Treg Therapy Research Articles

IMMUNE DYSREGULATION IN AUTOIMMUNE HEPATITIS: THE ROLE OF AUTOANTIBODIES, T-REG CELLS, AND NOVEL THERAPEUTIC APPROACHES

Introduction. The liver is a crucial immunological tolerogenic organ with a unique ability to generate effective immune responses against hepatotropic pathogens while maintaining both local and systemic immune tolerance to self and foreign antigens. Autoimmune hepatitis (AIH) is an immune-mediated inflammatory liver disease caused by the loss of tolerance to liver-specific antigens, leading to autoimmune liver damage.Aim – to summarize current knowledge on the immunopathogenesis of AIH based on information from open sources.Material and methods. The selection of publications was conducted using databases such as PubMed, Clinical Key Elsevier, Cochrane Library, eBook Business Collection, and Google Scholar, which covered information on the immunopathogenesis of AIH. Literature search was performed using keywords: autoimmune diseases, autoimmune hepatitis, Kupffer cells, autoantibodies to liver microsomal antibodies, and mesenchymal stem cells.Research results. Liver immune regulation is mediated by unique cell populations, including T-reg cells, Kupffer cells, and NK cells, which interact to maintain immune tolerance and regulate inflammation. Deficiency or dysfunction of T-reg cells, along with the activation of Th17 cells, contribute to the development of inflammatory processes and autoimmune liver damage. The quantitative and qualitative composition of immune cells in the liver differs significantly from that in secondary lymphoid organs, such as lymph nodes, spleen, or peripheral blood. The CD8+/CD4+ ratio (3.5:1) of liver T cells contrasts with the 1:2 ratio observed in peripheral blood, lymph nodes, and spleen.Conclusions. AIH is a complex disease with a multifaceted immunopathogenesis. T-reg and MSC-based therapies show promising results in clinical trials; however, further research is needed to optimize these methods and ensure their safe and effective application.

Activation in Autoimmunity: Mechanisms and Clinical Implications

Autoimmunity arises when the immune system mistakenly targets the body’s own tissues, leading to chronic inflammatory diseases that can affect multiple organs. The central event in autoimmunity is the inappropriate activation of immune cells, which normally tolerate self-antigens. This review examines the mechanisms underlying immune activation in autoimmunity, focusing on the loss of immune tolerance, molecular mimicry, bystander activation, and epitope spreading. Genetic predispositions and environmental triggers, such as infections, play key roles in this process, contributing to the dysregulation of immune responses. Specific immune cells, including autoreactive T cells, B cells producing autoantibodies, and dendritic cells presenting self-antigens, drive disease pathogenesis by perpetuating inflammation and tissue damage. Dysfunctions in regulatory T cells (Tregs) further exacerbate immune activation by failing to suppress autoreactive lymphocytes. Clinically, understanding these activation mechanisms is crucial for developing diagnostic biomarkers and targeted therapies. Current treatments focus on modulating immune responses through biologic agents that block pro-inflammatory cytokines (e.g., TNF-α, IL-6), deplete B cells (e.g., rituximab), or restore immune regulation (e.g., Treg therapies). Emerging therapies aim to restore immune tolerance, offering hope for more effective and personalized interventions. This review highlights the critical role of immune activation in autoimmunity and discusses therapeutic strategies to prevent or reverse this process. The ongoing exploration of these mechanisms is key to improving outcomes for patients with autoimmune diseases. Keywords: Autoimmunity, immune tolerance, molecular mimicry, T cells, autoantibodies, targeted therapy

Identification of a novel PDC-E2 epitope in primary biliary cholangitis: Application for engineered Treg therapy

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease, characterized by progressive destruction of small intrahepatic bile ducts and portal inflammation. Treatment options are limited, with reliance on liver transplantation in advanced cases. The adaptive immune response is implicated in disease pathogenesis by the presence of anti-mitochondrial antibodies targeting the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) in 90–95 % of patients and T cells infiltrating the portal tracts. Here, we examined T cell responses to peptides derived from PDC-E2, with a focus on CD4 T cell responses restricted to HLA Class II DRB4∗01:01, an allele found in 62 % of PBC patients, to uncover PDC-E2 epitopes that could be used for engineered regulatory T cell (Treg; EngTreg) therapy. Using an activation-induced marker assay and single cell RNA-sequencing, we found clonal expansion of CD4 T cells reactive to PDC-E2 epitopes among both T conventional (Tconv) and Tregs. Those T cell receptor (TCR) repertoires were non-overlapping and private and included TCRs specific for a novel PDC-E2 epitope restricted to DRB4∗01:01. CD4 Tconv cells reactive to the PDC-E2 novel epitope showed phenotypic heterogeneity skewed towards T follicular helper cells. Using a TCR specific for this novel PDC-E2 epitope, we created an EngTreg that suppressed PDC-E2-specific polyclonal CD4 Tconv cells from PBC patients. This study advances knowledge of PDC-E2-specific T cell responses and introduces a novel PDC-E2 epitope recognized by both Tconv and Tregs. Generation of EngTreg specific for this epitope provides therapeutic potential for PBC.

Repetitive transcranial magnetic stimulation alleviates motor impairment in Parkinson’s disease: association with peripheral inflammatory regulatory T-cells and SYT6

BackgroundRepetitive transcranial magnetic stimulation (rTMS) has been used to treat various neurological disorders. However, the molecular mechanism underlying the therapeutic effect of rTMS on Parkinson’s disease (PD) has not been fully elucidated. Neuroinflammation like regulatory T-cells (Tregs) appears to be a key modulator of disease progression in PD. If rTMS affects the peripheral Tregs in PD remains unknown.MethodsHere, we conducted a prospective clinical study (Chinese ClinicalTrials. gov: ChiCTR 2100051140) involving 54 PD patients who received 10-day rTMS (10 Hz) stimulation on the primary motor cortex (M1) region or sham treatment. Clinical and function assessment as well as flow cytology study were undertaken in 54 PD patients who were consecutively recruited from the department of neurology at Zhujiang hospital between September 2021 and January 2022. Subsequently, we implemented flow cytometry analysis to examine the Tregs population in spleen of MPTP-induced PD mice that received rTMS or sham treatment, along with quantitative proteomic approach reveal novel molecular targets for Parkinson's disease, and finally, the RNA interference method verifies the role of these new molecular targets in the treatment of PD.ResultsWe demonstrated that a 10-day rTMS treatment on the M1 motor cortex significantly improved motor dysfunction in PD patients. The beneficial effects persisted for up to 40 days, and were associated with an increase in peripheral Tregs. There was a positive correlation between Tregs and motor improvements in PD cases. Similarly, a 10-day rTMS treatment on the brains of MPTP-induced PD mice significantly ameliorated motor symptoms. rTMS reversed the downregulation of circulating Tregs and tyrosine hydroxylase neurons in these mice. It also increased anti-inflammatory mediators, deactivated microglia, and decreased inflammatory cytokines. These effects were blocked by administration of a Treg inhibitor anti-CD25 antibody in MPTP-induced PD mice. Quantitative proteomic analysis identified TLR4, TH, Slc6a3 and especially Syt6 as the hub node proteins related to Tregs and rTMS therapy. Lastly, we validated the role of Treg and rTMS-related protein syt6 in MPTP mice using the virus interference method.ConclusionsOur clinical and experimental studies suggest that rTMS improves motor function by modulating the function of Tregs and suppressing toxic neuroinflammation. Hub node proteins (especially Syt6) may be potential therapeutic targets.Trial registrationChinese ClinicalTrials, ChiCTR2100051140. Registered 15 December 2021, https://www.chictr.org.cn/bin/project/edit?pid=133691Graphical rTMS is a safe and non-invasive method for Parkinson's disease. In this study, we showed the proportion of CD4+CD25+CD127- regulatory T-cells (Tregs) in the peripheral blood was significantly increased after rTMS treatment. Similar effects of rTMS treatment were verified in MPTP-induced PD mice. Proteomic analysis and RNA interference analyses identified TLR4, TH, Slc6a3 and especially Syt6 as hub node proteins that can be modulated by rTMS therapy in PD.

MOG-specific CAR Tregs: a novel approach to treat multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS) with the immune system attacking myelin sheaths leading to neuronal death. While several disease-modifying therapies are available to treat MS, these therapies are not universally effective and do not stop disease progression. More personalized long-term treatment options that target specific aspects of the disease, such as reducing relapse frequency, delaying disability accumulation, and addressing symptoms that impact daily functioning, as well as therapies that can promote neuroprotection and repair are needed. Chimeric Antigen Receptor (CAR) Tcell therapies have revolutionized cancer treatment by intravenously (IV) administering a defined dose of T cells with high specificity provided by the CAR. An autologous CAR T cell therapy using suppressive regulatory T cells (Tregs) inducing long-lasting tolerance would be the ideal treatment for patients. Hence, we expanded the application of CAR-T cells by introducing a CAR into Tregs to treat MS patients. We developed a myelin oligodendrocyte glycoprotein (MOG)-specific CAR Treg cell therapy for patients with MS. MOG is expressed on the outer membrane of the myelin sheath, the insulating layer the forms around nerves, making it an ideal target for CAR Treg therapy. Our lead candidate is a 2nd generation CAR, composed of an anti-MOG scFv screened from a large human library. In vitro, we demonstrated CAR-dependent functionality and showed efficacy in vivo using a passive EAE mouse model. Additionally, the MOG-CAR Tregs have very low tonic signaling with a desirable signal-to-noise ratio resulting in a highly potent CAR. In summary our data suggest that MOG-CAR Tregs are a promising MS treatment option with the potential to induce long-lasting tolerance in patients.

IL23R-specific CAR Tregs for the treatment of Crohn's disease.

Regulatory T cells (Tregs) are key regulators in maintaining tissue homeostasis. Disrupted immune homeostasis is associated with Crohn's disease (CD) pathogenesis. Thus, Treg therapy represents a promising long-acting treatment to restore immune balance in the diseased intestine. CAR (Chimeric Antigen Receptor) T-cell therapy has revolutionized cancer treatment. This innovative approach also provides the opportunity to improve therapy for CD. By targeting a disease-relevant protein, Interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-CAR for treating active CD. Intestinal IL23R expression from active CD was verified by immunohistochemical analysis. Phenotypic and functional characteristics of IL23R-CAR Tregs were assessed using in vitro assays and their migration capacity was monitored in a xenograft tumor model. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR Treg activation against colon biopsy-derived cells from active CD patients. Our study showed that IL23R-CAR displayed negligible tonic signalling and strong signal-to-noise ratio. IL23R-CAR Tregs maintained regulatory phenotype during in vitro expansion, even when chronically exposed to proinflammatory cytokines and target antigen. IL23R engagement on IL23R-CAR Tregs triggered CAR-specific activation and significantly enhanced their suppressive activity. Also, IL23R-CAR Tregs migrated to IL23R-expressing tissue in humanized mice. Finally, IL23R-CAR Tregs elicited a specific activation against colon biopsy-derived cells from active CD, suggesting an efficient CAR engagement in active CD. Molecular profiling of CD patient biopsies also revealed transcriptomic and proteomic patterns associated with IL23R-CAR activation. Overall, our results demonstrate that IL23R-CAR Tregs represent a promising therapy for active CD.

The potential of autologous regulatory T cell (Treg) therapy to prevent Cardiac Allograft Vasculopathy (CAV) in paediatric heart transplant recipients.

Paediatric heart transplant is an established treatment for end stage heart failure in children, however patients have to commit to lifelong medical surveillance and adhere to daily immunosuppressants to minimise the risk of rejection. Compliance with immunosuppressants can be burdensome with their toxic side effects and need for frequent blood monitoring especially in children. Though the incidence of early rejection episodes has significantly improved overtime, the long-term allograft health and survival is determined by Cardiac Allograft Vasculopathy (CAV) which affects a vast number of post-transplant patients. Once CAV has set in, there is no medical or surgical treatment to reverse it and graft survival is significantly compromised across all age groups. Current treatment strategies include novel immunosuppressant agents and drugs to lower blood lipid levels to address the underlying immunological pathophysiology and to manage traditional cardiac risk factors. Translational researchers are seeking novel immunological approaches that can lead to permanent acceptance of the allograft such as using regulatory T cell (Tregs) immunotherapy. Clinical trials in the setting of graft versus host disease, autoimmunity and kidney and liver transplantation using Tregs have shown the feasibility and safety of this strategy. This review will summarise current knowledge of the latest clinical therapies for CAV and pre-clinical evidence in support of Treg therapy for CAV. We will also discuss the different Treg sources and the considerations of translating this into a feasible immunotherapy in clinical practice in the paediatric population.

Graft-Specific Regulatory T Cells for Long-Lasting, Local Tolerance Induction.

Solid organ transplantation is hindered by immune-mediated chronic graft dysfunction and the side effects of immunosuppressive therapy. Regulatory T cells (Tregs) are crucial for modulating immune responses post-transplantation; however, the transfer of polyspecific Tregs alone is insufficient to induce allotolerance in rodent models. To enhance the efficacy of adoptive Treg therapy, we investigated different immune interventions in the recipients. By utilizing an immunogenic skin transplant model and existing transplantation medicine reagents, we facilitated the clinical translation of our findings. Specifically, antigen-specific Tregs were used. Our study demonstrated that combining the available induction therapies with drug-induced T-cell proliferation due to lymphopenia effectively increased the Treg/T effector ratios. This results in significant Treg accumulation within the graft, leading to long-term tolerance after the transfer of antigen-specific Tregs. Importantly, all the animals achieved operational tolerance, which boosted the presence of adoptively transferred Tregs within the graft. This protocol offers a means to establish tolerance by utilizing antigen-specific Tregs. These results have promising implications for future trials involving adoptive Treg therapy in organ transplantation.

Electrostimulation suppresses allograft rejection via promoting lymphatic regulatory T cell migration mediated by lymphotoxin - lymphotoxin receptor β signaling

Conventional immunosuppressants that suppress allograft rejection cause various side effects. Although regulatory T cells (Tregs) are essential for allograft survival, the limited efficacy of Treg therapy demands improvement. Thus, it is imperative to seek new approaches to enhancing Treg suppression. Low-intensity electrostimulation (ES) has been shown to exert antiinflammatory effects without causing major adverse reactions. However, it remains unknown whether and how ES regulates alloimmunity. Here, we found that regional ES delayed murine skin allograft rejection and promoted long-term allograft survival induced by an mTOR inhibitor, rapamycin. ES also extended islet allograft survival. Mechanistically, ES enhanced the expression of lymphotoxin α (LTα) on Tregs after transplantation. Blockade of lymphotoxin β receptor–mediated nonclassical NFκB signaling suppressed lymphatic Treg migration and largely reversed the effects of ES on allograft survival. Moreover, ES failed to extend allograft survival when recipients lacked LTα/lymph nodes or if transferred Tregs lacked LTα. Therefore, ES promoted the lymphatic migration of CD4+Foxp3+ Tregs by upregulating their surface expression of LTα. Finally, ES augmented expression of LTα on murine or human Tregs, but not conventional T cells, while promoting their calcium influx in vitro. This ES-mediated upregulation of LTα relied on calcium influx. Thus, our findings have unveiled novel mechanisms underlying ES-mediated immunoregulation.

Late Treatment With Autologous Expanded Regulatory T-cell Therapy After Alemtuzumab Induction Is Safe and Facilitates Immunosuppression Minimization in Living Donor Renal Transplantation.

The TWO Study (Transplantation Without Overimmunosuppression) aimed to investigate a novel approach to regulatory T-cell (Treg) therapy in renal transplant patients, using a delayed infusion protocol at 6 mo posttransplant to promote a Treg-skewed lymphocyte repopulation after alemtuzumab induction. We hypothesized that this would allow safe weaning of immunosuppression to tacrolimus alone. The COVID-19 pandemic led to the suspension of alemtuzumab use, and therefore, we report the unique cohort of 7 patients who underwent the original randomized controlled trial protocol. This study presents a unique insight into Treg therapy combined with alemtuzumab and is therefore an important proof of concept for studies in other diseases that are considering lymphodepletion. Living donor kidney transplant recipients were randomized to receive autologous polyclonal Treg at week 26 posttransplantation, coupled with weaning doses of tacrolimus, (Treg therapy arm) or standard immunosuppression alone (tacrolimus and mycophenolate mofetil). Primary outcomes were patient survival and rejection-free survival. Successful cell manufacturing and cryopreservation until the 6-mo infusion were achieved. Patient and transplant survival was 100%. Acute rejection-free survival was 100% in the Treg-treated group at 18 mo after transplantation. Although alemtuzumab caused a profound depletion of all lymphocytes, including Treg, after cell therapy infusion, there was a transient increase in peripheral Treg numbers. The study establishes that delayed autologous Treg therapy is both feasible and safe, even 12 mo after cell production. The findings present a new treatment protocol for Treg therapy, potentially expanding its applications to other indications.

A phase Ib, open-label study of add-on therapy with CK0804 in participants with myelofibrosis and suboptimal response to ruxolitinib.

6580 Background: Suboptimal response to JAK2 inhibitor ruxolitinib in patients with myelofibrosis (MF) remains a major unmet medical need. Deregulated inflammatory pathways including CXCR4/CXCR12 axis might limit the therapeutic efficacy of ruxolitinib and contribute to disease progression. In preclinical studies, cord blood-derived, CXCR4 enriched T regulatory cells [CK0804; T-regs] showed ability to suppress inflammatory cytokines playing major role in MF (Huang et al., Cytotherapy, 2023). Methods: This phase Ib study evaluates the safety and activity of up to 6 doses of CK0804 (non-HLA matched, Cryopreserved) T-regs therapy, fixed dose of 100 million cells, added every 28 days to ruxolitinib. Patients with MF on ruxolitinib for ≥12 weeks and stable dose for ≥8 weeks, who have palpable splenomegaly, symptoms, or grade 2 cytopenia are eligible. Primary objective is safety, secondary objective is overall response per IWG-MRT criteria at 24 weeks. Present analysis includes cohort of initial run-in safety phase. Results: Nine patients of median age 68 years (range, 55-84) were enrolled, 44% were males. Median blood counts [range] showed while blood cells 10.6 [3.1-70.4] x10^9/L, hemoglobin 8.8 [7.8-11.5] g/dL, platelets 177 [148-311] x10^9/L. Three patients were transfusion dependent. Median spleen volume was 1449 cubic centimeters (176-5609), 7 patients (78%) had clinically worsening splenomegaly. Median symptoms score (MPN-SAF TSS) was 23 (20-40). Six (67%) had diploid karyotype, 5 (56%) had JAK2 mutation and 8 (89%) had additional no-driver mutations. Median duration of prior ruxolitinib was 35 months (range, 10-132), all patients were on ≥ 10 mg twice daily dose with no dose change throughout the study. Four patients received all six doses of CK0804, three patients have ongoing treatments. One patient experienced infusion reaction to second dose of CK0804 likely due to the excipient dimethylsulfoxide and withdrew consent. One patient died of unrelated cause prior to the infusion 6. There were no nonhematologic or hematologic adverse events. Two transfusion dependent patients who were evaluable for response had decreased monthly need for transfusions by the end of the sixth cycle: 4 to 2.8 units and 1.2 to 0.8 units, respectively. All patients noticed symptoms improvement; median best decline in MPN-SAF TSS was -38% (range, -20% to -71%); with ≥ 50% reduction in 3 patients. One patient achieved ≥ 35% spleen volume reduction at week 12. Longitudinal analysis of markers of inflammation is in progress and will be presented at the conference. Conclusions: This preliminary analysis of run-in phase of study evaluating CXCR4 enriched T regs cell therapy as addition to ruxolitinib shows initial safety with no myelosuppressive adverse events and promising clinical activity. Active enrollment of participants to the expansion cohort is ongoing. Clinical trial information: NCT05423691 .

Elucidating inflammation-induced cell fate decisions in primary human Tregs

Abstract Adoptive regulatory T-cell (Treg) therapy has remarkable efficacy in promoting immune tolerance in preclinical models of transplantation and autoimmune disease. However, lineage-tracing studies have revealed that Tregs can undergo epigenetic reprogramming in chronically inflamed tissue environments, resulting in the acquisition of proinflammatory functions and the capacity to exacerbate autoimmunity. Despite intense interests in developing Treg therapy for humans, inflammation-induced human Treg lineage-decommitment remains poorly understood. Here, we present a robust in vitro model of IL6, IL1β, and IL23-driven human Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3 conserved non-coding sequence (CNS)-2 enhancer re-methylation, diminished in vitro suppressive function, and elevated proinflammatory cytokine expression. Single-cell transcriptomic and chromatin accessibility analyses of human Tregs undergoing destabilization revealed a gradual closing of a Treg-specific element (A) and a reciprocal opening of a competing element (B) at the IRF4 locus. CRISPR-mediated excision of the B element (IRF4ΔB) preserved Treg identity and enhanced in vitro suppressive activity following prolonged inflammatory exposure. Ongoing experiments aim to evaluate the in vivo function and stability of IRF4ΔB Tregs in a xenogeneic graft-versus-host model. These findings may facilitate the design of Treg therapeutics with greater potency and improved safety.

Genetic screening to identify novel targets enhancing regulatory T-cell (Treg) phenotype.

Abstract Tregs possess immunosuppressive properties that can restore and maintain immune homeostasis via various suppressive mechanisms. A marker of Tregs is the transcription factor Forkhead box P3 (FoxP3), which regulates Treg development and function. Autoimmune patients have shown dysfunction or reduction in FoxP3+ Tregs, highlighting the critical role of these cells in preventing pathogenic immune responses. There is great interest in developing medical interventions against autoimmune diseases by enhancing Treg function and/or numbers. Currently, a major limitation to generate efficient Treg therapies is the lack of selective targets as starting points for drug discovery. To identify novel Treg targets, Evotec developed a CRISPR/Cas9-based arrayed screening platform using human CD4+ T-cells. About 8,000 genes were screened with a FACS read-out and the effect of gene knockout (KO) on the frequency of FoxP3+ Tregs was assessed. We identified ~450 targets where the KO led to an increase of FoxP3+ cells within the CD4+ T-cell compartment, indicating their potential for drug development starting points. In addition to FoxP3, the effect of KO on CD4+ conventional as well as Treg viability, proliferation and markers such as, CTLA-4, PD-1 or CD25 was monitored. The hits are being confirmed and the impact of the target KO on Treg function and phenotype assessed in a panel of in vitro assays. The goal is to identify novel Treg targeting product candidates to treat autoimmune diseases.

Polyclonally Derived Alloantigen-Specific T Regulatory Cells Exhibit Target-Specific Suppression and Capture MHC Class II from Dendritic Cells.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs invitro and explored the fine specificity of their suppressive function and their mechanism of action invitro and invivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function invivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

Rapamycin rescues loss of function in blood-brain barrier-interacting Tregs.

In autoimmunity, FOXP3+ Tregs skew toward a proinflammatory, nonsuppressive phenotype and are, therefore, unable to control the exaggerated autoimmune response. This largely affects the success of autologous Treg therapy, which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the CNS during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss of function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential and a proinflammatory Th1/17 signature, and they upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less-suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In summary, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive proinflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.

Blocking Microglial Proliferation by CSF-1R Inhibitor Does Not Alter the Neuroprotective Effects of Adoptive Regulatory T Cells in 3xTg Alzheimer's Disease Mice.

Alzheimer's disease (AD) is a chronic neurodegenerative disease that causes cognitive impairment. Neuroinflammation induced by activated microglia exacerbates AD. Regulatory T cells (Tregs) play roles in limiting neuroinflammation by converting microglial polarization. Therefore, adoptive Treg therapy is considered an attractive option for neurodegenerative disorders. However, the mechanism underlying Treg therapy via microglial modulation is not fully understood. In this study, we sought to determine whether adoptively transferred Tregs were effective when microglia proliferation was inhibited by using GW2580, which is an inhibitor of CSF1R. We found that inhibition of microglial proliferation during Treg transfer did not alter the therapeutic effects of Tregs on cognitive deficits and the accumulation of Aβ and pTAU in 3xTg-AD mice. The expression of pro- and anti-inflammatory markers in the hippocampus of 3xTg mice showed that GW2580 did not affect the inhibition of neuroinflammation by Treg transfer. Additionally, adoptively transferred Tregs were commonly detected in the brain on day 7 after transfer and their levels decreased slowly over 100 days. Our findings suggest that adoptively transferred Tregs can survive longer than 100 days in the brain, suppressing microglial activation and thus alleviating AD pathology. The present study provides valuable evidence to support the prolonged efficacy of adoptive Treg therapy in AD.

ROS-responsive drug-releasing injectable microgels for ameliorating myocardial infarction

Despite of the recent advances in regulatory T cell (Treg) therapy, a limited number of available cells and specificity at the desired tissue site have severely compromised their efficacy. Herein, an injectable drug-releasing (MTK-TK-drug) microgel system in response to in situ stimulation by reactive oxygen species (ROS) was constructed with a coaxial capillary microfluidic system and UV curing. The spherical microgels with a size of 150 μm were obtained. The MTK-TK-drug microgels efficiently converted the pro-inflammatory Th17 cells into anti-inflammatory regulatory T cells (Treg) cells in vitro, and the ROS-scavenging materials synergistically enhanced the effect by modulating the inflammation microenvironment. Thus, the microgels significantly reduced cardiomyocyte apoptosis and decreased the inflammatory response in the early stages of post-myocardial infarction (MI) in vivo, thereby reducing fibrosis, promoting vascularization, and preserving cardiac function. Overall, our results indicate that the MTK-TK-drug microgels can attenuate the inflammatory response and improve MI therapeutic effects in vivo.

The Role of Regulatory T cells (Tregs) in Tumorigenesis: A Comprehensive Literature Review

Introduction: Regulatory T cells (Tregs) are a subpopulation of CD4+ T lymphocytes that contribute to immune homeostasis by suppressing excessive immune activation. However, these immunosuppressive properties can lead to the suppression of anti-tumor immune responses. Depletion or blocking of Tregs through therapeutics has emerged as a possible method for enhancing anti-tumor immunity. However, the lack of selective targeting of Tregs in the tumor microenvironment is a significant limitation to the effectiveness of Treg therapies. Therefore, this investigation aims to review current literature on how Tregs suppress the antitumor immune response and how they can be targeted to promote anti-tumor immunity. Methods: This review examines recent literature on Tregs in the tumor microenvironment, focusing on both cell-contact dependent and independent mechanisms. Clinical trial studies were also included to assess therapeutic targeting of Tregs. The PubMed database was systematically searched for English articles from 2010 to present, supplemented by manual searches without date restrictions. Boolean expressions ensured comprehensive study retrieval. Results: The involvement of Tregs in the development of multiple cancer types is evident, and targeting these cells could potentially enhance the efficacy of antitumor immunity. In addition, we compiled a list of the novel approaches currently being used for Treg targeting in the context of cancer. Discussion: This review has identified the most promising targets for Treg-based therapies, opening avenues for accelerating the development of innovative cancer treatments. Conclusion: Our literature review offers insights into the complex interplay between the immune system and cancer. The understanding of this interaction is not just an endpoint but could potentially act as a steppingstone towards new scientific discoveries.

OP02 IL23R-CAR-Tregs: creating a therapeutic breakthrough for Crohn’s Disease

Abstract Background Regulatory T cells (Tregs) are key immunomodulators to maintain immune homeostasis and self-tolerance, including in the intestine. Disrupted immune homeostasis is associated with Crohn’s Disease (CD) pathogenesis. As a living drug, Treg therapy is a promising treatment for restoring immune balance and achieving durable tissue tolerance in the diseased intestine. By targeting a disease-relevant protein, Interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-chimeric antigen receptor (CAR) to create IL23R-CAR-Tregs for treating CD. Methods IL23R protein expression in CD intestine was verified by immunohistochemical analysis. Tonic signaling and CAR-specific activation were quantified during the IL23R-CAR screening. The IL23R-CAR-Treg phenotype was assessed by monitoring the expression of FoxP3 and Helios transcription factors, and the demethylation status of FoxP3 Treg-specific demethylated region (TSDR). The suppressive function of IL23R-CAR-Tregs was evaluated using in vitro assays and a colitis mouse model. The functionality of IL23R-CAR-Tregs were further assessed using intestinal biopsy-derived cells from CD patients in an in vitro activation assay. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR-Treg activation. Results Our highly potent IL23R-CAR lead candidate displays negligible tonic signalling and a strong signal-to-noise ratio. The resulting IL23R-CAR-Tregs maintain their regulatory phenotype with high FoxP3 and Helios expression and TSDR demethylation during in vitro expansion, CAR-specific stimulation, and in the presence of proinflammatory cytokines. In accordance with their regulatory phenotype, IL23R-CAR-Tregs exhibit CAR-dependent suppressive activity, promote a tolerogenic environment, and protect mice from colitis. The expression of IL23R was also confirmed in the intestinal mucosa from CD patients. Importantly, IL23R-CAR-Tregs elicit activation following exposure to intestinal biopsy-derived cells from CD, demonstrating the engagement and potency of IL23R-CAR towards their target in CD. We also identified molecular profiles that correlated with IL23R-CAR activation and, more broadly, showed their potential to monitor IL23R-CAR-Treg activity in CD patients. Conclusion Our results demonstrate that an IL23R-CAR-Treg product could be a promising therapy for CD patients, with the potential long-term benefits of immune tolerance.

Therapeutic Effects of Aβ-Specific Regulatory T Cells in Alzheimer's Disease: A Study in 5xFAD Mice.

The aging global population is placing an increasing burden on healthcare systems, and the social impact of Alzheimer's disease (AD) is on the rise. However, the availability of safe and effective treatments for AD remains limited. Adoptive Treg therapy has been explored for treating neurodegenerative diseases, including AD. To facilitate the clinical application of Treg therapy, we developed a Treg preparation protocol and highlighted the therapeutic effects of Tregs in 5xFAD mice. CD4+CD25+ Tregs, isolated after Aβ stimulation and expanded using a G-rex plate with a gas-permeable membrane, were adoptively transferred into 5xFAD mice. Behavioral analysis was conducted using Y-maze and passive avoidance tests. Additionally, we measured levels of Aβ, phosphorylated tau (pTAU), and nitric oxide synthase 2 (NOS2) in the hippocampus. Real-time RT-PCR was employed to assess the mRNA levels of pro- and anti-inflammatory markers. Our findings indicate that Aβ-specific Tregs not only improved cognitive function but also reduced Aβ and pTAU accumulation in the hippocampus of 5xFAD mice. They also inhibited microglial neuroinflammation. These effects were observed at doses as low as 1.5 × 103 cells/head. Collectively, our results demonstrate that Aβ-specific Tregs can mitigate AD pathology in 5xFAD mice.