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Objective To evaluate the prognosis and safety of patients with advanced pancreatic ductal adenocarcinoma (PDAC) who received I-125 seed implantation in treatment with anti-PD-1 monoclonal antibody+chemotherapy. Methods A retrospective analysis was conducted on patients with stage Ⅳ metastatic PDAC who received anti-PD-1 combined chemotherapy treatment at Yixing Hospital, Jiangsu University from Jan 2021 to Jun 2023. Patients were divided into two groups based on whether they received I-125 seed implantation: the I-125 seed implantation+anti-PD-1 monoclonal antibody+ Chemotherapy group (IPC group) and the anti-PD-1 monoclonal antibody+chemotherapy group (PC group）. The follow-up period ranged from 2 to 24 months, with a median follow-up time of 9 months. The prognosis of patients was analysed in combination with peripheral blood biomarkers. The peripheral lymphocyte subsets of patients in different treatment groups were preliminarily analysed by flow cytometry. Results A total of 13 patients were included, with 5 in the IPC group and 8 in the PC group. Progression-free survival (PFS) and overall survival (OS) in the IPC group were significantly longer than those in the PC group. The treatment in the IPC group was relatively safe, adverse reactions were controllable. The neutrophil-lymphocyte ratio (NLR) and CD4/CD8 ratio indicated that the prognosis of the IPC patients was better. The levels of regulatory T cells (Treg) and active regulatory T cells (aTreg) cells in the IPC patients were reduced after treatment compared with those of the PC patients. Conclusion The addition of I-125 seed implantation can improve the prognosis of patients with advanced PDAC who receive anti-PD-1 monoclonal antibody+chemotherapy, the post-treatment levels of patients’ circulating aTreg cells are reduced, and the combination therapy has good safety. [ABSTRACT FROM AUTHOR]

Abstract Background Postmenopausal osteoporosis (PMO) results from a reduction in bone mass and microarchitectural deterioration in bone tissue due to estrogen deficiency, which may increase the incidence of fragility fractures. In recent years, the “gut-immune response-bone” axis has been proposed as a novel potential approach in the prevention and treatment of PMO. Studies on ovariectomized murine model indicated the reciprocal role of Th17 cells and Treg cells in the aetiology of osteoporosis. However, the relationship among gut microbiota, immune cells and bone metabolic indexes remains unknown in PMO. Methods A total of 77 postmenopausal women were recruited for the study and divided into control (n = 30), osteopenia (n = 19), and osteoporosis (n = 28) groups based on their T score. The frequency of Treg and Th17 cells in lymphocytes were analyzed by flow cytometry. The serum levels of interleukin (IL)-10, 17 A, 1β, 6, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) were determined via enzyme-linked immunosorbent assay. Additionally, 16S rRNA gene V3-V4 region sequencing analysis was performed to investigate the gut microbiota of the participants. Results The results demonstrated decreased bacterial richness and diversed intestinal composition in PMO. In addition, significant differences of relative abundance of the gut microbial community in phylum and genus levels were found, mainly including increased Bacteroidota, Proteobacteria, and Campylobacterota, as well as reduced Firmicutes, Butyricicoccus, and Faecalibacterium. Intriugingly, in the osteoporosis group, the concentration of Treg cells and associated IL-10 in peripheral circulation was negatively regulated, while other chronic systemic proinflammatory cytokines and Th17 cells showed opposite trends. Moreover, significantly elevated plasma lipopolysaccharide (LPS) in patients with osteoporosis indicated that disrupted intestinal integrity and permeability. A correlation analysis showed close relationships between gut bacteria and inflammation. Conclusions Collectively, these observations will lead to a better understanding of the relationship among bone homeostasis, the microbiota, and circulating immune cells in PMO. The elevated LPS levels of osteoporosis patients which not only indicate a breach in intestinal integrity but also suggest a novel biomarker for assessing osteoporosis risk linked to gut health.

Immune checkpoint blockade (ICB) therapeutics are highly effective in cancer immunotherapy, but gastrointestinal toxicity limited the application. Intestinal microbiota plays a crucial role in ICB-associated colitis. 2’-Fucosyllactose (2’FL) is most abundance prebiotic in human milk that can reshape gut microbiota and exert immune regulatory effect. The study aimed to determine the effects of 2’FL on ICB-associated colitis and to uncover the mediating mechanism. ICB-associated colitis was induced by the ipilimumab and dextran sulfate sodium. Oral administration of 2’FL (0.6 g/(kg’day)) ameliorated ICB-induced colitis by enhancing regulatory T cells (Treg) and the M2/M1 ratio of macrophages in colon. 2’FL treatment also increased the expression of tight junction proteins (zonula occludens-1 (ZO-1) and mucin 2 (MUC2)) and antioxidant stress indicators (superoxide dismutase (SOD) and catalase (CAT)). In addition, administration of 2’FL increased the abundance of Bifidobacterium and Lactobacillus, and elevated the levels of microbial metabolites, such as indole-3-lactic acid (ILA), which activated the aryl hydrocarbon receptor ligands (AHR) pathway. The protective effect of 2’FL was abolished upon depletion of gut microbiota, and ILA treatment partially simulated the protective effect of 2’FL. Notably, 2’FL did not exhibit inhibition of antitumor immunity. These f indings suggest that 2’FL could serve as a potential protective strategy for ICB-associated colitis by modulating the intestinal microbiota and bacterial metabolites.

Background: Postmenopausal osteoporosis (PMO) results from a reduction in bone mass and microarchitectural deterioration in bone tissue due to estrogen deficiency, which may increase the incidence of fragility fractures. In recent years, the "gut-immune response-bone" axis has been proposed as a novel potential approach in the prevention and treatment of PMO. Studies on ovariectomized murine model indicated the reciprocal role of Th17 cells and Treg cells in the aetiology of osteoporosis. However, the relationship among gut microbiota, immune cells and bone metabolic indexes remains unknown in PMO. Methods: A total of 77 postmenopausal women were recruited for the study and divided into control (n = 30), osteopenia (n = 19), and osteoporosis (n = 28) groups based on their T score. The frequency of Treg and Th17 cells in lymphocytes were analyzed by flow cytometry. The serum levels of interleukin (IL)-10, 17 A, 1β, 6, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) were determined via enzyme-linked immunosorbent assay. Additionally, 16S rRNA gene V3-V4 region sequencing analysis was performed to investigate the gut microbiota of the participants. Results: The results demonstrated decreased bacterial richness and diversed intestinal composition in PMO. In addition, significant differences of relative abundance of the gut microbial community in phylum and genus levels were found, mainly including increased Bacteroidota, Proteobacteria, and Campylobacterota, as well as reduced Firmicutes, Butyricicoccus, and Faecalibacterium. Intriugingly, in the osteoporosis group, the concentration of Treg cells and associated IL-10 in peripheral circulation was negatively regulated, while other chronic systemic proinflammatory cytokines and Th17 cells showed opposite trends. Moreover, significantly elevated plasma lipopolysaccharide (LPS) in patients with osteoporosis indicated that disrupted intestinal integrity and permeability. A correlation analysis showed close relationships between gut bacteria and inflammation. Conclusions: Collectively, these observations will lead to a better understanding of the relationship among bone homeostasis, the microbiota, and circulating immune cells in PMO. The elevated LPS levels of osteoporosis patients which not only indicate a breach in intestinal integrity but also suggest a novel biomarker for assessing osteoporosis risk linked to gut health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. Methods In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. Results The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the “healthy” phenotype of neutrophils. Conclusions Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Replacement of diseased organs with transplanted healthy donor ones remains the best and often only treatment option for end-stage organ disease. Immunosuppressants have decreased the incidence of acute rejection, but long-term survival remains limited. The broad action of current immunosuppressive drugs results in global immune impairment, increasing the risk of cancer and infections. Hence, achievement of allograft tolerance, in which graft function is maintained in the absence of global immunosuppression, has long been the aim of transplant clinicians and scientists. Regulatory T cells (Treg) are a specialized subset of immune cells that control a diverse array of immune responses, can prevent allograft rejection in animals, and have recently been explored in early phase clinical trials as an adoptive cellular therapy in transplant recipients. It has been established that allograft residency by Tregs can promote graft acceptance, but whether intragraft Treg functional diversification and spatial organization contribute to this process is largely unknown. In this review, we will explore what is known regarding the properties of intragraft Tregs during allograft acceptance and rejection. We will summarize recent advances in understanding Treg tissue residency through spatial, transcriptomic and high-dimensional cytometric methods in both animal and human studies. Our discussion will explore properties of intragraft Tregs in mediating operational tolerance to commonly transplanted solid organs. Finally, given recent developments in Treg cellular therapy, we will review emerging knowledge of whether and how these adoptively transferred cells enter allografts in humans. An understanding of the properties of intragraft Tregs will help lay the foundation for future therapies that will promote immune tolerance. [ABSTRACT FROM AUTHOR]

We enrolled healthy subjects that received 2 to 4 injections of mRNA-based vaccination to prevent COVID-19 months to a year from the last vaccine boost, and we found numerous SARS-CoV-2 spike-specific regulatory T cell (Treg) that developed T cell memory as effector memory T cells (TEM) and central memory T cells (TCM). CD4+ CD25high Treg expressed the chemokine receptor CCR6 in a considerable percentage, suggesting T cell homing to the vascular endothelium, lung and gut epithelial cells and brain. Treg phenotype was different than peripherally-induced Treg (pTreg) that revert from pro-inflammatory T cells under repeated stimulatory conditions, suggesting that SARS-CoV-2 spike-specific Treg differentiated from naïve T cells in tissues where the SARS-CoV-2 spike proteins were synthetized. Twenty two of 22 subjects studied responded to vaccination developing a spike-specific CD4+ T helper (Th)1 response, and 20 of 22 developing a spike-specific CD8+ cytotoxic T cells (CTL) response. However, in vaccine recipients the expansion of spike-specific pro-inflammatory T cells was less significant than the expansion of spike-specific Treg. Effector (TEM) and central memory (TCM) Treg were numerous as early as after two vaccine doses, with no significant differences following additional vaccine boosts. In co-culture experiments under stimulatory conditions, Treg regulated naïve T cell differentiation toward a pro-inflammatory phenotype and suppressed interferon (IFN)γ production by SARS-CoV-2-specific CD4 + Th1 cells.

Regulatory T cells (Treg) are known to be critical for the maintenance of immune homeostasis by suppressing the activation of auto-or allo-reactive effector T cells through a diverse repertoire of molecular mechanisms. Accordingly, therapeutic strategies aimed at enhancing Treg numbers or potency in the setting of autoimmunity and allogeneic transplants have been energetically pursued and are beginning to yield some encouraging outcomes in early phase clinical trials. Less well recognized from a translational perspective, however, has been the mounting body of evidence that Treg directly modulate most aspects of innate immune response under a range of different acute and chronic disease conditions. Recognizing this aspect of Treg immune modulatory function provides a bridge for the application of Treg-based therapies to common medical conditions in which organ and tissue damage is mediated primarily by inflammation involving myeloid cells (mononuclear phagocytes, granulocytes) and innate lymphocytes (NK cells, NKT cells, gd T cells and ILCs). In this review, we comprehensively summarize pre-clinical and human research that has revealed diverse modulatory effects of Treg and specific Treg subpopulations on the range of innate immune cell types. In each case, we emphasize the key mechanistic insights and the evidence that Treg interactions with innate immune effectors can have significant impacts on disease severity or treatment. Finally, we discuss the opportunities and challenges that exist for the application of Treg-based therapeutic interventions to three globally impactful, inflammatory conditions: type 2 diabetes and its end-organ complications, ischemia reperfusion injury and atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. Methods: In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. Results: The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the "healthy" phenotype of neutrophils. Conclusions: Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response. [ABSTRACT FROM AUTHOR]

Replacement of diseased organs with transplanted healthy donor ones remains the best and often only treatment option for end-stage organ disease. Immunosuppressants have decreased the incidence of acute rejection, but long-term survival remains limited. The broad action of current immunosuppressive drugs results in global immune impairment, increasing the risk of cancer and infections. Hence, achievement of allograft tolerance, in which graft function is maintained in the absence of global immunosuppression, has long been the aim of transplant clinicians and scientists. Regulatory T cells (Treg) are a specialized subset of immune cells that control a diverse array of immune responses, can prevent allograft rejection in animals, and have recently been explored in early phase clinical trials as an adoptive cellular therapy in transplant recipients. It has been established that allograft residency by Tregs can promote graft acceptance, but whether intragraft Treg functional diversification and spatial organization contribute to this process is largely unknown. In this review, we will explore what is known regarding the properties of intragraft Tregs during allograft acceptance and rejection. We will summarize recent advances in understanding Treg tissue residency through spatial, transcriptomic and high-dimensional cytometric methods in both animal and human studies. Our discussion will explore properties of intragraft Tregs in mediating operational tolerance to commonly transplanted solid organs. Finally, given recent developments in Treg cellular therapy, we will review emerging knowledge of whether and how these adoptively transferred cells enter allografts in humans. An understanding of the properties of intragraft Tregs will help lay the foundation for future therapies that will promote immune tolerance.

Simple Summary: Immune dysfunction is a major feature of chronic spinal cord injuries (SCIs). It is associated with many of the complications observed in these patients, such as increased vulnerability to infections and other medical challenges. The role of regulatory T cells (Tregs) after SCI is beginning to be more acutely understood; however, little is known about their implications at chronic stages. By using flow cytometry, we characterized circulating Tregs from chronic SCI patients according to the time of initial injury (1–5 years; 5–15 years; and >15 years). Our results demonstrate significant changes in the immunological phenotypes of these cells, especially in patients with long periods of evolution (5–15 years and >15 years with chronic SCI). Despite the fact that a deeper understanding of the immune dysfunction caused by chronic SCI is still required, the characterization of circulating leukocytes such as Tregs and other populations can open up the possibility of finding translational biomarkers or therapeutic approaches that may aid in the clinical management of chronic SCI. Spinal cord injury (SCI) is a progressive and complex neurological disorder accompanied by multiple systemic challenges. Peripheral immune dysfunction is a major event occurring after SCI, especially in its chronic phase. Previous works have demonstrated significant changes in different circulating immune compartments, including in T cells. However, the precise characterization of these cells remains to be fully unraveled, particularly when considering important variants such as the time since the initial injury. In the present work, we aimed to study the level of circulating regulatory T cells (Tregs) in SCI patients depending on the duration of evolution. For this purpose, we studied and characterized peripheral Tregs from 105 patients with chronic SCI using flow cytometry, with patients classified into three major groups depending on the time since initial injury: short period chronic (SCI-SP, <5 years since initial injury); early chronic (SCI-ECP, from 5–15 years post-injury) and late chronic SCI (SCI-LCP, more than 15 years post-injury. Our results show that both the SCI-ECP and SCI-LCP groups appeared to present increased proportions of CD4+ CD25+/low Foxp3+ Tregs in comparison to healthy subjects, whereas a decreased number of these cells expressing CCR5 was observed in SCI-SP, SCI-ECP, and SCI-LCP patients. Furthermore, an increased number of CD4+ CD25+/high/low Foxp3 with negative expression of CD45RA and CCR7 was observed in SCI-LCP patients when compared to the SCI-ECP group. Taken together, these results deepen our understanding of the immune dysfunction reported in chronic SCI patients and how the time since initial injury may drive this dysregulation. [ABSTRACT FROM AUTHOR]

We enrolled healthy subjects that received 2 to 4 injections of mRNA-based vaccination to prevent COVID-19 months to a year from the last vaccine boost, and we found numerous SARS-CoV-2 spike-specific regulatory T cell (Treg) that developed T cell memory as effector memory T cells (TEM) and central memory T cells (TCM). CD4+ CD25high Treg expressed the chemokine receptor CCR6 in a considerable percentage, suggesting T cell homing to the vascular endothelium, lung and gut epithelial cells and brain. Treg phenotype was different than peripherally-induced Treg (pTreg) that revert from pro-inflammatory T cells under repeated stimulatory conditions, suggesting that SARS-CoV-2 spike-specific Treg differentiated from naïve T cells in tissues where the SARS-CoV-2 spike proteins were synthetized. Twenty two of 22 subjects studied responded to vaccination developing a spike-specific CD4+ T helper (Th)1 response, and 20 of 22 developing a spike-specific CD8+ cytotoxic T cells (CTL) response. However, in vaccine recipients the expansion of spike-specific pro-inflammatory T cells was less significant than the expansion of spike-specific Treg. Effector (TEM) and central memory (TCM) Treg were numerous as early as after two vaccine doses, with no significant differences following additional vaccine boosts. In co-culture experiments under stimulatory conditions, Treg regulated naïve T cell differentiation toward a pro-inflammatory phenotype and suppressed interferon (IFN)γ production by SARS-CoV-2-specific CD4 + Th1 cells. [ABSTRACT FROM AUTHOR]

BackgroundRegulatory T cells (Tregs) play an important role in the maintenance of immune homeostasis and the establishment of immune tolerance. Since Tregs do not secrete endogenous IL-2, they are especially dependent on external IL-2. IL-2 deficiency leads to lower Treg numbers, instability of the Treg phenotype and loss of immune regulation. After organ transplantation, patients are treated with calcineurin inhibitors (CNIs), which further limits available IL-2. Application of low-dose IL-2 expands Tregs but also activates NK and CD8+ T cells. It was recently shown that graft-specific Tregs recognizing mismatched MHC I molecules via a chimeric antigen receptor were far more potent than polyclonal Tregs in the regulation of immune responses after solid organ transplantation in a humanized mouse model.MethodsTherefore, our aim was to enhance the function and stability of transferred CAR-Tregs via expression of membrane-associated IL-2 (mbIL-2).ResultsmbIL-2 promoted higher survival, phenotypic stability, and function among CAR-Tregs than observed in clinical trials. The cells were also more stable under inflammatory conditions. In a preclinical humanized mouse model, we demonstrated that mbIL-2 CAR Tregs survive better in the Treg niche than control CAR Tregs and are even resistant to CNI therapy without affecting other Tregs, thus acting mainly in cis.DiscussionThe functional and phenotypic improvements observed after membrane-attached IL-2 expression in CAR-Tregs will be important step for enhancing CAR-Treg therapies currently being tested in clinical trials for use after kidney and liver transplantation as well as in autoimmune diseases.

CD4+ regulatory T cells (Tregs) respond to environmental cues to permit or suppress inflammation, and atherosclerosis weakens Treg suppression and promotes plasticity. However, the effects of smoking plus morphine (SM + M) on Treg plasticity remain unknown. To determine whether SM + M promotes Treg plasticity to T helper 17 (Th17) cells, we analyzed the RNA sequencing data from SM, M, and SM + M treated Tregs and performed knowledge-based and IPA analysis. We demonstrated that (1) SM + M, M, and SM upregulated the transcripts of cytokines, chemokines, and clusters of differentiation (CDs) and modulated the transcripts of kinases and phosphatases in Tregs; (2) SM + M, M, and SM upregulated the transcripts of immunometabolism genes, trained immunity genes, and histone modification enzymes; (3) SM + M increased the transcripts of Th17 transcription factor (TF) RORC and Tfh factor CXCR5 in Tregs; M increased the transcripts of T helper cell 1 (Th1) TF RUNX3 and Th1-Th9 receptor CXCR3; and SM inhibited Treg TGIF1 transcript; (4) six genes upregulated in SM + M Tregs were matched with the top-ranked Th17 pathogenic genes; and 57, 39 genes upregulated in SM + M Tregs were matched with groups II and group III Th17 pathogenic genes, respectively; (5) SM + M upregulated the transcripts of 70 IPA-TFs, 11 iTregs-specific TFs, and 4 iTregs-Th17 shared TFs; and (6) SM + M, M, and SM downregulated Treg suppression TF Rel (c-Rel); and 35 SM + M downregulated genes were overlapped with Rel−/− Treg downregulated genes. These results provide novel insights on the roles of SM + M in reprogramming Treg transcriptomes and Treg plasticity to Th17 cells and novel targets for future therapeutic interventions involving immunosuppression in atherosclerotic cardiovascular diseases, autoimmune diseases, transplantation, and cancers. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic cancer is one of the most lethal cancer types and its high refractoriness to therapies, including immunotherapy, has often been associated with the predominantly immune suppressive tumor microenvironment that characterizes pancreatic tumors. Regulatory T cells (Tregs) are generally considered as drivers of immune suppression in cancers. However, an increasing number of reports suggest a paradoxical association between tumor infiltration by Tregs and improved patient prognosis, in particular in gastrointestinal cancers. Here we show that Treg infiltration in pancreatic ductal adenocarcinomas (PDAC) is associated with better overall survival of patients. Pancreatic ductal adenocarcinoma (PDAC) is considered to be a poorly immunogenic cancer type that combines a low mutation burden with a strong immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are major drivers of immune suppression but their prognostic role, particularly in gastrointestinal malignancies, remains controversial. Lymphocytic infiltration in 122 PDAC samples was assessed by multispectral immunofluorescence with anti-Keratin, -CD3, -CD8, -FOXP3 and -CD163 antibodies. Differential infiltration by Tregs was analyzed in the context of transcriptomic profiles that were available for 65 tumors. High infiltration of CD3+CD8− (mainly CD4+) T cells and, especially, of the subset expressing FOXP3 (Tregs) was associated with improved patient survival, whilst cytotoxic CD3+CD8+ T cell infiltration did not have an impact on overall survival. Transcriptomic analysis revealed three signatures in PDAC tumors comprising of epithelial-mesenchymal transition (EMT)/stromal, metabolic, and secretory/pancreatic signature. However, none of these signatures explained differences in Treg infiltration. We show that Tregs associate with improved overall survival in PDAC patients. This effect was independent of cytotoxic T cell infiltration and the transcriptomic profiles of their respective tumors. These findings provide a new layer of complexity in the study of PDAC tumor microenvironment that must be considered when developing immunotherapeutic interventions for this disease. [ABSTRACT FROM AUTHOR]

Refractory or recurrent immune thrombocytopenia (ITP) patients suffer from the dual threat of high mortality and drug toxicity in addition to a very poor quality of life. Previous studies have shown that high mobility group box 1 (HMGB1) can promote the development of rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases. However, there is still a lack of research on the role of HMGB1 in the pathogenesis of ITP and whether it can be used as a predictor of efficacy and prognosis. 20 patients of adult ITP with splenectomy were chosen as the experimental group, while 19 adults underwent splenectomy for traumatic splenic rupture without other diseases as the control group. We measured the expression of HMGB1, RORγt and Foxp3 in spleen tissues by immunohistochemistry. Another 50 patients, of which 20 were newly diagnosed without treatment and 30 were refractory ITP, and 25 healthy controls were enrolled to analyse the expression levels and mRNA levels of HMGB1, RORγt and Foxp3 in peripheral blood by Western blot and RT-qPCR. The expression of HMGB1, IL-17 and IL-10 in serum was assayed by ELISA. PBMCs from newly diagnosed ITP patients were cultured in vitro which stimulated with recombinant humanHMGB1 (rHMGB1) and its inhibitors, in which the expressions of RORγt and Foxp3 were measured. The expression of HMGB1 in the spleen with refractory ITP was significantly higher, while Foxp3 was decreased. A significant negative correlation was found between HMGB1 and Foxp3, and the overexpression of HMGB1 was significantly correlated with poor efficacy after splenectomy. The expression of HMGB1 and IL-17 increased and showed a positive correlation in serum, while IL-10 decreased and was negatively correlated with HMGB1. In PBMCs, the expression of HMGB1 and RORγt increased, while Foxp3 decreased, and the differences were more obvious in the refractory chronic ITP group. In a coculture system with PBMCs of untreated ITP patients, rHMGB1 increased RORγt expression and decreased Foxp3 expression, while an antiHMGB1 antibody partially corrected the above changes. Our results suggest that HMGB1 is associated with the imbalance of Treg/Th17 cells and is involved in the pathogenesis of ITP. [ABSTRACT FROM AUTHOR]

Abstract Background Long non-coding RNAs (lncRNAs) are extensively intricate in the tumorigenesis and metastasis of various cancer types. Nevertheless, the detailed molecular mechanisms of lncRNA in non-small cell lung cancer (NSCLC) still remain mainly undetermined. Methods qPCR was performed to verify LINC00301 expression in NSCLC clinical specimens or cell lines. Fluorescence in situ hybridization (FISH) was conducted to identify the localization of LINC00301 in NSCLC cells. Chromatin immunoprecipitation (ChIP) was subjected to validate the binding activity between FOXC1 and LINC00301 promoters. RNA immunoprecipitation (RIP) was performed to explore the binding activity between LINC00301 and EZH2. RNA pull-down followed by dot-blot, protein domain mapping, and RNA electrophoresis mobility shift assay (EMSA) were conducted to identify the detailed binding regions between LINC00301 and EZH2. Alpha assay was conducted to quantitatively assess the interaction between LINC00301 and EZH2. Results LINC00301 is highly expressed in NSCLC and closely corelated to its prognosis by analyzing the relationship between differentially expressed lncRNAs and prognosis in NSCLC samples. in vitro and in vivo experiments revealed that LINC00301 facilitates cell proliferation, releases NSCLC cell cycle arrest, promotes cell migration and invasion, and suppresses cell apoptosis in NSCLC. In addition, LINC00301 increases regulatory T cell (Treg) while decreases CD8+ T cell population in LA-4/SLN-205-derived tumors through targeting TGF-β. The transcription factor FOXC1 mediates LINC00301 expression in NSCLC. Bioinformatics prediction and in vitro experiments indicated that LINC00301 (83–123 nucleotide [nt]) can directly bind to the enhancer of zeste homolog 2 (EZH2) (612–727 amino acid [aa]) to promote H3K27me3 at the ELL protein-associated factor 2 (EAF2) promoter. EAF2 directly binds and stabilizes von Hippel–Lindau protein (pVHL), so downregulated EAF2 augments hypoxia-inducible factor 1 α (HIF1α) expression by regulating pVHL in NSCLC cells. Moreover, we also found that LINC00301 could function as a competing endogenous RNA (ceRNA) against miR-1276 to expedite HIF1α expression in the cytoplasm of NSCLC cells. Conclusions In summary, our present research revealed the oncogenic roles of LINC00301 in clinical specimens as well as cellular and animal experiments, illustrating the potential roles and mechanisms of the FOXC1/LINC00301/EZH2/EAF2/pVHL/HIF1α and FOXC1/LINC00301/miR-1276/HIF1α pathways, which provides novel insights and potential theraputic targets to NSCLC.

Summary: Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth. We also show that T-reg-cell-specific inducible deletion of Tim-3 enhances anti-tumor immunity. Enhancement of Treg cell function by Tim-3 is strongly correlated with increased expression of interleukin-10 (IL-10) and a shift to a more glycolytic metabolic phenotype. Our data demonstrate that Tim-3+ Treg cells may be a relevant therapeutic target cell type for the treatment of cancer.

Gut-associated lymphoid tissue (GALT) is crucial for the maintenance of the intestinal homeostasis, but it is also the potential site of the activation of autoreactive cells and initiation/propagation of autoimmune diseases in the gut and in the distant organs. Type 3 innate lymphoid cells (ILC3) residing in the GALT integrate signals from food ingredients and gut microbiota metabolites in order to control local immunoreactivity. Notably, ILC3 secrete IL-17 and GM-CSF that activate immune cells in combating potentially pathogenic microorganisms. ILC3 also produce IL-22 that potentiates the strength and integrity of epithelial tight junctions, production of mucus and antimicrobial peptides thus enabling the proper function of the intestinal barrier. The newly discovered function of small intestine ILC3 is the secretion of IL-2 and the promotion of regulatory T cell (Treg) generation and function. Since the intestinal barrier dysfunction, together with the reduction in small intestine ILC3 and Treg numbers are associated with the pathogenesis of type 1 diabetes (T1D), the focus of this article is intestinal ILC3 modulation for the therapy of T1D. Of particular interest is free fatty acids receptor 2 (FFAR2), predominantly expressed on intestinal ILC3, that can be stimulated by available selective synthetic agonists. Thus, we propose that FFAR2-based interventions by boosting ILC3 beneficial functions may attenuate autoimmune response against pancreatic β cells during T1D. Also, it is our opinion that treatments based on ILC3 stimulation by functional foods can be used as prophylaxis in individuals that are genetically predisposed to develop T1D.

Regulatory T cells (Tregs) are key immunosuppressive cells that promote tumor growth by hindering the effector immune response. Tregs utilize multiple suppressive mechanisms to inhibit pro-inflammatory responses within the tumor microenvironment (TME) by inhibition of effector function and immune cell migration, secretion of inhibitory cytokines, metabolic disruption and promotion of metastasis. In turn, Tregs are being targeted in the clinic either alone or in combination with other immunotherapies, in efforts to overcome the immunosuppressive TME and increase anti-tumor effects. However, it is now appreciated that Tregs not only suppress cells intratumorally via direct engagement, but also serve as key interactors in the peritumor, stroma, vasculature and lymphatics to limit anti-tumor immune responses prior to tumor infiltration. We will review the suppressive mechanisms that Tregs utilize to alter immune and non-immune cells outside and within the TME and discuss how these mechanisms collectively allow Tregs to create and promote a physical and biological barrier, resulting in an immune-excluded or limited tumor microenvironment.

Regulatory T cells (Tregs) are key immunosuppressive cells that promote tumor growth by hindering the effector immune response. Tregs utilize multiple suppressive mechanisms to inhibit pro-inflammatory responses within the tumor microenvironment (TME) by inhibition of effector function and immune cell migration, secretion of inhibitory cytokines, metabolic disruption and promotion of metastasis. In turn, Tregs are being targeted in the clinic either alone or in combination with other immunotherapies, in efforts to overcome the immunosuppressive TME and increase anti-tumor effects. However, it is now appreciated that Tregs not only suppress cells intratumorally via direct engagement, but also serve as key interactors in the peritumor, stroma, vasculature and lymphatics to limit anti-tumor immune responses prior to tumor infiltration. We will review the suppressive mechanisms that Tregs utilize to alter immune and non-immune cells outside and within the TME and discuss how these mechanisms collectively allow Tregs to create and promote a physical and biological barrier, resulting in an immune-excluded or limited tumor microenvironment. [ABSTRACT FROM AUTHOR]

Gut-associated lymphoid tissue (GALT) is crucial for the maintenance of the intestinal homeostasis, but it is also the potential site of the activation of autoreactive cells and initiation/propagation of autoimmune diseases in the gut and in the distant organs. Type 3 innate lymphoid cells (ILC3) residing in the GALT integrate signals from food ingredients and gut microbiota metabolites in order to control local immunoreactivity. Notably, ILC3 secrete IL-17 and GM-CSF that activate immune cells in combating potentially pathogenic microorganisms. ILC3 also produce IL-22 that potentiates the strength and integrity of epithelial tight junctions, production of mucus and antimicrobial peptides thus enabling the proper function of the intestinal barrier. The newly discovered function of small intestine ILC3 is the secretion of IL-2 and the promotion of regulatory T cell (Treg) generation and function. Since the intestinal barrier dysfunction, together with the reduction in small intestine ILC3 and Treg numbers are associated with the pathogenesis of type 1 diabetes (T1D), the focus of this article is intestinal ILC3 modulation for the therapy of T1D. Of particular interest is free fatty acids receptor 2 (FFAR2), predominantly expressed on intestinal ILC3, that can be stimulated by available selective synthetic agonists. Thus, we propose that FFAR2-based interventions by boosting ILC3 beneficial functions may attenuate autoimmune response against pancreatic β cells during T1D. Also, it is our opinion that treatments based on ILC3 stimulation by functional foods can be used as prophylaxis in individuals that are genetically predisposed to develop T1D. [ABSTRACT FROM AUTHOR]

Persistent liver inflammation can lead to cirrhosis, which associates with significant morbidity and mortality worldwide. There are no curative treatments beyond transplantation, followed by long-term immunosuppression. The global burden of end stage liver disease has been increasing and there is a shortage of donor organs, therefore new therapies are desperately needed. Harnessing the power of the immune system has shown promise in certain autoimmunity and cancer settings. In the context of the liver, regulatory T cell (Treg) therapies are in development. The hypothesis is that these specialized lymphocytes that dampen inflammation may reduce liver injury in patients with chronic, progressive diseases, and promote transplant tolerance. Various strategies including intrinsic and extracorporeal expansion of Treg cells, aim to increase their abundance to suppress immune responses. We recently discovered that hepatocytes engulf and delete Treg cells by enclysis. Herein, we propose that inhibition of enclysis may potentiate existing regulatory T cell therapeutic approaches in patients with autoimmune liver diseases and in patients receiving a transplant. Moreover, in settings where the abundance of Treg cells could hinder beneficial immunity, such us in chronic viral infection or liver cancer, enhancement of enclysis could result in transient, localized reduction of Treg cell numbers and tip the balance towards antiviral and anti-tumor immunity. We describe enclysis as is a natural process of liver immune regulation that lends itself to therapeutic targeting, particularly in combination with current Treg cell approaches.

Persistent liver inflammation can lead to cirrhosis, which associates with significant morbidity and mortality worldwide. There are no curative treatments beyond transplantation, followed by long-term immunosuppression. The global burden of end stage liver disease has been increasing and there is a shortage of donor organs, therefore new therapies are desperately needed. Harnessing the power of the immune system has shown promise in certain autoimmunity and cancer settings. In the context of the liver, regulatory T cell (Treg) therapies are in development. The hypothesis is that these specialized lymphocytes that dampen inflammation may reduce liver injury in patients with chronic, progressive diseases, and promote transplant tolerance. Various strategies including intrinsic and extracorporeal expansion of Treg cells, aim to increase their abundance to suppress immune responses. We recently discovered that hepatocytes engulf and delete Treg cells by enclysis. Herein, we propose that inhibition of enclysis may potentiate existing regulatory T cell therapeutic approaches in patients with autoimmune liver diseases and in patients receiving a transplant. Moreover, in settings where the abundance of Treg cells could hinder beneficial immunity, such us in chronic viral infection or liver cancer, enhancement of enclysis could result in transient, localized reduction of Treg cell numbers and tip the balance towards antiviral and anti-tumor immunity. We describe enclysis as is a natural process of liver immune regulation that lends itself to therapeutic targeting, particularly in combination with current Treg cell approaches. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a T cell-dependent, B-cell mediated autoimmune disease caused by antibodies against the nicotinic acetylcholine receptor or other components of the post-synaptic muscle endplate at the neuromuscular junction. These specific antibodies serve as excellent biomarkers for diagnosis, but do not adequately substitute for clinical evaluations to predict disease severity or treatment response. Several immunoregulatory cell populations are implicated in the pathogenesis of MG. The immunophenotype of these populations has been well-characterized in human peripheral blood. CD4+FoxP3+ regulatory T cells (Tregs) are functionally defective in MG, but there is a lack of consensus on whether they show numerical perturbations. Myeloid-derived suppressor cells (MDSCs) have also been explored in the context of MG. Adoptive transfer of CD4+FoxP3+ Tregs or MDSCs suppresses ongoing experimental autoimmune MG (EAMG), a rodent model of MG, suggesting a protective role of both populations in this disease. An imbalance between follicular Tregs and follicular T helper cells is found in untreated MG patients, correlating with disease manifestations. There is an inverse correlation between the frequency of circulating IL-10–producing B cells and clinical status in MG patients. Taken together, both functional and numerical defects in various populations of immunoregulatory cells in EAMG and human MG have been demonstrated, but how they relate to pathogenesis and whether these cells can serve as biomarkers of disease activity in humans deserve further exploration.

Retinal dehydrogenase (RALDH) enzymatic activities catalyze the conversion of vitamin A to its metabolite Retinoic acid (RA) in intestinal dendritic cells (DCs) and promote immunological tolerance. However, precise understanding of the exogenous factors that act as initial trigger of RALDH activity in these cells is still evolving. By using germ-free (GF) mice raised on an antigen free (AF) elemental diet, we find that certain components in diet are critically required to establish optimal RALDH expression and activity, most prominently in small intestinal CD103+CD11b+ DCs (siLP-DCs) right from the beginning of their lives. Surprisingly, systematic screens using modified diets devoid of individual dietary components indicate that proteins, starch and minerals are dispensable for this activity. On the other hand, in depth comparison between subtle differences in dietary composition among different dietary regimes reveal that adequate glucose concentration in diet is a critical determinant for establishing RALDH activity specifically in siLP-DCs. Consequently, pre-treatment of siLP-DCs, and not mesenteric lymph node derived MLNDCs with glucose, results in significant enhancement in the in vitro generation of induced Regulatory T (iTreg) cells. Our findings reveal previously underappreciated role of dietary glucose concentration in establishing regulatory properties in intestinal DCs, thereby extending a potential therapeutic module against intestinal inflammation.

MicroRNAs (miRNAs) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally. Several hundred miRNAs exist in the mammalian genome and regulate developmental processes, cell cycle, and survival.In this review, we highlight general modes of miRNA function and relate them to how such regulation can be beneficial for immune homeostasis and the prevention of autoimmune diseases. We highlight examples of experimentally verified miRNA function and their target genes in the immune system and place them in context of concepts relevant to an understanding of autoimmune pathogenesis. Where available, we refer to clinical correlations. Finally, we speculate how emerging knowledge about miRNA function in the immune system might be used diagnostically and therapeutically.

Myasthenia gravis (MG) is a T cell-dependent, B-cell mediated autoimmune disease caused by antibodies against the nicotinic acetylcholine receptor or other components of the post-synaptic muscle endplate at the neuromuscular junction. These specific antibodies serve as excellent biomarkers for diagnosis, but do not adequately substitute for clinical evaluations to predict disease severity or treatment response. Several immunoregulatory cell populations are implicated in the pathogenesis of MG. The immunophenotype of these populations has been well-characterized in human peripheral blood. CD4+FoxP3+ regulatory T cells (Tregs) are functionally defective in MG, but there is a lack of consensus on whether they show numerical perturbations. Myeloid-derived suppressor cells (MDSCs) have also been explored in the context of MG. Adoptive transfer of CD4+FoxP3+ Tregs or MDSCs suppresses ongoing experimental autoimmune MG (EAMG), a rodent model of MG, suggesting a protective role of both populations in this disease. An imbalance between follicular Tregs and follicular T helper cells is found in untreated MG patients, correlating with disease manifestations. There is an inverse correlation between the frequency of circulating IL-10–producing B cells and clinical status in MG patients. Taken together, both functional and numerical defects in various populations of immunoregulatory cells in EAMG and human MG have been demonstrated, but how they relate to pathogenesis and whether these cells can serve as biomarkers of disease activity in humans deserve further exploration. [ABSTRACT FROM AUTHOR]

Retinal dehydrogenase (RALDH) enzymatic activities catalyze the conversion of vitamin A to its metabolite Retinoic acid (RA) in intestinal dendritic cells (DCs) and promote immunological tolerance. However, precise understanding of the exogenous factors that act as initial trigger of RALDH activity in these cells is still evolving. By using germ-free (GF) mice raised on an antigen free (AF) elemental diet, we find that certain components in diet are critically required to establish optimal RALDH expression and activity, most prominently in small intestinal CD103+CD11b+ DCs (siLP-DCs) right from the beginning of their lives. Surprisingly, systematic screens using modified diets devoid of individual dietary components indicate that proteins, starch and minerals are dispensable for this activity. On the other hand, in depth comparison between subtle differences in dietary composition among different dietary regimes reveal that adequate glucose concentration in diet is a critical determinant for establishing RALDH activity specifically in siLP-DCs. Consequently, pre-treatment of siLP-DCs, and not mesenteric lymph node derived MLNDCs with glucose, results in significant enhancement in the in vitro generation of induced Regulatory T (iTreg) cells. Our findings reveal previously underappreciated role of dietary glucose concentration in establishing regulatory properties in intestinal DCs, thereby extending a potential therapeutic module against intestinal inflammation. [ABSTRACT FROM AUTHOR]

In T cells, as in all other cells of the body, sphingolipids form important structural components of membranes. Due to metabolic modifications, sphingolipids additionally play an active part in the signaling of cell surface receptors of T cells like the T cell receptor or the co-stimulatory molecule CD28. Moreover, the sphingolipid composition of their membranes crucially affects the integrity and function of subcellular compartments such as the lysosome. Previously, studying sphingolipid metabolism has been severely hampered by the limited number of analytical methods/model systems available. Besides well-established high resolution mass spectrometry new tools are now available like novel minimally modified sphingolipid subspecies for click chemistry as well as recently generated mouse mutants with deficiencies/overexpression of sphingolipid-modifying enzymes. Making use of these tools we and others discovered that the sphingolipid sphingomyelin is metabolized to ceramide to different degrees in distinct T cell subpopulations of mice and humans. This knowledge has already been translated into novel immunomodulatory approaches in mice and will in the future hopefully also be applicable to humans. In this paper we are, thus, summarizing the most recent findings on the impact of sphingolipid metabolism on T cell activation, differentiation, and effector functions. Moreover, we are discussing the therapeutic concepts arising from these insights and drugs or drug candidates which are already in clinical use or could be developed for clinical use in patients with diseases as distant as major depression and chronic viral infection.

In T cells, as in all other cells of the body, sphingolipids form important structural components of membranes. Due to metabolic modifications, sphingolipids additionally play an active part in the signaling of cell surface receptors of T cells like the T cell receptor or the co-stimulatory molecule CD28. Moreover, the sphingolipid composition of their membranes crucially affects the integrity and function of subcellular compartments such as the lysosome. Previously, studying sphingolipid metabolism has been severely hampered by the limited number of analytical methods/model systems available. Besides well-established high resolution mass spectrometry new tools are now available like novel minimally modified sphingolipid subspecies for click chemistry as well as recently generated mouse mutants with deficiencies/overexpression of sphingolipid-modifying enzymes. Making use of these tools we and others discovered that the sphingolipid sphingomyelin is metabolized to ceramide to different degrees in distinct T cell subpopulations of mice and humans. This knowledge has already been translated into novel immunomodulatory approaches in mice and will in the future hopefully also be applicable to humans. In this paper we are, thus, summarizing the most recent findings on the impact of sphingolipid metabolism on T cell activation, differentiation, and effector functions. Moreover, we are discussing the therapeutic concepts arising from these insights and drugs or drug candidates which are already in clinical use or could be developed for clinical use in patients with diseases as distant as major depression and chronic viral infection. [ABSTRACT FROM AUTHOR]

Summary: 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 Tregin 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 Tregin 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Novel homozygous missense IL2Rα gene mutation. • Cytofluorimetric analysis as diagnostic tool of CD25 deficiency. • Homology Modeling as method to confirm IL2Rα mutated protein instability. [ABSTRACT FROM AUTHOR]

Abstract Background Allogeneic hematopoietic stem cell transplantation (HSCT) is potentially curative in a variety of hematological malignancies. Graft-vs.-host disease (GvHD) remains a life-threatening complication. Standard treatment is high-dose (HD) corticosteroids. Steroid-refractory (SR) GvHD is associated with poor prognosis. At present, second-line treatment is ill-defined and includes a number of agents. Novel insights into the pathophysiology of acute GvHD (aGvHD) highlight the relevant role of the host inflammatory response governed by several kinase families, including Janus kinases (JAK)1/2. Ruxolitinib, a JAK1/2 inhibitor approved for intermediate-2/high-risk myelofibrosis, was recently employed in SR-GvHD with encouraging overall response rates. Clinical experience however remains limited. Case presentation A 51-year-old male with refractory anemia with excess blast type-2 underwent a myeloablative allogeneic HSCT from a 9/10 HLA-matched unrelated donor after conditioning with busulfan and cyclophosphamide. GvHD prophylaxis consisted of cyclosporine, methotrexate, and thymoglobulin. CD34+ cells/kg infused were 8.69 × 106 kg. On day 29, the patient developed overall grade IV aGvHD with biopsy proven stage IV gastrointestinal (GI) GvHD refractory to HD corticosteroids. Patient conditions rapidly deteriorated and became critical despite the addition of mycophenolate mofetil and budesonide. On day 33, Ruxolitinib was started, and on day 39 the patient clinical conditions gradually improved. Complete resolution of aGvHD was also confirmed by histology on day 54. Conclusions At 5 months from HSCT, the patient is well and in continuous hematological complete remission without flare of GvHD. Ruxolitinib was discontinued on day 156. Ruxolitinib is feasible and effective in SR-aGvHD though large prospective clinical trials are warranted.

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.

Pediatric slow progressors (PSP) are rare ART-naïve, HIV-infected children who maintain high CD4 T-cell counts and low immune activation despite persistently high viral loads. Using a well-defined cohort of PSP, we investigated the role of regulatory T-cells (TREG) and of IL-7 homeostatic signaling in maintaining normal-for-age CD4 counts in these individuals. Compared to children with progressive disease, PSP had greater absolute numbers of TREG, skewed toward functionally suppressive phenotypes. As with immune activation, overall T-cell proliferation was lower in PSP, but was uniquely higher in central memory TREG (CM TREG), indicating active engagement of this subset. Furthermore, PSP secreted higher levels of the immunosuppressive cytokine IL-10 than children who progressed. The frequency of suppressive TREG, CM TREG proliferation, and IL-10 production were all lower in PSP who go on to progress at a later time-point, supporting the importance of an active TREG response in preventing disease progression. In addition, we find that IL-7 homeostatic signaling is enhanced in PSP, both through preserved surface IL-7receptor (CD127) expression on central memory T-cells and increased plasma levels of soluble IL-7receptor, which enhances the bioactivity of IL-7. Combined analysis, using a LASSO modeling approach, indicates that both TREG activity and homeostatic T-cell signaling make independent contributions to the preservation of CD4 T-cells in HIV-infected children. Together, these data demonstrate that maintenance of normal-for-age CD4 counts in PSP is an active process, which requires both suppression of immune activation through functional TREG, and enhanced T-cell homeostatic signaling.

Type 1 diabetes (T1D) is an autoimmune disease in which a strong inflammatory response causes the death of insulin-producing pancreatic β-cells, while inefficient regulatory mechanisms allow that response to become chronic. Ethyl pyruvate (EP), a stable pyruvate derivate and certified inhibitor of an alarmin–high mobility group box 1 (HMGB1), exerts anti-oxidant and anti-inflammatory properties in animal models of rheumatoid arthritis and encephalomyelitis. To test its therapeutic potential in T1D, EP was administered intraperitoneally to C57BL/6 mice with multiple low-dose streptozotocin (MLDS)-induced T1D. EP treatment decreased T1D incidence, reduced the infiltration of cells into the pancreatic islets and preserved β-cell function. Apart from reducing HMGB1 expression, EP treatment successfully interfered with the inflammatory response within the local pancreatic lymph nodes and in the pancreas. Its effect was restricted to boosting the regulatory arm of the immune response through up-regulation of tolerogenic dendritic cells (CD11c+CD11b−CD103+) within the pancreatic infiltrates and through the enhancement of regulatory T cell (Treg) levels (CD4+CD25highFoxP3+). These EP-stimulated Treg displayed enhanced suppressive capacity reflected in increased levels of CTLA-4, secreted TGF-β, and IL-10 and in the more efficient inhibition of effector T cell proliferation compared to Treg from diabetic animals. Higher levels of Treg were a result of increased differentiation and proliferation (Ki67+ cells), but also of the heightened potency for migration due to increased expression of adhesion molecules (CD11a and CD62L) and CXCR3 chemokine receptor. Treg isolated from EP-treated mice had the activated phenotype and T-bet expression more frequently, suggesting that they readily suppressed IFN-γ-producing cells. The effect of EP on Treg was also reproduced in vitro. Overall, our results show that EP treatment reduced T1D incidence in C57BL/6 mice predominantly by enhancing Treg differentiation, proliferation, their suppressive capacity, and recruitment into the pancreas.

Background/Aims: Regulatory T cells (Treg) are critical regulators of asthma. Crocetin is isolated from Chinese herb saffron and is a natural carotenoid dicarboxylic acid with anti-inflammatory potential. However, the effects of Crocetin on asthma as well as the underlying mechanisms have not been studied. Methods: We used Crocetin to treat mice with established ovalbumin (OVA)-induced asthma. We purified CD4+CD25+ Treg cells by flow cytometry and analyzed the levels of two immunoregulatory proteins Foxp3 and tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) in Treg cells. We depleted either Foxp3 or TIPE2 in mouse lung through lentivirus-mediated delivery of shRNA, and analyzed their effects on severity of asthma and Treg cells after Crocetin treatment. Results: Crocetin treatment significantly reduced the severity of an ovalbumin (OVA)-induced asthma in mice. Moreover, Crocetin significantly increased the levels of TIPE2 and Foxp3 in Treg cells and the number of Treg cells. Depletion of Foxp3 abolished the increased in Treg cells, and the effects of Crocetin on the severity of asthma, without affecting TIPE2 levels in Treg cells. On the other hand, depletion of TIPE2 abolished both the increased in Treg cells and the effects of Crocetin on the severity of asthma, through suppressing Foxp3. Conclusion: Crocetin may activate Foxp3 through TIPE2 in asthma-associated Treg cells to mitigate the severity of asthma.

Background: Many studies showed that regulatory T cells (Tregs) have immunosuppressive effects on immune responses in transplantation and autoimmune disease. Silymarin (isolated from milk thistle or silybum marianum plant) is a flavolignan complex with anti-inflammatory, hepatoprotective, antioxidant and immunomodulatory activities. Previous studies in our group revealed inhibition effect of silymarin on mammalian target of rapamycin (mTOR) activity in activated T cells. Among immunosuppressive drugs, rapamycin can inhibit mTOR, results in Foxp3 expression, Tregs expansion and conventional T cells inhibition. In this study, the effect of silymarin on in-vitro generation of CD4+Foxp3+ cells, in comparison with rapamycin, was evaluated. Methods: Naïve CD4+ T cells were separated from healthy individuals’ peripheral blood mononuclear cells (PBMCs) and activated with monoclonal antibody anti-CD3 and anti-CD28 for 18 hours in Roswell Park Memorial Institute (RPMI) complete medium. Then, incubation was continued with adding Interlukin-2 (IL-2) and silymarin or its control, dimethyl sulfoxide (DMSO), or cultured in present or absent of rapamycin for 3 days. Cells were harvested and stained with anti-CD4 and anti-FoxP3 antibodies for flow cytometry. Findings: Silymarin increased CD4+Foxp3+ T cells compared with its control, DMSO, and with rapamycin after three days of culture of naïve T cells (P 0.05). Conclusion: Given the importance of replacement less harmful medicine and Tregs role in regulating immune system, silymarin, as aTreg generation drug, can be used in the treatment of autoimmune diseases and even in organ transplantation.

Pediatric slow progressors (PSP) are rare ART-naïve, HIV-infected children who maintain high CD4 T-cell counts and low immune activation despite persistently high viral loads. Using a well-defined cohort of PSP, we investigated the role of regulatory T-cells (TREG) and of IL-7 homeostatic signaling in maintaining normal-for-age CD4 counts in these individuals. Compared to children with progressive disease, PSP had greater absolute numbers of TREG, skewed toward functionally suppressive phenotypes. As with immune activation, overall T-cell proliferation was lower in PSP, but was uniquely higher in central memory TREG (CM TREG), indicating active engagement of this subset. Furthermore, PSP secreted higher levels of the immunosuppressive cytokine IL-10 than children who progressed. The frequency of suppressive TREG, CM TREG proliferation, and IL-10 production were all lower in PSP who go on to progress at a later time-point, supporting the importance of an active TREG response in preventing disease progression. In addition, we find that IL-7 homeostatic signaling is enhanced in PSP, both through preserved surface IL-7receptor (CD127) expression on central memory T-cells and increased plasma levels of soluble IL-7receptor, which enhances the bioactivity of IL-7. Combined analysis, using a LASSO modeling approach, indicates that both TREG activity and homeostatic T-cell signaling make independent contributions to the preservation of CD4 T-cells in HIV-infected children. Together, these data demonstrate that maintenance of normal-for-age CD4 counts in PSP is an active process, which requires both suppression of immune activation through functional TREG, and enhanced T-cell homeostatic signaling. [ABSTRACT FROM AUTHOR]