Acute urticaria can be a presenting symptom of anaphylaxis characterized by transient red swellings or fulminant wheals, often accompanied by severe pruritus. Numerous studies have substantiated the important involvement of regulatory T cells (Tregs) in the occurrence of allergic diseases and autoimmune diseases. However, the role of Tregs in the pathogenesis of acute urticaria is unclear. In this study, we found that the frequency of Tregs in peripheral blood mononuclear cells (PBMCs) was decreased in patients with acute urticaria compared with normal controls by flow cytometry. Analysis of Assay for transposase-accessible chromatin with sequencing (ATAC-seq) data identified 28 differentially accessible regions comparing Tregs from healthy individuals and patients with acute urticaria, all showing increased chromatin accessibility in the Tregs from acute urticaria. IL-1b was highly expressed in sera of patients with acute urticaria and the level of IL-1b was moderately positively related to white blood cell count. The elevated expression of IL-1b may be due to the diminished immune-suppressive function following the decline of Tregs in this study. We found that IL1B gene expression was also significantly increased in the skin lesions of both chronic spontaneous urticaria and solar urticaria compared to healthy controls. IL1B might play a key role in the development of acute urticaria and IL1B could be a potential prognostic biomarker and therapeutic target in urticaria.

T-cell dysfunction is a hallmark of chronic lymphocytic leukemia (CLL), but the extent to which individual CD4+ or CD8+ T-cell subpopulations influence specific clinical events remains unclear. To address this knowledge gap, we utilised high-dimensional mass cytometry to profile circulating CD4+ and CD8+ T-cells in pre-treatment samples from a well-defined cohort of CLL patients undergoing initial therapy as part of a clinical trial.

Pre-treatment blood samples from 138 CLL patients receiving initial chemoimmunotherapy containing bendamustine or chlorambucil in the NCRI RIAltO trial (NCT01678430; EudraCT 2011-000919-22) were subjected to deep immunophenotyping by mass cytometry using a bespoke panel of 37 antibodies. T-cell clusters were identified through unsupervised clustering and related to treatment outcomes. Additionally, a randomly selected cohort of 30 CLL patients underwent T-cell stimulation with anti-CD3/CD28 microbeads, followed by cytokine analysis using a separate 36-antibody panel, which included seven cytokines.

Seventeen CD4+ and 22 CD8+ T-cell clusters were identified in a discovery cohort of 79 patients. Three of these clusters, measured as a proportion of their parental CD4+ or CD8+ populations, correlated with a reduced risk of grade ≥3 infection, grade ≥3 second primary malignancy (SPM) and death, respectively. Three corresponding T-cell subpopulations prospectively defined by non-redundant markers and Boolean gating (ICOS+HLA-DR+PD1+TIGIT+Tbet+CD4+ T-helper cells; CD27+CD28-PD1+Tbet+Eomes+CD8+ cells; and CD27+CD28-GrymB+Tbet+Eomes+CD8+ terminal effector cells) showed the same clinical correlations as the clusters on which they were based. With the exception of SPM for which there were insufficient events, these correlations were confirmed in a separate validation cohort of 59 patients. In-vitro stimulation of a subset of CLL patients in the discovery cohort showed an enrichment of primed and polyfunctional cells in all three Tbet+ T-cell subpopulations of interest.

Our study provides new insights into the potential for Tbet+ T-cell subpopulations to influence and predict specific clinical events in CLL. This, in turn, raises the possibility that these respective subpopulations could play an important role in controlling infection, solid tumours and CLL itself.

https://www.clinicaltrials.gov/, identifier NCT01678430; https://www.isrctn.com/ISRCTN09988575, identifier EudraCT 2011-000919-22.

Organ transplantation is a lifesaving procedure, with 50,000 transplants happening every year in the United States. However, many patients harbor antibodies and B cells directed against allogeneic human leukocyte antigen (HLA) molecules, notably HLA-A2, greatly decreasing their likelihood of receiving a compatible organ. Moreover, antibody-mediated rejection is a significant contributor to chronic transplant rejection. Current strategies to desensitize patients non- specifically target circulating antibodies and B cells, resulting in poor efficacy and complications. Regulatory T cells (Tregs) are immune cells dedicated to suppressing specific immune responses by interacting with both innate and adaptive immune cells. Here, we genetically modified human Tregs with a chimeric anti-HLA antibody receptor (CHAR) consisting of an extracellular HLA-A2 protein fused to a CD28-CD3zeta intracellular signaling domain, driving Treg activation upon recognition of anti-HLA-A2 antibodies on the surface of alloreactive B cells. We find that HLA-A2 CHAR Tregs get activated specifically by anti-HLA-A2 antibody-producing cells. Of note, HLA-A2 CHAR activation does not negatively affect Treg stability, as measured by expression of the Treg lineage transcription factors FOXP3 and HELIOS. Interestingly, HLA-A2 CHAR Tregs are not cytotoxic towards anti-HLA-A2 antibody-producing cells, unlike HLA-A2 CHAR modified conventional CD4 + T cells. Importantly, HLA-A2 CHAR Tregs recognize and significantly suppress high affinity IgG antibody production by B cells from HLA-A2 sensitized patients. Altogether, our results provide proof-of-concept of a new strategy to specifically inhibit alloreactive B cells to desensitize transplant recipients.

Regulatory T cells (Tregs) mediate tissue homeostasis and repair. The function of the interleukin-7 receptor α (IL-7Rα) in nonlymphoid tissue Tregs is still unknown, although low expression of IL-7Rα is a widely accepted marker for Tregs. Here, we show that IL-33R (ST2)-expressing Tregs in the visceral adipose tissue (VAT) express the IL-7Rα at high levels. Treg-specific IL-7Rα-deficient mice exhibited reduced adipose ST2+ Tregs and impaired glucose tolerance, whereas IL-7Rα was dispensable for Tregs in lymphoid tissues. Mice deficient in thymic stromal lymphopoietin (TSLP), an additional ligand for IL-7Rα, displayed a modest decrease in adipose ST2+ Tregs and a reduced accumulation of adipose eosinophils, accompanied by slightly impaired glucose tolerance. In the VAT, mesothelial cells expressed IL-7, whereas adipose stem cells and folate receptor β-expressing tissue-resident macrophages expressed TSLP. Thus, this study indicates the significance of IL-7Rα signaling in the maintenance of VAT Tregs and glucose homeostasis, revealing a novel role for IL-7 and TSLP in immunometabolism.

Regulatory T cells (Tregs) are important in maintaining tolerance and are key players in immunity. In aging, increased Treg function along with low-grade inflammation has been reported. This dichotomy of enhanced Treg function along with inflammation highlights the importance of understanding Treg biology and communication patterns in the very old. In this proof-of-concept study, we demonstrate that aged Tregs (85 years) do not significantly communicate with CD4+ and CD8+ T effectors when compared with healthy < 66-year-olds. Of note was the enhanced communication of aged Tregs with CD3+CD8+CD56+CD161- NK-like T-cell populations, which are important in antitumor and chronic viral diseases in older individuals. We found that in turn this population of killer-like T cells showed diminished cytotoxic characteristics, and killer receptor expression. Taken together, our proof-of-concept study delineates the biology of Tregs and identifies previously undefined communication patterns in the very old.

Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors have been used to redirect the specificity of Tregs, using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using human leukocyte antigen (HLA)-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein that integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε knockout Tregs via repetitive CD3/CD28 stimulation in a good manufacturing practice-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2+ skin grafts from rejection compared with polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.

Primary glomerular diseases (PGDs), including nephrotic syndrome (NS), membranous nephropathy (MN), and IgA nephropathy (IgAN), are complex renal conditions influenced by immune system dysregulation. Although associations between immune cell phenotypes and PGDs have been observed, the precise causal relationships have not been fully elucidated.

Utilizing genetic association data from genome-wide association studies (GWASs), we investigated 731 immunophenotypes in relation to PGDs. A bidirectional two-sample Mendelian randomization (MR) approach, primarily employing inverse variance weighting (IVW), was conducted to establish causality. MR-Egger, weighted median, simple mode, and weighted mode were used as complementary methods to reinforce the robustness and validity of the results. Sensitivity analyses further validated the sensitivity and stability of our results.

We identified 38 immunophenotypes suggestively related to IgAN, with 20 as risk factors and 18 as protective effects. Six immunophenotypes remained significant after Bonferroni correction: The percentage of CD25hi among T cells; the percentage of CD25hi CD45RA- CD4 not T regulatory (Treg) among T cells; the percentage of CD25hi CD45RA- CD4 not Treg within the CD4+ T cell population; CX3CR1 expression on monocytes; CD40 expression on monocytes; and CD64 expression on CD14+ CD16- monocytes. In the validation analysis of IgAN, CD3 expression on effector memory CD4+ T cells further confirmed the predisposing risk role of effector memory T cells in the development of IgAN. Additionally, the MR analysis demonstrated suggestive associations between 25 immunophenotypes and MN (8 risk factors and 17 protective factors), as well as between 22 immunophenotypes and NS (10 risk factors and 12 protective factors). Last, by intersecting the immunophenotypes showing suggestive associations with PGDs, we identified two common immunophenotypes shared by IgAN and MN, three by IgAN and NS, and one by MN and NS.

This genetic-level investigation uncovers causal associations between immunophenotypes and PGDs, providing valuable insights into the immunological underpinnings of PGDs. Our findings suggest potential targets for treatment strategies, thereby facilitating more personalized and effective therapeutic approaches in PGDs management.

Gonadotropins, such as follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG), are widely used to induce ovarian hyperovulation during in vitro fertilization and embryo transfer (IVF-ET) for the treatment of infertility. However, the effects of repeated administration of these gonadotropins on immune function, particularly on T cell development in the thymus, remain poorly understood. This study investigated the effects of repeated administration of pregnant mare serum gonadotropin (PMSG) and hCG on thymic T cell development in mice. Histological analysis revealed structural changes in the thymus, including a blurred boundary between the medulla and cortex and reduced vascularization after repeated administration of PMSG and hCG. Quantitative real-time PCR showed increased expression of adipogenesis-related genes [phosphoenolpyruvate carboxykinase (PEPCK), adipocyte fatty acid-binding protein 2 (aP2), peroxisome proliferator-activated receptor gamma (PPARγ)] but no significant changes in thymic epithelial cell-related genes [autoimmune regulator (AIRE), epithelial V-like antigen (EVA), interleukin 7 (IL-7)]. Flow cytometry revealed a decrease in CD4+CD8+ T cells and an increase in CD4-CD8-T cells with altered CD25/CD44 subsets. In addition, CD4+ and CD8+ T cells in the spleen were significantly reduced. These findings suggest that repeated gonadotropin exposure may disrupt thymic T cell development and peripheral T cell populations, potentially impairing immune function. Further research is needed to elucidate the underlying mechanisms and broader immunologic consequences of gonadotropin use in infertility treatment.

Global per capita alcohol consumption is increasing, posing significant socioeconomic and medical challenges also due to alcohol-related traumatic injuries but also its biological effects. Trauma as a leading cause of death in young adults, is often associated with an increased risk of complications, such as sepsis and multiple organ failure, due to immunological imbalances. Regulatory T cells play a crucial role in maintaining immune homeostasis by regulating the inflammatory response. Since it is crucial to understand the effects of alcohol in healthy volunteers, in order to refer findings from trauma cohorts, this study investigates the time- and dose-dependent modulation of CD4+ lymphocytes and their subsets following acute alcohol consumption, considering both general and sex-specific variations.

Twelve female and ten male healthy volunteers consumed twelve alcohol mixed drinks over four hours to achieve a blood alcohol level of 1.0‰. Blood samples were collected before and at various time points (2, 4, 6, 24 and 48 h) post-consumption for flow cytometric analyses of the phenotype and activation makers CD4/CD25/CD127 of CD4+ T cells and their subtypes.

CD4+ lymphocytes significantly decreased at 4 h and increased at 6 h post-alcohol consumption. Naïve CD25-CD127+ T cells significantly decreased from 2 to 24 h in women and 2 to 48 h in men, while CD25+CD127+ effector T cells significantly increased during the same period. Natural CD25+CD127- regulatory T cells increased significantly at 4 and 6 h, with a higher increase in men. Induced regulatory T cells (CD4+CD25highCD127-) significantly increased at 2 h for all volunteers, with lower proportions of natural and induced regulatory T cells in women.

Acute alcohol consumption induces immune modulation persisting for days, impacting T cell subsets differently in men and women. The prolonged modulation in men may contribute to slightly poorer clinical outcomes, emphasizing the need to consider these effects in trauma patients with acute alcohol intoxication.

Anti-programmed cell death protein 1 (anti-PD-1) antibodies have achieved revolutionary success in cancer therapy. However, the impact of anti-PD-1 therapy on host humoral immunity in humans during cancer immunotherapy requires further investigation.

We evaluated immunoglobulin titers by ELISA and screened the immune landscape of immune cells from 25 healthy donors and 50 cases including 25 new-onset hepatocellular carcinoma (HCC) patients prior to systemic treatment and 25 HCC patients undergoing anti-PD-1 therapy by multicolor flow cytometry. Flow or beads sorted cells were cultured ex vivo for proliferation and functional analysis.

Anti-PD-1 therapy significantly increased the levels of IgG and IgA in the periphery of HCC patients. Anti-PD-1 treatment led to an increase in plasmablasts and a notable rise in circulating T follicular regulatory (cTfr) cells, while changes in circulating B cells, T follicular helper cells, or regulatory T cells were not significant. Anti-PD-1 therapy also influenced the proliferation and function of cTfr cells, promoting the differentiation of CD38+ cTfr cells. We observed that the CD38+ Tfr cell subset in the peripheral blood can promote plasmablast differentiation, associated with altered antibody production.

Together, these data demonstrate the immunomodulatory role of PD-1 in restricting the differentiation and function of human cTfr cells and in regulating humoral immunity.

Chimeric Antigen Receptor (CAR)-T cell therapy has rapidly emerged as a groundbreaking approach in cancer treatment, particularly for hematologic malignancies. However, the application of CAR-T cell therapy in solid tumors remains challenging. This review summarized the development of CAR-T technologies, emphasized the challenges and solutions in CAR-T cell therapy for solid tumors. Also, key innovations were discussed including specialized CAR-T, combination therapies and the novel use of CAR-Treg, CAR-NK and CAR-M cells. Besides, CAR-based cell therapy have extended its reach beyond oncology to autoimmune disorders. We reviewed preclinical experiments and clinical trials involving CAR-T, Car-Treg and CAAR-T cell therapies in various autoimmune diseases. By highlighting these cutting-edge developments, this review underscores the transformative potential of CAR technologies in clinical practice.

The reasons for the low frequency of anti-Ro/SS-A antibody in patients with HTLV-1-associated myelopathy complicated with Sjögren's syndrome (SS) are unclear. In this study, we investigated whether HTLV-1-infected T cells can act directly on B cells and suppress B cells' production of antibodies, including anti-Ro/SS-A antibody. For this purpose, we established an in vitro T-cell-free B-cell antibody production system. The productions of total IgG and anti-cytomegalovirus IgG in B cells from healthy subjects and those of total IgG and anti-Ro/SS-A IgG in B cells from SS patients were significantly suppressed by the addition of HTLV-1-positive T-cell lines (MT-2 and HCT-5). Our analysis of co-cultured B cells identified no sign of HTLV-1 infection and revealed that MT-2 and HCT-5 cells act on the early stages of B-cell differentiation, not the activation stage. MT-2 and HCT-5 cells constitutively expressed CD70, ICAM-1, LAP (TGF-β), and PD-L1/2, but blocking monoclonal antibodies to these molecules or PD-L1/2 receptor PD-1 had no significant canceling effect on B-cell IgG production regarding their suppressive activity. Importantly, autologous CD4+CD25+CD127low Treg cells had no inhibitory effect on B-cell IgG production. These results demonstrate that HTLV-1-positive T cells can directly suppress B-cell antibody production through mechanisms that differ from Treg functions.

Regulatory T cells (Tregs) have emerged as crucial players in maintaining maternal-fetal tolerance and promoting successful pregnancy outcomes. This review examines the importance of these cells in pregnancy, drawing on human and animal-based studies, with a focus on their role in bovine fertility. Tregs employ various mechanisms to mediate maternal-fetal tolerance, including regulation of effector T-cell responses, interactions with innate immune cells in the uterine microenvironment, and modulation of trophoblast function. In humans, Treg dynamics during normal pregnancy and alterations in pregnancy complications provide compelling evidence for their involvement in maintaining fetal-maternal harmony. Animal models, particularly mouse studies, have further elucidated the importance of Tregs in preventing fetal rejection and promoting successful pregnancy outcomes. The review also explores the characterization of bovine Tregs, highlighting their similarities and unique features compared to human and rodent counterparts. Recent studies have indicated the presence and potential significance of Tregs in the bovine uterine environment during early pregnancy. Translational applications of Treg research in livestock fertility are discussed, with a focus on immunomodulatory strategies for enhancing Treg function, such as antigen-specific tolerance induction, pharmacological targeting of Treg pathways, and cell-based therapies using autologous or allogeneic Tregs. The review concludes by emphasizing the potential impact of Treg-based strategies on the livestock industry and the broader implications for human reproductive health. Future research directions are outlined, underscoring the need for further investigations into the role of Tregs in bovine reproductive tissues and their relationship with fertility outcomes.

T cell development occurs in the thymus in both mice and humans. Upon entry into the thymus, bone marrow-derived blood-borne progenitors receive instructive signals, including Notch signaling, to eliminate their potential to develop into alternative immune lineages while committing to the T cell fate. Upon T-lineage commitment, developing T cells receive further instructional cues to generate different T cell sublineages, which together possess diverse immunological functions to provide host immunity. Over the years, numerous studies have contributed to a greater understanding of key thymic signals that govern T cell differentiation and subset generation. Here, we review these critical signaling factors that govern the different stages of both mouse and human T cell development, while also focusing on the transcriptional changes that mediate T cell identity and diversity.

Classic regulatory T (Treg) cells expressing CD4 and the hallmark transcription factor FOXP3 are integral to the prevention of multi-system autoimmunity. However, immune-mediated arthritis is often associated with increased numbers of Treg cells in the inflamed joints. To understand these seemingly conflicting observations, which we collectively describe as 'the Treg paradox', we provide an overview of Treg cell biology with a focus on Treg cell heterogeneity, function and dysfunction in arthritis. We discuss how the inflamed environment constrains the immunosuppressive activity of Treg cells while also promoting the differentiation of TH17-like Treg cell, exTreg cell (effector T cells that were formerly Treg cells), and osteoclastogenic Treg cell subsets that mediate tissue injury. We present a new framework to understand Treg cells in joint inflammation and define potential strategies for Treg cell-directed interventions in human inflammatory arthritis.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28-activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFN-γ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

Objectives: Aplastic anemia (AA) is one of the immune-mediated bone marrow failure disorders caused by multiple factors, including the inability of CD4 + CD25 + regulatory T cells (Tregs) to negatively regulate cytotoxic T lymphocytes (CTLs). Dioscin is a natural steroid saponin that has a similar structure to steroid hormones. The purpose of this study is to look into the effect of Dioscin on the functions of CD4 + CD25+ Tregs in the AA mouse model and explore its underlying mechanism.Methods: To begin with, bone marrow failure was induced through total body irradiation and allogeneic lymphocyte infusion using male Balb/c mice. After 14 consecutive days of Dioscin orally administrated, the AA mouse model was tested for complete blood counts, HE Staining of the femur, Foxp3, IL-10 and TGF-β. Then CD4 + CD25+ Tregs were isolated from splenic lymphocytes of the AA mouse model, Tregs and the biomarkers and cytokines of Tregs were measured after 24 h of Dioscin intervention treatment in vitro.Results: Dioscin promotes the expression of Foxp3, IL-10, IL-35 and TGF-β, indicating its Tregs-promoting properties. Mechanistically, the administration of Dioscin resulted in the alteration of CD152, CD357, Perforin and CD73 on the surface of Tregs, and restored the expression of Foxp3.Conclusion: Dioscin markedly attenuated bone marrow failure, and promoted Tregs differentiation, suggesting the maintenance of theimmune balance effect of Dioscin. Dioscin attenuates pancytopenia and bone marrow failure via its Tregs promotion properties.

Regenerative medicine is a rapidly expanding field harnessing human pluripotent stem cell (hPSC)-derived cells and tissues to treat many diseases, including type 1 diabetes. However, graft immune protection remains a key challenge. Chimeric antigen receptor (CAR) technology confers new specificities to effector T cells and immunosuppressive regulatory T cells (Tregs). One challenge in CAR design is identifying target molecules unique to the cells of interest. Here, we employ combinatorial genetic engineering to confer CAR-Treg-mediated localized immune protection to stem cell-derived cells. We engineered hPSCs to express truncated epidermal growth factor receptor (EGFRt), a biologically inert and generalizable target for CAR-Treg homing and activation, and generated CAR-Tregs recognizing EGFRt. Strikingly, CAR-Tregs suppressed innate and adaptive immune responses in vitro and prevented EGFRt-hPSC-derived pancreatic beta-like cell (sBC [stem cell-derived beta cell]) graft immune destruction in vivo. Collectively, we provide proof of concept that hPSCs and Tregs can be co-engineered to protect hPSC-derived cells from immune rejection upon transplantation.

Regulatory T-cell (Treg) detection in peripheral blood, based on flow cytometry, is invaluable for diagnosis and treatment of immune-mediated diseases. However, there is a lack of reliable methods to verify the performance, which is pivotal toward standardization of the Tregs assay.

To conduct standardization studies and verify the performance of 3 commercially available reagent sets for the Tregs assay based on flow cytometry and agreement analysis for Treg detection across the different reagent sets.

The analytical performance of Tregs assay using reagent sets supplied by 3 manufacturers was evaluated after establishing the gating strategy and determining the optimal antibody concentration. Postcollection sample stability was evaluated, as well as the repeatability, reproducibility, reportable range, linearity, and assay carryover. Agreement between the different assays was assessed via Bland-Altman plots and linear regression analysis. The relationship between the frequency of CD4+CD25+CD127low/- Tregs and CD4+CD25+Foxp3+ Tregs was evaluated.

The postcollection sample stability was set at 72 hours after collection at room temperature. The accuracy, repeatability, reproducibility, and accuracy all met the requirements for clinical analysis. Excellent linearity, with R2 ≥0.9 and no assay carryover, was observed. For reportable range, a minimum of 1000 events in the CD3+CD4+ gate was required for Tregs assay. Moreover, the results for Tregs labeled by antibodies from the 3 manufacturers were in good agreement. The percentage of CD4+CD25+CD127low/- Tregs was closely correlated with CD4+CD25+Foxp3+ Tregs.

This is the first study to evaluate systematically the measurement performance of Tregs in peripheral blood by flow cytometry, which provides a practical solution to verifying the performance of flow cytometry-based immune monitoring projects in clinical practice.

Opioid addiction presents a relevant health challenge, with chronic heroin use linked to detrimental effects on various aspects of physical, mental, and sociological health. Opioid maintenance therapy (OMT), particularly using methadone, is the primary treatment option for heroin addiction. Previous studies using blood samples from current heroin addicts and OMT patients have shown immunomodulatory effects of heroin and methadone on T cell function. However, various additional factors beyond heroin and methadone affect these results, including the consumption of other substances, a stressful lifestyle, comorbid psychological and somatic disorders, as well as additional medications. Therefore, we here investigated the direct effects of heroin and methadone on purified human T cells in vitro. Our results reveal that both, heroin and methadone directly suppress Tcell activation and proliferation. Strikingly, this inhibitory effect was markedly stronger in the presence of methadone, correlating with a decrease in secretion of pro-inflammatory cytokines. While heroin did not interfere with the in vitro differentiation and expansion of regulatory Tcells (Tregs), methadone significantly impaired the proliferation of Tregs. Overall, our findings suggest a direct inhibitory impact of both opioids on effector T cell function in vitro, with methadone additionally interfering with Treg induction and expansion in contrast to heroin.

Regulatory T cells (Treg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of Treg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited Treg cell products. Here, we examined different Treg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each Treg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory Treg cell populations, including a hitherto unidentified naïve-like memory Treg cell subset, outperformed late-differentiated effector memory-like Treg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional Treg cell products could be achieved by depleting the small effector memory-like Treg cell subset before manufacturing. Considering Treg cell subset composition appears critical to maintain lineage stability in the final cell product.

Immune cell-mediated inflammation is important in normal tissue regeneration but can be pathologic in diabetic wounds. Limited literature exists on the role of CD4+ T cells in normal or diabetic wound repair; however, the imbalance of CD4+ Th17/Tregs has been found to promote inflammation in other diabetic tissues. Here, using human tissue and murine transgenic models, we identified that the histone methyltransferase Mixed-lineage-leukemia-1 (MLL1) directly regulates the Th17 transcription factor RORγ via an H3K4me3 mechanism and increases expression of Notch receptors and downstream Notch signaling. Furthermore, we found that Notch receptor signaling regulates CD4+ Th cell differentiation and is critical for normal wound repair, and loss of upstream Notch pathway mediators or receptors in CD4+ T cells resulted in the loss of CD4+ Th cell differentiation in wounds. In diabetes, MLL1 and Notch-receptor signaling was upregulated in wound CD4+ Th cells, driving CD4+ T cells toward the Th17 cell phenotype. Treatment of diabetic wound CD4+ T cells with a small molecule inhibitor of MLL1 (MI-2) yielded a significant reduction in CD4+ Th17 cells and IL-17A. This is the first study to our knowledge to identify the MLL1-mediated mechanisms responsible for regulating the Th17/Treg balance in normal and diabetic wounds and to define the complex role of Notch signaling in CD4+ T cells in wounds, where increased or decreased Notch signaling both result in pathologic wound repair. Therapeutic targeting of MLL1 in diabetic CD4+ Th cells may decrease pathologic inflammation through regulation of CD4+ T cell differentiation.

It is crucial to decipher the modulation of regulatory T cells (Tregs) in tumor microenvironment (TME) induced by chemotherapy, which may contribute to improving the efficacy of neoadjuvant chemoimmunotherapy in resectable non-small cell lung cancer (NSCLC). We retrospectively collected specimens from patients with II-III NSCLC, constituting two cohorts: a neoadjuvant chemotherapy (NAC) cohort (N = 141) with biopsy (N = 58) and postoperative specimens (N = 141), and a surgery-only cohort (N = 122) as the control group. Then, the cell density (Dens), infiltration score (InS), and Treg-cell proximity score (TrPS) were conducted using a panel of multiplex fluorescence staining (Foxp3, CD4, CD8, CK, CD31, ɑSMA). Subsequently, the association of Tregs with cancer microvessels (CMVs) and cancer-associated fibroblasts (CAFs) was analyzed. Patients with NAC treatment have a higher density of Tregs in both paired (P < 0.001) and unpaired analysis (P = 0.022). Additionally, patients with NAC treatment showed higher infiltration score (paired, P < 0.001; unpaired, P = 0.014) and more CD8+T cells around Tregs (paired/unpaired, both P < 0.001). Subgroup analysis indicated that tumors with a diameter of ≤ 5 cm exhibited increase in both Dens(Treg) and InS(Treg), and gemcitabine, pemetrexed and taxel enhanced Dens(Treg) and TrPS(CD8) following NAC. Multivariate analysis identified that the Dens(Tregs), InS(Tregs) and TrPS(CD8) were significantly associated with better chemotherapy response [OR = 8.54, 95%CI (1.69, 43.14), P = 0.009; OR = 7.14, 95%CI (1.70, 30.08), P = 0.024; OR = 5.50, 95%CI (1.09, 27.75), P = 0.039, respectively] and positive recurrence-free survival [HR = 3.23, 95%CI (1.47, 7.10), P = 0.004; HR = 2.70; 95%CI (1.27, 5.72); P = 0.010; HR = 2.55, 95%CI (1.21, 5.39), P = 0.014, respectively]. Moreover, TrPS(CD8) and TrPS(CD4) were negatively correlated with the CMVs and CAFs. These discoveries have deepened our comprehension of the immune-modulating impact of chemotherapy and underscored that the modified spatial landscape of Tregs after chemotherapy should be taken into account for personalized immunotherapy, aiming to ultimately improve clinical outcomes in patients with NSCLC.

Regulatory T cells (Tregs) are crucial for preserving tolerance in the body, rendering Treg immunotherapy a promising treatment option for both organ transplants and autoimmune diseases. Presently, organ transplant recipients must undergo lifelong immunosuppression to prevent allograft rejection, while autoimmune disorders lack definitive cures. In the last years, there has been notable advancement in comprehending the biology of both antigen-specific and polyclonal Tregs. Clinical trials involving Tregs have demonstrated their safety and effectiveness. To maximize the efficacy of Treg immunotherapy, it is essential for these cells to migrate to specific target tissues, maintain stability within local organs, bolster their suppressive capabilities, and ensure their intended function's longevity. In pursuit of these goals, the utilization of biomaterials emerges as an attractive supportive strategy for Treg immunotherapy in addressing these challenges. As a result, the prospect of employing biomaterial-enhanced Treg immunotherapy holds tremendous promise as a treatment option for organ transplant recipients and individuals grappling with autoimmune diseases in the near future. This paper introduces strategies based on biomaterial-assisted Treg immunotherapy to enhance transplant medicine and autoimmune treatments.

Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity.

We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs.

We showed that Tregs could be enriched to 87- 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136-732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A.

This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

Objective Chimeric antigen receptor (CAR) T cell therapy is an emerging and effective therapy for relapsed or refractory diffuse large B cell lymphoma (R/R DLBCL). The characteristic toxicities of CAR T cell therapy include cytokine release syndrome (CRS) and prolonged cytopenia. We investigated the factors associated with these complications after CAR T cell therapy by analyzing lymphocyte subsets following CAR T cell infusion. Methods We retrospectively analyzed peripheral blood samples on days 7, 14, and 28 after tisagenlecleucel (tisa-cel) infusion by flow cytometry at our institution between June 2020 and September 2022. Patients Thirty-five patients with R/R DLBCL who received tisa-cel therapy were included. Results A flow cytometry-based analysis of blood samples from these patients revealed that the proportion of CD4+CD25+CD127+ T cells (hereafter referred to as "activated CD4+ T cells" ) among the total CD4+ T cells on day 7 after tisa-cel infusion correlated with the duration of CRS (r=0.79, p<0.01). In addition, a prognostic analysis of the overall survival (OS) using time-dependent receiver operating characteristic curves indicated a significantly more favorable OS and progression-free survival of patients with a proportion of activated CD4+ T cells among the total CD4+ T cells <0.73 (p=0.01, and p<0.01, respectively). Conclusion These results suggest that the proportion of activated CD4+ T cells on day 7 after tisa-cel infusion correlates with the CRS duration and predicts clinical outcomes after CAR T cell therapy. Further studies with a larger number of patients are required to validate these observations.

Latent human cytomegalovirus (hCMV) infection can pose a serious threat of reactivation and disease occurrence in immune-compromised individuals. Although T cells are at the core of the protective immune response to hCMV infection, a detailed characterization of different T cell subsets involved in hCMV immunity is lacking. Here, in an unbiased manner, we characterized over 8000 hCMV-reactive peripheral memory T cells isolated from seropositive human donors, at a single-cell resolution by analysing their single-cell transcriptomes paired with the T cell antigen receptor (TCR) repertoires. The hCMV-reactive T cells were highly heterogeneous and consisted of different developmental and functional memory T cell subsets such as, long-term memory precursors and effectors, T helper-17, T regulatory cells (TREGs) and cytotoxic T lymphocytes (CTLs) of both CD4 and CD8 origin. The hCMV-specific TREGs, in addition to being enriched for molecules known for their suppressive functions, showed enrichment for the interferon response signature gene sets. The hCMV-specific CTLs were of two types, the pre-effector- and effector-like. The co-clustering of hCMV-specific CD4-CTLs and CD8-CTLs in both pre-effector as well as effector clusters suggest shared transcriptomic signatures between them. The huge TCR clonal expansion of cytotoxic clusters suggests a dominant role in the protective immune response to CMV. The study uncovers the heterogeneity in the hCMV-specific memory T cells revealing many functional subsets with potential implications in better understanding of hCMV-specific T cell immunity. The data presented can serve as a knowledge base for designing vaccines and therapeutics.

Regulatory T cells (Tregs), a suppressive subpopulation of T cells, are potent mediators of peripheral tolerance, responsible for immune homeostasis. Many autoimmune diseases exhibit disruptions in Treg function or quantity, resulting in an imbalance between protective and pathogenic immune cells. Selective expansion or manipulation of Tregs is a promising therapeutic approach for autoimmune diseases. However, the extensive diversity of Treg subpopulations and the multiple approaches used for Treg identification leads to high complexity, making it difficult to develop a successful treatment capable of modulating Tregs. In this review, we describe the suppressive mechanisms, subpopulations, classification, and identification methodology for Tregs, and their role in the pathogenesis of autoimmune diseases.

Regulatory T cells (Tregs) are a type of lymphocyte that is key to maintaining immunological self-tolerance, with great potential for therapeutic applications. A long-standing challenge in the study of Tregs is that the only way they can be unambiguously identified is by using invasive intracellular markers. Practically, the purification of live Tregs is often compromised by other cell types since only surrogate surface markers can be used. We present here a non-invasive method based on Raman spectroscopy that can detect live unaltered Tregs by coupling optical detection with machine learning implemented with regularized logistic regression. We demonstrate the validity of this approach first on murine cells expressing a surface Foxp3 reporter, and then on peripheral blood human T cells. By including methods to account for sample purity, we could generate reliable models that can identify Tregs with an accuracy higher than 80%, which is already comparable with typical sorting purities achievable with standard methods that use proxy surface markers. We could also demonstrate that it is possible to reliably detect Tregs in fully independent donors that are not part of the model training, a key milestone for practical applications.

Uveal melanoma (UM) is the most common cancer of the eye. The loss of chromosome 3 (M3) is associated with a high risk of metastases. M3 tumors are more infiltrated by T-lymphocytes than low-risk disomic-3 (D3) tumors, contrasting with other tumor types in which T cell infiltration correlates with better prognosis. Whether these T cells represent an antitumor response and how these T cells would be primed in the eye are both unknown. Herein, we characterized the T cells infiltrating primary UMs. CD8+ and Treg cells were more abundant in M3 than in D3 tumors. CD39+PD-1+CD8+ T cells were enriched in M3 tumors, suggesting specific responses to tumor antigen (Ag) as confirmed using HLA-A2:Melan-A tetramers. scRNAseq-VDJ analysis of T cells evidenced high numbers of proliferating CD39+PD1+CD8+ clonal expansions, suggesting in situ antitumor Ag responses. TCRseq and tumor-Ag tetramer staining characterized the recirculation pattern of the antitumor responses in M3 and D3 tumors. Thus, tumor-Ag responses occur in localized UMs, raising the question of the priming mechanisms in the absence of known lymphatic drainage.

To control immune responses, regulatory CD4+CD25+Foxp3+ T cells (Treg) maintain their wide and diverse repertoire through continuous arrival of recent thymic emigrants (RTE). However, during puberty, the activity of RTE starts to decline as a natural process of thymic involution, introducing consequences, not completely described, to the repertoire. Type 1 diabetes (T1D) patients show quantitative and qualitative impairments on the Treg cells. Our aim was to evaluate peripheral Treg and RTE cell frequencies, in T1D patients from two distinct age groups (young and adults) and verify if HLA phenotypes are concomitant associated. To this, blood samples from Brazilian twenty established T1D patients (12 young and 8 adults) and twenty-one healthy controls (11 young and 10 adults) were analyzed, by flow cytometry, to verify the percentages of CD4, Treg (CD4+CD25+Foxp3+) and the subsets of CD45RA+ (naive) and CD31+(RTE) within then. Furthermore, the HLA typing was also set. We observed that the young established T1D patients feature decreased frequencies in total Treg cells and naive RTE within Treg cells. Significant prevalence of HLA alleles, associated with risk, in T1D patients, was also identified. Performing a multivariate analysis, we confirmed that the cellular changes described offers significant variables that distinct T1D patients from the controls. Our data collectively highlight relevant aspects about homeostasis imbalances in the Treg cells of T1D patients, especially in young, and disease prognosis; that might contribute for future therapeutic strategies involving Treg cells manipulation.

BCG vaccination affects other diseases beyond tuberculosis by unknown-potentially immunomodulatory-mechanisms. Recent studies have shown that BCG vaccination administered during overt type 1 diabetes (T1D) improved glycemic control and affected immune and metabolic parameters. Here, we comprehensively characterized Ghanaian T1D patients with or without routine neonatal BCG vaccination to identify vaccine-associated alterations. Ghanaian long-term T1D patients (n = 108) and matched healthy controls (n = 214) were evaluated for disease-related clinical, metabolic, and immunophenotypic parameters and compared based on their neonatal BCG vaccination status. The majority of study participants were BCG-vaccinated at birth and no differences in vaccination rates were detected between the study groups. Notably, glycemic control metrics, i.e., HbA1c and IDAA1c, showed significantly lower levels in BCG-vaccinated as compared to unvaccinated patients. Immunophenotype comparisons identified higher expression of the T cell activation marker CD25 on CD8+ T cells from BCG-vaccinated T1D patients. Correlation analysis identified a negative correlation between HbA1c levels and CD25 expression on CD8+ T cells. In addition, we observed fractional increases in glycolysis metabolites (phosphoenolpyruvate and 2/3-phosphoglycerate) in BCG-vaccinated T1D patients. These results suggest that neonatal BCG vaccination is associated with better glycemic control and increased activation of CD8+ T cells in T1D patients.

Janus kinase [JAK] inhibitors [JAKinibs] are effective small molecule therapies for treating Crohn's disease [CD] and ulcerative colitis [UC], collectively known as inflammatory bowel disease [IBD]. By preventing JAKs from phosphorylating signal transducer and activator of transcription proteins, JAKinibs disrupt cytokine signalling pathways that promote inflammation. Despite considerable overlap in the JAKs they target, first- and second-generation JAKinibs display different clinical efficacies in CD and UC.

We conducted a comparative phosflow study of four JAKinibs [filgotinib, upadacitinib, tofacitinib, and deucravacitinib] to observe subtle mechanistic differences that may dictate their clinical behaviour. Resected mesenteric lymph node [MLN] cells from 19 patients [9 CD, 10 UC] were analysed by flow cytometry in the presence or absence of different cytokine stimuli and titrated JAKinibs.

We found a higher potency of the JAK 1/3-preferential inhibitor, tofacitinib, for JAK 3-dependent cytokine signalling pathways in comparison to filgotinib, but a higher potency of the JAK 1-preferential inhibitors, filgotinib and upadacitinib, for JAK 3-independent cytokine signalling pathways. Deucravacitinib, a TYK2-preferential inhibitor, demonstrated a much narrower selectivity by inhibiting only IL-10 and IFN-β pathways, albeit more potently than the other JAKinibs. Additionally, we found some differences in the sensitivity of immune cells from CD versus UC, and patients with versus without a CD-associated NOD2 polymorphism, to phosphorylate signal transducer and activator of transcriptions in response to specific cytokine stimulation.

Despite their similarities, differences exist in the relative potencies of different JAKinibs against distinct cytokine families, to explain their clinical efficacy.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

Recently, increased interest in the use of Tregs as adoptive cell therapy for the treatment of autoimmune diseases and transplant rejection had led to several advances in the field. However, Treg cell therapies, while constantly advancing, indiscriminately suppress the immune system without the permanent stabilization of certain diseases. Genetically modified Tregs hold great promise towards solving these problems, but, challenges in identifying the most potent Treg subtype, accompanied by the ambiguity involved in identifying the optimal Treg source, along with its expansion and engineering in a clinical-grade setting remain paramount. This review highlights the recent advances in methodologies for the development of genetically engineered Treg cell-based treatments for autoimmune, inflammatory diseases, and organ rejection. Additionally, it provides a systematized guide to all the recent progress in the field and informs the readers of the feasibility and safety of engineered adoptive Treg cell therapy, with the aim to provide a framework for researchers involved in the development of engineered Tregs.