjögren’s syndrome

To the Editor: Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by the infiltration of lymphocytes into dysfunctional exocrine glands. Previous study discovered that excessive follicular helper T (Tfh) cells supported autoantibody production in several autoimmune diseases.[1] Our previous study showed that activation of Tfh is positively associated with higher titers of autoantibodies and more severe manifestation in systemic lupus erythematosus (SLE) patients. The pathogenesis role of Tfh cells in autoimmune diseases has been verified.[2] However, the results from pSS studies were inconsistent and controversial. To provide support for further research in pSS, this study focused on describing the profile of the main T helper cells, particularly Tfh cells, in patients with active pSS. In this study, 57 active pSS patients and 52 age- and sex-matched healthy controls (HCs) were enrolled. Patients completed the European League Against Rheumatism (EULAR) Sjögren’s Syndrome Patient Reported Index (ESSPRI) to measure symptoms.[3] And the ESSPRI scores were divided into two categories, low (ESSPRI <5) and high (ESSPRI ≥5). Serological tests, including tests of antinuclear antibody (ANA), anti-Ro/SSA antibody (anti-SSA), anti-La/SSB antibody (anti-SSB), anti-α-fodrin antibody, rheumatoid factor (RF), complement 3 (C3), C4, immunoglobulin A (IgA), IgG, and IgM were performed in all patients. All clinical test were performed at the Clinical Laboratory at Peking University People’s Hospital. Liver insufficiency was defined if the alanine aminotransferase (ALT) and aspartate transferase (AST) of patients were abnormal. Peripheral blood was obtained from pSS patients and circulating Tfh (cTfh) cells were characterized using flow cytometry based on the expression of C-X-C motif chemokine receptor 5 (CXCR5) and programmed death protein-1 (PD-1). Additionally, the expression levels of the surface chemokine receptors, C-C motif chemokine receptor 6 (CCR6), and CXCR3 were used to classify cTfh into Tfh1 (CCR6−CXCR3+), Tfh2 (CCR6−CXCR3−), and Tfh17 (CCR6+CXCR3−).[4] Helper T (Th) cells were also detected including CCR7lowTh1, CCR7lowTh2, and CCR7lowTh17. Immune cell populations were compared between pSS patients and HCs using nonparametric Mann−Whitney test. Categorical variables were examined using the two-tailed Fisher’s exact test while correlations between immune cell populations and blood parameters or clinical manifestations were evaluated using Spearman’s correlation analysis. Statistical analyses were performed using SPSS v.22.0 software (SPSS Inc., Armonk, NY, USA). Two-sided P values <0.05 were considered statistically significant. The study was approved by the Ethical Committee of Peking University People’s Hospital (2020PHB359-01) and all participants provided written consent. Among patients with pSS, systemic manifestations included leukopenia (19/57, 33.3%), pulmonary (23/57, 40.4%) or articular involvement (18/57, 31.6%), and renal involvement (5/57, 8.8%), anemia (10/57, 17.5%), thrombocytopenia (11/57, 19.3%); parotid gland enlargement (25/57, 43.9%), and neurologic (2/57, 3.5%) and cutaneous involvement (1/57, 1.8%) [Supplementary Table 1, https://links.lww.com/CM9/C136]. Compared to HCs, pSS patients showed higher proportion of cTfh cells (P = 0.018, Figure 1A). Data also demonstrated that the proportions of CCR7lowTh17 cells were markedly increased in pSS patients as compared to HCs (P = 0.010), CCR7lowTh1 and CCR7lowTh2 shared the similar tendency despite reaching no significant difference [Supplementary Table 2, Figure 1B–D, https://links.lww.com/CM9/C136].Figure 1: Profiles of cTfh cells in patients with pSS. (A) The proportion of Tfh cells in pSS patients was higher than that in the control group. (B–D) the proportions of CCR7lowTh1, CCR7lowTh2, and CCR7lowTh17 cells were increased in pSS as compared to the healthy group. (E) A higher proportion of Tfh17 was associated with increased IgA. (F) CCR7lowTh2 increased in the presence of decreased C3. (G) The correlations between proportions of Tfh, Tfh1, Tfh2, Tfh17, Th1, Th2, Th17, Treg cells, and clinical characters in pSS patients; (H) Correlation of Tfh% (red), Tfh1% (black), Tfh2% (yellow), and Tfh17% (blue), and ESSDAI score in pSS patients. ANA: Anti-nucleosome antibodies; C3: Complement 3; CD: Cluster of differentiation; cTfh: Circulating Tfh; ESR: Erythrocyte sedimentation rate; ESSDAI: European League Against Rheumatism Sjögren’s Syndrome Disease Activity Index; HC: Healthy control; HGB: Hemoglobin; IgA: Immunoglobulin A; IgG: Immunoglobulin G; pSS: Primary Sjögren’s syndrome; Tfh: Follicular helper T; Th1: Helper T cell 1; Th2: Helper T cell 2; Th17: Helper T cell 17; Treg: Regulatory T cell.The correlations between cTfh subpopulations with other peripheral laboratory markers were analyzed, and results showed that there were positive correlations between white blood cell (WBC) count and cTfh (r = 0.348, P = 0.008), Tfh1 (r = 0.329, P = 0.012), and Tfh2 (r = 0.295, P = 0.026) cells, and negative correlations between Tfh2 cells and platelets (r = −0.330, P = 0.012) [Supplementary Table 3, https://links.lww.com/CM9/C136]. Besides, a positive correlation was found between Tfh17 cells and IgA levels (r = 0.291, P = 0.046, Figure 1E), and a negative correlation between C3 and CCR7lowTh2 cells (r = –0.392, P = 0.009, Figure 1F). In addition, there is a significant correlation between Tfh17 cells and the presence of anti-SSB antibodies [Figure 1G]. And the correlations between other T cell subsets and clinical characters in pSS patients are shown in Figure 1G. We hypothesized that Tfh subtypes serve as potent indicators in disease activation of pSS. The proportion of Tfh was positively associated with higher EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) scores [Tfh: P = 0.015, r2 = 0.3784; Figure 1H], as Tfh1 and Tfh17 shared the similar tendency in pSS patients, but it failed to reach statistical significance. The proportion of Tfh17 cells revealed a dramatic decline in patients with high ESSPRI scores compared to the low group (2.90 ± 1.68 vs. 4.32 ± 2.18, P = 0.023, Supplementary Table 4, https://links.lww.com/CM9/C136). In addition, Tfh, Tfh1, and Tfh17 cells were increased in patients with refractory glandular swelling [Supplementary Figure 1, https://links.lww.com/CM9/C136]. However, there was no significant association between Tfh cell populations and other clinical indicators studied [Supplementary Table 3, https://links.lww.com/CM9/C136]. The blood of pSS patients exhibited significant increase in the proportion of Tfh and Tfh17 cells Figure 1A and D. We also found a significant correlation between increased levels of circulating Tfh cells and laboratory parameters in pSS patients. Notably, as the levels of Tfh and Tfh17 cells increased, hemoglobin (HGB) levels showed a tendency to decrease [Figure 1G]. This suggests a close link between changes in Tfh cell subsets and disease activation in pSS patients. A significant correlation between Tfh17 cells and the presence of anti-SSB antibodies suggests that these specific cell subtypes, which are believed to be T effector cells, play a significant role in the progression and manifestation of pSS. These insights contribute to a better understanding of the complex immunological landscape of pSS and its implications for disease management. There still existed several limitations in our study. First, small sample size and disease spectrum diversity among our participants could conceal specific patterns. Second, the cross-sectional design of our study restricted our ability to comprehend the temporal dynamics and progression of immune changes in pSS. Third, lacking patients with other autoimmune disorders including SLE, rheumatoid arthritis (RA) also limits our ability to contextualize our findings within the broader spectrum of immune responses. Future studies with larger and more diverse cohorts and longitudinal follow-ups are needed to validate and expand upon our findings. In summary, our results draw the profile of circulating T cell subsets in pSS patients. The aberrant cTfh subpopulations were associated with the disease activity of pSS patients. These findings provide a novel insight into the potential therapeutic approach in clinical practice. Acknowledgements We acknowledge the patients, research nurses, and clinicians who supported this study. Funding This study was supported by grants from the National Key R&D Program of China (Nos. 2022YFE0131700 and 2022YFC3602000), the National Natural Science Foundation of China (No. 82071813), the Beijing Sci-Tech Program (No. Z191100006619114), and Clinical Medicine Plus X-Young scholars Project of Peking University (No. PKU2020LCXQ018). Conflicts of interest None.