Small extracellular vesicles derived from PD-L1-modified mesenchymal stem cell promote Tregs differentiation and prolong allograft survival.

We aimed to explore whether programmed cell death protein-1 ligand (PD-L1) modification on small extracellular vesicles (sEVs) could promote T regulatory cells (Tregs) differentiation. In this study, it was confirmed that under physiological conditions, PD-L1 expression was minimal in the MSCs and absent in the MSC-sEVs. A vector harboring the PD-L1 gene was constructed and transfected into bone marrow mesenchymal stem cells (BM-MSCs). By extracting the sEVs of these modified BM-MSCs and monitoring the expression of the PD-L1 protein, however, PD-L1 expression was substantially increased in the MSCs and concentrated in the sEVs. Then, the rat naïve CD4 + T cells were cocultured with the sEVs derived from the PD-L1-modified MSCs (sEVsPD-L1). By flow cytometry, a higher percentage of Tregs and anti-inflammatory downstream cytokines (including IL-2, IFN-γ, TGF-ß, IL-10) was detected in the sEVsPD-L1 group than that in the control group treated by either sEVs in wild type, modified by empty vector, or blank control. Suppressive effect on CD4 + T cell proliferation serves as additional evidence to support the immunoregulation capacity of sEVsPD-L1. The animal model of vascularized composite allograft further confirmed that PD-L1-modified sEVs induce an immune tolerance, by clinically observation, histopathology, T cell fate and cell product. In conclusion, sEVsPD-L1 efficiently promotes Treg cell differentiation in vitro and in vivo, which suggests their therapeutic potential in the treatment of allograft rejection.

Diversity of T Helper and Regulatory T Cells and Their Contribution to the Pathogenesis of Allergic Diseases.

T helper (Th) and regulatory T (Treg) cells represent important effectors of adaptive immunity. They mediate communication between the immune system and tissue sites and thereby coordinate effective defense against environmental threats or maintain tolerance, respectively. Since the discovery of two prototypic T helper cells, Th1 and Th2, additional phenotypic and functional distinct subsets have been described ranging from Th17, Th22, Th9, and T follicular helper cells. The same holds true for regulatory T cells that represent a family with functionally distinct subsets characterized by co-expression of the transcription factors T-bet, Gata3, or RORγt. Here, we summarize the current knowledge on differentiation and function of T helper and regulatory T cell subsets and discuss their lineage stability versus plasticity towards other subsets. In addition, we highlight the direct and indirect contribution of each subset to the pathology of allergies and indicate novel therapies for specific targeting the effector functions of T helper and regulatory T cells.

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein.

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

Reduced PD-1 expression on circulating follicular and conventional FOXP3+ Treg cells in children with new onset type 1 diabetes and autoantibody-positive at-risk children.

Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 diabetes (T1D), but the contribution of follicular regulatory T (Treg) cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5+) and conventional (CXCR5-) Treg cells in the blood of children with new-onset T1D, and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood follicular and conventional Treg cells were higher in frequency in children with new onset T1D, but expressed reduced amounts of PD-1 as compared to AAb-negative children. Interestingly, the proportion of circulating FOXP3+ Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to AAb-negative controls, suggesting its potential use as a biomarker of disease progression. Follicular Treg cells were reduced in frequency in the spleens of prediabetic NOD mice as they became older and turned diabetic. Interestingly, PD-1 expression declined also on circulating follicular and conventional Treg cells in prediabetic NOD mice as they aged. Together, these findings show that the frequency of circulating follicular and conventional Treg cells and their levels of PD-1 change with disease progression in children at-risk for developing T1D and in NOD mice.

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells.

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed-infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T-cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi-maturation of human monocytes-derived DCs (moDCs), and elevated levels of IL-10, IL-27 and IL-6 but not IL-12p70 and TNF-α. Consistently, HMOS-conditioned human moDCs promoted Treg generation from naïve CD4+ T cells. Interestingly, HMOS limited LPS-induced maturation of human moDCs, while maintained IL-10 and IL-27 secretion and reduced LPS-induced production of IL-12p70, IL-6 and TNF-α. Furthermore, HMOS+LPS-stimulated DCs induced a higher frequency of Tregs and increased IL-10 production, while a reduction in Tbet+Th1 frequency and IFN-γ production was detected as compared to LPS-DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC-SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.

Regulatory T Cells in the Tumor Microenvironment.

Regulatory T cells (Tregs) are an immunosuppressive subpopulation of CD4+ T cells that are endowed with potent suppressive activity and function to limit immune activation and maintain homeostasis. These cells are identified by the hallmark transcription factor FOXP3 and the high-affinity interleukin-2 (IL-2) receptor chain CD25. Tregs can be recruited to and persist within the tumor microenvironment (TME), acting as a potent barrier to effective antitumor immunity. This chapter will discuss [i] the history and hallmarks of Tregs; [ii] the recruitment, development, and persistence of Tregs within the TME; [iii] Treg function within TME; asnd [iv] the therapeutic targeting of Tregs in the clinic. This chapter will conclude with a discussion of likely trends and future directions.

Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.

Myeloid-derived suppressor cells (MDSCs) represent a heterogenous population of immature myeloid cells capable of modulating immune responses. In the context of cancer, MDSCs are abnormally produced and recruited to the tumor microenvironment (TME) to aid in the establishment of an immunosuppressive TME that facilitates tumor escape. Additionally, MDSCs contribute to non-immunologic aspects of tumor biology, including tumor angiogenesis and metastasis. The clinical significance of MDSCs has recently been appreciated as numerous studies have suggested a correlation between circulating and intratumoral MDSC frequencies and tumor stage, progression, and treatment resistance. In this chapter, we review MDSC characterization, development, expansion, and mechanisms that facilitate immunosuppression and tumor progression. Furthermore, we highlight studies demonstrating the clinical significance of MDSCs in various disease states in addition to strategies that modulate various aspects of MDSC biology for therapeutic gain.

Ethyl Pyruvate Promotes Proliferation of Regulatory T Cells by Increasing Glycolysis.

Ethyl pyruvate (EP), a stable form of pyruvate, has shown beneficial effects in animal models of shock, ischemia/reperfusion injury, and sepsis due to its potent anti-oxidant and anti-inflammatory properties. Our recent study demonstrated that EP application prevented the clinical manifestation of type 1 diabetes in mice by augmenting regulatory T cell (Treg) number and function. Our present study shows that EP increases Treg proliferation and suppressive function (perforin and IL-10 expression) during in vitro differentiation from conventional CD4+CD25- T cells. Enhanced expansion of Treg after EP treatment correlated with increased ATP levels and relied on increased glycolysis. Inhibition of oxidative phosphorylation did not attenuate EP stimulatory effects, suggesting that this metabolic pathway was not mandatory for EP-driven Treg proliferation. Moreover, EP lowered the expression of carnitine palmitoyltransferase I, an enzyme involved in fatty acid oxidation. Further, the stimulatory effect of EP on Treg proliferation was not mediated through inhibition of the mTOR signaling pathway. When given in vivo either intraperitoneally or orally to healthy C57BL/6 mice, EP increased the number of Treg within the peritoneal cavity or gut-associated lymphoid tissue, respectively. In conclusion, EP promotes in vitro Treg proliferation through increased glycolysis and enhances Treg proliferation when administered in vivo.

CD25 deficiency: A new conformational mutation prevents the receptor expression on cell surface.

CD25 deficiency is a very rare autosomal recessive disorder that shows a clinical phenotype highly overlapping IPEX syndrome with an increased susceptibility to viral, bacterial, and fungal infections. It is due to mutations in the IL2Rα gene that codes for the α subunit of the IL2 receptor complex. Here we report the characterization of a novel IL2Rα gene mutation leading to a severe protein conformational alteration that abrogates its cell surface expression in a child presenting with early-onset IPEX-like disorder. Cytofluorimetric analysis revealed the total absence of CD25 cell surface expression and addressed IL2Rα molecular investigation. The early clinical and molecular diagnosis of CD25 deficiency in this patient promptly led to hematopoietic stem cell transplantation (HSCT), allowing complete resolution of the symptoms and definitive cure of the disease.

From stability to dynamics: understanding molecular mechanisms of regulatory T cells through Foxp3 transcriptional dynamics.

Studies on regulatory T cells (Treg ) have focused on thymic Treg as a stable lineage of immunosuppressive T cells, the differentiation of which is controlled by the transcription factor forkhead box protein 3 (Foxp3). This lineage perspective, however, may constrain hypotheses regarding the role of Foxp3 and Treg in vivo, particularly in clinical settings and immunotherapy development. In this review, we synthesize a new perspective on the role of Foxp3 as a dynamically expressed gene, and thereby revisit the molecular mechanisms for the transcriptional regulation of Foxp3. In particular, we introduce a recent advancement in the study of Foxp3-mediated T cell regulation through the development of the Timer of cell kinetics and activity (Tocky) system, and show that the investigation of Foxp3 transcriptional dynamics can reveal temporal changes in the differentiation and function of Treg in vivo. We highlight the role of Foxp3 as a gene downstream of T cell receptor (TCR) signalling and show that temporally persistent TCR signals initiate Foxp3 transcription in self-reactive thymocytes. In addition, we feature the autoregulatory transcriptional circuit for the Foxp3 gene as a mechanism for consolidating Treg differentiation and activating their suppressive functions. Furthermore, we explore the potential mechanisms behind the dynamic regulation of epigenetic modifications and chromatin architecture for Foxp3 transcription. Lastly, we discuss the clinical relevance of temporal changes in the differentiation and activation of Treg .

LAP+ Treg is a better biomarker than total Treg in viral myocarditis.

Latency associated peptide (LAP) is a protein expressed on the membrane of some regulatory T cells (Treg). LAP+ Treg have a greater immunomodulatory effect than that of their negative counterparts. In this study, we presented the data on the proportion of LAP+ Treg out of CD4+ cells in mice with viral myocarditis, which we believed was more sensitive and specific than that of the ratio of total Treg in CD4+ cells. Comparing with the previously recognized total Treg, LAP+ Treg was a better biomarker on myocardial inflammation.

Fibroblast activation proteins-α suppress tumor immunity by regulating T cells and tumor-associated macrophages.

Fibroblast activation protein-α (FAPα) is a type-II cell-surface-bound integral transmembrane serine protease and selectively overexpressed by tumor-associated stromal fibroblasts (TAFs), which are the main components in the tumor microenvironment, in >90% of malignant epithelial carcinomas. FAPα regulates the immunosuppression of tumor cells in the tumor microenvironment. Regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) are the major immunosuppressive cells in the tumor microenvironment. However, the effect of FAPα on Tregs and TAMs is unknown. The non-enzymatic function of FAPα on Treg and TAM was investigated. In this study, we confirm that FAPα can promote the generation of Tregs and TAMs, which suggests that FAPα plays a immunosuppressive role in the tumor microenvironment and provides evidence for FAP α as a potent immunotherapeutic target for cancer.

Spectrum of Phenotypes Associated with Mutations in LRBA.

To date, several germline mutations have been identified in the LRBA gene in patients suffering from a variety of clinical symptoms. These mutations abolish the expression of the LRBA protein, leading to autoimmunity, chronic diarrhea, B-cell deficiency, hypogammaglobulinemia, functional T-cell defects and aberrant autophagy. We review the clinical and laboratory features of patients with LRBA mutations and present five novel mutations in eight patients suffering from a multitude of clinical features.

Adoptive immunotherapy with the use of regulatory T cells and virus-specific T cells derived from cord blood.

Cord blood transplantation, an alternative to traditional stem cell transplants (bone marrow or peripheral blood stem cell transplantation), is an attractive option for patients lacking suitable stem cell transplant donors. Cord blood units have also proven to be a valuable donor source for the development of cellular therapeutics. Virus-specific T cells and regulatory T cells are two cord blood-derived products that have shown promise in early-phase clinical trials to prevent and/or treat viral infections and graft-versus-host disease, respectively. We describe how current strategies that use cord blood-derived regulatory T cells and virus-specific T cells have been developed to improve outcomes for cord blood transplant recipients.

Adriamycin-induced nephropathy in rats: functional and cellular effects characterized by MRI.

PURPOSE: To assess with magnetic resonance imaging (MRI) adriamycin-induced nephropathy in living rats, an established model for proteinuric renal disease was used. MATERIALS AND METHODS: Functional information of contrast agent clearance was obtained with dynamic contrast-enhanced (DCE) imaging following intravenous Gd-DOTA administration. Perfusion data were obtained with a bolus tracking technique comprising intravenous injection of superparamagnetic iron oxide (SPIO) nanoparticles. Cellular information was derived from anatomical images acquired 24 hours after SPIO. Treatment with the transforming growth factor-ß123 (TGF-ß1,2,3 ) antibody, 1D11, started 1 week after adriamycin. Histology was performed at week 6 post-adriamycin. RESULTS: Tracer washout rates derived by DCE-MRI decreased by 65.5% with respect to baseline at week 6 post-adriamycin. The impaired kidney function agreed with glomerulopathy, nephropathy and fibrosis revealed histologically (picrosirius collagen staining in adriamycin-treated rats increased by 125.8% [P = 0.005] with respect to controls). Perfusion was reduced by 16.1%. Images acquired 24 hours after SPIO presented contrast changes that correlated inversely with the histologically determined iron content (R = -0.74, P = 2.6 × 10(-4) ). In adriamycin-challenged animals, iron was found in macrophages and in sclerotic tubuli, only in areas where macrophages were present. Treatment with 1D11 did not improve the adriamycin-induced renal injury. CONCLUSION: MRI provides longitudinal functional and cellular (macrophage infiltration) information that correlates with nephropathy development in adriamycin-challenged rats.

Regulatory T cells and immune escape in HCC: understanding the tumor microenvironment and advancing CAR-T cell therapy.

Liver cancer, which most commonly manifests as hepatocellular carcinoma (HCC), is the sixth most common cancer in the world. In HCC, the immune system plays a crucial role in the growth and proliferation of tumor cells. HCC achieve immune escape through the tumor microenvironment, which significantly promotes the development of this cancer. Here, this article introduces and summarizes the functions and effects of regulatory T cells (Tregs) in the tumor microenvironment, highlighting how Tregs inhibit and regulate the functions of immune and tumor cells, cytokines, ligands and receptors, etc, thereby promoting tumor immune escape. In addition, it discusses the mechanism of CAR-T therapy for HCC and elaborate on the relationship between CAR-T and Tregs.

Regulatory T cells administration reduces anxiety-like behavior in mice submitted to chronic restraint stress.

Background: Major depression disorder (MDD) and anxiety are common mental disorders that significantly affect the quality of life of those who suffer from them, altering the person's normal functioning. From the biological perspective, the most classical hypothesis explaining their occurrence relies on neurotransmission and hippocampal excitability alterations. However, around 30% of MDD patients do not respond to medication targeting these processes. Over the last decade, the involvement of inflammatory responses in depression and anxiety pathogenesis has been strongly acknowledged, opening the possibility of tackling these disorders from an immunological point of view. In this context, regulatory T cells (Treg cells), which naturally maintain immune homeostasis by suppressing inflammation could be promising candidates for their therapeutic use in mental disorders. Methods: To test this hypothesis, C57BL/6 adult male mice were submitted to classical stress protocols to induce depressive and anxiety-like behavior; chronic restriction stress (CRS), and chronic unpredictable stress (CUS). Some of the stressed mice received a single adoptive transfer of Treg cells during stress protocols. Mouse behavior was analyzed through the open field (OFT) and forced swim test (FST). Blood and spleen samples were collected for T cell analysis using cell cytometry, while brains were collected to study changes in microglia by immunohistochemistry. Results: Mice submitted to CRS and CUS develop anxiety and depressive-like behavior, and only CRS mice exhibit lower frequencies of circulating Treg cells. Adoptive transfer of Treg cells decreased anxiety-like behavior in the OFT only in CRS model, but not depressive behavior in FST in neither of the two models. In CRS mice, Treg cells administration lowered the number of microglia in the hippocampus, which increased due this stress paradigm, and restored its arborization. However, in CUS mice, Treg cells administration increased microglia number with no significant effect on their arborization. Conclusion: Our results for effector CD4+ T cells in the spleen and microglia number and morphology in the hippocampus add new evidence in favor of the participation of inflammatory responses in the development of depressive and anxiety-like behavior and suggest that the modulation of key immune cells such as Treg cells, could have beneficial effects on these disorders.

atRA Attenuates High Salt-Driven EAE Mainly Through Suppressing Th17-Like Regulatory T Cell Response Mediated by the Inhibition of IL-23R Signaling Pathway.

High salt diet (HSD) is implicated in numerous disorders, which boosts Th17 cell development and weakens immunosuppressive function of regulatory T cells (Treg cells) Treg cells, leading to the exacerbation of EAE. However, little is known regarding the harness of excessive proinflammatory responses evoked by HSD. Here we show that atRA, a key vitamin A metabolite with multifaceted immunoregulatory properties has the potential in inhibiting the proinflammatory reaction of high salt. Treatment with atRA in vivo elicited the Treg generation in cervical and axillary lymph nodes (CALs), and in CNS of experimental autoimmune encephalomyelitis (EAE). Meanwhile, the proportion of Th17-like Treg cells (RORγt-positive or GM-CSF-positive Treg cells) decreased in CALs. atRA also inhibited IL-17A expression in CD4+ effector T cells. In-vitro mechanistic studies showed that atRA inhibit IL-23R but not SGK1 expression in Treg cells and this results in maintained immunosuppressive function of Treg cells even in the presence of IL-6 and high salt. Furthermore, treatment of EAE with anti-IL-23R mAb attenuated HSD-provoked EAE progress. This was associated with a reduction in the number of CNS-infiltrating Th17 cells and an increase of CAL-Treg cells. Mechanically, treatment with atRA significantly promoted LP-CD103+CD11c+ dendritic cells, a subgroup of cells most closely involved in endogenous retinoic acid metabolism, and enhanced intestinal Aldh1a1 and Rdh10 expression from HSD-fed EAE mice. Interestingly, anti-IL-23R mAb administration also reduced IL-23R expression in Treg cells, along with the increased proportion of LP-CD103+CD11c+ dendritic cells and Rdh10 mRNA expression. In conclusion, administration of atRA might be a way to combat the proinflammatory effects of HSD. Meanwhile, systematic inhibition of IL-23R also had a moderate therapeutic potential in inhibiting inflammatory effects of high salt, which may serve as a basis for the identification of novel therapeutic strategies against HSD-driven autoimmune disorders.

Advancing autoimmune Rheumatic disease treatment: CAR-T Cell Therapies - Evidence, Safety, and future directions.

INTRODUCTION: Despite advancements in managing autoimmune rheumatic diseases (ARDs) with existing treatments, many patients still encounter challenges such as inadequate responses, difficulty in maintaining remission, and side effects. Chimeric Antigen Receptor (CAR) T-cell therapy, originally developed for cancer, has now emerged as a promising option for cases of refractory ARDs. METHODS: A search of the literature was conducted to compose a narrative review exploring the current evidence, potential safety, limitations, potential modifications, and future directions of CAR-T cells in ARDs. RESULTS: CAR-T cell therapy has been administered to patients with refractory ARDs, including systemic lupus erythematosus, antisynthetase syndrome, and systemic sclerosis, demonstrating significant improvement. Notable responses include enhanced clinical symptoms, reduced serum autoantibody titers, and sustained remissions in disease activity. Preclinical and in vitro studies using both animal and human samples also support the efficacy and elaborate on potential mechanisms of CAR-T cells against antineutrophil cytoplasmic antibody-associated vasculitis and rheumatoid arthritis. While cautious monitoring of adverse events, such as cytokine release syndrome, is crucial, the therapy appears to be highly tolerable. Nevertheless, challenges persist, including cost, durability due to potential CAR-T cell exhaustion, and manufacturing complexities, urging the development of innovative solutions to further enhance CAR-T cell therapy accessibility in ARDs. CONCLUSIONS: CAR-T cell therapy for refractory ARDs has demonstrated high effectiveness. While no significant warning signs are currently reported, achieving a balance between therapeutic efficacy and safety is vital in adapting CAR-T cell therapy for ARDs. Moreover, there is significant potential for technological advancements to enhance the delivery of this treatment to patients, thereby ensuring safer and more effective disease control for patients.