Mesenchymal Stromal Cells Attenuate Primary Sjogren's Syndrome by Modulating PD-1+CXCR5− T Peripheral Helper Cells via Galectin-1

Mesenchymal Stromal Cells Attenuate Primary Sjogren's Syndrome by Modulating PD-1+CXCR5− T Peripheral Helper Cells via Galectin-1 | 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 Mesenchymal Stromal Cells Attenuate Primary Sjogren's Syndrome by Modulating PD-1+CXCR5− T Peripheral Helper Cells via Galectin-1 Shiyi Zhang, Fanzhang Yin, Rui Chai, Tao Li, Yingying Gao, Xiaoxiang Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6173991/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Oct, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Objective To investigate the role of PD-1 + CXCR5 - peripheral helper T (Tph) cells in patients with primary Sjogren's syndrome (pSS) and to assess the therapeutic efficacy of mesenchymal stromal cells (MSC) in pSS patients through modulating Tph cells. Methods We measured the frequencies and numbers of T cell subsets, including Tph cells, follicular helper T (Tfh) cells, and Treg cells, as well as the levels of IL-21 in patients with pSS and healthy controls (HC). Additionally, we analyzed their correlations with the levels of serological indicators (IgG, C3, C4) and the score of the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). Immunofluorescence technique was employed to assess the infiltration of lymphocytes in labial gland tissues. Before and after mesenchymal stromal cell transplantation (MSCT). The alterations in Tph, Tfh, and Treg cell subsets were -evaluated using Fluorescence-activated cell sorting (FACS). Meanwhile, proteomic analysis of peripheral blood samples was conducted to identify the key proteins associated with Tph cells, and these proteins were subsequently validated in vitro. Results Compared with CXCR5 - PD-1 + CD4 + Tph cells and the Tph/Treg ratio were significantly higher in pSS patients than in the HC group. The proportion of circulating Tph cells and Tph/Treg ratio were significantly positively correlated with the ESSDAI score. MSC treatment effectively increased the levels of C3 and C4, while reducing the levels of IgG, the EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI) score, and the ESSDAI score. MSC significantly reduced the proportion of Tph cells and the Tph/Treg ratio, while increased the proportion of Treg cells, which contributed to restoring immune homeostasis. Proteomic analysis and co-culture experiments of MSC and peripheral blood mononuclear cells (PBMC) indicated that MSC may ameliorate the immune imbalance in pSS patients by up-regulating galectin-1 (Gal-1), thereby inhibiting Tph cell-associated inflammatory responses. Conclusion Our study established a link between increased Tph cell counts and increased Tph/Treg ratios with enhanced disease activity in pSS patients. MSC therapy, which reduces the number of Tph cells by inducing the expression of galectin-1, emerges as a promising therapeutic approach for pSS. Primary Sjögren's syndrome Mesenchymal stromal (stem) cell peripheral helper T cell IL-21 Galectin-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Primary Sjögren’s syndrome (pSS) manifests as a chronic autoimmune disorder characterized by the preferential involvement of lacrimal and salivary glands, resulting in the cardinal symptoms of dry mouth and dry eye[ 1 , 2 ]. The pathogenesis involves of pSS extensive lymphocytic infiltration that disrupts gland function[ 3 ]. At present, the complex interplay of immune dysregulation, B cell hyperactivity, and autoantibody production, are pivotal in pSS pathogenesis[ 4 ]. The precise mechanisms have not been fully elucidated. Follicular helper T (Tfh) cells, a subset of CD4 + T cells that express inducible T-cell co-stimulator (ICOS), PD-1, and CXCR5[ 5 , 6 ], are implicated in the progression of pSS[ 7 , 8 ]. Tfh cells support effector B cells and augment autoimmunity[ 9 ] by producing interleukin-21 (IL-21) and other cytokines[ 10 , 11 ]. It has been proved that Tfh cells are associated with the titer of anti-SSA, anti-SSB antibodies [ 12 ]. Tph, a novel subset of helper T cells, which distinct from Tfh, has been identified in the synovial fluid (SF) and peripheral blood of patients with rheumatoid arthritis (RA)[ 13 ]. Like Tfh cells, Tph cells express CXCL13, IL-21, CD40L, and ICOS but lack CXCR5 and exhibit low B-cell lymphoma 6 (Bcl-6) expression [ 14 ].Additionally, Tph cells express the chemokine receptors CCR2, CCR5, CXCR3 and CX3CR, which are crucial for their migration to inflammatory sites[ 15 ]. Circulating Tph (cTph) cells have been proven to be associated with various autoimmune diseases, including RA[ 13 ], systemic lupus erythematosus(SLE)[ 16 ], IgG4-related disease (IgG4-RD)[ 17 ], type 1 diabetes (T1D) [ 18 ] and pSS[ 19 ].cTph cells drive B-cells hyperactivity[ 20 , 21 ], contributing to the pathogenesis of these diseases. However, the potential of therapeutic interventions targeting Tph cells to ameliorate the disease has not been fully explored. In contrast, regulatory T (Treg) cells appear to inhibit B-cell responses by suppressing the activities of Tfh or Tph cells, which prevent the development of pSS[ 22 ]. Therefore, restoring the balance among Tph, Treg and Tph cells may be an effective measure for the treatment of pSS. Mesenchymal stromal (stem) cells (MSC) have been shown to possess remarkable immunomodulatory properties by interacting with a variety of immune cell populations, such as Tfh cells and Treg cells [ 23 , 24 ]. MSC can mitigate the aberrant activation of immune cells that drive the disease progression, thereby reducing the inflammatory burden on the salivary and lacrimal glands[ 25 ], which has emerged as a promising treatment for pSS patients[ 26 ]. Moreover, MSC promote the repair and regeneration of damaged glandular tissue, consequently improving glandular function and alleviating the symptoms of dryness [ 27 ]. Preliminary studies in animal models of pSS have revealed a decrease in lymphocytic infiltration, a reduction in pro-inflammatory cytokines, and an enhancement of glandular function after MSC treatment [ 23 ]. A clinical study involving 24 pSS patients has confirmed the therapeutic efficacy of MSC treatment, with improvements in salivary flow rate, alleviation of oral dryness, and a decrease in anti-SSA/anti-SSB levels, all without serious adverse events[ 28 ]. Although the effects of MSC in pSS patients were being explored, the specific mechanisms by which MSC regulate Tph cells in these patients are yet to be fully investigated. This study investigates the roles of Tph, Tfh, and Treg cells, and the Tph/Treg ratio in pSS pathogenesis, and the impact of MSC therapy on Tph cells and the Tph/Treg ratio. Materials and Methods Patients and Controls From September 2020 to December 2023, hospitalized pSS patients were selected from the Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, affiliated with Nanjing University Medical School. Among them, 10 patients underwent MSC transplantation. All participants fulfilled the revised 2002 American–European criteria [ 29 ], with exclusions for other autoimmune diseases. The disease activity was assessed using the ESSDAI scores[ 30 ]. The European Sjögren's Syndrome Patient Reporting Index (ESSPRI) was used to measure the severity of symptoms[ 31 ]. The demographic and clinical characteristics of pSS patients are presented in Supplementary Table 1. Informed consent was obtained from all participants. The study was approved by the Ethics Committee of Nanjing Drum Tower Hospital, affiliated with Nanjing University Medical School, in compliance with the Declaration of Helsinki. After MSC transplantation, patients were followed up for 6 months, with clinical evaluations performed at 6 months post-treatment. ESSDAI and ESSPRI scores were measured pre-MSCT and at the 6-month follow-up visit to assess changes in disease activity and symptom severity. The primary outcome measures included the changes in ESSDAI scores, the Tph/Treg ratio, and other immune cell subsets pre-MSCT and post-MSCT, as well as the correlation between these immune parameters and clinical outcomes. Flow Cytometry Peripheral blood mononuclear cells (PBMC) were extracted from pSS patients and HC. The following fluorescence-conjugated mouse anti-human antibodies (BioLegend, San Diego, CA, USA) were applied on a FACS Calibur cytometer with Diva software (BD Biosciences; San Jose, CA, USA): BV510 anti-CD4, APC anti-CXCR5, BV421 anti-PD1, BV605 anti-CCR2, BV711 anti-CD25, PE anti-FOXP3, PerCP/Cyanine5.5 anti-lL-21, PE anti-CD19, AF488 anti-CD27 and PerCP/Cyanine5.5 anti-CD38, or relevant isotype controls. Data analysis was conducted using FlowJo 10.4. Quantitative Proteomics A total of 8 serum samples from pSS patients, as well as their serum collected 24 hours post-MSCT, were collected for proteomic analysis. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Tandem Mass Tags (TMT) are popular labeling technologies for quantitative proteomics. They label peptides by combining with the amino group of amino acid terminals and lysine residues, allowing comparison between samples labeled with reagents of different molecular weights. The workflow includes sample preparation, protein hydrolysis, High-Performance Liquid Chromatography (HPLC) classification, Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) detection, database comparison, and bioinformatics analysis. Immunofluorescent Staining of Tissue Labial gland specimens from 12 pSS patients were sectioned into 4–5 µm slices, deparaffinized, and rehydrated. Antigen retrieval was done using citrate buffer and microwave heating. Sections were blocked by PBS with 1% BSA, then incubated with primary antibodies (CD4, CCR2, CXCR5, FOXP3) at 4°C. After washed with PBS, fluorescently labeled secondary antibodies were added and incubated in the dark. Nuclear staining was performed with DAPI, followed by PBS washes. Sections were mounted with anti-fade reagent and examined using a fluorescence microscope. Images were analyzed with ImageJ to quantify marker intensity and distribution. Coculture of MSC and PBMC The membrane used had a pore size of 0.4 µm (Corning, USA) to prevent cell passage. PBMC were cultured in the lower chamber of a 24-well plate with RPMI-1640, 1×10 6 PBMC, anti-CD3 (1µg/ml), and anti-CD28 (0.5µg/ml) at 37℃ and 5% CO2 for 2 hours. The Transwell chamber was then added, and 100 µL of liquid was placed in the upper chamber. Experimental groups included blank medium, DF12 with MSC (1×10 5 /well), Gal-1 inhibitor Thiodigalactoside (TDG) (50µM), and Gal-1 recombinant protein (3µM). After 3 days of incubation at 37℃ and 5% CO2, PBMC and the supernatant from the lower chamber were collected. Enzyme-Linked Immunosorbent Assay Equilibrate strips to room temperature. Add 100 µL of samples or standards, incubate at 37°C for 40 minutes, and wash five times. Add 50 µL of antibody solution, incubate at 37°C for 20 minutes, and wash five times. Add 100 µL of enzyme solution, incubate in the dark at 37°C for 10 minutes, and wash five times. Add 100 µL of substrate solution, incubate in the dark at 37°C for 15 minutes, then add 100 µL of stop solution. Measure OD450 within 30 minutes and plot against standards. In this experiment, we measured the levels of CCL2 (Jonln, China) and gal-1 (Jonln, China) to evaluate their expression in the samples. Statistical Analysis The data were presented as mean ± standard deviation(‾x ± SD). Two-tailed unpaired Student’s t -tests were used for parametric data analysis and the Mann–Whitney U test was used for non-parametric data analysis. We used the GraphPad Prism 9.0 software for statistical analysis, P -values < 0.05 were set as statistically significant. Results Tph Cells and Tph/Treg Ratio Were Elevated in pSS Patients. Compared with HC, pSS patients exhibited a significant increase in in the proportion of CXCR5 − PD-1 + CD4 + Tph cells (32.78 ± 11.41% vs. 16.04 ± 5.24%) (Fig. 1 b) and a markedly higher level of CCR2 expression in Tph cells (Fig. 1 c). The IL-21 + CD4 + T cells was also higher in pSS patients (0.17 ± 0.12% vs. 0.04 ± 0.04%) (Fig. 1 d) and was positively correlated with the proportion of circulating Tph cells ( R = 0.553, P < 0.05), but not with the proportion of circulating Tfh cells ( R = -0.088, P = 0.729) (Fig. 1 e). Notably, the Tph/Treg ratio was significantly elevated in pSS patients (25.18 ± 19.64 vs. 4.98 ± 2.42) (Figure. 1b). In contrast, the proportion of circulating Tfh cells were significantly reduced in pSS patients (2.35 ± 1.57% vs. 4.22 ± 1.33%), as were Treg cells (1.97 ± 1.14% vs. 3.43 ± 0.72%) (Fig. 1 a, b). In the salivary glands of pSS patients, an infiltration of CD4 + T cells marked by PD-1 and CCR2 expression was observed (Fig. 1 f). Strikingly, a pronounced increase in PD-1 + CCR2 + CD4 + Tph cells was found to be positively correlated with the severity of lymphocytic infiltration (Fig. 1 f). Additionally, CXCR5 + PD1 + CD4 + Tfh cells (Fig. 1 g) and FOXP3 + CD4 + Treg cells (Fig. 1 h) were also identified in the salivary glands of pSS patients. The number of Tph cells (Fig. 1 i), Tfh cells (Fig. 1 j) and Treg cells (Fig. 1 k) were increased with the degree of lymphocyte infiltration. Moreover, the proportion of these T cells were increased with the degree of lymphocyte infiltration, too (Fig. 1 l). Tph Cells and Tph/Treg Ratio Were Associated with Disease Activity Next, we evaluated the relationship between circulating Tph cells and disease activity parameters of pSS patients. We found a positive correlation between the proportion of circulating with ESSDAI scores ( R = 0.613, P < 0.05) (Fig. 2 a). Additionally, we also discovered a positive correlation between the Tph/Treg ratio and ESSDAI ( R = 0.479, P < 0.05) (Fig. 2 b). In contrast, the proportions Tfh was negatively correlated with ESSDAI scores ( R = -0.497, P < 0.05), while Treg cells did not exhibit a significant correlation ( R = -0.418, P = 0.085) (Supplement Fig. 2 ). Serum analysis revealed that the proportion of Tph cell was significantly negatively correlated with both C3 ( R = -0.511, P < 0.05) and IgG levels ( R = 0.589, P < 0.05) (Fig. 2 a). However, no such associations were observed for the proportion of Tfh or Treg cells with C3 and IgG (Supplement Fig. 2 ). Additionally, there were no correlations were found between the proportions of Tph cells or the proportion of other subsets and C4 levels (Fig. 2 a). Furthermore, the proportion of CD19 + CD11c + B cells in pSS was significantly higher than in HC (0.34 ± 0.26% vs. 0.20 ± 0.14%) (Fig. 2 c-d). Moreover, significant positive association was found between Tph and CD19 + CD11c + B cells. However, no significant correlations were found between Tfh cells, Treg cells, Tph/Treg ratio and CD19 + CD11c + B cells (Fig. 2 e). MSC Treatment Improves Clinical Symptoms in pSS Patients and Modulates Immune Response by Targeting Tph Cells Given the remarkable efficacy of MSC in various autoimmune diseases, peripheral blood samples were collected from 10 pSS patients pre- and 24 hours post- MSC transplantation (MSCT). The demographic and clinical information of patients is presented in Supplement Table 3. We found that MSC treatment could significantly increase the levels of C3 and C4 and significantly reduced the levels of IgG, as well as the ESSPRI scores and ESSDAI scores in pSS patients (Fig. 3 a). These results demonstrate that MSCT effectively improves clinical symptoms and immune profiles in pSS patients. Subsequently, we evaluated the impact of MSC treatment on T cell subsets. Our results demonstrated a statistically significant reduction in the proportions of Tph cell and the Tph/Treg ratio after MSC transplantation. Concurrently, there was a significant increase in the proportion of Treg cells (Fig. 3 b), which was consistent with our previous studies. MSC Inhibited Tph Cells in PBMC of pSS Patients through the Gal-1 Protein To further explore the mechanism, peripheral blood samples from pSS patients pre- and post-MSC transplantation were collected for proteomic analysis. Proteomic analysis identified three upregulated and five downregulated proteins in the serum of pSS patients pre- and post-MSC transplantation including gal-1, which will be further validated in subsequent experiments (Fig. 4 a, Supplement Table 2). Subsequently, Gene Ontology (GO) analysis was conducted on these differentially expressed proteins (Supplement Fig. 3 ). The results revealed that the functions enriched in the GO analysis included lymphocyte activation, B cell activation in the immune response, and so on. Previous studies have provided evidence that the upregulated Galectin-1 (Gal-1) suppresses pro-inflammatory mediators such as CCL2[ 32 ], while another significantly reduced downregulated protein Mannan-binding lectin serine protease 2 (MASP2), which is integral to the complement pathway[ 33 ], consistent with our observed inverse correlation between Tph cell proportions and serum C3 levels (Fig. 2 a). Subsequently, ELISA analysis was conducted on four pSS patients and confirmed an increase in Gal-1 levels and a decrease in CCL2 levels after MSC transplantation (Fig. 4 b). Specifically, when compared with the HC groups, Gal-1 levels were significantly lower (15.09 ± 7.23% vs. 47.07 ± 33.24%) in pSS patients, while CCL2 levels were significantly higher (1015.80 ± 1714.46% vs. 290.77 ± 137.02%) (Fig. 4 c). To elucidate the therapeutic mechanism of MSC, we conducted a transwell co-culture experiment using PBMC from pSS patient and MSC. The experimental conditions included PBMC alone, MSC + PBMC, recombinant Gal-1 + PBMC, and MSC + PBMC with Gal-1 inhibitor TDG. In both the MSC + PBMC and Gal-1 + PBMC groups, the numbers of Tph cells were significantly lower than in the PBMC alone group (Fig. 4 d-e). There was no significant change in the proportion of Tfh cells, Treg cells and Tph/Treg in all treatment groups, and the differences between all groups did not reach statistical significance. Additionally, ELISA analysis of the supernatants revealed elevated Gal-1 levels and decreased CCL2 levels in the MSC + PBMC and Gal-1 + PBMC groups (Fig. 4 f). These results suggest that MSC modulate Tph cells in pSS patients via the Gal-1 protein. Discussion In this study, we demonstrated that the elevated proportion of Tph cells in pSS patients was correlated with the ESSDAI score. It was also linked to serum C3 and IgG levels, as well as the proportion of CD19 + CD11c + B cells. The results suggest a connection between Tph cells and IL-21. This indicates a mechanism through which Tph cells may be involved in the pathogenesis of pSS disease. Tph cells have been shown to be increased in seropositive RA patients, and associated with disease activity[ 13 ]. In RA, Tph cells have also been found to induce production of CXCL13 in synovial tissue, recruiting CXCR5 + naive B cells and Tfh cells[ 34 ]. Moreover, Tph cells promote B cell differentiation through IL-21 and signaling lymphocytic activation molecule family member 5 (SLAMF5) interactions [ 13 ]. Studies have found that Tph cells are significantly enriched in both the peripheral blood and salivary glands with germinal centers in SS patients, express ICOS, serve as the primary source of IL-21[ 35 ]. Our findings of increased Tph cells in pSS patients indicate an immune dysfunction and suggest their potential as biomarkers for pSS activity. In addition to the role of Tph cells, our findings showed a significantly elevated Tph/Treg ratio in pSS patients, which correlated with higher ESSDAI scores. This elevated Tph/Treg ratio highlights an imbalance that is likely to exacerbate immune activation and tissue damage in pSS. Therefore, the Tph/Treg ratio in pSS patients may represent another important indicator of disease severity and immune dysfunction, further supporting the notion that Tph cells act as drivers of inflammation in pSS. Dry mouth is a cardinal symptom of Sjögren's syndrome, which is mainly caused by T lymphocyte infiltration and the subsequent destruction of exocrine glands [ 23 ]. Although the specific T cell subsets that drive pSS pathogenesis remain elusive, studies have identified CXCR5 − CD4 + PD-1 hi T cells within the germinal centers of salivary glands (SGs) in pSS patients, which co-express IL-21 and IFN-γ[ 24 ]. Additionally, the increased expression of CCR2 protein and CCL2 in SGs suggests that Tph cells, which are characterized by CCR2 expression, play a role in the immunopathogenesis of disease [ 13 ]. Our immunofluorescence analysis of the salivary gland tissue from pSS patient disclosed a correlation between the intensity of lymphocytic infiltration and the abundance of CD4 + PD-1 + CCR2 + Tph cells, CD4 + CXCR5 + PD-1 + Tfh cells, and CD4 + FOXP3 + Treg cells. This finding indicates that higher levels of lymphocytic infiltration are associated with increased presence of these T cell subsets. Notably, Tph cells in the salivary glands were found to express CCR2, which may potentially mediate their migration to these salivary glands. This observation highlights the role of Tph cells in the local pathogenesis of pSS and the significance of chemokines in immune cell migration and tissue-specific infiltration. Moreover, we also found that CD4 + CXCR5 + PD-1 + T cells and CD4 + Foxp3 + T cells in salivary gland, consistent with previous findings[ 12 , 13 ]. MSC has been increasingly recognized for their therapeutic potential in pSS due to their robust immunomodulatory capabilities, particularly by upregulating Tregs and downregulating Th1, Th17, or Tfh cells[ 36 – 38 ]. Additional, MSC can differentiate into salivary epithelial cells[ 39 ], offering a promising alternative treatment for pSS. In our study, Tph cells and the Tph/Treg ratio, which are known to play a pivotal role in autoimmune diseases, were found to be decreased in pSS patients following MSC transplantation. This suggests an improvement in the immunoregulatory milieu. Gal-1, β-galactoside-binding lectin critical for cell death, cell adhesion, and immune responses[ 40 ], was found to be elevated after MSC treatment. The increase of Gal-1 implies that MSC may exert their anti-inflammatory effects by secreting this lectin, thus having a positive impact on the immune environment in pSS patients. Furthermore, our research indicated a decrease in CCL2 levels following MSC treatment. This decrease may potentially reduce the migration and activation of CCR2 + Tph cells, thereby mitigating the inflammatory process and improving the immunopathological state of pSS. By upregulating Gal-1, MSC may modulate the CCL2/CCR2 axis, presenting a novel mechanism for curbing inflammatory responses[ 41 , 42 ]. The intricate interplay between Gal-1 and Tph cells suggests that MSC therapy could influence immune regulation through multiple pathways. Our in vitro experiments substantiated these findings, demonstrating a significant decrease in the proportions of Tph cells when exposed to MSC co-culture and treatment with recombinant Gal-1 protein. The use of the Gal-1 inhibitor TDG reversed this regulatory effect, underscoring the essential role of Gal-1 in MSC-mediated immunomodulation. ELISA results further confirmed that MSC treatment and the addition of Gal-1 significantly elevated Gal-1 levels and reduced CCL2 levels, indicating a regulatory influence on Tph cells and inflammatory responses through the modulation of Gal-1. Further study is needed to comprehensively understand the ways by which MSC regulate the immune environment via Gal-1 and other potential molecular mechanisms. Understanding how these pathways synergize within the immune system could potentially improve therapeutic approaches for autoimmune diseases. Moreover, exploring the impacts of MSC on other key immune cell subsets, such as Tregs, effector T cells (Teffs), and B cells, will provide a more comprehensive perspective on the immunomodulatory functions of MSC. Conclusions This study identifies PD-1⁺CXCR5⁻ peripheral helper T cells as key contributors to immune dysregulation in pSS. Elevated Tph cells and the Tph/Treg ratio correlated with disease activity and B cell activation. MSCT reduced Tph cells and restored immune balance. Further research found that Gal-1 plays a crucial role in this process. These findings highlight MSC therapy as a promising strategy for pSS treatment, and Tph cells can be used as an effective index to evaluate the disease activity in pSS patients. Abbreviations Bcl-6: B-cell lymphoma 6; ESSDAI: EULAR Sjögren's Syndrome Disease Activity Index; ESSPRI: EULAR Sjögren’s Syndrome Patient Reported Index; FACS: Fluorescence-activated cell sorting; Gal-1: Galectin-1; HC: Healthy Control; ICOS: Inducible T-cell co-stimulator; IgG4-RD: IgG4-related disease; MASP2: Mannan-binding lectin serine protease 2; MSC: Mesenchymal stromal cell; MSCT: Mesenchymal stromal cell transplantation; PBMC: Peripheral blood mononuclear cell; pSS: Primary Sjögren’s syndrome; RA: Rheumatoid arthritis; SLE: Systemic lupus erythematosus; Tfh: Follicular helper T cell; Tph: Peripheral helper T cell; Treg: Regulatory T cell. Declarations Funding This work was supported by the National Key R&D Program of China (Grant No. 2020YFA0710800), the Natural Science Foundation of Jiangsu Province (Grant No. BK20240121), and the Clinical Trials Funding from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (Grant No. 2021-LCYJ-PY-16). Ethics approval and consent to participate The studies involving human participants were reviewed and approved by the Ethics Committee of Nanjing Drum Tower Hospital. Title of the approved project: Regulation of lipid metabolism and T cell via mTOR by mesenchymal stem cells. Approval number: 2021-035. Date of approval: Jan. 21, 2021. All participants gave their written informed consent approved by the Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School in accordance with the Declaration of Helsinki. Consent for publication Not applicable. Acknowledgments We used the Large Language Model—ChatGPT—in the drafting of this paper for grammar and language refinement. Competing Interests The authors declare no conflicts of interest. Authors’ Contributions LS and LG designed the study. SZ, FY, and TL collected the data. YG and XC performed the experiments and statistical analysis. RC, GY, and XT analyzed the data. SZ, LG, and BT wrote the manuscript. 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SAT0252 The number of treg cells in peripheral blood in pss patients is decreased and low dose IL-2 can promote its proliferation. Annals of the Rheumatic Diseases 2017; 76(Suppl 2):869. Ramos-Casals M, Cervera R, Font J, Garcia-Carrasco M, Espinosa G, Reino S, Pallares L, Ingelmo M. Young onset of primary Sjogren's syndrome: clinical and immunological characteristics. Lupus 1998; 7(3):202-206. Iwamoto N, Kawakami A, Arima K, Nakamura H, Kawashiri SY, Tamai M, Kita J, Okada A, Koga T, Kamachi M et al . Regulation of disease susceptibility and mononuclear cell infiltration into the labial salivary glands of Sjogren's syndrome by monocyte chemotactic protein-1. Rheumatology (Oxford) 2010; 49(8):1472-1478. Aluri HS, Samizadeh M, Edman MC, Hawley DR, Armaos HL, Janga SR, Meng Z, Sendra VG, Hamrah P, Kublin CL et al . Delivery of Bone Marrow-Derived Mesenchymal Stem Cells Improves Tear Production in a Mouse Model of Sjogren's Syndrome. Stem Cells Int 2017; 2017(3134543. Chen W, Yu Y, Ma J, Olsen N, Lin J. Mesenchymal Stem Cells in Primary Sjogren's Syndrome: Prospective and Challenges. Stem Cells Int 2018; 2018(4357865. Veernala I, Jaffet J, Fried J, Mertsch S, Schrader S, Basu S, Vemuganti GK, Singh V. Lacrimal gland regeneration: The unmet challenges and promise for dry eye therapy. Ocul Surf 2022; 25(129-141. Xu J, Wang D, Liu D, Fan Z, Zhang H, Liu O, Ding G, Gao R, Zhang C, Ding Y et al . Allogeneic mesenchymal stem cell treatment alleviates experimental and clinical Sjogren syndrome. Blood 2012; 120(15):3142-3151. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, Daniels TE, Fox PC, Fox RI, Kassan SS et al . Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002; 61(6):554-558. Seror R, Ravaud P, Bowman SJ, Baron G, Tzioufas A, Theander E, Gottenberg JE, Bootsma H, Mariette X, Vitali C et al . EULAR Sjogren's syndrome disease activity index: development of a consensus systemic disease activity index for primary Sjogren's syndrome. Ann Rheum Dis 2010; 69(6):1103-1109. Seror R, Ravaud P, Mariette X, Bootsma H, Theander E, Hansen A, Ramos-Casals M, Dorner T, Bombardieri S, Hachulla E et al . EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI): development of a consensus patient index for primary Sjogren's syndrome. Ann Rheum Dis 2011; 70(6):968-972. Starossom SC, Mascanfroni ID, Imitola J, Cao L, Raddassi K, Hernandez SF, Bassil R, Croci DO, Cerliani JP, Delacour D et al . Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration. Immunity 2012; 37(2):249-263. Stengaard-Pedersen K, Thiel S, Gadjeva M, Moller-Kristensen M, Sorensen R, Jensen LT, Sjoholm AG, Fugger L, Jensenius JC. Inherited deficiency of mannan-binding lectin-associated serine protease 2. N Engl J Med 2003; 349(6):554-560. Kobayashi S, Murata K, Shibuya H, Morita M, Ishikawa M, Furu M, Ito H, Ito J, Matsuda S, Watanabe T et al . A distinct human CD4+ T cell subset that secretes CXCL13 in rheumatoid synovium. Arthritis Rheum 2013; 65(12):3063-3072. Pontarini E, Murray-Brown WJ, Croia C, Lucchesi D, Conway J, Rivellese F, Fossati-Jimack L, Astorri E, Prediletto E, Corsiero E et al . Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjogren's syndrome with ectopic germinal centres and MALT lymphoma. Ann Rheum Dis 2020; 79(12):1588-1599. Bolandi Z, Mokhberian N, Eftekhary M, Sharifi K, Soudi S, Ghanbarian H, Hashemi SM. Adipose derived mesenchymal stem cell exosomes loaded with miR-10a promote the differentiation of Th17 and Treg from naive CD4(+) T cell. Life Sci 2020; 259(118218. Riazifar M, Mohammadi MR, Pone EJ, Yeri A, Lässer C, Segaliny AI, McIntyre LL, Shelke GV, Hutchins E, Hamamoto A et al . Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders. ACS Nano 2019; 13(6):6670-6688. Zhang W, Lin J, Shi P, Su D, Cheng X, Yi W, Yan J, Chen H, Cheng F. Small Extracellular Vesicles Derived From MSCs Have Immunomodulatory Effects to Enhance Delivery of ASO-210 for Psoriasis Treatment. Frontiers in Cell and Developmental Biology 2022; 10( Park YJ, Koh J, Kwon JT, Park YS, Yang L, Cha S. Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes. PLoS One 2017; 12(2):e0169677. Rubinstein N, Alvarez M, Zwirner NW, Toscano MA, Ilarregui JM, Bravo A, Mordoh J, Fainboim L, Podhajcer OL, Rabinovich GA. Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection; A potential mechanism of tumor-immune privilege. Cancer Cell 2004; 5(3):241-251. Fajka-Boja R, Urbán VS, Szebeni GJ, Czibula Á, Blaskó A, Kriston-Pál É, Makra I, Hornung Á, Szabó E, Uher F et al . Galectin-1 is a local but not systemic immunomodulatory factor in mesenchymal stromal cells. Cytotherapy 2016; 18(3):360-370. Moadab F, Khorramdelazad H, Abbasifard M. Role of CCL2/CCR2 axis in the immunopathogenesis of rheumatoid arthritis: Latest evidence and therapeutic approaches. Life Sci 2021; 269(119034. 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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-6173991","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436803063,"identity":"6891e4a0-102f-4212-8cde-071d676096a5","order_by":0,"name":"Shiyi Zhang","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Shiyi","middleName":"","lastName":"Zhang","suffix":""},{"id":436803064,"identity":"42a0d1b6-523a-47b8-89b9-71c0d63bc326","order_by":1,"name":"Fanzhang Yin","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Fanzhang","middleName":"","lastName":"Yin","suffix":""},{"id":436803065,"identity":"111b7315-4cb2-4920-b335-137e1a3eac10","order_by":2,"name":"Rui Chai","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Chai","suffix":""},{"id":436803066,"identity":"70f2c955-8b84-46be-83cd-538356ff65fe","order_by":3,"name":"Tao Li","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Li","suffix":""},{"id":436803067,"identity":"f5cf90c9-419f-4f84-9070-58ce81086169","order_by":4,"name":"Yingying Gao","email":"","orcid":"","institution":"nantong daxue di er fushu yiyuan: Nantong City No 1 People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Gao","suffix":""},{"id":436803068,"identity":"330037bb-4a57-4f6d-90f0-351947fb6641","order_by":5,"name":"Xiaoxiang Chen","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Rheumatology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxiang","middleName":"","lastName":"Chen","suffix":""},{"id":436803069,"identity":"bb97613c-d34a-4e23-a5fd-324992ffc75c","order_by":6,"name":"Genghong Yao","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Genghong","middleName":"","lastName":"Yao","suffix":""},{"id":436803070,"identity":"e96472a5-c63a-455f-9d74-2b32f4acfe91","order_by":7,"name":"Xiaojun Tang","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Tang","suffix":""},{"id":436803071,"identity":"75b4f82f-e8ec-4058-bca1-2496ee11ed11","order_by":8,"name":"Xiaojuan Han","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Han","suffix":""},{"id":436803072,"identity":"c42b5d28-f0d6-461a-963e-69477ad34804","order_by":9,"name":"Betty P. Tsao","email":"","orcid":"","institution":"Medical University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Betty","middleName":"P.","lastName":"Tsao","suffix":""},{"id":436803073,"identity":"1d5442dc-b294-4b16-a169-f34a1a3789e9","order_by":10,"name":"Linyu Geng","email":"","orcid":"","institution":"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Linyu","middleName":"","lastName":"Geng","suffix":""},{"id":436803074,"identity":"3d3ee638-3358-4cc6-9556-19b94e09a3d1","order_by":11,"name":"Lingyun Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACPmY4k/kAgwSIPkBACxtCC1sCkVoQTB4DCE1QCzvv4de8bXfsDW73fHtg2cYgx3cjgfFzAV6H8aVZ87Y9S9xw5+x2A8k2BmPJGwnM0jPwauExM+ZtO5xgcCN3mwRQS+KGGwlAQSK02BvcyHkG0lJPjBbjx0AtjBtu5LCBtACtI8IWxjnnDifOvJFmJiFxTsJw5pmHzdL4tPDznzH+8KbssD3fjeRn0hJlNvJ8x5MPfsanBWSRFEwBswQ4Mhkb8GsAKvz4A8pi/EBI7SgYBaNgFIxIAACcHkSmqyilSwAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing University Medical School","correspondingAuthor":true,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-03-07 01:19:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6173991/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6173991/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04631-9","type":"published","date":"2025-10-06T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82055765,"identity":"495ba993-dcef-49d5-a957-6ed3a437a759","added_by":"auto","created_at":"2025-05-06 10:26:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":975044,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in lymphocyte subsets in peripheral blood and salivary gland tissue of pSS patients and healthy controls. a. Flow cytometry plots illustrating the differences in the proportions of Tph cells, Tfh cells, and Treg cells in peripheral blood of pSS patients (n=18) and healthy controls (n=20). b. Statistical differences in the proportions of Tph cells, Tfh cells, Treg cells and Tph/Treg between pSS patients (n=18) and healthy controls (n=20). c. Differences in CCR2 expression on Tfh and Tph cells. d. Differences in IL-21 expression in CD4\u003csup\u003e+\u003c/sup\u003e T cells between pSS patients (n=18) and healthy controls (n=20). e. Correlation analysis of Tph cells, Tfh cells, and Treg cells with IL-21 in the peripheral blood of pSS patients (n=18). f-h. Immunofluorescence images of Tph cells (f), Tfh cells (g) and Treg cells (h) in salivary gland tissue with different grades of lymphocyte infiltration in pSS patients (n=12). i-k. Statistical analysis of the number of Tph (i), Tfh (j), and Treg (k) cells in salivary gland tissue with different grades of lymphocyte infiltration in pSS patients (n=12). l. Changes in the proportion of Tph,Tfh,treg cells in salivary gland tissue with different grades of lymphocyte infiltration in pSS patients (n=12). (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; ns, not significant; \u003cem\u003eR\u003c/em\u003e, correlation coefficient).\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-6173991/v1/e2b8cb2da67b8c2deca87889.png"},{"id":82053999,"identity":"277c07bc-3620-42c8-8653-f1379e6333e4","added_by":"auto","created_at":"2025-05-06 10:18:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2584790,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of Tph cells and Tph/Treg with Disease Activity in pSS patients.\u003c/p\u003e\n\u003cp\u003ea. Correlation of the proportions of Tph cells with the results of laboratory detection. b. Correlation of the proportions of Tph/Treg ratio with the results of laboratory. c. Differences in the proportions of CD19\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB cells in peripheral blood of pSS patients (n=18) and healthy controls (n=20). d. Statistical comparisons in CD19\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB cells between patients with pSS and healthy controls (n=20). e. Correlation analysis between the proportions of Tph cells, Tfh cells, Treg cells, the Tph/Treg ratio and CD19\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB cells. (\u003cem\u003eR\u003c/em\u003e, correlation coefficient.)\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-6173991/v1/7e5608c2b699fb90f2d7e4c5.png"},{"id":82053982,"identity":"139311ee-c5df-4b12-b31d-24265208ec65","added_by":"auto","created_at":"2025-05-06 10:18:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2904602,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in the proportions of Tph, Tfh, Treg and the Tph/Treg ratio in the peripheral blood of pSS patients pre-MSCT and post-MSCT.\u003c/p\u003e\n\u003cp\u003ea. Statistical comparisons of the levels of C3, C4, IgG, and the scores of ESSPRI and ESSDAI in pSS patients (n=10) pre-MSCT and post-MSCT. b. Flow cytometry analysis showing the differences in the proportions of Tph, Tfh, Treg cells and the Tph/Treg ratio in the peripheral blood of pSS patients (n=10) pre-MSCT and post-MSCT. (*, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ns, not significant.)\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-6173991/v1/a311aa4012279fd1c29450de.png"},{"id":82055770,"identity":"68658358-85e1-4033-a7e5-5cb38567f075","added_by":"auto","created_at":"2025-05-06 10:26:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2690310,"visible":true,"origin":"","legend":"\u003cp\u003eMSC Inhibits Tph Cells in PBMC of pSS Patients through the Gal-1 Protein.\u003c/p\u003e\n\u003cp\u003ea. Volcano plot of differentially expressed proteins in the peripheral blood of pSS patients (n=8) pre-MSCT and post-MSCT. b. Changes in the levels of Gal-1 and CCL2 levels in the peripheral blood of pSS patients (n=8) before and after MSC transplantation therapy. c. Changes in the levels of Gal-1 and CCL2 levels in the peripheral blood of pSS patients (n=24) and healthy controls (n=24). d. Flow cytometry plots showing the proportions of Tph cells, Tfh cells, Treg cells and the Tph/Treg ratio after in vitro culture of PBMC from pSS patients (n=3) in each group. e. Statistical comparisons of the proportions of Tph cells, Tfh cells, Treg cells and the Tph/Treg ratio among the groups after in vitro culture of PBMC from pSS patients (n=3). f. ELISA analysis the levels of Gal-1 and CCL2 in the supernatants of in vitro cultured PBMC from pSS patients (n=3) in each group. (*, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; ns, not statistically significant.)\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-6173991/v1/93039710fd0402c5522a91a4.png"},{"id":93419908,"identity":"6e8a5a78-73f3-49e2-b7af-ed30ec68982f","added_by":"auto","created_at":"2025-10-13 16:08:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9539149,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6173991/v1/3cd5ae25-f94c-4171-aa27-7bf75b1fa620.pdf"},{"id":82054002,"identity":"d1071298-b909-4a5a-9916-239e77b492b8","added_by":"auto","created_at":"2025-05-06 10:18:02","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14379325,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementtableandfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6173991/v1/8c7a77ae487da1fab05b4bfa.docx"}],"financialInterests":"","formattedTitle":"Mesenchymal Stromal Cells Attenuate Primary Sjogren's Syndrome by Modulating PD-1+CXCR5− T Peripheral Helper Cells via Galectin-1","fulltext":[{"header":"Background","content":"\u003cp\u003ePrimary Sj\u0026ouml;gren\u0026rsquo;s syndrome (pSS) manifests as a chronic autoimmune disorder characterized by the preferential involvement of lacrimal and salivary glands, resulting in the cardinal symptoms of dry mouth and dry eye[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The pathogenesis involves of pSS extensive lymphocytic infiltration that disrupts gland function[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. At present, the complex interplay of immune dysregulation, B cell hyperactivity, and autoantibody production, are pivotal in pSS pathogenesis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The precise mechanisms have not been fully elucidated.\u003c/p\u003e \u003cp\u003eFollicular helper T (Tfh) cells, a subset of CD4\u003csup\u003e+\u003c/sup\u003e T cells that express inducible T-cell co-stimulator (ICOS), PD-1, and CXCR5[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], are implicated in the progression of pSS[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Tfh cells support effector B cells and augment autoimmunity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] by producing interleukin-21 (IL-21) and other cytokines[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has been proved that Tfh cells are associated with the titer of anti-SSA, anti-SSB antibodies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Tph, a novel subset of helper T cells, which distinct from Tfh, has been identified in the synovial fluid (SF) and peripheral blood of patients with rheumatoid arthritis (RA)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Like Tfh cells, Tph cells express CXCL13, IL-21, CD40L, and ICOS but lack CXCR5 and exhibit low B-cell lymphoma 6 (Bcl-6) expression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].Additionally, Tph cells express the chemokine receptors CCR2, CCR5, CXCR3 and CX3CR, which are crucial for their migration to inflammatory sites[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Circulating Tph (cTph) cells have been proven to be associated with various autoimmune diseases, including RA[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], systemic lupus erythematosus(SLE)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], IgG4-related disease (IgG4-RD)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], type 1 diabetes (T1D) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and pSS[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].cTph cells drive B-cells hyperactivity[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], contributing to the pathogenesis of these diseases. However, the potential of therapeutic interventions targeting Tph cells to ameliorate the disease has not been fully explored. In contrast, regulatory T (Treg) cells appear to inhibit B-cell responses by suppressing the activities of Tfh or Tph cells, which prevent the development of pSS[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, restoring the balance among Tph, Treg and Tph cells may be an effective measure for the treatment of pSS.\u003c/p\u003e \u003cp\u003eMesenchymal stromal (stem) cells (MSC) have been shown to possess remarkable immunomodulatory properties by interacting with a variety of immune cell populations, such as Tfh cells and Treg cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. MSC can mitigate the aberrant activation of immune cells that drive the disease progression, thereby reducing the inflammatory burden on the salivary and lacrimal glands[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which has emerged as a promising treatment for pSS patients[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, MSC promote the repair and regeneration of damaged glandular tissue, consequently improving glandular function and alleviating the symptoms of dryness [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Preliminary studies in animal models of pSS have revealed a decrease in lymphocytic infiltration, a reduction in pro-inflammatory cytokines, and an enhancement of glandular function after MSC treatment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A clinical study involving 24 pSS patients has confirmed the therapeutic efficacy of MSC treatment, with improvements in salivary flow rate, alleviation of oral dryness, and a decrease in anti-SSA/anti-SSB levels, all without serious adverse events[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although the effects of MSC in pSS patients were being explored, the specific mechanisms by which MSC regulate Tph cells in these patients are yet to be fully investigated.\u003c/p\u003e \u003cp\u003eThis study investigates the roles of Tph, Tfh, and Treg cells, and the Tph/Treg ratio in pSS pathogenesis, and the impact of MSC therapy on Tph cells and the Tph/Treg ratio.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Controls\u003c/h2\u003e \u003cp\u003eFrom September 2020 to December 2023, hospitalized pSS patients were selected from the Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, affiliated with Nanjing University Medical School. Among them, 10 patients underwent MSC transplantation. All participants fulfilled the revised 2002 American\u0026ndash;European criteria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], with exclusions for other autoimmune diseases. The disease activity was assessed using the ESSDAI scores[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The European Sj\u0026ouml;gren's Syndrome Patient Reporting Index (ESSPRI) was used to measure the severity of symptoms[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The demographic and clinical characteristics of pSS patients are presented in Supplementary Table\u0026nbsp;1. Informed consent was obtained from all participants. The study was approved by the Ethics Committee of Nanjing Drum Tower Hospital, affiliated with Nanjing University Medical School, in compliance with the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003eAfter MSC transplantation, patients were followed up for 6 months, with clinical evaluations performed at 6 months post-treatment. ESSDAI and ESSPRI scores were measured pre-MSCT and at the 6-month follow-up visit to assess changes in disease activity and symptom severity. The primary outcome measures included the changes in ESSDAI scores, the Tph/Treg ratio, and other immune cell subsets pre-MSCT and post-MSCT, as well as the correlation between these immune parameters and clinical outcomes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003ePeripheral blood mononuclear cells (PBMC) were extracted from pSS patients and HC. The following fluorescence-conjugated mouse anti-human antibodies (BioLegend, San Diego, CA, USA) were applied on a FACS Calibur cytometer with Diva software (BD Biosciences; San Jose, CA, USA): BV510 anti-CD4, APC anti-CXCR5, BV421 anti-PD1, BV605 anti-CCR2, BV711 anti-CD25, PE anti-FOXP3, PerCP/Cyanine5.5 anti-lL-21, PE anti-CD19, AF488 anti-CD27 and PerCP/Cyanine5.5 anti-CD38, or relevant isotype controls. Data analysis was conducted using FlowJo 10.4.\u003c/p\u003e\n\u003ch3\u003eQuantitative Proteomics\u003c/h3\u003e\n\u003cp\u003eA total of 8 serum samples from pSS patients, as well as their serum collected 24 hours post-MSCT, were collected for proteomic analysis. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Tandem Mass Tags (TMT) are popular labeling technologies for quantitative proteomics. They label peptides by combining with the amino group of amino acid terminals and lysine residues, allowing comparison between samples labeled with reagents of different molecular weights. The workflow includes sample preparation, protein hydrolysis, High-Performance Liquid Chromatography (HPLC) classification, Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) detection, database comparison, and bioinformatics analysis.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescent Staining of Tissue\u003c/h3\u003e\n\u003cp\u003eLabial gland specimens from 12 pSS patients were sectioned into 4\u0026ndash;5 \u0026micro;m slices, deparaffinized, and rehydrated. Antigen retrieval was done using citrate buffer and microwave heating. Sections were blocked by PBS with 1% BSA, then incubated with primary antibodies (CD4, CCR2, CXCR5, FOXP3) at 4\u0026deg;C. After washed with PBS, fluorescently labeled secondary antibodies were added and incubated in the dark. Nuclear staining was performed with DAPI, followed by PBS washes. Sections were mounted with anti-fade reagent and examined using a fluorescence microscope. Images were analyzed with ImageJ to quantify marker intensity and distribution.\u003c/p\u003e\n\u003ch3\u003eCoculture of MSC and PBMC\u003c/h3\u003e\n\u003cp\u003eThe membrane used had a pore size of 0.4 \u0026micro;m (Corning, USA) to prevent cell passage. PBMC were cultured in the lower chamber of a 24-well plate with RPMI-1640, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e PBMC, anti-CD3 (1\u0026micro;g/ml), and anti-CD28 (0.5\u0026micro;g/ml) at 37℃ and 5% CO2 for 2 hours. The Transwell chamber was then added, and 100 \u0026micro;L of liquid was placed in the upper chamber. Experimental groups included blank medium, DF12 with MSC (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well), Gal-1 inhibitor Thiodigalactoside (TDG) (50\u0026micro;M), and Gal-1 recombinant protein (3\u0026micro;M). After 3 days of incubation at 37℃ and 5% CO2, PBMC and the supernatant from the lower chamber were collected.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay\u003c/h2\u003e \u003cp\u003eEquilibrate strips to room temperature. Add 100 \u0026micro;L of samples or standards, incubate at 37\u0026deg;C for 40 minutes, and wash five times. Add 50 \u0026micro;L of antibody solution, incubate at 37\u0026deg;C for 20 minutes, and wash five times. Add 100 \u0026micro;L of enzyme solution, incubate in the dark at 37\u0026deg;C for 10 minutes, and wash five times. Add 100 \u0026micro;L of substrate solution, incubate in the dark at 37\u0026deg;C for 15 minutes, then add 100 \u0026micro;L of stop solution. Measure OD450 within 30 minutes and plot against standards. In this experiment, we measured the levels of CCL2 (Jonln, China) and gal-1 (Jonln, China) to evaluate their expression in the samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation(\u0026oline;x\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests were used for parametric data analysis and the Mann\u0026ndash;Whitney U test was used for non-parametric data analysis. We used the GraphPad Prism 9.0 software for statistical analysis, \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were set as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTph Cells and Tph/Treg Ratio Were Elevated in pSS Patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared with HC, pSS patients exhibited a significant increase in in the proportion of CXCR5\u003csup\u003e\u0026minus;\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Tph cells (32.78\u0026thinsp;\u0026plusmn;\u0026thinsp;11.41% vs. 16.04\u0026thinsp;\u0026plusmn;\u0026thinsp;5.24%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and a markedly higher level of CCR2 expression in Tph cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The IL-21\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells was also higher in pSS patients (0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12% vs. 0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and was positively correlated with the proportion of circulating Tph cells (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.553, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not with the proportion of circulating Tfh cells (\u003cem\u003eR\u003c/em\u003e = -0.088, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.729) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Notably, the Tph/Treg ratio was significantly elevated in pSS patients (25.18\u0026thinsp;\u0026plusmn;\u0026thinsp;19.64 vs. 4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.42) (Figure. 1b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, the proportion of circulating Tfh cells were significantly reduced in pSS patients (2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57% vs. 4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33%), as were Treg cells (1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14% vs. 3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). In the salivary glands of pSS patients, an infiltration of CD4\u003csup\u003e+\u003c/sup\u003e T cells marked by PD-1 and CCR2 expression was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Strikingly, a pronounced increase in PD-1\u003csup\u003e+\u003c/sup\u003eCCR2\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Tph cells was found to be positively correlated with the severity of lymphocytic infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Additionally, CXCR5\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Tfh cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg) and FOXP3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Treg cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) were also identified in the salivary glands of pSS patients. The number of Tph cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), Tfh cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej) and Treg cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek) were increased with the degree of lymphocyte infiltration. Moreover, the proportion of these T cells were increased with the degree of lymphocyte infiltration, too (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTph Cells and Tph/Treg Ratio Were Associated with Disease Activity\u003c/h2\u003e \u003cp\u003eNext, we evaluated the relationship between circulating Tph cells and disease activity parameters of pSS patients. We found a positive correlation between the proportion of circulating with ESSDAI scores (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.613, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Additionally, we also discovered a positive correlation between the Tph/Treg ratio and ESSDAI (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.479, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, the proportions Tfh was negatively correlated with ESSDAI scores (\u003cem\u003eR\u003c/em\u003e = -0.497, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while Treg cells did not exhibit a significant correlation (\u003cem\u003eR\u003c/em\u003e = -0.418, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085) (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Serum analysis revealed that the proportion of Tph cell was significantly negatively correlated with both C3 (\u003cem\u003eR\u003c/em\u003e = -0.511, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and IgG levels (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.589, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). However, no such associations were observed for the proportion of Tfh or Treg cells with C3 and IgG (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, there were no correlations were found between the proportions of Tph cells or the proportion of other subsets and C4 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Furthermore, the proportion of CD19\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB cells in pSS was significantly higher than in HC (0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26% vs. 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d). Moreover, significant positive association was found between Tph and CD19\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB cells. However, no significant correlations were found between Tfh cells, Treg cells, Tph/Treg ratio and CD19\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC Treatment Improves Clinical Symptoms in pSS Patients and Modulates Immune Response by Targeting Tph Cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the remarkable efficacy of MSC in various autoimmune diseases, peripheral blood samples were collected from 10 pSS patients pre- and 24 hours post- MSC transplantation (MSCT). The demographic and clinical information of patients is presented in Supplement Table\u0026nbsp;3. We found that MSC treatment could significantly increase the levels of C3 and C4 and significantly reduced the levels of IgG, as well as the ESSPRI scores and ESSDAI scores in pSS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These results demonstrate that MSCT effectively improves clinical symptoms and immune profiles in pSS patients. Subsequently, we evaluated the impact of MSC treatment on T cell subsets. Our results demonstrated a statistically significant reduction in the proportions of Tph cell and the Tph/Treg ratio after MSC transplantation. Concurrently, there was a significant increase in the proportion of Treg cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), which was consistent with our previous studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC Inhibited Tph Cells in PBMC of pSS Patients through the Gal-1 Protein\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further explore the mechanism, peripheral blood samples from pSS patients pre- and post-MSC transplantation were collected for proteomic analysis. Proteomic analysis identified three upregulated and five downregulated proteins in the serum of pSS patients pre- and post-MSC transplantation including gal-1, which will be further validated in subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Supplement Table\u0026nbsp;2). Subsequently, Gene Ontology (GO) analysis was conducted on these differentially expressed proteins (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results revealed that the functions enriched in the GO analysis included lymphocyte activation, B cell activation in the immune response, and so on.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have provided evidence that the upregulated Galectin-1 (Gal-1) suppresses pro-inflammatory mediators such as CCL2[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], while another significantly reduced downregulated protein Mannan-binding lectin serine protease 2 (MASP2), which is integral to the complement pathway[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], consistent with our observed inverse correlation between Tph cell proportions and serum C3 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Subsequently, ELISA analysis was conducted on four pSS patients and confirmed an increase in Gal-1 levels and a decrease in CCL2 levels after MSC transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Specifically, when compared with the HC groups, Gal-1 levels were significantly lower (15.09\u0026thinsp;\u0026plusmn;\u0026thinsp;7.23% vs. 47.07\u0026thinsp;\u0026plusmn;\u0026thinsp;33.24%) in pSS patients, while CCL2 levels were significantly higher (1015.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1714.46% vs. 290.77\u0026thinsp;\u0026plusmn;\u0026thinsp;137.02%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eTo elucidate the therapeutic mechanism of MSC, we conducted a transwell co-culture experiment using PBMC from pSS patient and MSC. The experimental conditions included PBMC alone, MSC\u0026thinsp;+\u0026thinsp;PBMC, recombinant Gal-1\u0026thinsp;+\u0026thinsp;PBMC, and MSC\u0026thinsp;+\u0026thinsp;PBMC with Gal-1 inhibitor TDG. In both the MSC\u0026thinsp;+\u0026thinsp;PBMC and Gal-1\u0026thinsp;+\u0026thinsp;PBMC groups, the numbers of Tph cells were significantly lower than in the PBMC alone group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). There was no significant change in the proportion of Tfh cells, Treg cells and Tph/Treg in all treatment groups, and the differences between all groups did not reach statistical significance. Additionally, ELISA analysis of the supernatants revealed elevated Gal-1 levels and decreased CCL2 levels in the MSC\u0026thinsp;+\u0026thinsp;PBMC and Gal-1\u0026thinsp;+\u0026thinsp;PBMC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). These results suggest that MSC modulate Tph cells in pSS patients via the Gal-1 protein.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that the elevated proportion of Tph cells in pSS patients was correlated with the ESSDAI score. It was also linked to serum C3 and IgG levels, as well as the proportion of CD19\u0026thinsp;+\u0026thinsp;CD11c\u0026thinsp;+\u0026thinsp;B cells. The results suggest a connection between Tph cells and IL-21. This indicates a mechanism through which Tph cells may be involved in the pathogenesis of pSS disease. Tph cells have been shown to be increased in seropositive RA patients, and associated with disease activity[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In RA, Tph cells have also been found to induce production of CXCL13 in synovial tissue, recruiting CXCR5\u003csup\u003e+\u003c/sup\u003e naive B cells and Tfh cells[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, Tph cells promote B cell differentiation through IL-21 and signaling lymphocytic activation molecule family member 5 (SLAMF5) interactions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies have found that Tph cells are significantly enriched in both the peripheral blood and salivary glands with germinal centers in SS patients, express ICOS, serve as the primary source of IL-21[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our findings of increased Tph cells in pSS patients indicate an immune dysfunction and suggest their potential as biomarkers for pSS activity. In addition to the role of Tph cells, our findings showed a significantly elevated Tph/Treg ratio in pSS patients, which correlated with higher ESSDAI scores. This elevated Tph/Treg ratio highlights an imbalance that is likely to exacerbate immune activation and tissue damage in pSS. Therefore, the Tph/Treg ratio in pSS patients may represent another important indicator of disease severity and immune dysfunction, further supporting the notion that Tph cells act as drivers of inflammation in pSS.\u003c/p\u003e \u003cp\u003eDry mouth is a cardinal symptom of Sj\u0026ouml;gren's syndrome, which is mainly caused by T lymphocyte infiltration and the subsequent destruction of exocrine glands [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although the specific T cell subsets that drive pSS pathogenesis remain elusive, studies have identified CXCR5\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003ehi\u003c/sup\u003e T cells within the germinal centers of salivary glands (SGs) in pSS patients, which co-express IL-21 and IFN-γ[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, the increased expression of CCR2 protein and CCL2 in SGs suggests that Tph cells, which are characterized by CCR2 expression, play a role in the immunopathogenesis of disease [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our immunofluorescence analysis of the salivary gland tissue from pSS patient disclosed a correlation between the intensity of lymphocytic infiltration and the abundance of CD4\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003eCCR2\u003csup\u003e+\u003c/sup\u003e Tph cells, CD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003e Tfh cells, and CD4\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003e Treg cells. This finding indicates that higher levels of lymphocytic infiltration are associated with increased presence of these T cell subsets. Notably, Tph cells in the salivary glands were found to express CCR2, which may potentially mediate their migration to these salivary glands. This observation highlights the role of Tph cells in the local pathogenesis of pSS and the significance of chemokines in immune cell migration and tissue-specific infiltration. Moreover, we also found that CD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003e T cells and CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e T cells in salivary gland, consistent with previous findings[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMSC has been increasingly recognized for their therapeutic potential in pSS due to their robust immunomodulatory capabilities, particularly by upregulating Tregs and downregulating Th1, Th17, or Tfh cells[\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additional, MSC can differentiate into salivary epithelial cells[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], offering a promising alternative treatment for pSS. In our study, Tph cells and the Tph/Treg ratio, which are known to play a pivotal role in autoimmune diseases, were found to be decreased in pSS patients following MSC transplantation. This suggests an improvement in the immunoregulatory milieu. Gal-1, β-galactoside-binding lectin critical for cell death, cell adhesion, and immune responses[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], was found to be elevated after MSC treatment. The increase of Gal-1 implies that MSC may exert their anti-inflammatory effects by secreting this lectin, thus having a positive impact on the immune environment in pSS patients. Furthermore, our research indicated a decrease in CCL2 levels following MSC treatment. This decrease may potentially reduce the migration and activation of CCR2\u003csup\u003e+\u003c/sup\u003e Tph cells, thereby mitigating the inflammatory process and improving the immunopathological state of pSS. By upregulating Gal-1, MSC may modulate the CCL2/CCR2 axis, presenting a novel mechanism for curbing inflammatory responses[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The intricate interplay between Gal-1 and Tph cells suggests that MSC therapy could influence immune regulation through multiple pathways. Our in vitro experiments substantiated these findings, demonstrating a significant decrease in the proportions of Tph cells when exposed to MSC co-culture and treatment with recombinant Gal-1 protein. The use of the Gal-1 inhibitor TDG reversed this regulatory effect, underscoring the essential role of Gal-1 in MSC-mediated immunomodulation. ELISA results further confirmed that MSC treatment and the addition of Gal-1 significantly elevated Gal-1 levels and reduced CCL2 levels, indicating a regulatory influence on Tph cells and inflammatory responses through the modulation of Gal-1.\u003c/p\u003e \u003cp\u003eFurther study is needed to comprehensively understand the ways by which MSC regulate the immune environment via Gal-1 and other potential molecular mechanisms. Understanding how these pathways synergize within the immune system could potentially improve therapeutic approaches for autoimmune diseases. Moreover, exploring the impacts of MSC on other key immune cell subsets, such as Tregs, effector T cells (Teffs), and B cells, will provide a more comprehensive perspective on the immunomodulatory functions of MSC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identifies PD-1⁺CXCR5⁻ peripheral helper T cells as key contributors to immune dysregulation in pSS. Elevated Tph cells and the Tph/Treg ratio correlated with disease activity and B cell activation. MSCT reduced Tph cells and restored immune balance. Further research found that Gal-1 plays a crucial role in this process. These findings highlight MSC therapy as a promising strategy for pSS treatment, and Tph cells can be used as an effective index to evaluate the disease activity in pSS patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBcl-6: B-cell lymphoma 6; ESSDAI: EULAR Sj\u0026ouml;gren\u0026apos;s Syndrome Disease Activity Index; ESSPRI: EULAR Sj\u0026ouml;gren\u0026rsquo;s Syndrome Patient Reported Index; FACS: Fluorescence-activated cell sorting; Gal-1: Galectin-1; HC: Healthy Control; ICOS: Inducible T-cell co-stimulator; IgG4-RD: IgG4-related disease; MASP2: Mannan-binding lectin serine protease 2; MSC: Mesenchymal stromal cell; MSCT: Mesenchymal stromal cell transplantation; PBMC: Peripheral blood mononuclear cell; pSS: Primary Sj\u0026ouml;gren\u0026rsquo;s syndrome; RA: Rheumatoid arthritis; SLE: Systemic lupus erythematosus; Tfh: Follicular helper T cell; Tph: Peripheral helper T cell; Treg: Regulatory T cell.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (Grant No. 2020YFA0710800), the Natural Science Foundation of Jiangsu Province (Grant No. BK20240121), and the Clinical Trials Funding from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (Grant No. 2021-LCYJ-PY-16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by the Ethics Committee of Nanjing Drum Tower Hospital. Title of the approved project: Regulation of lipid metabolism and T cell via mTOR by mesenchymal stem cells. Approval number: 2021-035. Date of approval: Jan. 21, 2021. All participants gave their written informed consent approved by the Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School in accordance with the Declaration of Helsinki.\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\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used the Large Language Model—ChatGPT—in the drafting of this paper for grammar and language refinement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLS and LG designed the study. SZ, FY, and TL collected the data. YG and XC performed the experiments and statistical analysis. RC, GY, and XT analyzed the data. SZ, LG, and BT wrote the manuscript. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the present study are not publicly available out of concern for patient privacy, as data from medical records is considered sensitive, but de-identified datasets are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrito-Zeron P, Baldini C, Bootsma H, Bowman SJ, Jonsson R, Mariette X, Sivils K, Theander E, Tzioufas A, Ramos-Casals M. Sjogren syndrome. Nat Rev Dis Primers 2016; 2(16047.\u003c/li\u003e\n\u003cli\u003eBarrera MJ, Bahamondes V, Sepulveda D, Quest AF, Castro I, Cortes J, Aguilera S, Urzua U, Molina C, Perez P\u003cem\u003e et al\u003c/em\u003e. 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Int J Mol Sci 2023; 24(15):\u003c/li\u003e\n\u003cli\u003eMiao M, Hao Z, Guo Y, Zhang X, Zhang S, Wu X, Xu D, Gao C, Li X. SAT0252 The number of treg cells in peripheral blood in pss patients is decreased and low dose IL-2 can promote its proliferation. Annals of the Rheumatic Diseases 2017; 76(Suppl 2):869.\u003c/li\u003e\n\u003cli\u003eRamos-Casals M, Cervera R, Font J, Garcia-Carrasco M, Espinosa G, Reino S, Pallares L, Ingelmo M. Young onset of primary Sjogren\u0026apos;s syndrome: clinical and immunological characteristics. Lupus 1998; 7(3):202-206.\u003c/li\u003e\n\u003cli\u003eIwamoto N, Kawakami A, Arima K, Nakamura H, Kawashiri SY, Tamai M, Kita J, Okada A, Koga T, Kamachi M\u003cem\u003e et al\u003c/em\u003e. Regulation of disease susceptibility and mononuclear cell infiltration into the labial salivary glands of Sjogren\u0026apos;s syndrome by monocyte chemotactic protein-1. 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Life Sci 2021; 269(119034.\u003c/li\u003e\n\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Primary Sjögren's syndrome, Mesenchymal stromal (stem) cell, peripheral helper T cell, IL-21, Galectin-1","lastPublishedDoi":"10.21203/rs.3.rs-6173991/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6173991/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of PD-1\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003e-\u003c/sup\u003e peripheral helper T (Tph) cells in patients with primary Sjogren's syndrome (pSS) and to assess the therapeutic efficacy of mesenchymal stromal cells (MSC) in pSS patients through modulating Tph cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe measured the frequencies and numbers of T cell subsets, including Tph cells, follicular helper T (Tfh) cells, and Treg cells, as well as the levels of IL-21 in patients with pSS and healthy controls (HC). Additionally, we analyzed their correlations with the levels of serological indicators (IgG, C3, C4) and the score of the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). Immunofluorescence technique was employed to assess the infiltration of lymphocytes in labial gland tissues. Before and after mesenchymal stromal cell transplantation (MSCT). The alterations in Tph, Tfh, and Treg cell subsets were -evaluated using Fluorescence-activated cell sorting (FACS). Meanwhile, proteomic analysis of peripheral blood samples was conducted to identify the key proteins associated with Tph cells, and these proteins were subsequently validated in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with CXCR5\u003csup\u003e-\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+ \u003c/sup\u003eTph cells and the Tph/Treg ratio were significantly higher in pSS patients than in the HC group. The proportion of circulating Tph cells and Tph/Treg ratio were significantly positively correlated with the ESSDAI score. MSC treatment effectively increased the levels of C3 and C4, while reducing the levels of IgG, the EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI) score, and the ESSDAI score. MSC significantly reduced the proportion of Tph cells and the Tph/Treg ratio, while increased the proportion of Treg cells, which contributed to restoring immune homeostasis. Proteomic analysis and co-culture experiments of MSC and peripheral blood mononuclear cells (PBMC) indicated that MSC may ameliorate the immune imbalance in pSS patients by up-regulating galectin-1 (Gal-1), thereby inhibiting Tph cell-associated inflammatory responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study established a link between increased Tph cell counts and increased Tph/Treg ratios with enhanced disease activity in pSS patients. MSC therapy, which reduces the number of Tph cells by inducing the expression of galectin-1, emerges as a promising therapeutic approach for pSS.\u003c/p\u003e","manuscriptTitle":"Mesenchymal Stromal Cells Attenuate Primary Sjogren's Syndrome by Modulating PD-1+CXCR5− T Peripheral Helper Cells via Galectin-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 10:17:56","doi":"10.21203/rs.3.rs-6173991/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-01T10:51:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T09:54:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-25T10:05:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-03-24T03:55:40+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2025-03-17T16:18:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4763d6f4-cf18-42af-8e3e-38fa189658b8","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T16:04:33+00:00","versionOfRecord":{"articleIdentity":"rs-6173991","link":"https://doi.org/10.1186/s13287-025-04631-9","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-10-06 15:57:57","publishedOnDateReadable":"October 6th, 2025"},"versionCreatedAt":"2025-05-06 10:17:56","video":"","vorDoi":"10.1186/s13287-025-04631-9","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04631-9","workflowStages":[]},"version":"v1","identity":"rs-6173991","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6173991","identity":"rs-6173991","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}