Therapeutic Potential of Targeting Tfr/Tfh Cell Balance by Low-Dose-IL-2 in Active SLE: A Post-Hoc Analysis from a Double-Blind RCT Study

Therapeutic Potential of Targeting Tfr/Tfh Cell Balance by Low-Dose-IL-2 in Active SLE: A Post-Hoc Analysis from a Double-Blind RCT Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Therapeutic Potential of Targeting Tfr/Tfh Cell Balance by Low-Dose-IL-2 in Active SLE: A Post-Hoc Analysis from a Double-Blind RCT Study Miao Miao, Xian Xiao, Jiayi Tian, Yunzhi Zhufeng, Ruiling Feng, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-138443/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective: To investigate the regulation of T follicular regulatory (Tfr) and T follicular (Tfh) cell subtypes by low-dose IL-2 in systemic lupus erythematosus (SLE) in a randomized, double-blind, placebo-controlled clinical trial. Methods: A post-hoc analysis was performed in a randomized cohort of SLE patients (n=60) receiving low-dose IL-2 therapy (n=30) or placebo (n=30), along with standard of care treatment. The primary end point was attainment of SLE responder index-4 (SRI-4) at week 12 in the trial. Twenty three healthy controls were enrolled for T cell subsets detection at the same time with the trial. The t-stochastic neighbor embedding (tSNE) analysis of CD4 T subsets based on immune cells flow cytometry markers was performed to distinguish Tfh, Tfh1, Tfh2, Tfh17 and Tfr cell subsets. Results: Compared with HC, the frequency of Tfr (CXCR5 + PD-1 low Treg and CXCR5 + PD-1 high Treg) cells was significantly reduced, while the pro-inflammatory Tfh cells were increased in patients with SLE. The imbalanced Tfh cell was associated with several pathogenic factors (anti-dsDNA antibodies (r=0.309, P=0.027) and serum IL-17 (r=0.328, P=0.021)) and SLE Disease Activity Index (SLEDAI) score (r=0.273, P=0.052). Decreased CXCR5 + PD-1 low Treg/Tfh and CXCR5 + PD-1 low Treg/Tfh17 were both associated with increased immunoglobulin M (IgM) (r=-0.448, P=0.002 and r=-0.336, P=0.024 respectively). Efficacy of low-dose IL-2 therapy was associated with a restored Tfr/Tfh cell balance. Conclusion: These data supports the hypothesis that promotion of Tfr associated with decreased disease activities, and that low-dose IL-2 therapy can recover Tfr/Tfh immune balance. Trial registration number ClinicalTrials.gov Registries (NCT02465580). Rheumatology systemic lupus erythematosus T follicular helper cell T follicular regulatory cell low-dose interleukin-2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Key Messages Deregulation between Tfr and Tfh subsets associated with severity of SLE. Low-dose IL-2 therapy was efficient in patients with SLE. Low-dose IL-2 therapy elevates the Tfr/Tfh ratio, which might be a novel concept to design the therapeutic regimen. Introduction Systemic lupus erythematosus (SLE) is characterized by the breakdown of immune tolerance leading to auto-reactive immune responses and consequently, tissue and organ damages. Over the past decades, extensive studies on regulatory T (Treg) cells have revealed that these cells can maintain tolerance and regulate immune responses [ 1 , 2 ], while T follicular helper cells (Tfh) play an important role in the production of autoantibodies and pro-inflammatory cytokines in SLE [ 3 – 5 ]. Moreover, the imbalance of the immune response between pro-inflammatory and anti-inflammatory cells is central in SLE pathogenesis. T follicular regulatory (Tfr) cells share features with Tfh and conventional Treg cells, and can inhibit Tfh cells and germinal center (GC) responses with a significant impact in humoral immunity [ 6 – 8 ]. Previous studies have suggested that Tfr cells can be identified as CXCR5 + PD-1 low Treg and CXCR5 + PD1 high Treg according to the surface marker CXCR5 and programmed cell death protein 1 (PD-1) [ 9 – 13 ]. The function of the two subsets of Tfr remains unclear. Imbalance or disfunction of Tfr subsets may directly or indirectly affect B cells, leading to expansion of overactive B cells which contributes to various immune-related clinical diseases [ 14 , 15 ]. So far, the role of balance between Tfh and Tfr subsets in SLE is still controversial due to the heterogeneity of the disease, cohort size, and methods of studies [ 16 , 17 ]. Efficacious treatments, including low-dose Interleukin 2 (IL-2), might promote Tfr cell responses, and inhibit Tfh cell development in SLE [ 18 , 19 ]. However, it is not well understood how these circulating Tfh-like cell subsets, including Tfr and Tfh subsets, are involved in the disease. To date, the balance of these new subsets has not been addressed, neither how these cells respond to SLE treatment. Here, we identified an imbalanced profile of Tfh cell subsets in SLE, including newly described anti-inflammatory CXCR5 + PD-1 low Treg, CXCR5 + PD-1 high Treg and the pro-inflammatory Tfh and Tfh17; and investigated the change of these subtypes by low-dose IL-2 treatment in a randomized, double-blind, placebo-controlled study in SLE. Participants And Methods Participants This was a post-hoc analysis of data from an RCT clinical study (NCT02465580) of low-dose IL-2 in SLE patients. Full details of study designs and inclusion/exclusion criteria for each completed study have previously been published [ 1 , 2 ]. Studies were conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonization Guidelines for Good Clinical Practice. Besides, 23 healthy controls (HC) were enrolled during the same time of RCT study. Table 1 summarized baseline characteristics. Written informed consents were obtained from these healthy controls. The experimental protocol followed the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of Peking University People’s Hospital (Beijing, China). Table 1 Baseline characteristics of SLE patients and healthy controls (HC) in this study Characteristics SLE (n = 60) HC (n = 23) P value Age, year, mean ± SD 30.84 ± 9.48 29.83 ± 9.72 0.474 Female/Male 56/4 21/2 > 0.99 Duration, months, mean ± SD 65.15 ± 58.65 - - Medications - - Prednisone dose, mg/day, median (range) 13.5 (0, 50) - - Hydroxychloroquine 57 (95) - - Cyclophosphamide 4 (6.67) - - Azathioprine 5 (8.33) - - Cyclosporine 5 (8.33) - - Mycophenolate Mofetil 17 (28.33) - - Tacrolimus 2 (3.33) - - Leflunomide 4 (6.67) - - Thalidomide 1 (1.67) - - Methotrexate 1 (1.67) - - Interleukin-2 30(50) - - For a continuous variable, median (range) or mean ± SD. For a categorical variable, count (percentage). SLE, Systemic lupus erythematosus. Flow Cytometric Analysis Single-cell suspensions from peripheral blood in SLE patients and HC were analyzed by multicolor flow cytometry (FACSAria II; BD Biosciences, Franklin Lakes, NJ, USA). t-stochastic neighbor embedding (tSNE) analysis of CD4 T subsets based on immune cells flow cytometry markers was shown in Fig. 1 . Data were also analyzed using FlowJo v10 software (Tree Star, Ashland, OR, USA) (Figure S1). The absolute number of CD4 T cell subsets was calculated by multiplying proportion of CD4 T cell subsets in lymphocytes by absolute lymphocyte number determined with an automated hematology analyzer. Detailed protocol of trial has been published online [ 18 ]. Cytometric Beads Array (cba) Analysis Of Serum Cytokines Serum levels of IL-2, IL-17 and other inflammatory cytokines were determined by human Th1/Th2/Th17 14-plex (QuantoBio, Beijing, China) according to the manufacturer’s instructions. Statistical analysis Data were expressed as the median and range for non-normally distributed data, while mean ± standard deviation (SD.) for normally distributed data. The Student’s unpaired or paired t test was performed to compare two groups for parametric data, and the Mann-Whitney U test or Wilcoxon rank sum test was performed for nonparametric data. Relationships between variables were analyzed by Spearman’s rank test. Statistical analyses were performed using SPSS v.22.0 or R v.3.6.3 software. Two-sided P values < 0.1 were considered statistically significant. Results Characteristics of SLE patients Given recent studies showing the imbalances in the effector and regulatory Tfh cells compartment in SLE patients [3, 4], and in light of our finding that low-dose IL-2 treatment significantly influences Tfh subtypes, we recruited a small cohort of healthy controls (HC) (n=23, Table 1) for comparative analysis. The demographic and clinical manifestations of these patients were shown in Table 1. There was no significant difference between patients and HCs regarding age or gender. 98% of the SLE patients were positive in anti-dsDNA tests, 42% had renal involvement and 48% had skin manifestations. Imbalanced Tfr/Tfh in SLE As shown in Table 2, regulatory T cells including Treg, CXCR5 + PD-1 low Treg, CXCR5 + PD-1 high Treg cells were significantly reduced in SLE patients than those in HC (P=0.087, P=0.033, P<0.001, P<0.001, respectively). In contrast, the effector Tfh cells were significantly increased in SLE patients than in HC (p=0.081). Besides, Tfr subsets: Tfh subsets ratios in SLE were dramatically decreased, including CXCR5 + PD-1 low Treg/Tfh (P=0.043), CXCR5 + PD-1 high Treg/Tfh (p<0.001), CXCR5 + PD-1 low Treg/Tfh17 (P=0.052) and CXCR5 + PD-1 high Treg/Tfh17 (P<0.001). High Tfh associated with decreased serum IL-2 As show in Figure 2, an expanded inflammatory Tfh cell compartment was correlated with higher serum IL-17 levels (r=0.328, P=0.021) and increased frequency of Tfh17 correlated with lower serological IL-2 (r=-0.295, P=0.04) (Figure 2). There was a reduction of CXCR5/PD-1 double positive subset (CXCR5 + PD1 high Treg), which was related with IL-10 elevation (r=0.243, P=0.093, Figure 3). Table 2. Difference of CD4 T subsets between HC and SLE, and between active and remission group. Variables HC (n=23) SLE P , HC VS. Active P , Remission VS. Active Active (n=60) (Before therapy) Remission (n=59) (After therapy) Proportion (percentage in lymphocyte, %) Treg 1.22 (1, 1.47) 0.95 (0.65, 1.53) 1.6 (0.97, 2.49) 0.087 0.001 CXCR5 + PD-1 low Treg 0.09 (0.04, 0.14) 0.06 (0.03, 0.1) 0.13 (0.06, 0.25) 0.033 <0.001 CXCR5 + PD-1 high Treg 0.09 (0.07, 0.13) 0.02 (0.01, 0.04) 0.11 (0.06, 0.19) <0.001 <0.001 Tfh 0.28 (0.17, 0.43) 0.39 (0.24, 0.56) 0.19 (0.09, 0.37) 0.081 0.002 Tfh1 0.61 (0.38, 0.99) 0.2 (0.08, 0.36) 0.17 (0.09, 0.28) <0.001 0.42 Tfh2 1.06 (0.58, 1.5) 0.42 (0.21, 0.6) 0.36 (0.19, 0.68) <0.001 0.367 Tfh17 0.45 (0.15, 0.65) 0.47 (0.27, 0.87) 0.44 (0.2, 0.83) 0.345 0.851 Absolute number (cells/L) Treg 25.51 (19.98, 30.78) 11.94 (6, 20.81) 20.51 (9.17, 29.06) 0.035 0.014 CXCR5 + PD-1 low Treg 2.98 (1.71, 3.1) 0.67 (0.24, 1.21) 1.33 (0.8, 3.54) 0.037 0.005 CXCR5 + PD-1 high Treg 2.99 (1.18, 3.57) 0.24 (0.06, 0.56) 1.5 (0.71, 2.27) 0.001 <0.001 Tfh 7.29 (4.34, 8.92) 4.6 (2.58, 10.28) 2.84 (0.81, 6.04) 0.625 0.008 Tfh1 14.26 (12.37, 18.22) 2.11 (0.95, 5.47) 1.92 (0.68, 4.28) 0.002 0.427 Tfh2 19.31 (14.66, 24.22) 4.43 (2.11, 11.2) 4.04 (1.73, 11.88) 0.002 0.751 Tfh17 8.82 (7.25, 12.22) 5.39 (2.33, 14.38) 4.67 (2.47, 10.36) 0.642 0.664 Ratios Treg/Tfh 3.69 (3.04, 8.49) 2.49 (1.89, 4.5) 8.93 (4.04, 18.29) 0.002 <0.001 CXCR5 + PD-1 low Treg/Tfh 0.3 (0.21, 0.57) 0.16 (0.08, 0.31) 0.61 (0.4, 1.32) 0.002 <0.001 CXCR5 + PD-1 high Treg/Tfh 0.46 (0.21, 0.69) 0.05 (0.01, 0.1) 0.54 (0.24, 1.3) <0.001 <0.001 Treg/Tfh17 3.87 (2.27, 5.71) 2.29 (1.23, 4.21) 3.89 (2.01, 6.62) 0.047 <0.001 CXCR5 + PD-1 low Treg/Tfh17 0.25 (0.17, 0.34) 0.11 (0.05, 0.22) 0.28 (0.18, 0.52) 0.002 <0.001 CXCR5 + PD-1 high Treg/Tfh17 0.3 (0.14, 0.64) 0.03 (0.01, 0.09) 0.23 (0.12, 0.45) <0.001 <0.001 SLE, systemic lupus erythematosus. HC, healthy controls. Data are median (IQR). Imbalanced Tfh and Tfr cell association with disease activity in SLE Treg cells were decreased and associated with elevated ESR (r=-0.382, P<0.01) and Safety of Estrogens in Lupus Erythematosus National Assessment version of the SLE Disease Activity Index (SLEDAI) (r=-0.245, P=0.089, Figure 2). At the same time, the decrease of CXCR5 + PD-1 low Treg was associated with increased ESR and anti-dsDNA antibodies production (Figure 2, 3). In contrast, an increased inflammatory Tfh cell compartment was found and was correlated with elevated SLEDAI, titer of anti-AnuA, anti-dsDNA antibodies, serum IL-17 and decreased C3 (Figure 2, 3). Upon further analysis of the correlation between regulatory and effector subsets, we found decreased Treg/Tfh ratio in severe patients with higher SLEDAI score, higher titers of anti-AnuA and anti-dsDNA antibodies (Figure 3). In addition, there was a reduced CXCR5 + PD1 low Treg/Tfh in this group of severe patient. Figure 3 showed that the frequency of Tfh1 and Tfh2 was positively correlated with the number of total B cells and switched memory B (CD19 + IgD - CD27 + ) cells. CXCR5 + PD-1 low Treg/Tfh17 was negatively correlated with switched memory B cells and plasma B cells (r=-0.341, P=0.027, Figure 3). And decreased CXCR5 + PD-1 low Treg/Tfh and CXCR5 + PD-1 low Treg/Tfh17 were both associated with increased serum level of IgM. Low-dose IL-2 therapy increased Tfr/Tfh ratio in SLE patients In this RCT of low-dose IL-2 therapy in SLE, low-dose IL-2 significantly increased Tregs [10]. With effective treatment, British Isles Lupus Assessment Group (BILAG), SLEDAI, SLE Responder Index-4 (SRI-4), physician’s global assessment (PGA), myositis, fever, alopecia, vasculitis, arthritis, oral ulcer and rash were all improved at week 12 (Figure 4) [10]. After 3 cycles of low-dose IL-2 therapy, the frequency of CXCR5 + PD-1 low Treg cells and CXCR5 + PD-1 high Treg cells in lymphocyte was significantly increased at week 12 compared to placebo control (0.06 (0.03, 0.1) vs. 0.13 (0.06, 0.25), p <0.001 and 0.02 (0.01, 0.04) vs. 0.11 (0.06, 0.19), P<0.001 respectively) (Table 2, Figure 4). Similarly, the absolute number of these Treg cells (CXCR5 + PD-1 high Treg and CXCR5 + PD-1 low Treg) were significantly increased after the treatment of low-dose IL-2 (0.67 (0.24, 1.21) vs. 1.33 (0.8, 3.54), P=0.005 and 0.24 (0.06, 0.56) vs. 1.5 (0.71, 2.27), P<0.001 respectively) (Table 2, Figure 4). Besides, compared to baseline, Tfr subsets: Tfh subsets ratios in SLE were dramatically increased, including CXCR5 + PD-1 low Treg/Tfh (P<0.001), CXCR5 + PD-1 high Treg/Tfh (p<0.001), CXCR5 + PD-1 low Treg/Tfh17 (P<0.001) and CXCR5 + PD-1 high Treg/Tfh17 (P<0.001). Discussion Increasing evidence indicates that Tfh cells are important in the pathogenesis of SLE. Tfh cells are recognized as a distinct T-cell subset, which provides help for GC formation, B-cell affinity maturation, and immunoglobulin class switching, as an indispensable part of adaptive immunity. Our previous work showed that patients with SLE have an increased number of peripheral Tfh cells, which positively correlates with autoantibody titers (anti- dsDNA antibodies) and disease activity, as measured by the SLEDAI. Others have reported that the aberrant expression of Tfh cells is a common feature in mouse models of SLE, suggesting its contribution in the development of autoimmune diseases [ 4 , 5 ]. Besides Tfh cells, a subset of Treg cells, named Tfr cells, have been identified. These cells share common characteristics with Tfh and conventional Treg cells, and can inhibit GC responses, regulating the number of Tfh and GC B cells [ 6 – 8 ]. Therefore, it is generally believed that Tfr cells constrain the B-cell “help” provided by Tfh cells to maintain immune homeostasis. An aberrant or disordered Tfh/ Tfr balance may result in the break of tolerance, excessive B-cell proliferation, antibody production, and the development of autoimmune diseases. A recent study showed the importance of the Tfr/ Tfh balance in autoimmune responses in BXD2 mice, which display spontaneous autoreactive GC formation [ 21 ]. In addition, intravenous immunoglobulin administration to mice with collagen-induced arthritis augments the number of Tfr cells and represses the subsequent maturation of GC B cells [ 22 ], which also supportes the idea of a critical role for Tfr cells in autoimmune diseases. In our study, we found a deficiency of Tfr cell subsets, including CXCR5 + PD-1 low Treg, and CXCR5 + PD-1 high Treg; and increased Tfh cells in the peripheral blood of SLE patients. The shifted balance between circulating CXCR5 + PD-1 low Treg and Tfh cells correlated not only with reduced serum IL-2, IL-10 and increased IL-21 levels in patients; but also with clinical SLE parameters, e.g. ESR, anti-dsDNA antibodies and disease activity (SLEDAI Scores). These findings are consistent with previous studies in vitro, in which Tfh and CXCR5 + PD-1 low Treg or CXCR5 + PD1 high Treg cells can antagonize B cell function, production of high-affinity antibodies and the memory B cell differentiation [ 22 ]. CXCR5 + PD-1 low Treg cells play an important immunosuppressive function by curbing self-reactive auto-antibodies development within the GC during an inflammatory immune response [ 5 ]. Therefore, deregulation of the Tfr and Tfh cell compartments is associated with disease severity, B cell frequency and antibody production in SLE. There have been several relatively successful attempts to reduce the severity of SLE in humans via blockade of Tfh-cell differentiation and activity. Studies using monoclonal antibodies against ICOSL inhibited the development of Tfh and GC B cells resulting in decreased anti-dsDNA antibodies and improved kidney function in both human and mouse [ 23 ]. For years, SLE therapy has relied on broad spectrum immunosuppressants; however, a growing body of work shows that a targeted increase of regulatory T cells may be a more attractive therapy [ 18 , 19 , 24 , 25 ]. IL-2 is essential for the development and maintenance of Treg cells, which prevent the development of autoimmune disease. Low-dose IL-2 can promote Tregs by activating the transcription factor STAT5, which binds to the Foxp3 locus and promotes Foxp3 expression without activation of effector T cells. More recently, IL-2 has been shown to be essential for the inhibition of Tfh cell development. Thus, in this study, we asked if low-dose IL-2 therapy might also elevate the Tfr/Tfh ratio, exploring a novel concept for rational therapeutic design. Our previous studies had proven a deficient Treg cell compartment and decreased IL-2 levels in circulation of SLE, and the efficacy of low-dose IL-2 treatment. But there was no study addressing the impact of low-dose IL-2 on Tfr : Tfh balance. After effective therapy, especially low-dose IL-2 therapy, the imbalanced Tfr and Tfh subsets were reversed accompanying improvement of disease activity. Furthermore, Tfr subsets were all increased regardless of output measurement; proportion and absolute number. Although we didn’t see a significant change in Th17 frequency, the ratios of CXCR5 + PD-1 low Treg/Tfh17 and CXCR5 + PD-1 high Treg/Tfh17 were significantly decreased compared to those in healthy controls. Besides, we didn’t see any obvious difference between CXCR5 + PD-1 low Treg and CXCR5 + PD-1 high Treg, perhaps reflecting a functional overlap of these two subsets. Conclusions In summary, our findings indicate that imbalance of Tfh and Tfr is important for SLE severity; and low-dose IL-2 ameliorates lupus autoimmunity favoring Tfr cell expansion. Our study added to these findings by demonstrating that low-dose IL-2 therapy selectively activates and expands Tfr cells, while demonstrating clinical efficacy in SLE. Further studies are needed to better understand how to explore Tfh cell or Tfr cell signatures to stratify patients, and guide the design of novel treatment regiments for SLE in future clinical trials. Abbreviations Tfr: T follicular regulatory; Tfh: T follicular; IL-2: interleukin-2; SLE: systemic lupus erythematosus; tSNE: t-stochastic neighbor embedding; HC: healthy controls; IL-17: interleukin-17; SLEDAI: SLE Disease Activity Index; IgM: immunoglobulin M; Treg: regulatory T; GC: germinal center; SD: standard deviation; SRI-4: SLE responder index-4; Anti-AnuA: anti-nucleosome antibody; Anti-dsDNA: anti-double-stranded DNA antibody; C3: complement 3; BILAG: British Isles Lupus Assessment Group; PGA: physician’s global assessment; ESR: erythrocyte sedimentation rate. PD-1: programmed cell death protein 1. Declarations Acknowledgements We acknowledge the patients, research nurses and clinicians who have helped support this study. Author’s contributions JH had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. JH, XS, ZL and MM conceived of and designed the study. MM, XX, JT, YZ, RZ, RF, JC, XZ, BH and YJ acquired the data. MM, XX, JT, YZ and RF analyzed and interpreted the data. All authors were involved in drafting the article or making critical revisions for important intellectual content, and all authors read and approved the submitted manuscript. Funding The National Natural Science Foundation of China (31870879, 81971520), Peking-Tsinghua Center for Life Sciences, Peking University Clinical Scientist Program (BMU2019LCKXJ004) and Clinical Medicine Plus X-Young scholars Project of Peking University (PKU2020LCXQ018) supported by the Fundamental Research Funds for the Central Universities. Ethics approval and consent to participate This study was approved the Human Ethics Committee of Peking University People’s Hospital (Beijing, China) and all subjects provided written informed consent. Consent for publication Not applicable. Disclosure statement The authors have declared no conflicts of interest. 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Supplementary Files supplementaryfigure2020.11.29.pptx Cite Share Download PDF Status: Under Review Version 1 posted Review # 2 received at journal 19 Mar, 2021 Editorial decision: Major revision 19 Mar, 2021 Review # 1 received at journal 07 Mar, 2021 Reviewer # 2 agreed at journal 04 Mar, 2021 Reviewers invited by journal 04 Mar, 2021 Reviewer # 1 agreed at journal 04 Mar, 2021 Editor assigned by journal 20 Dec, 2020 Submission checks completed at journal 20 Dec, 2020 Editor invited by journal 20 Dec, 2020 First submitted to journal 17 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-138443","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":7295948,"identity":"ae1d20d0-33a7-471f-ac23-908c0c27e8c8","order_by":0,"name":"Miao Miao","email":"","orcid":"https://orcid.org/0000-0001-8927-4128","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Miao","suffix":""},{"id":7295949,"identity":"ce7addd7-5e45-40e6-8b95-4591d0ea607c","order_by":1,"name":"Xian Xiao","email":"","orcid":"","institution":"Peking University People's Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Xiao","suffix":""},{"id":7295950,"identity":"16e339db-e595-4853-b7b1-b3f7ae3866c2","order_by":2,"name":"Jiayi Tian","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Tian","suffix":""},{"id":7295951,"identity":"dc579099-51f8-423f-a95c-9b8c7f0277bf","order_by":3,"name":"Yunzhi Zhufeng","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunzhi","middleName":"","lastName":"Zhufeng","suffix":""},{"id":7295952,"identity":"e2fd8eb8-945c-47c7-bfbf-5a9d495a229a","order_by":4,"name":"Ruiling Feng","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruiling","middleName":"","lastName":"Feng","suffix":""},{"id":7295953,"identity":"6851afe3-e85b-431d-8164-39d2284ffb3e","order_by":5,"name":"Ruijun Zhang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruijun","middleName":"","lastName":"Zhang","suffix":""},{"id":7295954,"identity":"aa18e8ff-75a0-44a1-a5da-8ac765771962","order_by":6,"name":"Jiali Chen","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Chen","suffix":""},{"id":7295955,"identity":"5ca6a55b-0f62-4da5-8842-d6d8e1275a72","order_by":7,"name":"Xiaoying Zhang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Zhang","suffix":""},{"id":7295956,"identity":"41c04661-f9d2-4777-a040-d42187404925","order_by":8,"name":"Bo Huang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Huang","suffix":""},{"id":7295957,"identity":"6bc79d87-441a-40fa-afe5-16ff77fa14d0","order_by":9,"name":"Yuebo Jin","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuebo","middleName":"","lastName":"Jin","suffix":""},{"id":7295958,"identity":"65bf88ab-4156-439d-b577-22aa71bd13e2","order_by":10,"name":"Xiaolin Sun","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Sun","suffix":""},{"id":7295959,"identity":"c723a090-9e47-483a-917f-81306db3ebde","order_by":11,"name":"Jing He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie3PsQrCQAyA4bi0S0o3uaLQV6g4qCD4KldcKzh2koLiVJwrvoSjY8qBLudeqKDiCwguIh20g472RsH7t0A+SAB0uh/MA+AEwNBwBdE1VCfdps0MP02kGikL+06CbWHNFEjHjM5i/GDo5fGVrAhcu07fSS8mLpaLFzns1+RsoLVc8YrDMs6FFb8IjdbUksC9vIocT28SnMifq5AMuMA7QycJgFIlIsvDIoY2br00kkzhl50c3rCYDAxzerkVYd+1GxUEADnU5p+JVa2XmQRQqCzqdDrd3/YEkAlOkDz/MTYAAAAASUVORK5CYII=","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"He","suffix":""},{"id":7295960,"identity":"8acf30ad-b34b-47a2-8f5c-efdfcdb11c0e","order_by":12,"name":"Zhanguo Li","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhanguo","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2020-12-30 17:16:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-138443/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-138443/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4733127,"identity":"6ccbc6f9-a41a-4696-9e5f-7c29c7359507","added_by":"auto","created_at":"2021-01-05 23:31:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":260641,"visible":true,"origin":"","legend":"tSNE analysis of CD4 T subsets based on immune cells flow cytometry markers. tSNE, t-stochastic neighbor embedding.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-138443/v1/67e9f10a49d9b39bd7196871.png"},{"id":4733066,"identity":"57d1d48f-8ca2-4ee0-aec9-36a4c171ef0a","added_by":"auto","created_at":"2021-01-05 23:28:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69586,"visible":true,"origin":"","legend":"The correlations between CD4 T cell subsets and clinical characteristics in SLE patients. SLEDAI, SLE Disease Activity Index. ESR, erythrocyte sedimentation rate. C3, complement 3. C4, complement 4. IFN-α, interferon-α. IL-2, Interleukin-2. IL-21, Interleukin-21. IL-7, Interleukin-7. IFN-γ, interferon-γ. IL-17, Interleukin-17. IL-10, Interleukin-10. TGF-β, tumor necrosis factor-β. **, P\u003c0.01. *, P\u003c0.05. ▲, P\u003c0.1. #, absolute number. %, percentage in lymphocyte. ","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-138443/v1/4523d2b2f6da30e51fa3e735.png"},{"id":4733129,"identity":"4c808c42-5097-4782-b2ee-171b0efabe7b","added_by":"auto","created_at":"2021-01-05 23:31:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70241,"visible":true,"origin":"","legend":"Correlations between CD4 T cell subsets, B cells and immunoglobulins in SLE. B, B cell. Plasma B, Plasma B cell. Trans B, class-switched memory B cells. IgA, immunoglobulin A. IgG, immunoglobulin G. IgM, immunoglobulin M. ANuA, anti-nucleosome antibodies. dsDNA, Anti-double stranded DNA antibodies. *, P\u003c0.05. **,P\u003c0.01. ▲, P\u003c0.1. #, absolute number. %, percentage in LY. ","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-138443/v1/08a463dee135b2974520e0e2.png"},{"id":4733224,"identity":"0dce0749-fbd7-4c31-8e03-9a299dea599d","added_by":"auto","created_at":"2021-01-05 23:34:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102002,"visible":true,"origin":"","legend":"The response of clinical and immune cells to Low-dose IL-2 (n=30) and traditional (n=30) therapy in SLE. (A) Relative change of disease activity value and patient number. (B) Relative change of ratios at baseline and week 10. (C) Relative change of Treg subsets at baseline and week 10. (D) Relative change of Tfh subsets at baseline and week 10. #, absolute number. %, proportion in lymphocyte. BILAG, British Isles Lupus Assessment Group. SLEDAI, Safety of Estrogens in Lupus Erythematosus National Assessment version of the SLE Disease Activity Index. SRI-4, SLE Responder Index-4. PGA, physician’s global assessment. ","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-138443/v1/85fee3854085560034fb0b97.png"},{"id":13643236,"identity":"b348075d-ffc1-4a5f-ba57-388989a47e61","added_by":"auto","created_at":"2021-09-17 09:10:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":933712,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-138443/v1/114cc780-4dbb-4cd4-8f6b-ff3206d57104.pdf"},{"id":4733130,"identity":"c50db188-e841-453e-b5b2-a874f3315fa3","added_by":"auto","created_at":"2021-01-05 23:31:47","extension":"pptx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":188313,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure2020.11.29.pptx","url":"https://assets-eu.researchsquare.com/files/rs-138443/v1/e799e1d50f1e91bcd121f31b.pptx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTherapeutic Potential of Targeting Tfr/Tfh Cell Balance by Low-Dose-IL-2 in Active SLE: A Post-Hoc Analysis from a Double-Blind RCT Study\u003c/p\u003e","fulltext":[{"header":"Key Messages","content":"\u003col\u003e\n\u003cli\u003eDeregulation between Tfr and Tfh subsets associated with severity of SLE.\u003c/li\u003e\n\u003cli\u003eLow-dose IL-2 therapy was efficient in patients with SLE.\u003c/li\u003e\n\u003cli\u003eLow-dose IL-2 therapy elevates the Tfr/Tfh ratio, which might be a novel concept to design the therapeutic regimen.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":" \u003cp\u003eSystemic lupus erythematosus (SLE) is characterized by the breakdown of immune tolerance leading to auto-reactive immune responses and consequently, tissue and organ damages. Over the past decades, extensive studies on regulatory T (Treg) cells have revealed that these cells can maintain tolerance and regulate immune responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], while T follicular helper cells (Tfh) play an important role in the production of autoantibodies and pro-inflammatory cytokines in SLE [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, the imbalance of the immune response between pro-inflammatory and anti-inflammatory cells is central in SLE pathogenesis.\u003c/p\u003e \u003cp\u003eT follicular regulatory (Tfr) cells share features with Tfh and conventional Treg cells, and can inhibit Tfh cells and germinal center (GC) responses with a significant impact in humoral immunity [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have suggested that Tfr cells can be identified as CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003e Treg and CXCR5\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003ehigh\u003c/sup\u003e Treg according to the surface marker CXCR5 and programmed cell death protein 1 (PD-1) [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The function of the two subsets of Tfr remains unclear. Imbalance or disfunction of Tfr subsets may directly or indirectly affect B cells, leading to expansion of overactive B cells which contributes to various immune-related clinical diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. So far, the role of balance between Tfh and Tfr subsets in SLE is still controversial due to the heterogeneity of the disease, cohort size, and methods of studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEfficacious treatments, including low-dose Interleukin 2 (IL-2), might promote Tfr cell responses, and inhibit Tfh cell development in SLE [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, it is not well understood how these circulating Tfh-like cell subsets, including Tfr and Tfh subsets, are involved in the disease. To date, the balance of these new subsets has not been addressed, neither how these cells respond to SLE treatment. Here, we identified an imbalanced profile of Tfh cell subsets in SLE, including newly described anti-inflammatory CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg, CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg and the pro-inflammatory Tfh and Tfh17; and investigated the change of these subtypes by low-dose IL-2 treatment in a randomized, double-blind, placebo-controlled study in SLE.\u003c/p\u003e "},{"header":"Participants And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eThis was a post-hoc analysis of data from an RCT clinical study (NCT02465580) of low-dose IL-2 in SLE patients. Full details of study designs and inclusion/exclusion criteria for each completed study have previously been published [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies were conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonization Guidelines for Good Clinical Practice. Besides, 23 healthy controls (HC) were enrolled during the same time of RCT study. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarized baseline characteristics. Written informed consents were obtained from these healthy controls. The experimental protocol followed the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of Peking University People\u0026rsquo;s Hospital (Beijing, China).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of SLE patients and healthy controls (HC) in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSLE (n\u0026thinsp;=\u0026thinsp;60)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHC (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, year, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.84\u0026thinsp;\u0026plusmn;\u0026thinsp;9.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.83\u0026thinsp;\u0026plusmn;\u0026thinsp;9.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.474\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale/Male\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56/4\u003c/p\u003e \u003c/td\u003e 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\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrednisone dose, mg/day, median (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.5 (0, 50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydroxychloroquine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57 (95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclophosphamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (6.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzathioprine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (8.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclosporine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (8.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMycophenolate Mofetil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (28.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTacrolimus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (3.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeflunomide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (6.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThalidomide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethotrexate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInterleukin-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30(50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eFor a continuous variable, median (range) or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. For a categorical variable, count (percentage). SLE, Systemic lupus erythematosus.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eFlow Cytometric Analysis\u003c/h2\u003e\n \u003cp\u003eSingle-cell suspensions from peripheral blood in SLE patients and HC were analyzed by multicolor flow cytometry (FACSAria II; BD Biosciences, Franklin Lakes, NJ, USA). t-stochastic neighbor embedding (tSNE) analysis of CD4 T subsets based on immune cells flow cytometry markers was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Data were also analyzed using FlowJo v10 software (Tree Star, Ashland, OR, USA) (Figure S1). The absolute number of CD4 T cell subsets was calculated by multiplying proportion of CD4 T cell subsets in lymphocytes by absolute lymphocyte number determined with an automated hematology analyzer. Detailed protocol of trial has been published online [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003ch2\u003e Cytometric Beads Array (cba) Analysis Of Serum Cytokines\u003c/h2\u003e \u003cp\u003eSerum levels of IL-2, IL-17 and other inflammatory cytokines were determined by human Th1/Th2/Th17 14-plex (QuantoBio, Beijing, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as the median and range for non-normally distributed data, while mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD.) for normally distributed data. The Student\u0026rsquo;s unpaired or paired t test was performed to compare two groups for parametric data, and the Mann-Whitney U test or Wilcoxon rank sum test was performed for nonparametric data. Relationships between variables were analyzed by Spearman\u0026rsquo;s rank test. Statistical analyses were performed using SPSS v.22.0 or R v.3.6.3 software. Two-sided P values\u0026thinsp;\u0026lt;\u0026thinsp;0.1 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results ","content":"\u003cp\u003e\u003cstrong\u003eCharacteristics of SLE patients \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven recent studies showing the imbalances in the effector and regulatory Tfh cells compartment in SLE patients [3, 4], and in light of our finding that low-dose IL-2 treatment significantly influences Tfh subtypes, we recruited a small cohort of healthy controls (HC) (n=23, Table 1) for comparative analysis.\u003c/p\u003e\n\u003cp\u003eThe demographic and clinical manifestations of these patients were shown in Table 1. There was no significant difference between patients and HCs regarding age or gender. 98% of the SLE patients were positive in anti-dsDNA tests, 42% had renal involvement and 48% had skin manifestations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImbalanced Tfr/Tfh in SLE \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, regulatory T cells including Treg, CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg, CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg cells were significantly reduced in SLE patients than those in HC (P=0.087, P=0.033, P\u0026lt;0.001, P\u0026lt;0.001, respectively). In contrast, the effector Tfh cells were significantly increased in SLE patients than in HC (p=0.081). Besides, Tfr subsets: Tfh subsets ratios in SLE were dramatically decreased, including CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh (P=0.043), CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh (p\u0026lt;0.001), CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17 (P=0.052) and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh17 (P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh Tfh associated with decreased serum IL-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs show in Figure 2, an expanded inflammatory Tfh cell compartment was correlated with higher serum IL-17 levels (r=0.328, P=0.021) and increased frequency of Tfh17 correlated with lower serological IL-2 (r=-0.295, P=0.04) (Figure 2). There was a reduction of CXCR5/PD-1 double positive subset (CXCR5\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003ehigh\u003c/sup\u003eTreg), which was related with IL-10 elevation (r=0.243, P=0.093, Figure 3).\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\" width=\"546\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Difference of CD4 T subsets between HC and SLE, and between active and remission group. \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e\u003cstrong\u003eHC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(n=23)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" width=\"182\"\u003e\n\u003cp\u003e\u003cstrong\u003eSLE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"46\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHC VS. Active\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"61\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e, Remission VS. Active\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u003cstrong\u003eActive (n=60)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Before therapy)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e\u003cstrong\u003eRemission (n=59)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(After therapy)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\" width=\"546\"\u003e\n\u003cp\u003e\u003cstrong\u003eProportion (percentage in lymphocyte, %)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Treg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.22 (1, 1.47)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.95 (0.65, 1.53)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e1.6 (0.97, 2.49)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e0.087\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.09 (0.04, 0.14)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.06 (0.03, 0.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.13 (0.06, 0.25)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e0.033\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.09 (0.07, 0.13)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.02 (0.01, 0.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.11 (0.06, 0.19)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.28 (0.17, 0.43)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.39 (0.24, 0.56)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.19 (0.09, 0.37)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e0.081\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.61 (0.38, 0.99)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.2 (0.08, 0.36)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.17 (0.09, 0.28)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.42\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh2\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.06 (0.58, 1.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.42 (0.21, 0.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.36 (0.19, 0.68)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.367\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh17\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.45 (0.15, 0.65)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.47 (0.27, 0.87)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.44 (0.2, 0.83)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e0.345\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.851\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\" width=\"546\"\u003e\n\u003cp\u003e\u003cstrong\u003eAbsolute number (cells/L)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Treg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e25.51 (19.98, 30.78)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e11.94 (6, 20.81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e20.51 (9.17, 29.06)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e2.98 (1.71, 3.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.67 (0.24, 1.21)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e1.33 (0.8, 3.54)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.037\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e2.99 (1.18, 3.57)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.24 (0.06, 0.56)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e1.5 (0.71, 2.27)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e7.29 (4.34, 8.92)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e4.6 (2.58, 10.28)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e2.84 (0.81, 6.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.625\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e14.26 (12.37, 18.22)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e2.11 (0.95, 5.47)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e1.92 (0.68, 4.28)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.427\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh2\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e19.31 (14.66, 24.22)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e4.43 (2.11, 11.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e4.04 (1.73, 11.88)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.751\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Tfh17\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e8.82 (7.25, 12.22)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e5.39 (2.33, 14.38)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e4.67 (2.47, 10.36)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.642\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.664\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003eRatios\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Treg/Tfh\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e3.69 (3.04, 8.49)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e2.49 (1.89, 4.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e8.93 (4.04, 18.29)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.3 (0.21, 0.57)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.16 (0.08, 0.31)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e0.61 (0.4, 1.32)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.46 (0.21, 0.69)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.05 (0.01, 0.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e0.54 (0.24, 1.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Treg/Tfh17\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e3.87 (2.27, 5.71)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e2.29 (1.23, 4.21)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e3.89 (2.01, 6.62)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.047\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.25 (0.17, 0.34)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.11 (0.05, 0.22)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e0.28 (0.18, 0.52)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh17\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.3 (0.14, 0.64)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.03 (0.01, 0.09)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"87\"\u003e\n\u003cp\u003e0.23 (0.12, 0.45)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"46\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\" width=\"546\"\u003e\n\u003cp\u003eSLE, systemic lupus erythematosus. HC, healthy controls. Data are median (IQR).\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eImbalanced Tfh and Tfr cell association with disease activity in SLE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreg cells were decreased and associated with elevated ESR (r=-0.382, P\u0026lt;0.01) and Safety of Estrogens in Lupus Erythematosus National Assessment version of the SLE Disease Activity Index (SLEDAI) (r=-0.245, P=0.089, Figure 2). At the same time, the decrease of CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg was associated with increased ESR and anti-dsDNA antibodies production (Figure 2, 3). In contrast, an increased inflammatory Tfh cell compartment was found and was correlated with elevated SLEDAI, titer of anti-AnuA, anti-dsDNA antibodies, serum IL-17 and decreased C3 (Figure 2, 3).\u003c/p\u003e\n\u003cp\u003eUpon further analysis of the correlation between regulatory and effector subsets, we found decreased Treg/Tfh ratio in severe patients with higher SLEDAI score, higher titers of anti-AnuA and anti-dsDNA antibodies (Figure 3). In addition, there was a reduced CXCR5\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh in this group of severe patient.\u003c/p\u003e\n\u003cp\u003eFigure 3 showed that the frequency of Tfh1 and Tfh2 was positively correlated with the number of total B cells and switched memory B (CD19\u003csup\u003e+\u003c/sup\u003eIgD\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e) cells. CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17 was negatively correlated with switched memory B cells and plasma B cells (r=-0.341, P=0.027, Figure 3). And decreased CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17 were both associated with increased serum level of IgM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLow-dose IL-2 therapy increased \u003c/strong\u003e\u003cstrong\u003eTfr/Tfh ratio in SLE patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this RCT of low-dose IL-2 therapy in SLE, low-dose IL-2 significantly increased Tregs [10]. With effective treatment, British Isles Lupus Assessment Group (BILAG), SLEDAI, SLE Responder Index-4 (SRI-4), physician\u0026rsquo;s global assessment (PGA), myositis, fever, alopecia, vasculitis, arthritis, oral ulcer and rash were all improved at week 12 (Figure 4) [10]. After 3 cycles of low-dose IL-2 therapy, the frequency of CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg cells and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg cells in lymphocyte was significantly increased at week 12 compared to placebo control (0.06 (0.03, 0.1) vs. 0.13 (0.06, 0.25), \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 and 0.02 (0.01, 0.04) vs. 0.11 (0.06, 0.19), P\u0026lt;0.001 respectively) (Table 2, Figure 4). Similarly, the absolute number of these Treg cells (CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg) were significantly increased after the treatment of low-dose IL-2 (0.67 (0.24, 1.21) vs. 1.33 (0.8, 3.54), P=0.005 and 0.24 (0.06, 0.56) vs. 1.5 (0.71, 2.27), P\u0026lt;0.001 respectively) (Table 2, Figure 4). Besides, compared to baseline, Tfr subsets: Tfh subsets ratios in SLE were dramatically increased, including CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh (P\u0026lt;0.001), CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh (p\u0026lt;0.001), CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17 (P\u0026lt;0.001) and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh17 (P\u0026lt;0.001).\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eIncreasing evidence indicates that Tfh cells are important in the pathogenesis of SLE. Tfh cells are recognized as a distinct T-cell subset, which provides help for GC formation, B-cell affinity maturation, and immunoglobulin class switching, as an indispensable part of adaptive immunity. Our previous work showed that patients with SLE have an increased number of peripheral Tfh cells, which positively correlates with autoantibody titers (anti- dsDNA antibodies) and disease activity, as measured by the SLEDAI. Others have reported that the aberrant expression of Tfh cells is a common feature in mouse models of SLE, suggesting its contribution in the development of autoimmune diseases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides Tfh cells, a subset of Treg cells, named Tfr cells, have been identified. These cells share common characteristics with Tfh and conventional Treg cells, and can inhibit GC responses, regulating the number of Tfh and GC B cells [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, it is generally believed that Tfr cells constrain the B-cell \u0026ldquo;help\u0026rdquo; provided by Tfh cells to maintain immune homeostasis. An aberrant or disordered Tfh/ Tfr balance may result in the break of tolerance, excessive B-cell proliferation, antibody production, and the development of autoimmune diseases. A recent study showed the importance of the Tfr/ Tfh balance in autoimmune responses in BXD2 mice, which display spontaneous autoreactive GC formation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, intravenous immunoglobulin administration to mice with collagen-induced arthritis augments the number of Tfr cells and represses the subsequent maturation of GC B cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which also supportes the idea of a critical role for Tfr cells in autoimmune diseases.\u003c/p\u003e \u003cp\u003eIn our study, we found a deficiency of Tfr cell subsets, including CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg, and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg; and increased Tfh cells in the peripheral blood of SLE patients. The shifted balance between circulating CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg and Tfh cells correlated not only with reduced serum IL-2, IL-10 and increased IL-21 levels in patients; but also with clinical SLE parameters, e.g. ESR, anti-dsDNA antibodies and disease activity (SLEDAI Scores). These findings are consistent with previous studies in vitro, in which Tfh and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg or CXCR5\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003ehigh\u003c/sup\u003eTreg cells can antagonize B cell function, production of high-affinity antibodies and the memory B cell differentiation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg cells play an important immunosuppressive function by curbing self-reactive auto-antibodies development within the GC during an inflammatory immune response [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, deregulation of the Tfr and Tfh cell compartments is associated with disease severity, B cell frequency and antibody production in SLE.\u003c/p\u003e \u003cp\u003eThere have been several relatively successful attempts to reduce the severity of SLE in humans via blockade of Tfh-cell differentiation and activity. Studies using monoclonal antibodies against ICOSL inhibited the development of Tfh and GC B cells resulting in decreased anti-dsDNA antibodies and improved kidney function in both human and mouse [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For years, SLE therapy has relied on broad spectrum immunosuppressants; however, a growing body of work shows that a targeted increase of regulatory T cells may be a more attractive therapy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-2 is essential for the development and maintenance of Treg cells, which prevent the development of autoimmune disease. Low-dose IL-2 can promote Tregs by activating the transcription factor STAT5, which binds to the Foxp3 locus and promotes Foxp3 expression without activation of effector T cells. More recently, IL-2 has been shown to be essential for the inhibition of Tfh cell development. Thus, in this study, we asked if low-dose IL-2 therapy might also elevate the Tfr/Tfh ratio, exploring a novel concept for rational therapeutic design.\u003c/p\u003e \u003cp\u003eOur previous studies had proven a deficient Treg cell compartment and decreased IL-2 levels in circulation of SLE, and the efficacy of low-dose IL-2 treatment. But there was no study addressing the impact of low-dose IL-2 on Tfr : Tfh balance. After effective therapy, especially low-dose IL-2 therapy, the imbalanced Tfr and Tfh subsets were reversed accompanying improvement of disease activity. Furthermore, Tfr subsets were all increased regardless of output measurement; proportion and absolute number. Although we didn\u0026rsquo;t see a significant change in Th17 frequency, the ratios of CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17 and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg/Tfh17 were significantly decreased compared to those in healthy controls. Besides, we didn\u0026rsquo;t see any obvious difference between CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg, perhaps reflecting a functional overlap of these two subsets.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn summary, our findings indicate that imbalance of Tfh and Tfr is important for SLE severity; and low-dose IL-2 ameliorates lupus autoimmunity favoring Tfr cell expansion. Our study added to these findings by demonstrating that low-dose IL-2 therapy selectively activates and expands Tfr cells, while demonstrating clinical efficacy in SLE. Further studies are needed to better understand how to explore Tfh cell or Tfr cell signatures to stratify patients, and guide the design of novel treatment regiments for SLE in future clinical trials.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cp\u003eTfr: T follicular regulatory; Tfh: T follicular; IL-2: interleukin-2; SLE: systemic lupus erythematosus; tSNE: t-stochastic neighbor embedding; HC: healthy controls; IL-17: interleukin-17; SLEDAI: SLE Disease Activity Index; IgM: immunoglobulin M; Treg: regulatory T; GC: germinal center; SD: standard deviation; SRI-4: SLE responder index-4; Anti-AnuA: anti-nucleosome antibody; Anti-dsDNA: anti-double-stranded DNA antibody; C3: complement 3; BILAG: British Isles Lupus Assessment Group; PGA: physician\u0026rsquo;s global assessment; ESR: erythrocyte sedimentation rate. PD-1: programmed cell death protein 1.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the patients, research nurses and clinicians who have helped support this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJH had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. JH, XS, ZL and MM conceived of and designed the study. MM, XX, JT, YZ, RZ, RF, JC, XZ, BH and YJ acquired the data. MM, XX, JT, YZ and RF analyzed and interpreted the data. All authors were involved in drafting the article or making critical revisions for important intellectual content, and all authors read and approved the submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Natural Science Foundation of China (31870879, 81971520), Peking-Tsinghua Center for Life Sciences, Peking University Clinical Scientist Program (BMU2019LCKXJ004) and Clinical Medicine Plus X-Young scholars Project of Peking University (PKU2020LCXQ018) supported by the Fundamental Research Funds for the Central Universities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved the Human Ethics Committee of Peking University People\u0026rsquo;s Hospital (Beijing, China) and all subjects provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Department of Rheumatology \u0026amp; Immunology, Peking University People's Hospital, Beijing, 100044, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSakaguchi S, Yamaguchi T, Nomura T, Ono M. 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Circulating T follicular regulatory and helper cells have memory-like properties. J Clin Invest. 2014;124:5191\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, et al. The ratio of circulating follicular T helper cell to follicular T regulatory cell is correlated with disease activity in systemic lupus erythematosus. Clin Immunol. 2017;183:46\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, et al. Increased circulating CD4 + CXCR5 + FoxP3 + follicular regulatory T cells correlated with severity of systemic lupus erythematosus patients. Int Immunopharmacol. 2018;56:261\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe J, Zhang R, Shao M, et al. Efficacy and safety of low-dose IL-2 in the treatment of systemic lupus erythematosus: a randomised, double-blind, placebo-controlled trial. Ann Rheum Dis. 2020;79(1):141\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe J, Zhang X, Wei Y, Sun X, Chen Y, et al. Low-dose interleukin-2 treatment selectively modulates CD4 + T cell subsets in patients with systemic lupus erythematosus. Nat Med. 2016;22:991\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurie RA, Petri MA, Wallace DJ, et al. Novel evidence-based systemic lupus erythematosus Responder index. Arthritis Rheum. 2009;61:1143\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Y, Li J, Yang P, Luo B, Wu Q, et al. Interleukin-21 promotes germinal center reaction by skewing the follicular regulatory T cell to follicular helper T cell balance in autoimmune BXD2 mice. Arthritis Rheumatol. 2014;66:2601\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SY, Jung YO, Ryu JG, et al. 2014. Intravenous immunoglobulin attenuates experimental autoimmune arthritis by inducing reciprocal regulation of Th17 and Treg cells in an interleukin-10-dependent manner. Arthritis Rheumatol. 2014;66:1768-78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin L, Waseem TC, Sahoo A, Bieerkehazhi S, Zhou H, Galkina EV, et al. Insights Into the Molecular Mechanisms of T Follicular Helper-Mediated Immunity and Pathology. Front Immunol. 2018;9:1884.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenzwajg M, Lorenzon R, Cacoub P, Pham HP, Pitoiset F, et al. Immunological and clinical effects of low-dose interleukin-2 across 11autoimmune diseases in a single, open clinical trial. Ann Rheum Dis. 2019;78:209\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHumrich JY, Spee-Mayer C, Siegert E, Bertolo M, Rose A, et al. Low-dose interleukin-2 therapy in refractory systemic lupus erythematosus: an investigator-initiated, single-centre phase 1 and 2a clinical trial. The Lancet Rheumatology. 2019;1(1):e44\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"arthritis-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arrt","sideBox":"Learn more about [Arthritis Research \u0026 Therapy](http://arthritis-research.biomedcentral.com/)","snPcode":"13075","submissionUrl":"https://submission.nature.com/new-submission/13075/3","title":"Arthritis Research \u0026 Therapy","twitterHandle":"@ArthritisRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"systemic lupus erythematosus, T follicular helper cell, T follicular regulatory cell, low-dose interleukin-2","lastPublishedDoi":"10.21203/rs.3.rs-138443/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-138443/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To investigate the regulation of T follicular regulatory (Tfr) and T follicular (Tfh) cell subtypes by low-dose IL-2 in systemic lupus erythematosus (SLE) in a randomized, double-blind, placebo-controlled clinical trial.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA post-hoc analysis was performed in a randomized cohort of SLE patients (n=60) receiving low-dose IL-2 therapy (n=30) or placebo (n=30), along with standard of care treatment. The primary end point was attainment of SLE responder index-4 (SRI-4) at week 12 in the trial. Twenty three healthy controls were enrolled for T cell subsets detection at the same time with the trial. The t-stochastic neighbor embedding (tSNE) analysis of CD4 T subsets based on immune cells flow cytometry markers was performed to distinguish Tfh, Tfh1, Tfh2, Tfh17 and Tfr cell subsets.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCompared with HC, the frequency of Tfr (CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003eTreg) cells was significantly reduced, while the pro-inflammatory Tfh cells were increased in patients with SLE. The imbalanced Tfh cell was associated with several pathogenic factors (anti-dsDNA antibodies (r=0.309, P=0.027) and serum IL-17 (r=0.328, P=0.021)) and SLE Disease Activity Index (SLEDAI) score (r=0.273, P=0.052). Decreased CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh and CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003elow\u003c/sup\u003eTreg/Tfh17 were both associated with increased immunoglobulin M (IgM) (r=-0.448, P=0.002 and r=-0.336, P=0.024 respectively). Efficacy of low-dose IL-2 therapy was associated with a restored Tfr/Tfh cell balance.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThese data supports the hypothesis that promotion of Tfr associated with decreased disease activities, and that low-dose IL-2 therapy can recover Tfr/Tfh immune balance.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTrial registration number\u003c/strong\u003e ClinicalTrials.gov Registries (NCT02465580).\u003c/p\u003e","manuscriptTitle":"Therapeutic Potential of Targeting Tfr/Tfh Cell Balance by Low-Dose-IL-2 in Active SLE: A Post-Hoc Analysis from a Double-Blind RCT Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-05 23:28:45","doi":"10.21203/rs.3.rs-138443/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-03-20T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2021-03-20T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-08T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-03-05T01:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-03-05T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-03-05T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-21T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-20T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-20T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-12-18T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"arthritis-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arrt","sideBox":"Learn more about [Arthritis Research \u0026 Therapy](http://arthritis-research.biomedcentral.com/)","snPcode":"13075","submissionUrl":"https://submission.nature.com/new-submission/13075/3","title":"Arthritis Research \u0026 Therapy","twitterHandle":"@ArthritisRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"abe765c7-2cab-499f-8177-b81aa2e8e162","owner":[],"postedDate":"January 5th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1745772,"name":"Rheumatology"}],"tags":[],"updatedAt":"2021-05-21T20:53:55+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-05 23:28:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-138443","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-138443","identity":"rs-138443","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}