TIM3-Mediated Differentiation of IL-10-Producing CD25+ B Cells by Expanded Regulatory T Cells

TIM3-Mediated Differentiation of IL-10-Producing CD25+ B Cells by Expanded Regulatory T Cells | 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 TIM3-Mediated Differentiation of IL-10-Producing CD25+ B Cells by Expanded Regulatory T Cells Rowa Y Alhabbab, Daniela Mastronicola, Giovanna Lombardi, Cristiano Scottá This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6135682/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Dec, 2025 Read the published version in Journal of Molecular Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Cell-based immunotherapy utilizing regulatory T cells (Tregs) has recently advanced into clinical applications, demonstrating promising results in phase I/II trials to prevent transplant rejection and treat autoimmune diseases. We have completed a clinical trial in renal transplant patients in which the significant biological effect was the increase of B cells with a regulatory phenotype in the blood of kidney transplant patients. The mechanisms by which Tregs regulate B cells and the specific molecules involved in this process remained poorly understood. In this study, we employed an in vitro system of co-culture of peripherally purified B cells and expanded Tregs to show that Tregs can induce a population of memory B cells that express IL-10 and CD25. This subset of B cells has been previously identified as one of humans' regulatory B cell populations. Notably, these expanded Tregs’ regulation of B cells was found to be independent of IL-10 and reliant on direct cell contact. We established that TIM3 expression by Tregs was crucial for the induction of IL-10-producing CD25 + memory B cells. Our findings suggest that TIM3 is a critical molecule for the induction of regulatory B cells by Tregs, indicating that TIM3 expression by adoptively transferred Tregs is vital in diseases where B cells play a pathogenic role. Regulatory T cells B cells TIM3 CD25 IL-10 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message Expanded Tregs induce IL-10+ CD25+ B cells. TIM3 expression on Tregs is crucial for IL-10+ B cell induction. Tregs require direct cell contact to regulate B cells. Blocking TIM3 reduces IL-10+ B cells but increases IFN-γ, TNF-α, IL-17. Tregs enhance regulatory B cell differentiation, promoting tolerance. INTRODUCTION Regulatory T cells (Tregs) are a small but crucial subset of CD4 + T cells that prevent autoimmune diseases and maintain immune homeostasis [ 1 ]. Numerous studies have demonstrated that impairments in the function or number of Tregs are associated with the development of various autoimmune disorders [ 2 , 3 ]. Tregs exert their immunosuppressive effects by limiting the activation and proliferation of other immune cells through multiple mechanisms. These include the production of anti-inflammatory cytokines such as TGF-β, IL-35, and IL-10 and the expression of membrane-bound molecules like CD39, TIGIT, LAG-3, and CTLA-4. Additionally, Tregs can modulate the function of antigen-presenting cells through cell contact-dependent mechanisms, which alter the capacity of these cells for co-stimulation and antigen presentation. Their high expression of CD25 allows Tregs to sequester local IL-2, thereby limiting the expansion and function of effector T cells by depriving them of this critical growth factor [ 4 ]. Given their regulatory characteristics, Tregs have emerged as an attractive population for immunotherapy. In recent years, Tregs have been successfully isolated and expanded ex vivo in large numbers. Several phase I/II clinical trials have been conducted with promising results, some still ongoing [ 5 ], while others have been completed and shown some biological efficacy [ 6 , 7 ]. Notably, our research has demonstrated that the infusion of polyclonal Tregs in kidney transplant patients leads to a dose-dependent increase in B cells with regulatory phenotype in the blood of treated individuals, suggesting that Tregs can influence B cell fate towards a more regulatory phenotype [ 8 ] and, more recently, an increase in another population of regulatory B cells (Bregs) has been seen in the first three renal transplant patients treated with Tregs in the TWO Study [ 9 ]. In humans, various B cell subsets have been identified that possess regulatory capacities through IL-10 production. These include transitional B cells (CD24 hi CD38 hi ) [ 10 ], CD19 + CD24 hi CD27 + B10 cells [ 11 ], plasmablasts (CD27 inter CD38 + ), TIM1 + Bregs [ 12 ], and CD25 + memory B cells [ 12 , 13 ]. IL-10-producing B cells have been shown to modulate T cell responses by suppressing T helper 1 (Th1) and Th17 cells while promoting Treg induction [ 10 , 14 ]. Furthermore, studies in autoimmune and transplant models indicate that the adoptive transfer of IL-10-producing B cells can improve disease outcomes [ 15 , 16 ]. Despite these insights, the mechanisms by which Tregs regulate B cells and the specific molecules involved in their crosstalk remain poorly understood. In this study, we demonstrate using an in vitro system that functionally enhanced ex vivo expanded Tregs are highly effective at inducing IL-10 + B cells, unlike freshly isolated Tregs. We found that TIM3 expression on expanded Tregs is essential for this effect. The induced IL-10 + B cells express CD25 and exhibit a memory phenotype (CD24 hi CD38 - ). Our findings extend the understanding of the critical role of TIM3 in Treg function, particularly in Treg therapies applied in conditions where B cells contribute to pathogenic processes. MATERIAL AND METHODS Human blood samples. All human blood samples were obtained from anonymous healthy donors with informed consent and full ethical authorization. Peripheral blood, collected as leukocyte-enriched blood cones, was supplied by the National Blood Service (NHS Blood and Transplantation, Tooting, London, UK). The Institutional Review Board of Guy's Hospital granted this study's ethical approval under reference number 09/H0707/86. Cell isolation and co-culture assays Peripheral blood mononuclear cells (PBMCs) were isolated by lymphoprep (Stemcell Technologies, UK) density gradient centrifugation. CD19 + B cells were enriched by negative selection via magnetic sorting (Miltenyi Biotec, UK). The purity of the B cells isolated with this protocol was always more than 95–98% by flow cytometry. To prepare activated γ-irradiated conventional CD4 + T cells (iTcells), CD4 + T cells were isolated by RosetteSep and incubated for 5 hours with a T cell activation cocktail (Cat. 423301, BioLegend, UK). The cells were then γ-irradiated and tested for CD40L expression by flow cytometry. They were cryopreserved at -80°C and thawed directly before being used. The iTcells were utilised at a 1:4 ratio with B cells. CD4 + T cells isolated by RosetteSep were also used for obtaining CD4 + CD25 + Tregs by CD25 microbeads magnetic enrichment (Miltenyi), and FACS sorted using antibodies specific for CD4, CD25, CD127 and CD45RA. Tregs were then either used directly in our co-culture setting with the negatively sorted B cells or expanded by using anti-CD3/CD28 beads (Miltenyi) in the presence of 100nM rapamycin (LC-laboratories) and 1000 IU/ml recombinant human IL-2 in X-vivo15 medium (Lonza) supplemented with 5% human AB serum (Biosera), as previously published [ 17 ]. Expanded Tregs (Treg exp ) were collected and co-cultured with B cells (at a 1:1 ratio) in the presence of anti-CD3/CD28 beads for 48 hours. Intracellular staining PMA, ionomycin and brefeldin A were added for the last 4 hours of co-cultures, and cytokines, including IFN-γ, TNF-α, IL-17 and IL-10, were measured by intracellular staining (ICC). Trans-well system and antibody neutralisation assay. Tregs and anti-CD3/CD28 beads were plated at the bottom of the well. The trans-well insert was placed on top, with B cells and iTcells (4:1 ratio). The plates were incubated for 48 hours. Anti-TIM3 blocking antibodies (10µg/ml) were added to the co-cultures, and the cells were incubated for 48 hours. PMA, ionomycin and brefeldin A were added for the last 4 hours of the culture, and cytokines were measured ICC by flow cytometry. tSNE analysis. Singlet live cells were gated using Flowjo10, and additional gates were applied as requested. Singlet live cells of all samples were down-sampled to 5000 events, and each group (non-stimulated, stimulated and 1stimulated-Bcells:1Tregs) was concatenated into one file. tSNE was run on the concatenated files, and grouped data were gated. Cell clusters were identified and overlapped by the gated population on the tSNE map. The MFI for each molecule within each cell population was then measured. Statistical analysis Comparisons between groups were performed using a T-test or two-way ANOVA and Tukey’s multiple comparisons as specified. Analyses were performed using GraphPad Prism software. RESULTS Expanded Tregs induce IL-10-expressing B cells. Building on the evidence that following the adoptive transfer of Tregs, B cells with regulatory phenotype are increasing in renal transplant patients [ 18 ]. This study focused on understanding how expanded human Tregs impact B cell phenotype and function. To do so, we established an in vitro system in which human Tregs and B cells were activated by irradiated allogeneic T cells. Tregs were enriched from blood, expanded ex vivo using a well-established protocol in our laboratory and adopted in our previous clinical trials [ 19 ]. Briefly, Tregs were purified from the peripheral blood of healthy volunteers by density gradient separation of PBMC followed by magnetic bead separation of CD4 + CD25 high T cells and FASC sorting (F-Tregs). Then, Tregs were stimulated with anti-CD3/CD28 beads (ratio cell/bead 1:1) and cultured for two weeks in the presence of IL-2 (1,000 IU/ml) and rapamycin (100 nM), as previously described, [ 20 , 21 ] to obtain a highly pure and suppressive expanded regulatory T cells (Treg exp ). At the end of the culture, the purity of Treg exp was verified by flow cytometry staining using the conventional markers associated with functional Tregs and compared to F-Tregs. As shown in Fig. 1 a, Treg exp increased CD25, FOXP3, CTLA4, and HELIOS expression levels while CD12s maintained low compared to freshly isolated Tregs [ 22 – 24 ]. Treg exp were co-cultured at varying ratios with CFSE-labelled conventional T cells (Teff) activated with anti-CD3/CD28 beads to assess their suppressive capacity. In these settings, the inhibition of Teff proliferation was measured via flow cytometry by CFSE dilution, and Treg exp suppressive capacity was compared to F-Tregs as previously described (Fig. 1 b) [ 20 , 21 ]. Treg exp were more suppressive than F-Tregs, further confirming the enhanced function of Treg exp . Similarly, B cells (1 x 10 7 ) were isolated from the peripheral blood of healthy volunteers by density gradient separation of PBMC followed by magnetic bead separation of CD19 + cells (purity > 95%). B cells were then activated by activated γ-irradiated conventional CD4 + T cells (iTcell) expressing high levels of CD40L (activated-B cells). To investigate the cytokines produced by B cells after 48-hour stimulation with only iTcells (baseline), we analysed the percentage of B cells producing IFN-γ, TNF-α, IL-17, and IL-10, using intracellular staining (see the gating strategy in Supplementary Fig. 1a ). The results showed that a fraction of activated-B cells were able to produce IFN-γ, TNF-α, and IL-17, but not IL-10 (Figs. 1 c-e). At the same time, to test the effect of Tregs, activated-B cells were co-cultured for 48 hours at a 1:1 ratio with either freshly isolated F-Tregs or Treg exp . Data in Fig. 1 c-e showed that in the presence of both F-Tregs and Treg exp , the percentages of B cells expressing IL-10 increased; however, the co-culture with Treg exp resulted in even higher percentages of B cells producing this cytokine. Moreover, the percentages of IFN-γ, IL-17, and TNF-α were significantly reduced in B cells upon co-culture with F-Tregs and Treg exp (Figs. 1 d-e). The expression of cytokines was also analysed in F-Tregs and Treg exp before and after the co-culture with activated B cells. Supplementary Fig. 1b-c shows that compared to F-Tregs, the percentage of Treg exp expressing IFN-γ, TNF-α, and IL-17 was very low. In both F-Tregs and Treg exp conditions, the percentage of TNF-α expressing cells was reduced following the co-culture with activated-B cells. IL-10 production was only detectable in F-Tregs, and the co-culture with activated-B cells reduced the percentage of cells producing this cytokine ( Supplementary Figs. 1b-c ). Treg exp induces a regulatory phenotype in the memory B cell subset. Following the evidence that the co-culture of activated B cells with Treg exp increased the percentages of IL-10-producing B cells, we sought to investigate the changes in the B cell subpopulations. B cells purified from the blood were stained with antibodies specific for CD24 and CD38 on CD19 + cells and analysed by flow cytometry. Three different subpopulations of B cells were identified: CD24 + CD38 − memory (CD24 + CD38 − ), transitional (CD24 + CD38 hi ) and mature (CD24 int CD38 int ) B cells (Fig. 2 a ) . The activation of B cells significantly increased the percentage of CD24 int CD38 int mature subset, and this effect was inhibited by the presence of Treg exp in the co-culture (Fig. 2 b). The other B cell subpopulations, with or without the co-culture with Treg exp , did not drastically change. The phenotypic characteristics of B cells at the end of the co-culture were also analysed by t-distributed stochastic neighbour embedding (tSNE); all data obtained from the co-culture conditions described above were combined to assess B cell subset distribution based on CD24 and CD38 expression along with the expression of IL-10 and CD25 (Fig. 2 c). The analysis confirmed that Treg exp mostly induced IL-10 production in B cells and that IL-10-producing B cells were clustered within the memory B cell subset (Fig. 2 d). Further analysis of CD25 expression, a molecule previously associated with a regulatory phenotype in B cells producing high levels of IL-10 [ 6 , 25 ], showed that co-culturing activated-B cells with Treg exp significantly increased this marker (Fig. 2 e). Among the three defined B cell subpopulations, memory B cells expressed the highest level of IL-10 and CD25 compared to the other subsets (Fig. 2 d-e). Of note, memory B cells were IgM hi , IgD low and CD27 low , suggesting that these B cells were memory precursors that can generate CD27 hi memory B cells ( Supplementary Fig. 2a-c ) [ 7 ]. All these findings support the idea that the presence of Treg exp induces the memory B cell subset to acquire a regulatory phenotype characterised by the high expression of CD25 and the production of IL-10. The expression of TIM3 on Treg exp mediates the induction of tolerogenic B cells. After observing that Treg exp may influence the differentiation of a memory B cell subset with anti-inflammatory properties, we explored the mechanisms behind this effect. To test whether Treg exp acts through direct cell-to-cell contact, we used trans-well inserts to physically separate stimulated Treg exp from stimulated B cells during a 48-hour co-culture period. When Treg exp were separated from B cells, the induction of IL-10 + B cells was prevented, and the reduction in IFN-γ and TNF-α B cells seen in co-cultures was also abolished (Fig. 3 a-c). These findings indicate that Treg exp required direct cell-to-cell contact to affect B-cell properties. To identify the mechanisms used by Treg exp , we examined the expression of molecules associated with their regulatory functions and compared them to F-Tregs [ 26 – 35 ]. F-Tregs and Treg exp were either left non-stimulated or activated with anti-CD3/CD28 beads, with or without activated-B cells at a 1:1 ratio for 48 hours. We evaluated the expression of CD134, TIM3, GARP, ICOS, CTLA-4, CD200, CD30, DR3, CD40L, and Galectin-9 (Gal-9) using flow cytometry. To visualise and confirm the most dominant molecule expressed by Treg exp and at lower levels by F-Tregs under different culture conditions, we normalised the mean fluorescence intensity (MFI) data, setting the lowest value to 0% and the highest to 100% (Fig. 4 a). Although Treg exp expressed high levels of CD134, TIM3, CTLA-4, and Gal-9, we found that the co-culture of Treg exp with B cells induced a very high expression of TIM3 and Gal-9 on Treg exp . However, while similar levels of Gal-9 were also expressed on F-Tregs, the exceptionally high expression of TIM3 was restricted only on Treg exp co-cultured with B cells (Fig. 4 a). The following analysis of the same samples using the tSNE algorithm (n = 5, 5000 events per sample) helped to visualise and identify the TIM3 distribution in the distinct cell clusters. Data in Fig. 4 b shows that the co-culture of B cells with Treg exp induced the whole population to express TIM3. Furthermore, Fig. 4 c shows that when TIM3 expression was compared between the two preparations of Tregs, Trege xp expressed considerably higher levels of TIM3 (MFI) compared to F-Tregs in all three different conditions (non-stimulated, stimulated and at 1:1 ratio with B cells). These results suggested that the expression of TIM3 by Treg exp played a crucial role in the induction of IL-10 + memory B cells. To confirm that TIM3 is involved in the crosstalk between Treg exp and B cells, the two cell types were co-cultured in the presence of an anti-TIM3 blocking antibody. Flow cytometry analysis showed a significant decrease in the percentages of IL-10 + B cells in the presence of anti-TIM3 (Fig. 5 a). However, blocking TIM3 did not affect the inhibition of IFN-γ and TNF-α in B cells (Fig. 5 a). In the same culture conditions, the inhibition of TIM3 increased the percentages of IFN-γ, TNF-α, and IL-17-producing Treg exp , with no effect on IL-10 production (Fig. 5 b). These results suggest that the crosstalk between Treg exp and B cells is complex and requires multiple signals. While TIM3 engagement on Treg exp plays a critical role in inducing IL-10 production in B cells, it is not necessary to inhibit proinflammatory cytokines such as IFN-γ and TNF-α. Conversely, blocking TIM3 signalling on Treg exp stimulates the production of IFN-γ, TNF-α, and IL-17 by Treg exp . DISCUSSION In this study, we utilised a novel in vitro co-culture system of human B cells and expanded Tregs to demonstrate the potent ability of TIM3-expressing Tregs to induce memory IL-10 + CD25 + B cells. Importantly, our findings highlight that TIM3 expression is crucial for this effect. Expanded Tregs offer significant advantages over freshly isolated Tregs, particularly in clinical applications. Expanded Tregs, similar to those utilized in clinical trials for liver and kidney transplantation (e.g., the ONE study and the THRIL study), exhibit enhanced functionality and stability. Notably, expanded Tregs express high levels of TIM3, making them particularly effective at modulating T-cell-mediated and B-cell responses. This dual capability positions them as ideal candidates for therapeutic strategies promoting tolerance in transplant settings. TIM3 is a molecule expressed on various immune cells, including B cells, T cells, monocytes, and dendritic cells. It plays a critical role in regulating immune responses and has been identified as a potential therapeutic target for several immune-related disorders, including cancer and sepsis. Recent literature has highlighted that tumours can exploit TIM3's expression on tumour cells to evade immune detection [ 36 ]. Additionally, TIM3’s involvement in sepsis underscores its multifaceted role in immune regulation [ 37 ]. B cells are central to immune tolerance induction, with various subsets capable of downregulating inflammatory responses associated with autoimmunity and transplant rejection, primarily through IL-10 production [ 10 , 15 ]. CD25 + B cells, initially recognised as a distinct subset, have been shown to differentiate upon stimulation via toll-like receptors [ 38 ]. These cells are now considered a regulatory B cell subset with memory characteristics that can enhance the Treg function [ 39 ]. Memory B cells, also known as B10 cells in humans, are particularly significant as IL-10 producers [ 40 , 41 ]. While naïve B cells can convert CD4 + CD25 − T cells into CD4 + CD25 + Tregs [ 42 , 43 ], our study uniquely demonstrates that memory B cells expressing IL-10 and CD25 can be induced by Tregs, specifically ex vivo expanded Tegs, emphasizing the critical role of TIM3 in this process. The interaction between expanded Tregs and B cells is complex and likely involves multiple communication mechanisms. The ligation of TIM3 with its ligand, galectin-9, is known to inhibit Th1 responses and promote peripheral tolerance [ 44 – 46 ]. TIM3 also plays a vital role in T cell exhaustion during chronic viral infections, where its inhibition enhances cytokine production specific to HCV and HIV [ 47 , 48 ]. In cancer, blocking TIM3 signalling has been shown to improve the function of tumour-infiltrating lymphocytes [ 49 ]. Furthermore, reduced TIM3 expression is associated with the development of autoimmune diseases [ 50 ]. Importantly, we found that blocking TIM3 during the co-culture of Tregs and B cells explicitly influences the induction of IL-10 production in B cells without affecting the Treg-mediated suppression of pro-inflammatory cytokines such as IFN-γ and TNF-α. These findings suggest a complex interplay between Tregs and B cells that may involve additional molecular mechanisms. Interestingly, the effect of TIM3 blockade on Tregs indicates that TIM3 also plays a role in regulating the expression of inflammatory cytokines in these cells. When TIM3 is inhibited, Tregs may begin to produce cytokines such as IFN-γ, TNF-α, and IL-17, which could compromise their regulatory function. Our results are consistent with previous studies in both human and murine models, demonstrating that blocking TIM3 on stimulated conventional T cells significantly increases IFN-γ secretion [ 51 , 52 ]. This underscores TIM’'s essential role in T cell immunoregulation. Furthermore, while TIM3-expressing Tregs are more potent suppressors than TIM3-negative Tregs, they are also typically enriched in IL-10 [ 52 , 53 ]. The discrepancies between these findings may stem from differences in the experimental models used, particularly the reliance on murine Tregs versus human cells. Tregs are well-established for suppressing inflammation through various mechanisms, including the production of IL-10, TGF-β, and IL-35, as well as through direct cell contact [ 4 ]. Their role in alleviating the severity of diseases such as autoimmunity and transplantation has been well documented [ 4 ]. Consequently, Tregs have emerged as promising candidates for cell-based immunotherapy. They can be isolated and expanded ex vivo in large quantities, shifting the balance between effector T cells and Tregs favouring the latter [ 54 ]. Our findings indicate that expanded Tregs expressing TIM3 are crucial for inducing CD25 + memory IL-10 + B cells contact-dependent, independent of IL-10. This suggests that TIM3 + Tregs enhance their suppressive capacity by promoting the differentiation of additional regulatory B cells, thereby creating a more tolerogenic environment. In conclusion, our study demonstrates that expanded Tregs, particularly those expressing TIM3, exhibit characteristics akin to exhaustion while maintaining robust suppressive functions. These Tregs effectively promote IL-10 expression in stimulated B cells with a memory phenotype. Leveraging TIM3 + Tregs could enhance their suppressive capacity and facilitate beneficial interactions with B cells, paving the way for innovative immune-based therapies. Declarations Acknowledgment We thank Brunel University of London, King’s College London and King Abdulaziz University for supporting this work. This research was supported by Lupus UK, Diabetes UK, King's Health Partners, MRC-Impact Accelerator 2022, BD Biosciences Research Program Award, Rosetrees Trust, National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London and the NIHR Clinical Research Facility. Funding This research was supported by Lupus UK, Diabetes UK (ref. 24/0006776), King's Health Partners, BD Biosciences Research Program (ref. 10/2017 Award), Rosetrees Trust (ref. CF-2021-2 107), National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London and the NIHR Clinical Research Facility. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data Availability Statement The data supporting this study's findings are available upon request from the corresponding authors. Ethical approval This study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was granted by the Institutional Review Board of Guy's Hospital under reference number 09/H0707/86. Consent to participate Informed consent was obtained from all individual participants included in the study. Author contributions Conceptualisation: RYA, GL and CS; Sample collection and methodology: RYA, CS and DM; Data curation: RYA and CS; Data analysis and interpretation: RYA, CS and DM; Investigation: RYA and CS; Data visualisation: RYA and CS; Resources and funding acquisition: RYA, GL and CS; Writing - Original draft: RYA and CS; Review and editing: RYA, GL and CS; Supervision: CS. References Yamaguchi T, Teraguchi S, Furusawa C, Machiyama H, Watanabe TM, Fujita H, Sakaguchi S, Yanagida T (2019) Theoretical modeling reveals that regulatory T cells increase T-cell interaction with antigen-presenting cells for stable immune tolerance. International Immunology 31: 743-753. DOI 10.1093/intimm/dxz043 Sharma VK, Kaveri SV, Bayry J (2015) Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits? Cellular & Molecular Immunology 13: 135-137. DOI 10.1038/cmi.2015.98 Eggenhuizen PJ, Ng BH, Ooi JD (2020) Treg Enhancing Therapies to Treat Autoimmune Diseases. International Journal of Molecular Sciences 21. DOI 10.3390/ijms21197015 Romano M, Fanelli G, Albany CJ, Giganti G, Lombardi G (2019) Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity. Frontiers in Immunology 10. DOI 10.3389/fimmu.2019.00043 Giganti G, Atif M, Mohseni Y, Mastronicola D, Grageda N, Povoleri GA, Miyara M, Scotta C (2021) Treg cell therapy: How cell heterogeneity can make the difference. Eur J Immunol 51: 39-55. DOI 10.1002/eji.201948131 Todo S, Yamashita K, Goto R, Zaitsu M, Nagatsu A, Oura T, Watanabe M, Aoyagi T, Suzuki T, Shimamura T, et al. (2016) A pilot study of operational tolerance with a regulatory T‐cell‐based cell therapy in living donor liver transplantation. Hepatology 64: 632-643. DOI 10.1002/hep.28459 Baeten P, Van Zeebroeck L, Kleinewietfeld M, Hellings N, Broux B (2021) Improving the Efficacy of Regulatory T Cell Therapy. Clinical Reviews in Allergy & Immunology 62: 363-381. DOI 10.1007/s12016-021-08866-1 Sawitzki B, Harden PN, Reinke P, Moreau A, Hutchinson JA, Game DS, Tang Q, Guinan EC, Battaglia M, Burlingham WJ, et al. (2020) Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials. The Lancet 395: 1627-1639. DOI 10.1016/s0140-6736(20)30167-7 Brook MO, Hester J, Petchey W, Rombach I, Dutton S, Bottomley MJ, Black J, Abdul-Wahab S, Bushell A, Lombardi G, et al. (2022) Transplantation Without Overimmunosuppression (TWO) study protocol: a phase 2b randomised controlled single-centre trial of regulatory T cell therapy to facilitate immunosuppression reduction in living donor kidney transplant recipients. BMJ Open 12. DOI 10.1136/bmjopen-2022-061864 Blair PA, Noreña LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, Mauri C (2010) CD19+CD24hiCD38hi B Cells Exhibit Regulatory Capacity in Healthy Individuals but Are Functionally Impaired in Systemic Lupus Erythematosus Patients. Immunity 32: 129-140. DOI 10.1016/j.immuni.2009.11.009 van der Vlugt LEPM, Mlejnek E, Ozir‐Fazalalikhan A, Janssen Bonas M, Dijksman TR, Labuda LA, Schot R, Guigas B, Möller GM, Hiemstra PS, et al. (2014) CD24hiCD27+ B cells from patients with allergic asthma have impaired regulatory activity in response to lipopolysaccharide. Clinical & Experimental Allergy 44: 517-528. DOI 10.1111/cea.12238 Alhabbab RY, Nova-Lamperti E, Aravena O, Burton HM, Lechler RI, Dorling A, Lombardi G (2019) Regulatory B cells: Development, phenotypes, functions, and role in transplantation. Immunological Reviews 292: 164-179. DOI 10.1111/imr.12800 Amu S, Brisslert M (2011) Phenotype and Function of CD25-Expressing B Lymphocytes Isolated from Human Umbilical Cord Blood. Clinical and Developmental Immunology 2011: 1-9. DOI 10.1155/2011/481948 Flores-Borja F, Bosma A, Ng D, Reddy V, Ehrenstein MR, Isenberg DA, Mauri C (2013) CD19+CD24hiCD38hi B Cells Maintain Regulatory T Cells While Limiting TH1 and TH17 Differentiation. Science Translational Medicine 5. DOI 10.1126/scitranslmed.3005407 Alhabbab R, Blair P, Elgueta R, Stolarczyk E, Marks E, Becker PD, Ratnasothy K, Smyth L, Safinia N, Sharif-Paghaleh E, et al. (2020) Author Correction: Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model. Scientific Reports 10. DOI 10.1038/s41598-020-62381-5 Alhabbab R, Blair P, Smyth LA, Ratnasothy K, Peng Q, Moreau A, Lechler R, Elgueta R, Lombardi G (2018) Galectin-1 is required for the regulatory function of B cells. Scientific Reports 8. DOI 10.1038/s41598-018-19965-z Aref S, Azmy E, El‐Gilany AH (2015) Upregulation of CD200 is associated with regulatory T cell expansion and disease progression in multiple myeloma. Hematological Oncology 35: 51-57. DOI 10.1002/hon.2206 McCallion O, Cross AR, Brook MO, Hennessy C, Ferreira R, Trzupek D, Mulley WR, Kumar S, Soares M, Roberts IS, et al. (2024) Regulatory T cell therapy is associated with distinct immune regulatory lymphocytic infiltrates in kidney transplants. Med. DOI 10.1016/j.medj.2024.11.014 Gorczynski R, Khatri I, Lee L, Boudakov I (2008) An Interaction between CD200 and Monoclonal Antibody Agonists to CD200R2 in Development of Dendritic Cells That Preferentially Induce Populations of CD4+CD25+ T Regulatory Cells. The Journal of Immunology 180: 5946-5955. DOI 10.4049/jimmunol.180.9.5946 Scotta C, Fanelli G, Hoong SJ, Romano M, Lamperti EN, Sukthankar M, Guggino G, Fazekasova H, Ratnasothy K, Becker PD, et al. (2015) Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells. Haematologica 101: 91-100. DOI 10.3324/haematol.2015.128934 Romano M, Fanelli G, Tan N, Nova-Lamperti E, McGregor R, Lechler RI, Lombardi G, Scottà C (2018) Expanded Regulatory T Cells Induce Alternatively Activated Monocytes With a Reduced Capacity to Expand T Helper-17 Cells. Frontiers in Immunology 9. DOI 10.3389/fimmu.2018.01625 Scotta C, Esposito M, Fazekasova H, Fanelli G, Edozie FC, Ali N, Xiao F, Peakman M, Afzali B, Sagoo P, et al. (2012) Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4+CD25+FOXP3+ T regulatory cell subpopulations. Haematologica 98: 1291-1299. DOI 10.3324/haematol.2012.074088 Liu W, Putnam AL, Xu-yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, de St. Groth BF, et al. (2006) CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. The Journal of Experimental Medicine 203: 1701-1711. DOI 10.1084/jem.20060772 Lowther DE, Goods BA, Lucca LE, Lerner BA, Raddassi K, van Dijk D, Hernandez AL, Duan X, Gunel M, Coric V, et al. (2016) PD-1 marks dysfunctional regulatory T cells in malignant gliomas. JCI Insight 1. DOI 10.1172/jci.insight.85935 Theil A, Tuve S, Oelschlägel U, Maiwald A, Döhler D, Oßmann D, Zenkel A, Wilhelm C, Middeke JM, Shayegi N, et al. (2015) Adoptive transfer of allogeneic regulatory T cells into patients with chronic graft-versus-host disease. Cytotherapy 17: 473-486. DOI 10.1016/j.jcyt.2014.11.005 Vu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A, Killeen N, Ishii N, Chang Li X (2007) OX40 costimulation turns off Foxp3+ Tregs. Blood 110: 2501-2510. DOI 10.1182/blood-2007-01-070748 Comte D, Karampetsou MP, Kis-Toth K, Yoshida N, Bradley SJ, Mizui M, Kono M, Solomon JR, Kyttaris VC, Tsokos GC (2016) Engagement of SLAMF3 enhances CD4+ T-cell sensitivity to IL-2 and favors regulatory T-cell polarization in systemic lupus erythematosus. Proceedings of the National Academy of Sciences 113: 9321-9326. DOI 10.1073/pnas.1605081113 Lucca LE, Dominguez-Villar M (2020) Modulation of regulatory T cell function and stability by co-inhibitory receptors. Nature Reviews Immunology 20: 680-693. DOI 10.1038/s41577-020-0296-3 Ren X, Ye F, Jiang Z, Chu Y, Xiong S, Wang Y (2007) Involvement of cellular death in TRAIL/DR5-dependent suppression induced by CD4+CD25+ regulatory T cells. Cell Death & Differentiation 14: 2076-2084. DOI 10.1038/sj.cdd.4402220 de Streel G, Bertrand C, Chalon N, Liénart S, Bricard O, Lecomte S, Devreux J, Gaignage M, De Boeck G, Mariën L, et al. (2020) Selective inhibition of TGF-β1 produced by GARP-expressing Tregs overcomes resistance to PD-1/PD-L1 blockade in cancer. Nature Communications 11. DOI 10.1038/s41467-020-17811-3 Li D-Y, Xiong X-Z (2020) ICOS+ Tregs: A Functional Subset of Tregs in Immune Diseases. Frontiers in Immunology 11. DOI 10.3389/fimmu.2020.02104 Yi M-H, Zhang E, Kim J-J, Baek H, Shin N, Kim S, Kim SR, Kim H-R, Lee SJ, Park JB, et al. (2016) CD200R/Foxp3-mediated signalling regulates microglial activation. Scientific Reports 6. DOI 10.1038/srep34901 Lam JH, Hong M, Koo S-L, Chua CWL, Lim KL, Wee F, Wan WK, Leow WQ, Yeo JG, Tan IBH, et al. (2021) CD30+OX40+ Treg is associated with improved overall survival in colorectal cancer. Cancer Immunology, Immunotherapy 70: 2353-2365. DOI 10.1007/s00262-021-02859-x Yu Y, Jiang P, Sun P, Su N, Lin F (2021) Analysis of therapeutic potential of preclinical models based on DR3/TL1A pathway modulation. Experimental and Therapeutic Medicine 22. DOI 10.3892/etm.2021.10125 Solanki K, Kumar A, Khan MS, Karthikeyan S, Atre R, Zhang KYJ, Bezsonov E, Obukhov AG, Baig MS (2023) Novel peptide inhibitors targeting CD40 and CD40L interaction: A potential for atherosclerosis therapy. Current Research in Structural Biology 6. DOI 10.1016/j.crstbi.2023.100110 Sauer N, Janicka N, Szlasa W, Skinderowicz B, Kołodzińska K, Dwernicka W, Oślizło M, Kulbacka J, Novickij V, Karłowicz-Bodalska K (2023) TIM-3 as a promising target for cancer immunotherapy in a wide range of tumors. Cancer Immunology, Immunotherapy 72: 3405-3425. DOI 10.1007/s00262-023-03516-1 Wang C, Liu J, Wu Q, Wang Z, Hu B, Bo L (2024) The role of TIM-3 in sepsis: a promising target for immunotherapy? Frontiers in Immunology 15. DOI 10.3389/fimmu.2024.1328667 Brisslert M, Bokarewa M, Larsson P, Wing K, Collins LV, Tarkowski A (2006) Phenotypic and functional characterization of human CD25+ B cells. Immunology 117: 548-557. DOI 10.1111/j.1365-2567.2006.02331.x Kessel A, Haj T, Peri R, Snir A, Melamed D, Sabo E, Toubi E (2012) Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells. Autoimmun Rev 11: 670-677. DOI 10.1016/j.autrev.2011.11.018 Hasan MM, Thompson-Snipes L, Klintmalm G, Demetris AJ, O'Leary J, Oh S, Joo H (2019) CD24hiCD38hi and CD24hiCD27+ Human Regulatory B Cells Display Common and Distinct Functional Characteristics. The Journal of Immunology 203: 2110-2120. DOI 10.4049/jimmunol.1900488 Ran Z, Yue-Bei L, Qiu-Ming Z, Huan Y (2020) Regulatory B Cells and Its Role in Central Nervous System Inflammatory Demyelinating Diseases. Frontiers in Immunology 11. DOI 10.3389/fimmu.2020.01884 Chu K-H, Chiang B-L (2012) Regulatory T Cells Induced by Mucosal B Cells Alleviate Allergic Airway Hypersensitivity. American Journal of Respiratory Cell and Molecular Biology 46: 651-659. DOI 10.1165/rcmb.2011-0246OC Hsu L-H, Li K-P, Chu K-H, Chiang B-L (2014) A B-1a cell subset induces Foxp3− T cells with regulatory activity through an IL-10-independent pathway. Cellular & Molecular Immunology 12: 354-365. DOI 10.1038/cmi.2014.56 Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R (2005) Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439: 682-687. DOI 10.1038/nature04444 Klibi J, Niki T, Riedel A, Pioche-Durieu C, Souquere S, Rubinstein E, Le Moulec S, Guigay J, Hirashima M, Guemira F, et al. (2009) Blood diffusion and Th1-suppressive effects of galectin-9–containing exosomes released by Epstein-Barr virus–infected nasopharyngeal carcinoma cells. Blood 113: 1957-1966. DOI 10.1182/blood-2008-02-142596 Sehrawat S, Suryawanshi A, Hirashima M, Rouse BT (2009) Role of Tim-3/Galectin-9 Inhibitory Interaction in Viral-Induced Immunopathology: Shifting the Balance toward Regulators. The Journal of Immunology 182: 3191-3201. DOI 10.4049/jimmunol.0803673 Jones RB, Ndhlovu LC, Barbour JD, Sheth PM, Jha AR, Long BR, Wong JC, Satkunarajah M, Schweneker M, Chapman JM, et al. (2008) Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. The Journal of Experimental Medicine 205: 2763-2779. DOI 10.1084/jem.20081398 Golden-Mason L, Palmer BE, Kassam N, Townshend-Bulson L, Livingston S, McMahon BJ, Castelblanco N, Kuchroo V, Gretch DR, Rosen HR (2009) Negative Immune Regulator Tim-3 Is Overexpressed on T Cells in Hepatitis C Virus Infection and Its Blockade Rescues Dysfunctional CD4+ and CD8+ T Cells. Journal of Virology 83: 9122-9130. DOI 10.1128/jvi.00639-09 Zhou Q, Munger ME, Veenstra RG, Weigel BJ, Hirashima M, Munn DH, Murphy WJ, Azuma M, Anderson AC, Kuchroo VK, et al. (2011) Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood 117: 4501-4510. DOI 10.1182/blood-2010-10-310425 Zhu C, Anderson AC, Kuchroo VK (2010) TIM-3 and Its Regulatory Role in Immune ResponsesNegative Co-Receptors and Ligands, pp. 1-15. Yang L, Anderson DE, Kuchroo J, Hafler DA (2008) Lack of TIM-3 Immunoregulation in Multiple Sclerosis. The Journal of Immunology 180: 4409-4414. DOI 10.4049/jimmunol.180.7.4409 Ju Y, Shang X, Liu Z, Zhang J, Li Y, Shen Y, Liu Y, Liu C, Liu B, Xu L, et al. (2014) The Tim-3/galectin-9 pathway involves in the homeostasis of hepatic Tregs in a mouse model of concanavalin A-induced hepatitis. Molecular Immunology 58: 85-91. DOI 10.1016/j.molimm.2013.11.001 Sakuishi K, Ngiow SF, Sullivan JM, Teng MWL, Kuchroo VK, Smyth MJ, Anderson AC (2014) TIM3+FOXP3+regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer. OncoImmunology 2. DOI 10.4161/onci.23849 Wieckiewicz J, Goto R, Wood KJ (2010) T regulatory cells and the control of alloimmunity: from characterisation to clinical application. Current Opinion in Immunology 22: 662-668. DOI 10.1016/j.coi.2010.08.011 Supplementary Files SF1.png Supplementary Figure 1. B cells induce small changes to Treg exp . a) Gating strategy used to separate Treg exp , iTcells and B cells in the coculture. b) representative FACS dot plots of IFN-g, TNFa, IL-17 and IL-10-producing F-Tregs and Treg exp following co-culture with B cells. c) Summary data showing the production of IFN-γ, TNF-α and IL-10 expression in F-Tregs and Treg exp co-cultured alone or with B cells. N=3 and N =4 for F-Treg: B cell and Treg exp : B cell co-culture, respectively. Statistics were calculated by t-test, ns- not significant, *P<0.05, **P<0.005, ***P=0.0005. SF2.png Supplementary Figure 2. Phenotypic characteristics of B cells co-cultured with Treg exp . Summary data of the expression (MFI) of IgM (a), IgD (b) and CD27 (c) on non-stimulated or activated B cells in the with or without Treg exp for 48 hrs. B cell subsets were identified using CD19, CD24, CD38, CD27, IgM and IgD expression. Data show mean ± SEM. Statistics were calculated by two-way ANOVA and Tukey’s multiple comparisons tests, ns- not significant, *P<0.05, **P<0.005, ***P=0.0005, (n=3). Cite Share Download PDF Status: Published Journal Publication published 27 Dec, 2025 Read the published version in Journal of Molecular Medicine → Version 1 posted Editorial decision: Major Revisions Needed 29 Jul, 2025 Reviewers agreed at journal 13 Mar, 2025 Reviewers invited by journal 12 Mar, 2025 Editor assigned by journal 04 Mar, 2025 First submitted to journal 03 Mar, 2025 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-6135682","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427763919,"identity":"1de188ac-f91f-4a0d-b43b-15ac3dae72fd","order_by":0,"name":"Rowa Y Alhabbab","email":"","orcid":"","institution":"King Abdulaziz University Faculty of Applied Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rowa","middleName":"Y","lastName":"Alhabbab","suffix":""},{"id":427763920,"identity":"a726a385-7daf-4156-9ff6-be58f9e46823","order_by":1,"name":"Daniela Mastronicola","email":"","orcid":"","institution":"King's College London Division of Transplantation Immunology and Mucosal Biology: King's College London Centre for Nephrology Urology \u0026 Transplantation","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Mastronicola","suffix":""},{"id":427763921,"identity":"2bbd9c5a-1392-43cc-a309-8c697598c5db","order_by":2,"name":"Giovanna Lombardi","email":"","orcid":"","institution":"King's College London Division of Transplantation Immunology and Mucosal Biology: King's College London Centre for Nephrology Urology \u0026 Transplantation","correspondingAuthor":false,"prefix":"","firstName":"Giovanna","middleName":"","lastName":"Lombardi","suffix":""},{"id":427763922,"identity":"e281d2c7-fd71-4b46-8bf6-b5c49199bd25","order_by":3,"name":"Cristiano Scottá","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-3942-5201","institution":"Brunel University London College of Health Medicine and Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Cristiano","middleName":"","lastName":"Scottá","suffix":""}],"badges":[],"createdAt":"2025-03-01 16:10:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6135682/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6135682/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00109-025-02606-0","type":"published","date":"2025-12-27T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78691321,"identity":"84e1a3b7-b889-4c72-9bad-ba150adaa404","added_by":"auto","created_at":"2025-03-17 16:18:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreg\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexp\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e reduces pro-inflammatory cytokine production and induces IL-10 expression in B cells. \u003c/strong\u003eTregs were purified from the peripheral blood of healthy volunteers and \u003cem\u003eex vivo\u003c/em\u003e expanded with anti-CD3/CD28 beads in the presence of a combination of high doses of IL-2 and rapamycin. a) Purity and phenotypic characteristics of Treg\u003csub\u003eexp\u003c/sub\u003e were assessed by flow cytometry after 5 days of culture. b) Treg\u003csub\u003eexp\u003c/sub\u003e suppressive ability was evaluated by co-culture with varying ratios of CFSE-labelled conventional T cells (Teff) activated with anti-CD3/CD28 beads. c) Schematic of the protocol used to activate B cells and co-culture with Tregs. d) Representative FACS plots of CD19\u003csup\u003e+\u003c/sup\u003e B cells producing IFN-g, TNFa, IL-17 and IL-10 following co-culture with F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e. e) Summary data showing the production of IFN-γ, TNF-α and IL-10 in CD19\u003csup\u003e+\u003c/sup\u003e B cells cultured alone or in the presence of F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e. N=3 and N =4 for F-Treg: B cell and Treg\u003csub\u003eexp\u003c/sub\u003e: B cell co-culture, respectively. Statistics were calculated by t-test, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/3d21add6da7055d51a2bab97.png"},{"id":78691325,"identity":"446f3d1e-b9f1-49c4-8d5b-66a5cf31c230","added_by":"auto","created_at":"2025-03-17 16:18:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":554628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-10-expressing B cells have a memory phenotype. \u003c/strong\u003eAnalysis of B cell phenotype following 48-hr co-culture with Tregs\u003csub\u003eexp\u003c/sub\u003e\u003cstrong\u003e. \u003c/strong\u003ea) Representative FACS dot plots showing the CD19\u003csup\u003e+\u003c/sup\u003e B cell subsets gating strategies. b) summary data showing the frequencies of B cell subsets with and without Tregs. c) tSNE map showing B cell subsets distribution and expression of IL-10 and CD25 within each B cell subsets. d-e) Summary data showing IL-10 and CD25 MFI among B cell subsets cultured with or without Treg\u003csub\u003eexp\u003c/sub\u003e. Statistics were calculated by two-way ANOVA and Tukey’s multiple comparisons tests, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005, (N=3).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/78536977c523dc8e2f696dd9.png"},{"id":78692609,"identity":"dfb7ee68-3adf-4230-8a9e-1e1114f6d412","added_by":"auto","created_at":"2025-03-17 16:26:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":526522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreg\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexp\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e requires cell-cell contact to induce IL-10-expressing B cells. \u003c/strong\u003eTreg\u003csub\u003eexp\u003c/sub\u003e and B cells were cultured together by a transwell permeable support in a 24-well plate for 48 hrs. PMA, ionomycin and brefeldin A were added to stimulate cytokine production for the last 4 hrs of culture. a, b and c) Representative FACS plots of CD19\u003csup\u003e+\u003c/sup\u003e B cell IFN-γ, TNF-α and IL-10 expression, and summary data showing IFN-γ, TNF-α and IL-10 expression by stimulated B cells alone and with Tregs in the presence and absence of trans-well insert. Statistics were calculated by two-way ANOVA and Tukey’s multiple comparisons tests, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005, (N=3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/11e384ba248dfc0c4d5f14a6.png"},{"id":78691328,"identity":"7b83e795-267b-4474-81c8-d0e9b1c24307","added_by":"auto","created_at":"2025-03-17 16:18:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreg\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexp\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e expresses high levels of TIM3. \u003c/strong\u003eF-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e phenotypic characteristics were analysed by flow cytometry. F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e were left non-stimulated or stimulated with anti-CD3/CD28 beads or co-cultured with activated B cells (B cells:Tregs:iTcells at ratio 4:4:1) for 48 hrs. Tregs were stained for markers previously associated with their suppressive ability.\u0026nbsp; a) Heatmap showing the normalized expression of the MFI data for each Treg-tested molecule. b) tSNE analysis of both F-Tregs and Treg\u003csub\u003eexp \u003c/sub\u003eanalysed in the conditions described above. The manual gating defined Tregs and B cells using CD4, CD25, CD127 and CD19 expression in non-stimulated, stimulated Tregs and Tregs co-cultured with stimulated B cells. Analysis was performed on 5,000 live singlet cells; samples were merged to generate a single tSNE map; TIM3 clusters were overlaid on tSNE maps to show TIM3 expression and distribution within Tregs and B cells. c) Summary data comparing MFI and percentage of TIM3 in F-Tregs and Treg\u003csub\u003eexp.\u003c/sub\u003e Statistical analysis was performed using Two-way ANOVA and Tukey’s multiple comparisons tests, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005, (N≥5).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/f7e3b4ac5c1bcbe2825afef8.png"},{"id":78691326,"identity":"001da4ba-101d-447b-a139-bd6132116d2b","added_by":"auto","created_at":"2025-03-17 16:18:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreg\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexp\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e induces IL-10-producing B cells through TIM3. \u003c/strong\u003eIntracellular staining of pro-inflammatory cytokines and IL-10 in B cells and Treg\u003csub\u003eexp\u003c/sub\u003e. Treg\u003csub\u003eexp\u003c/sub\u003e and B cells were cultured alone or together in the presence or absence of anti-TIM3 blocking antibody for 48 hrs. PMA, ionomycin and brefeldin A were added to stimulate cytokine production for the last 4 hrs of culture. a) Histograms show IFN-γ, TNF-α and IL-10 production (mean ± SEM) in B cells alone and with Tregs in the presence and absence of anti-TIM3 blocking antibodies. b) Data show IFN-γ, TNF-α, IL-17 and IL-10 production (mean ± SEM) in Treg\u003csub\u003eexp\u003c/sub\u003e alone and with B cells in the presence and absence of anti-TIM3 blocking antibodies. Statistics were calculated by two-way ANOVA and Tukey’s multiple comparisons tests, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005, (N=3). \u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/313056f68615e123707b2c0f.png"},{"id":99172213,"identity":"45619b02-14d3-4ed2-a64b-01292750a0b5","added_by":"auto","created_at":"2025-12-29 16:01:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3055869,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/67f0b258-2d4d-4f60-83d3-18d9021be821.pdf"},{"id":78692992,"identity":"e94158f3-af47-4e1d-97c6-d80cf901f834","added_by":"auto","created_at":"2025-03-17 16:34:30","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":930956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. B cells induce small changes to Treg\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexp\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. \u003c/strong\u003ea)\u003cstrong\u003e \u003c/strong\u003eGating strategy used to separate Treg\u003csub\u003eexp\u003c/sub\u003e, iTcells and B cells in the coculture. b) representative FACS dot plots of IFN-g, TNFa, IL-17 and IL-10-producing F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e following co-culture with B cells. c) Summary data showing the production of IFN-γ, TNF-α and IL-10 expression in F-Tregs and Treg\u003csub\u003eexp \u003c/sub\u003eco-cultured alone or\u003csub\u003e \u003c/sub\u003ewith B cells. N=3 and N =4 for F-Treg: B cell and Treg\u003csub\u003eexp\u003c/sub\u003e: B cell co-culture, respectively. Statistics were calculated by t-test, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005.\u003c/p\u003e","description":"","filename":"SF1.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/b4b70b43a3d0781e1841a352.png"},{"id":78691323,"identity":"8231140a-b638-40b8-82cf-3f5e8442b915","added_by":"auto","created_at":"2025-03-17 16:18:30","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":122478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Phenotypic characteristics of B cells co-cultured with Treg\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexp\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e Summary data of the expression (MFI) of IgM (a), IgD (b) and CD27 (c) on non-stimulated or activated B cells in the with or without Treg\u003csub\u003eexp\u003c/sub\u003e for 48 hrs. B cell subsets were identified using CD19, CD24, CD38, CD27, IgM and IgD expression. Data show mean ± SEM. Statistics were calculated by two-way ANOVA and Tukey’s multiple comparisons tests, ns- not significant, *P\u0026lt;0.05, **P\u0026lt;0.005, ***P=0.0005, (n=3). \u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"SF2.png","url":"https://assets-eu.researchsquare.com/files/rs-6135682/v1/07b5faa83e2272404d30306c.png"}],"financialInterests":"","formattedTitle":"TIM3-Mediated Differentiation of IL-10-Producing CD25+ B Cells by Expanded Regulatory T Cells","fulltext":[{"header":"Key message ","content":"\u003cul\u003e\n \u003cli\u003eExpanded Tregs induce IL-10+ CD25+ B cells.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTIM3 expression on Tregs is crucial for IL-10+ B cell induction.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTregs require direct cell contact to regulate B cells.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBlocking TIM3 reduces IL-10+ B cells but increases IFN-\u0026gamma;, TNF-\u0026alpha;, IL-17.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTregs enhance regulatory B cell differentiation, promoting tolerance.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eRegulatory T cells (Tregs) are a small but crucial subset of CD4\u003csup\u003e+\u003c/sup\u003e T cells that prevent autoimmune diseases and maintain immune homeostasis [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Numerous studies have demonstrated that impairments in the function or number of Tregs are associated with the development of various autoimmune disorders [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Tregs exert their immunosuppressive effects by limiting the activation and proliferation of other immune cells through multiple mechanisms. These include the production of anti-inflammatory cytokines such as TGF-\u0026beta;, IL-35, and IL-10 and the expression of membrane-bound molecules like CD39, TIGIT, LAG-3, and CTLA-4. Additionally, Tregs can modulate the function of antigen-presenting cells through cell contact-dependent mechanisms, which alter the capacity of these cells for co-stimulation and antigen presentation. Their high expression of CD25 allows Tregs to sequester local IL-2, thereby limiting the expansion and function of effector T cells by depriving them of this critical growth factor [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eGiven their regulatory characteristics, Tregs have emerged as an attractive population for immunotherapy. In recent years, Tregs have been successfully isolated and expanded \u003cem\u003eex vivo\u003c/em\u003e in large numbers. Several phase I/II clinical trials have been conducted with promising results, some still ongoing [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e], while others have been completed and shown some biological efficacy [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Notably, our research has demonstrated that the infusion of polyclonal Tregs in kidney transplant patients leads to a dose-dependent increase in B cells with regulatory phenotype in the blood of treated individuals, suggesting that Tregs can influence B cell fate towards a more regulatory phenotype [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e] and, more recently, an increase in another population of regulatory B cells (Bregs) has been seen in the first three renal transplant patients treated with Tregs in the TWO Study [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn humans, various B cell subsets have been identified that possess regulatory capacities through IL-10 production. These include transitional B cells (CD24\u003csup\u003ehi\u003c/sup\u003eCD38\u003csup\u003ehi\u003c/sup\u003e) [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e], CD19\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003ehi\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e B10 cells [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], plasmablasts (CD27\u003csup\u003einter\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e), TIM1\u003csup\u003e+\u003c/sup\u003e Bregs [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e], and CD25\u003csup\u003e+\u003c/sup\u003e memory B cells [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. IL-10-producing B cells have been shown to modulate T cell responses by suppressing T helper 1 (Th1) and Th17 cells while promoting Treg induction [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, studies in autoimmune and transplant models indicate that the adoptive transfer of IL-10-producing B cells can improve disease outcomes [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Despite these insights, the mechanisms by which Tregs regulate B cells and the specific molecules involved in their crosstalk remain poorly understood.\u003c/p\u003e\n\u003cp\u003eIn this study, we demonstrate using an \u003cem\u003ein vitro\u003c/em\u003e system that functionally enhanced \u003cem\u003eex vivo\u003c/em\u003e expanded Tregs are highly effective at inducing IL-10\u003csup\u003e+\u003c/sup\u003e B cells, unlike freshly isolated Tregs. We found that TIM3 expression on expanded Tregs is essential for this effect. The induced IL-10\u003csup\u003e+\u003c/sup\u003e B cells express CD25 and exhibit a memory phenotype (CD24\u003csup\u003ehi\u003c/sup\u003eCD38\u003csup\u003e-\u003c/sup\u003e). Our findings extend the understanding of the critical role of TIM3 in Treg function, particularly in Treg therapies applied in conditions where B cells contribute to pathogenic processes.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003e\u003cem\u003eHuman blood samples.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll human blood samples were obtained from anonymous healthy donors with informed consent and full ethical authorization. Peripheral blood, collected as leukocyte-enriched blood cones, was supplied by the National Blood Service (NHS Blood and Transplantation, Tooting, London, UK). The Institutional Review Board of Guy\u0026apos;s Hospital granted this study\u0026apos;s ethical approval under reference number 09/H0707/86.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCell isolation and co-culture assays\u003c/h2\u003e\n \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated by lymphoprep (Stemcell Technologies, UK) density gradient centrifugation.\u003c/p\u003e\n \u003cp\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells were enriched by negative selection via magnetic sorting (Miltenyi Biotec, UK). The purity of the B cells isolated with this protocol was always more than 95\u0026ndash;98% by flow cytometry.\u003c/p\u003eTo prepare activated \u0026gamma;-irradiated conventional CD4\u003csup\u003e+\u003c/sup\u003e T cells (iTcells), CD4\u003csup\u003e+\u003c/sup\u003e T cells were isolated by RosetteSep and incubated for 5 hours with a T cell activation cocktail (Cat. 423301, BioLegend, UK). The cells were then \u0026gamma;-irradiated and tested for CD40L expression by flow cytometry. They were cryopreserved at -80\u0026deg;C and thawed directly before being used. The iTcells were utilised at a 1:4 ratio with B cells.\u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells isolated by RosetteSep were also used for obtaining CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Tregs by CD25 microbeads magnetic enrichment (Miltenyi), and FACS sorted using antibodies specific for CD4, CD25, CD127 and CD45RA. Tregs were then either used directly in our co-culture setting with the negatively sorted B cells or expanded by using anti-CD3/CD28 beads (Miltenyi) in the presence of 100nM rapamycin (LC-laboratories) and 1000 IU/ml recombinant human IL-2 in X-vivo15 medium (Lonza) supplemented with 5% human AB serum (Biosera), as previously published [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Expanded Tregs (Treg\u003csub\u003eexp\u003c/sub\u003e) were collected and co-cultured with B cells (at a 1:1 ratio) in the presence of anti-CD3/CD28 beads for 48 hours.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIntracellular staining\u003c/h3\u003e\n\u003cp\u003ePMA, ionomycin and brefeldin A were added for the last 4 hours of co-cultures, and cytokines, including IFN-\u0026gamma;, TNF-\u0026alpha;, IL-17 and IL-10, were measured by intracellular staining (ICC).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrans-well system and antibody neutralisation assay.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTregs and anti-CD3/CD28 beads were plated at the bottom of the well. The trans-well insert was placed on top, with B cells and iTcells (4:1 ratio). The plates were incubated for 48 hours.\u003c/p\u003e\n\u003cp\u003eAnti-TIM3 blocking antibodies (10\u0026micro;g/ml) were added to the co-cultures, and the cells were incubated for 48 hours. PMA, ionomycin and brefeldin A were added for the last 4 hours of the culture, and cytokines were measured ICC by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003etSNE analysis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSinglet live cells were gated using Flowjo10, and additional gates were applied as requested. Singlet live cells of all samples were down-sampled to 5000 events, and each group (non-stimulated, stimulated and 1stimulated-Bcells:1Tregs) was concatenated into one file. tSNE was run on the concatenated files, and grouped data were gated. Cell clusters were identified and overlapped by the gated population on the tSNE map. The MFI for each molecule within each cell population was then measured.\u003c/p\u003e\n\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eComparisons between groups were performed using a T-test or two-way ANOVA and Tukey\u0026rsquo;s multiple comparisons as specified. Analyses were performed using GraphPad Prism software.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eExpanded Tregs induce IL-10-expressing B cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBuilding on the evidence that following the adoptive transfer of Tregs, B cells with regulatory phenotype are increasing in renal transplant patients [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This study focused on understanding how expanded human Tregs impact B cell phenotype and function. To do so, we established an \u003cem\u003ein vitro\u003c/em\u003e system in which human Tregs and B cells were activated by irradiated allogeneic T cells. Tregs were enriched from blood, expanded \u003cem\u003eex vivo\u003c/em\u003e using a well-established protocol in our laboratory and adopted in our previous clinical trials [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, Tregs were purified from the peripheral blood of healthy volunteers by density gradient separation of PBMC followed by magnetic bead separation of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003ehigh\u003c/sup\u003e T cells and FASC sorting (F-Tregs). Then, Tregs were stimulated with anti-CD3/CD28 beads (ratio cell/bead 1:1) and cultured for two weeks in the presence of IL-2 (1,000 IU/ml) and rapamycin (100 nM), as previously described, [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] to obtain a highly pure and suppressive expanded regulatory T cells (Treg\u003csub\u003eexp\u003c/sub\u003e). At the end of the culture, the purity of Treg\u003csub\u003eexp\u003c/sub\u003e was verified by flow cytometry staining using the conventional markers associated with functional Tregs and compared to F-Tregs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Treg\u003csub\u003eexp\u003c/sub\u003e increased CD25, FOXP3, CTLA4, and HELIOS expression levels while CD12s maintained low compared to freshly isolated Tregs [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Treg\u003csub\u003eexp\u003c/sub\u003e were co-cultured at varying ratios with CFSE-labelled conventional T cells (Teff) activated with anti-CD3/CD28 beads to assess their suppressive capacity. In these settings, the inhibition of Teff proliferation was measured via flow cytometry by CFSE dilution, and Treg\u003csub\u003eexp\u003c/sub\u003e suppressive capacity was compared to F-Tregs as previously described (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Treg\u003csub\u003eexp\u003c/sub\u003e were more suppressive than F-Tregs, further confirming the enhanced function of Treg\u003csub\u003eexp\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, B cells (1 x 10\u003csup\u003e7\u003c/sup\u003e) were isolated from the peripheral blood of healthy volunteers by density gradient separation of PBMC followed by magnetic bead separation of CD19\u003csup\u003e+\u003c/sup\u003e cells (purity\u0026thinsp;\u0026gt;\u0026thinsp;95%). B cells were then activated by activated γ-irradiated conventional CD4\u003csup\u003e+\u003c/sup\u003e T cells (iTcell) expressing high levels of CD40L (activated-B cells). To investigate the cytokines produced by B cells after 48-hour stimulation with only iTcells (baseline), we analysed the percentage of B cells producing IFN-γ, TNF-α, IL-17, and IL-10, using intracellular staining (see the gating strategy in \u003cb\u003eSupplementary Fig.\u0026nbsp;1a\u003c/b\u003e). The results showed that a fraction of activated-B cells were able to produce IFN-γ, TNF-α, and IL-17, but not IL-10 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e). At the same time, to test the effect of Tregs, activated-B cells were co-cultured for 48 hours at a 1:1 ratio with either freshly isolated F-Tregs or Treg\u003csub\u003eexp\u003c/sub\u003e. Data in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e showed that in the presence of both F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e, the percentages of B cells expressing IL-10 increased; however, the co-culture with Treg\u003csub\u003eexp\u003c/sub\u003e resulted in even higher percentages of B cells producing this cytokine. Moreover, the percentages of IFN-γ, IL-17, and TNF-α were significantly reduced in B cells upon co-culture with F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e).\u003c/p\u003e \u003cp\u003eThe expression of cytokines was also analysed in F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e before and after the co-culture with activated B cells. \u003cb\u003eSupplementary Fig.\u0026nbsp;1b-c\u003c/b\u003e shows that compared to F-Tregs, the percentage of Treg\u003csub\u003eexp\u003c/sub\u003e expressing IFN-γ, TNF-α, and IL-17 was very low. In both F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e conditions, the percentage of TNF-α expressing cells was reduced following the co-culture with activated-B cells. IL-10 production was only detectable in F-Tregs, and the co-culture with activated-B cells reduced the percentage of cells producing this cytokine (\u003cb\u003eSupplementary Figs.\u0026nbsp;1b-c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTreg\u003c/b\u003e \u003csub\u003e \u003cb\u003eexp\u003c/b\u003e \u003c/sub\u003e \u003cb\u003einduces a regulatory phenotype in the memory B cell subset.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the evidence that the co-culture of activated B cells with Treg\u003csub\u003eexp\u003c/sub\u003e increased the percentages of IL-10-producing B cells, we sought to investigate the changes in the B cell subpopulations. B cells purified from the blood were stained with antibodies specific for CD24 and CD38 on CD19\u003csup\u003e+\u003c/sup\u003e cells and analysed by flow cytometry. Three different subpopulations of B cells were identified: CD24\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e\u0026minus;\u003c/sup\u003e memory (CD24\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e\u0026minus;\u003c/sup\u003e), transitional (CD24\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003ehi\u003c/sup\u003e) and mature (CD24\u003csup\u003eint\u003c/sup\u003eCD38\u003csup\u003eint\u003c/sup\u003e) B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The activation of B cells significantly increased the percentage of CD24\u003csup\u003eint\u003c/sup\u003eCD38\u003csup\u003eint\u003c/sup\u003e mature subset, and this effect was inhibited by the presence of Treg\u003csub\u003eexp\u003c/sub\u003e in the co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The other B cell subpopulations, with or without the co-culture with Treg\u003csub\u003eexp\u003c/sub\u003e, did not drastically change.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phenotypic characteristics of B cells at the end of the co-culture were also analysed by t-distributed stochastic neighbour embedding (tSNE); all data obtained from the co-culture conditions described above were combined to assess B cell subset distribution based on CD24 and CD38 expression along with the expression of IL-10 and CD25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The analysis confirmed that Treg\u003csub\u003eexp\u003c/sub\u003e mostly induced IL-10 production in B cells and that IL-10-producing B cells were clustered within the memory B cell subset (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Further analysis of CD25 expression, a molecule previously associated with a regulatory phenotype in B cells producing high levels of IL-10 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], showed that co-culturing activated-B cells with Treg\u003csub\u003eexp\u003c/sub\u003e significantly increased this marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Among the three defined B cell subpopulations, memory B cells expressed the highest level of IL-10 and CD25 compared to the other subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e). Of note, memory B cells were IgM\u003csup\u003ehi\u003c/sup\u003e, IgD\u003csup\u003elow\u003c/sup\u003e and CD27\u003csup\u003elow\u003c/sup\u003e, suggesting that these B cells were memory precursors that can generate CD27\u003csup\u003ehi\u003c/sup\u003e memory B cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a-c\u003c/b\u003e) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. All these findings support the idea that the presence of Treg\u003csub\u003eexp\u003c/sub\u003e induces the memory B cell subset to acquire a regulatory phenotype characterised by the high expression of CD25 and the production of IL-10.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression of TIM3 on Treg\u003c/b\u003e \u003csub\u003e \u003cb\u003eexp\u003c/b\u003e \u003c/sub\u003e \u003cb\u003emediates the induction of tolerogenic B cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter observing that Treg\u003csub\u003eexp\u003c/sub\u003e may influence the differentiation of a memory B cell subset with anti-inflammatory properties, we explored the mechanisms behind this effect. To test whether Treg\u003csub\u003eexp\u003c/sub\u003e acts through direct cell-to-cell contact, we used trans-well inserts to physically separate stimulated Treg\u003csub\u003eexp\u003c/sub\u003e from stimulated B cells during a 48-hour co-culture period. When Treg\u003csub\u003eexp\u003c/sub\u003e were separated from B cells, the induction of IL-10\u003csup\u003e+\u003c/sup\u003e B cells was prevented, and the reduction in IFN-γ and TNF-α B cells seen in co-cultures was also abolished (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). These findings indicate that Treg\u003csub\u003eexp\u003c/sub\u003e required direct cell-to-cell contact to affect B-cell properties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the mechanisms used by Treg\u003csub\u003eexp\u003c/sub\u003e, we examined the expression of molecules associated with their regulatory functions and compared them to F-Tregs [\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. F-Tregs and Treg\u003csub\u003eexp\u003c/sub\u003e were either left non-stimulated or activated with anti-CD3/CD28 beads, with or without activated-B cells at a 1:1 ratio for 48 hours. We evaluated the expression of CD134, TIM3, GARP, ICOS, CTLA-4, CD200, CD30, DR3, CD40L, and Galectin-9 (Gal-9) using flow cytometry. To visualise and confirm the most dominant molecule expressed by Treg\u003csub\u003eexp\u003c/sub\u003e and at lower levels by F-Tregs under different culture conditions, we normalised the mean fluorescence intensity (MFI) data, setting the lowest value to 0% and the highest to 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough Treg\u003csub\u003eexp\u003c/sub\u003e expressed high levels of CD134, TIM3, CTLA-4, and Gal-9, we found that the co-culture of Treg\u003csub\u003eexp\u003c/sub\u003e with B cells induced a very high expression of TIM3 and Gal-9 on Treg\u003csub\u003eexp\u003c/sub\u003e. However, while similar levels of Gal-9 were also expressed on F-Tregs, the exceptionally high expression of TIM3 was restricted only on Treg\u003csub\u003eexp\u003c/sub\u003e co-cultured with B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe following analysis of the same samples using the tSNE algorithm (n\u0026thinsp;=\u0026thinsp;5, 5000 events per sample) helped to visualise and identify the TIM3 distribution in the distinct cell clusters. Data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows that the co-culture of B cells with Treg\u003csub\u003eexp\u003c/sub\u003e induced the whole population to express TIM3. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec shows that when TIM3 expression was compared between the two preparations of Tregs, Trege\u003csub\u003exp\u003c/sub\u003e expressed considerably higher levels of TIM3 (MFI) compared to F-Tregs in all three different conditions (non-stimulated, stimulated and at 1:1 ratio with B cells). These results suggested that the expression of TIM3 by Treg\u003csub\u003eexp\u003c/sub\u003e played a crucial role in the induction of IL-10\u003csup\u003e+\u003c/sup\u003e memory B cells.\u003c/p\u003e \u003cp\u003eTo confirm that TIM3 is involved in the crosstalk between Treg\u003csub\u003eexp\u003c/sub\u003e and B cells, the two cell types were co-cultured in the presence of an anti-TIM3 blocking antibody. Flow cytometry analysis showed a significant decrease in the percentages of IL-10\u003csup\u003e+\u003c/sup\u003e B cells in the presence of anti-TIM3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). However, blocking TIM3 did not affect the inhibition of IFN-γ and TNF-α in B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In the same culture conditions, the inhibition of TIM3 increased the percentages of IFN-γ, TNF-α, and IL-17-producing Treg\u003csub\u003eexp\u003c/sub\u003e, with no effect on IL-10 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results suggest that the crosstalk between Treg\u003csub\u003eexp\u003c/sub\u003e and B cells is complex and requires multiple signals. While TIM3 engagement on Treg\u003csub\u003eexp\u003c/sub\u003e plays a critical role in inducing IL-10 production in B cells, it is not necessary to inhibit proinflammatory cytokines such as IFN-γ and TNF-α. Conversely, blocking TIM3 signalling on Treg\u003csub\u003eexp\u003c/sub\u003e stimulates the production of IFN-γ, TNF-α, and IL-17 by Treg\u003csub\u003eexp\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we utilised a novel \u003cem\u003ein vitro\u003c/em\u003e co-culture system of human B cells and expanded Tregs to demonstrate the potent ability of TIM3-expressing Tregs to induce memory IL-10\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e B cells. Importantly, our findings highlight that TIM3 expression is crucial for this effect.\u003c/p\u003e \u003cp\u003eExpanded Tregs offer significant advantages over freshly isolated Tregs, particularly in clinical applications. Expanded Tregs, similar to those utilized in clinical trials for liver and kidney transplantation (e.g., the ONE study and the THRIL study), exhibit enhanced functionality and stability. Notably, expanded Tregs express high levels of TIM3, making them particularly effective at modulating T-cell-mediated and B-cell responses. This dual capability positions them as ideal candidates for therapeutic strategies promoting tolerance in transplant settings.\u003c/p\u003e \u003cp\u003eTIM3 is a molecule expressed on various immune cells, including B cells, T cells, monocytes, and dendritic cells. It plays a critical role in regulating immune responses and has been identified as a potential therapeutic target for several immune-related disorders, including cancer and sepsis. Recent literature has highlighted that tumours can exploit TIM3's expression on tumour cells to evade immune detection [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Additionally, TIM3\u0026rsquo;s involvement in sepsis underscores its multifaceted role in immune regulation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB cells are central to immune tolerance induction, with various subsets capable of downregulating inflammatory responses associated with autoimmunity and transplant rejection, primarily through IL-10 production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. CD25\u003csup\u003e+\u003c/sup\u003e B cells, initially recognised as a distinct subset, have been shown to differentiate upon stimulation via toll-like receptors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These cells are now considered a regulatory B cell subset with memory characteristics that can enhance the Treg function [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Memory B cells, also known as B10 cells in humans, are particularly significant as IL-10 producers [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. While na\u0026iuml;ve B cells can convert CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells into CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Tregs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], our study uniquely demonstrates that memory B cells expressing IL-10 and CD25 can be induced by Tregs, specifically \u003cem\u003eex vivo\u003c/em\u003e expanded Tegs, emphasizing the critical role of TIM3 in this process.\u003c/p\u003e \u003cp\u003eThe interaction between expanded Tregs and B cells is complex and likely involves multiple communication mechanisms. The ligation of TIM3 with its ligand, galectin-9, is known to inhibit Th1 responses and promote peripheral tolerance [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. TIM3 also plays a vital role in T cell exhaustion during chronic viral infections, where its inhibition enhances cytokine production specific to HCV and HIV [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In cancer, blocking TIM3 signalling has been shown to improve the function of tumour-infiltrating lymphocytes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Furthermore, reduced TIM3 expression is associated with the development of autoimmune diseases [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, we found that blocking TIM3 during the co-culture of Tregs and B cells explicitly influences the induction of IL-10 production in B cells without affecting the Treg-mediated suppression of pro-inflammatory cytokines such as IFN-γ and TNF-α. These findings suggest a complex interplay between Tregs and B cells that may involve additional molecular mechanisms.\u003c/p\u003e \u003cp\u003eInterestingly, the effect of TIM3 blockade on Tregs indicates that TIM3 also plays a role in regulating the expression of inflammatory cytokines in these cells. When TIM3 is inhibited, Tregs may begin to produce cytokines such as IFN-γ, TNF-α, and IL-17, which could compromise their regulatory function.\u003c/p\u003e \u003cp\u003eOur results are consistent with previous studies in both human and murine models, demonstrating that blocking TIM3 on stimulated conventional T cells significantly increases IFN-γ secretion [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This underscores TIM\u0026rsquo;'s essential role in T cell immunoregulation. Furthermore, while TIM3-expressing Tregs are more potent suppressors than TIM3-negative Tregs, they are also typically enriched in IL-10 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The discrepancies between these findings may stem from differences in the experimental models used, particularly the reliance on murine Tregs versus human cells.\u003c/p\u003e \u003cp\u003eTregs are well-established for suppressing inflammation through various mechanisms, including the production of IL-10, TGF-β, and IL-35, as well as through direct cell contact [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Their role in alleviating the severity of diseases such as autoimmunity and transplantation has been well documented [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, Tregs have emerged as promising candidates for cell-based immunotherapy. They can be isolated and expanded \u003cem\u003eex vivo\u003c/em\u003e in large quantities, shifting the balance between effector T cells and Tregs favouring the latter [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Our findings indicate that expanded Tregs expressing TIM3 are crucial for inducing CD25\u003csup\u003e+\u003c/sup\u003e memory IL-10\u003csup\u003e+\u003c/sup\u003e B cells contact-dependent, independent of IL-10. This suggests that TIM3\u003csup\u003e+\u003c/sup\u003e Tregs enhance their suppressive capacity by promoting the differentiation of additional regulatory B cells, thereby creating a more tolerogenic environment.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that expanded Tregs, particularly those expressing TIM3, exhibit characteristics akin to exhaustion while maintaining robust suppressive functions. These Tregs effectively promote IL-10 expression in stimulated B cells with a memory phenotype. Leveraging TIM3\u003csup\u003e+\u003c/sup\u003e Tregs could enhance their suppressive capacity and facilitate beneficial interactions with B cells, paving the way for innovative immune-based therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Brunel University of London, King\u0026rsquo;s College London and King Abdulaziz University for supporting this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was supported by Lupus UK, Diabetes UK, King\u0026apos;s Health Partners, MRC-Impact Accelerator 2022, BD Biosciences Research Program Award, Rosetrees Trust, National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy\u0026apos;s and St Thomas\u0026apos; NHS Foundation Trust and King\u0026apos;s College London and the NIHR Clinical Research Facility.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was\u0026nbsp;supported by Lupus UK, Diabetes UK (ref.\u0026nbsp;24/0006776), King\u0026apos;s Health Partners, BD Biosciences Research Program (ref. 10/2017 Award), Rosetrees Trust (ref. CF-2021-2 107), National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy\u0026apos;s and St Thomas\u0026apos; NHS Foundation Trust and King\u0026apos;s College London and the NIHR Clinical Research Facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eData\u0026nbsp;Availability\u0026nbsp;Statement\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings are available upon request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003eEthical\u0026nbsp;approval\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was granted by the Institutional Review Board of Guy\u0026apos;s Hospital under reference number 09/H0707/86.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualisation: RYA, GL and CS; Sample collection and methodology: RYA, CS and DM; Data curation: RYA and CS; Data analysis and interpretation: RYA, CS and DM; Investigation: RYA and CS; Data visualisation: RYA and CS; Resources and funding acquisition: RYA, GL and CS; Writing - Original draft: RYA and CS; Review and editing: RYA, GL and CS; Supervision: CS.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYamaguchi T, Teraguchi S, Furusawa C, Machiyama H, Watanabe TM, Fujita H, Sakaguchi S, Yanagida T (2019) Theoretical modeling reveals that regulatory T cells increase T-cell interaction with antigen-presenting cells for stable immune tolerance. International Immunology 31: 743-753. DOI 10.1093/intimm/dxz043\u003c/li\u003e\n\u003cli\u003eSharma VK, Kaveri SV, Bayry J (2015) Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits? Cellular \u0026amp; Molecular Immunology 13: 135-137. DOI 10.1038/cmi.2015.98\u003c/li\u003e\n\u003cli\u003eEggenhuizen PJ, Ng BH, Ooi JD (2020) Treg Enhancing Therapies to Treat Autoimmune Diseases. International Journal of Molecular Sciences 21. DOI 10.3390/ijms21197015\u003c/li\u003e\n\u003cli\u003eRomano M, Fanelli G, Albany CJ, Giganti G, Lombardi G (2019) Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity. Frontiers in Immunology 10. DOI 10.3389/fimmu.2019.00043\u003c/li\u003e\n\u003cli\u003eGiganti G, Atif M, Mohseni Y, Mastronicola D, Grageda N, Povoleri GA, Miyara M, Scotta C (2021) Treg cell therapy: How cell heterogeneity can make the difference. Eur J Immunol 51: 39-55. DOI 10.1002/eji.201948131\u003c/li\u003e\n\u003cli\u003eTodo S, Yamashita K, Goto R, Zaitsu M, Nagatsu A, Oura T, Watanabe M, Aoyagi T, Suzuki T, Shimamura T, et al. (2016) A pilot study of operational tolerance with a regulatory T‐cell‐based cell therapy in living donor liver transplantation. Hepatology 64: 632-643. DOI 10.1002/hep.28459\u003c/li\u003e\n\u003cli\u003eBaeten P, Van Zeebroeck L, Kleinewietfeld M, Hellings N, Broux B (2021) Improving the Efficacy of Regulatory T Cell Therapy. Clinical Reviews in Allergy \u0026amp; Immunology 62: 363-381. DOI 10.1007/s12016-021-08866-1\u003c/li\u003e\n\u003cli\u003eSawitzki B, Harden PN, Reinke P, Moreau A, Hutchinson JA, Game DS, Tang Q, Guinan EC, Battaglia M, Burlingham WJ, et al. (2020) Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials. The Lancet 395: 1627-1639. DOI 10.1016/s0140-6736(20)30167-7\u003c/li\u003e\n\u003cli\u003eBrook MO, Hester J, Petchey W, Rombach I, Dutton S, Bottomley MJ, Black J, Abdul-Wahab S, Bushell A, Lombardi G, et al. (2022) Transplantation Without Overimmunosuppression (TWO) study protocol: a phase 2b randomised controlled single-centre trial of regulatory T cell therapy to facilitate immunosuppression reduction in living donor kidney transplant recipients. BMJ Open 12. DOI 10.1136/bmjopen-2022-061864\u003c/li\u003e\n\u003cli\u003eBlair PA, Nore\u0026ntilde;a LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, Mauri C (2010) CD19+CD24hiCD38hi B Cells Exhibit Regulatory Capacity in Healthy Individuals but Are Functionally Impaired in Systemic Lupus Erythematosus Patients. Immunity 32: 129-140. DOI 10.1016/j.immuni.2009.11.009\u003c/li\u003e\n\u003cli\u003evan der Vlugt LEPM, Mlejnek E, Ozir‐Fazalalikhan A, Janssen Bonas M, Dijksman TR, Labuda LA, Schot R, Guigas B, M\u0026ouml;ller GM, Hiemstra PS, et al. (2014) CD24hiCD27+ B cells from patients with allergic asthma have impaired regulatory activity in response to lipopolysaccharide. Clinical \u0026amp; Experimental Allergy 44: 517-528. DOI 10.1111/cea.12238\u003c/li\u003e\n\u003cli\u003eAlhabbab RY, Nova-Lamperti E, Aravena O, Burton HM, Lechler RI, Dorling A, Lombardi G (2019) Regulatory B cells: Development, phenotypes, functions, and role in transplantation. Immunological Reviews 292: 164-179. DOI 10.1111/imr.12800\u003c/li\u003e\n\u003cli\u003eAmu S, Brisslert M (2011) Phenotype and Function of CD25-Expressing B Lymphocytes Isolated from Human Umbilical Cord Blood. Clinical and Developmental Immunology 2011: 1-9. DOI 10.1155/2011/481948\u003c/li\u003e\n\u003cli\u003eFlores-Borja F, Bosma A, Ng D, Reddy V, Ehrenstein MR, Isenberg DA, Mauri C (2013) CD19+CD24hiCD38hi B Cells Maintain Regulatory T Cells While Limiting TH1 and TH17 Differentiation. Science Translational Medicine 5. DOI 10.1126/scitranslmed.3005407\u003c/li\u003e\n\u003cli\u003eAlhabbab R, Blair P, Elgueta R, Stolarczyk E, Marks E, Becker PD, Ratnasothy K, Smyth L, Safinia N, Sharif-Paghaleh E, et al. (2020) Author Correction: Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model. Scientific Reports 10. DOI 10.1038/s41598-020-62381-5\u003c/li\u003e\n\u003cli\u003eAlhabbab R, Blair P, Smyth LA, Ratnasothy K, Peng Q, Moreau A, Lechler R, Elgueta R, Lombardi G (2018) Galectin-1 is required for the regulatory function of B cells. Scientific Reports 8. DOI 10.1038/s41598-018-19965-z\u003c/li\u003e\n\u003cli\u003eAref S, Azmy E, El‐Gilany AH (2015) Upregulation of CD200 is associated with regulatory T cell expansion and disease progression in multiple myeloma. Hematological Oncology 35: 51-57. DOI 10.1002/hon.2206\u003c/li\u003e\n\u003cli\u003eMcCallion O, Cross AR, Brook MO, Hennessy C, Ferreira R, Trzupek D, Mulley WR, Kumar S, Soares M, Roberts IS, et al. (2024) Regulatory T cell therapy is associated with distinct immune regulatory lymphocytic infiltrates in kidney transplants. Med. DOI 10.1016/j.medj.2024.11.014\u003c/li\u003e\n\u003cli\u003eGorczynski R, Khatri I, Lee L, Boudakov I (2008) An Interaction between CD200 and Monoclonal Antibody Agonists to CD200R2 in Development of Dendritic Cells That Preferentially Induce Populations of CD4+CD25+ T Regulatory Cells. The Journal of Immunology 180: 5946-5955. DOI 10.4049/jimmunol.180.9.5946\u003c/li\u003e\n\u003cli\u003eScotta C, Fanelli G, Hoong SJ, Romano M, Lamperti EN, Sukthankar M, Guggino G, Fazekasova H, Ratnasothy K, Becker PD, et al. (2015) Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells. Haematologica 101: 91-100. DOI 10.3324/haematol.2015.128934\u003c/li\u003e\n\u003cli\u003eRomano M, Fanelli G, Tan N, Nova-Lamperti E, McGregor R, Lechler RI, Lombardi G, Scott\u0026agrave; C (2018) Expanded Regulatory T Cells Induce Alternatively Activated Monocytes With a Reduced Capacity to Expand T Helper-17 Cells. Frontiers in Immunology 9. DOI 10.3389/fimmu.2018.01625\u003c/li\u003e\n\u003cli\u003eScotta C, Esposito M, Fazekasova H, Fanelli G, Edozie FC, Ali N, Xiao F, Peakman M, Afzali B, Sagoo P, et al. (2012) Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4+CD25+FOXP3+ T regulatory cell subpopulations. Haematologica 98: 1291-1299. DOI 10.3324/haematol.2012.074088\u003c/li\u003e\n\u003cli\u003eLiu W, Putnam AL, Xu-yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, de St. Groth BF, et al. (2006) CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. The Journal of Experimental Medicine 203: 1701-1711. DOI 10.1084/jem.20060772\u003c/li\u003e\n\u003cli\u003eLowther DE, Goods BA, Lucca LE, Lerner BA, Raddassi K, van Dijk D, Hernandez AL, Duan X, Gunel M, Coric V, et al. (2016) PD-1 marks dysfunctional regulatory T cells in malignant gliomas. JCI Insight 1. DOI 10.1172/jci.insight.85935\u003c/li\u003e\n\u003cli\u003eTheil A, Tuve S, Oelschl\u0026auml;gel U, Maiwald A, D\u0026ouml;hler D, O\u0026szlig;mann D, Zenkel A, Wilhelm C, Middeke JM, Shayegi N, et al. (2015) Adoptive transfer of allogeneic regulatory T cells into patients with chronic graft-versus-host disease. Cytotherapy 17: 473-486. DOI 10.1016/j.jcyt.2014.11.005\u003c/li\u003e\n\u003cli\u003eVu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A, Killeen N, Ishii N, Chang Li X (2007) OX40 costimulation turns off Foxp3+ Tregs. Blood 110: 2501-2510. DOI 10.1182/blood-2007-01-070748\u003c/li\u003e\n\u003cli\u003eComte D, Karampetsou MP, Kis-Toth K, Yoshida N, Bradley SJ, Mizui M, Kono M, Solomon JR, Kyttaris VC, Tsokos GC (2016) Engagement of SLAMF3 enhances CD4+ T-cell sensitivity to IL-2 and favors regulatory T-cell polarization in systemic lupus erythematosus. Proceedings of the National Academy of Sciences 113: 9321-9326. DOI 10.1073/pnas.1605081113\u003c/li\u003e\n\u003cli\u003eLucca LE, Dominguez-Villar M (2020) Modulation of regulatory T cell function and stability by co-inhibitory receptors. Nature Reviews Immunology 20: 680-693. DOI 10.1038/s41577-020-0296-3\u003c/li\u003e\n\u003cli\u003eRen X, Ye F, Jiang Z, Chu Y, Xiong S, Wang Y (2007) Involvement of cellular death in TRAIL/DR5-dependent suppression induced by CD4+CD25+ regulatory T cells. Cell Death \u0026amp; Differentiation 14: 2076-2084. DOI 10.1038/sj.cdd.4402220\u003c/li\u003e\n\u003cli\u003ede Streel G, Bertrand C, Chalon N, Li\u0026eacute;nart S, Bricard O, Lecomte S, Devreux J, Gaignage M, De Boeck G, Mari\u0026euml;n L, et al. (2020) Selective inhibition of TGF-\u0026beta;1 produced by GARP-expressing Tregs overcomes resistance to PD-1/PD-L1 blockade in cancer. Nature Communications 11. DOI 10.1038/s41467-020-17811-3\u003c/li\u003e\n\u003cli\u003eLi D-Y, Xiong X-Z (2020) ICOS+ Tregs: A Functional Subset of Tregs in Immune Diseases. Frontiers in Immunology 11. DOI 10.3389/fimmu.2020.02104\u003c/li\u003e\n\u003cli\u003eYi M-H, Zhang E, Kim J-J, Baek H, Shin N, Kim S, Kim SR, Kim H-R, Lee SJ, Park JB, et al. (2016) CD200R/Foxp3-mediated signalling regulates microglial activation. Scientific Reports 6. DOI 10.1038/srep34901\u003c/li\u003e\n\u003cli\u003eLam JH, Hong M, Koo S-L, Chua CWL, Lim KL, Wee F, Wan WK, Leow WQ, Yeo JG, Tan IBH, et al. (2021) CD30+OX40+ Treg is associated with improved overall survival in colorectal cancer. Cancer Immunology, Immunotherapy 70: 2353-2365. DOI 10.1007/s00262-021-02859-x\u003c/li\u003e\n\u003cli\u003eYu Y, Jiang P, Sun P, Su N, Lin F (2021) Analysis of therapeutic potential of preclinical models based on DR3/TL1A pathway modulation. Experimental and Therapeutic Medicine 22. DOI 10.3892/etm.2021.10125\u003c/li\u003e\n\u003cli\u003eSolanki K, Kumar A, Khan MS, Karthikeyan S, Atre R, Zhang KYJ, Bezsonov E, Obukhov AG, Baig MS (2023) Novel peptide inhibitors targeting CD40 and CD40L interaction: A potential for atherosclerosis therapy. Current Research in Structural Biology 6. DOI 10.1016/j.crstbi.2023.100110\u003c/li\u003e\n\u003cli\u003eSauer N, Janicka N, Szlasa W, Skinderowicz B, Kołodzińska K, Dwernicka W, Oślizło M, Kulbacka J, Novickij V, Karłowicz-Bodalska K (2023) TIM-3 as a promising target for cancer immunotherapy in a wide range of tumors. Cancer Immunology, Immunotherapy 72: 3405-3425. DOI 10.1007/s00262-023-03516-1\u003c/li\u003e\n\u003cli\u003eWang C, Liu J, Wu Q, Wang Z, Hu B, Bo L (2024) The role of TIM-3 in sepsis: a promising target for immunotherapy? Frontiers in Immunology 15. DOI 10.3389/fimmu.2024.1328667\u003c/li\u003e\n\u003cli\u003eBrisslert M, Bokarewa M, Larsson P, Wing K, Collins LV, Tarkowski A (2006) Phenotypic and functional characterization of human CD25+ B cells. Immunology 117: 548-557. DOI 10.1111/j.1365-2567.2006.02331.x\u003c/li\u003e\n\u003cli\u003eKessel A, Haj T, Peri R, Snir A, Melamed D, Sabo E, Toubi E (2012) Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells. Autoimmun Rev 11: 670-677. DOI 10.1016/j.autrev.2011.11.018\u003c/li\u003e\n\u003cli\u003eHasan MM, Thompson-Snipes L, Klintmalm G, Demetris AJ, O\u0026apos;Leary J, Oh S, Joo H (2019) CD24hiCD38hi and CD24hiCD27+ Human Regulatory B Cells Display Common and Distinct Functional Characteristics. The Journal of Immunology 203: 2110-2120. DOI 10.4049/jimmunol.1900488\u003c/li\u003e\n\u003cli\u003eRan Z, Yue-Bei L, Qiu-Ming Z, Huan Y (2020) Regulatory B Cells and Its Role in Central Nervous System Inflammatory Demyelinating Diseases. Frontiers in Immunology 11. DOI 10.3389/fimmu.2020.01884\u003c/li\u003e\n\u003cli\u003eChu K-H, Chiang B-L (2012) Regulatory T Cells Induced by Mucosal B Cells Alleviate Allergic Airway Hypersensitivity. American Journal of Respiratory Cell and Molecular Biology 46: 651-659. DOI 10.1165/rcmb.2011-0246OC\u003c/li\u003e\n\u003cli\u003eHsu L-H, Li K-P, Chu K-H, Chiang B-L (2014) A B-1a cell subset induces Foxp3\u0026minus; T cells with regulatory activity through an IL-10-independent pathway. Cellular \u0026amp; Molecular Immunology 12: 354-365. DOI 10.1038/cmi.2014.56\u003c/li\u003e\n\u003cli\u003eBarber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R (2005) Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439: 682-687. DOI 10.1038/nature04444\u003c/li\u003e\n\u003cli\u003eKlibi J, Niki T, Riedel A, Pioche-Durieu C, Souquere S, Rubinstein E, Le Moulec S, Guigay J, Hirashima M, Guemira F, et al. (2009) Blood diffusion and Th1-suppressive effects of galectin-9\u0026ndash;containing exosomes released by Epstein-Barr virus\u0026ndash;infected nasopharyngeal carcinoma cells. Blood 113: 1957-1966. DOI 10.1182/blood-2008-02-142596\u003c/li\u003e\n\u003cli\u003eSehrawat S, Suryawanshi A, Hirashima M, Rouse BT (2009) Role of Tim-3/Galectin-9 Inhibitory Interaction in Viral-Induced Immunopathology: Shifting the Balance toward Regulators. The Journal of Immunology 182: 3191-3201. DOI 10.4049/jimmunol.0803673\u003c/li\u003e\n\u003cli\u003eJones RB, Ndhlovu LC, Barbour JD, Sheth PM, Jha AR, Long BR, Wong JC, Satkunarajah M, Schweneker M, Chapman JM, et al. (2008) Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. The Journal of Experimental Medicine 205: 2763-2779. DOI 10.1084/jem.20081398\u003c/li\u003e\n\u003cli\u003eGolden-Mason L, Palmer BE, Kassam N, Townshend-Bulson L, Livingston S, McMahon BJ, Castelblanco N, Kuchroo V, Gretch DR, Rosen HR (2009) Negative Immune Regulator Tim-3 Is Overexpressed on T Cells in Hepatitis C Virus Infection and Its Blockade Rescues Dysfunctional CD4+ and CD8+ T Cells. Journal of Virology 83: 9122-9130. DOI 10.1128/jvi.00639-09\u003c/li\u003e\n\u003cli\u003eZhou Q, Munger ME, Veenstra RG, Weigel BJ, Hirashima M, Munn DH, Murphy WJ, Azuma M, Anderson AC, Kuchroo VK, et al. (2011) Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood 117: 4501-4510. DOI 10.1182/blood-2010-10-310425\u003c/li\u003e\n\u003cli\u003eZhu C, Anderson AC, Kuchroo VK (2010) TIM-3 and Its Regulatory Role in Immune ResponsesNegative Co-Receptors and Ligands, pp. 1-15.\u003c/li\u003e\n\u003cli\u003eYang L, Anderson DE, Kuchroo J, Hafler DA (2008) Lack of TIM-3 Immunoregulation in Multiple Sclerosis. The Journal of Immunology 180: 4409-4414. DOI 10.4049/jimmunol.180.7.4409\u003c/li\u003e\n\u003cli\u003eJu Y, Shang X, Liu Z, Zhang J, Li Y, Shen Y, Liu Y, Liu C, Liu B, Xu L, et al. (2014) The Tim-3/galectin-9 pathway involves in the homeostasis of hepatic Tregs in a mouse model of concanavalin A-induced hepatitis. Molecular Immunology 58: 85-91. DOI 10.1016/j.molimm.2013.11.001\u003c/li\u003e\n\u003cli\u003eSakuishi K, Ngiow SF, Sullivan JM, Teng MWL, Kuchroo VK, Smyth MJ, Anderson AC (2014) TIM3+FOXP3+regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer. OncoImmunology 2. DOI 10.4161/onci.23849\u003c/li\u003e\n\u003cli\u003eWieckiewicz J, Goto R, Wood KJ (2010) T regulatory cells and the control of alloimmunity: from characterisation to clinical application. Current Opinion in Immunology 22: 662-668. DOI 10.1016/j.coi.2010.08.011\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":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Regulatory T cells, B cells, TIM3, CD25, IL-10","lastPublishedDoi":"10.21203/rs.3.rs-6135682/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6135682/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCell-based immunotherapy utilizing regulatory T cells (Tregs) has recently advanced into clinical applications, demonstrating promising results in phase I/II trials to prevent transplant rejection and treat autoimmune diseases. We have completed a clinical trial in renal transplant patients in which the significant biological effect was the increase of B cells with a regulatory phenotype in the blood of kidney transplant patients. The mechanisms by which Tregs regulate B cells and the specific molecules involved in this process remained poorly understood. In this study, we employed an \u003cem\u003ein vitro\u003c/em\u003e system of co-culture of peripherally purified B cells and expanded Tregs to show that Tregs can induce a population of memory B cells that express IL-10 and CD25. This subset of B cells has been previously identified as one of humans' regulatory B cell populations. Notably, these expanded Tregs\u0026rsquo; regulation of B cells was found to be independent of IL-10 and reliant on direct cell contact. We established that TIM3 expression by Tregs was crucial for the induction of IL-10-producing CD25\u003csup\u003e+\u003c/sup\u003e memory B cells. Our findings suggest that TIM3 is a critical molecule for the induction of regulatory B cells by Tregs, indicating that TIM3 expression by adoptively transferred Tregs is vital in diseases where B cells play a pathogenic role.\u003c/p\u003e","manuscriptTitle":"TIM3-Mediated Differentiation of IL-10-Producing CD25+ B Cells by Expanded Regulatory T Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 16:18:25","doi":"10.21203/rs.3.rs-6135682/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-07-29T04:57:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-13T14:37:21+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-12T10:52:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-04T16:43:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2025-03-03T13:30:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"90ab3175-7710-424e-847e-141cee672488","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T15:58:57+00:00","versionOfRecord":{"articleIdentity":"rs-6135682","link":"https://doi.org/10.1007/s00109-025-02606-0","journal":{"identity":"journal-of-molecular-medicine","isVorOnly":false,"title":"Journal of Molecular Medicine"},"publishedOn":"2025-12-27 15:57:00","publishedOnDateReadable":"December 27th, 2025"},"versionCreatedAt":"2025-03-17 16:18:25","video":"","vorDoi":"10.1007/s00109-025-02606-0","vorDoiUrl":"https://doi.org/10.1007/s00109-025-02606-0","workflowStages":[]},"version":"v1","identity":"rs-6135682","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6135682","identity":"rs-6135682","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}