Mesenchymal Stem/Stromal Cells Derived from Dental Tissues Mediate the Immunoregulation of T Cells through the Purinergic Pathway.

Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3+ T cells and promoted the generation of CD4+CD25+FoxP3+CD39+CD73+ Tregs and Th17+CD39+ lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.

Correlation between Melatonin and Colostral Regulatory T Cells in Giardia lamblia Infection.

Giardiasis is a parasitic disease caused by Giardia lamblia (G. lamblia) that affects people worldwide. Still, few studies report on the immunoregulatory effects of the biomolecules of colostrum during interactions with G. lamblia. This study aimed to assess the concentrations of melatonin and cortisol hormones, the percentage of Treg cells, and the levels of cytokines IL-10 and TGF-ß in colostrum from mothers who tested positive for the parasite. This cross-sectional study analyzed colostrum samples from 25 puerperal. The samples were tested using an ELISA to determine if they were seropositive for G. lamblia and the type of antibody present (IgM and IgG). Based on the results, the samples were divided into three groups: a control group (N = 10) with no reaction to either IgM or IgG, a group seropositive for IgG (IgG+/IgM-; N = 8), and a group seropositive for IgM (IgM+/IgG-; N = 7). The concentrations of melatonin and cortisol were measured using the ELISA method. Additionally, cytokines IL-10 and TGF-ß and immunophenotyping were analyzed using flow cytometry. In the group that tested positive for IgM anti-G. lamblia, the concentration of melatonin was lower. However, in the colostrum from mothers who tested positive for IgG anti-G. lamblia, the level of this hormone had increased. The cortisol levels were similar between the groups, regardless of seropositivity. There was a higher percentage of Treg cells in the colostrum from mothers who tested positive for IgM anti-G. lamblia. TGF-ß levels also increased in the colostrum of mothers who tested positive for IgM anti-G. lamblia. In the seronegative group for G. lamblia, there was a positive correlation between melatonin concentration and the percentage of Treg cells. These data suggest that the increase in regulatory cells and cytokines and the reduction in melatonin in colostrum from mothers with recent giardia infection may contribute to the evolution and manifestation of the disease.

Identification of master regulator genes controlling pathogenic CD4+ T cell fate in inflammatory bowel disease through transcriptional network analysis.

Inflammatory bowel diseases (IBD) are a group of chronic inflammatory conditions of the gastrointestinal tract associated with multiple pathogenic factors, including dysregulation of the immune response. Effector CD4+ T cells and regulatory CD4+ T cells (Treg) are central players in maintaining the balance between tolerance and inflammation. Interestingly, genetic modifications in these cells have been implicated in regulating the commitment of specific phenotypes and immune functions. However, the transcriptional program controlling the pathogenic behavior of T helper cells in IBD progression is still unknown. In this study, we aimed to find master transcription regulators controlling the pathogenic behavior of effector CD4+ T cells upon gut inflammation. To achieve this goal, we used an animal model of IBD induced by the transfer of naïve CD4+ T cells into recombination-activating gene 1 (Rag1) deficient mice, which are devoid of lymphocytes. As a control, a group of Rag1-/- mice received the transfer of the whole CD4+ T cells population, which includes both effector T cells and Treg. When gut inflammation progressed, we isolated CD4+ T cells from the colonic lamina propria and spleen tissue, and performed bulk RNA-seq. We identified differentially up- and down-regulated genes by comparing samples from both experimental groups. We found 532 differentially expressed genes (DEGs) in the colon and 30 DEGs in the spleen, mostly related to Th1 response, leukocyte migration, and response to cytokines in lamina propria T-cells. We integrated these data into Gene Regulatory Networks to identify Master Regulators, identifying four up-regulated master gene regulators (Lef1, Dnmt1, Mybl2, and Jup) and only one down-regulated master regulator (Foxo3). The altered expression of master regulators observed in the transcriptomic analysis was confirmed by qRT-PCR analysis and found an up-regulation of Lef1 and Mybl2, but without differences on Dnmt1, Jup, and Foxo3. These two master regulators have been involved in T cells function and cell cycle progression, respectively. We identified two master regulator genes associated with the pathogenic behavior of effector CD4+ T cells in an animal model of IBD. These findings provide two new potential molecular targets for treating IBD.

Type 1 Diabetes Brazilian patients exhibit reduced frequency of recent thymic emigrants in regulatory CD4+CD25+Foxp3+T cells.

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

Effect of Dietary Supplementation with Omega-3 Fatty Acid on the Generation of Regulatory T Lymphocytes and on Antioxidant Parameters and Markers of Oxidative Stress in the Liver Tissue of IL-10 Knockout Mice.

INTRODUCTION: chronic low-grade inflammation, or inflammaging, emerges as a crucial element in the aging process and is associated with cardiovascular and neurological diseases, sarcopenia, and malnutrition. Evidence suggests that omega-3 fatty acids present a potential therapeutic agent in the prevention and treatment of inflammatory diseases, mitigating oxidative stress, and improving muscle mass, attributes that are particularly relevant in the context of aging. The objective of the present study was to evaluate the effectiveness of supplementation with omega-3 fish oil in improving the immune response and oxidative stress in knockout mice for interleukin IL-10 (IL-10-/-). MATERIAL AND METHODS: female C57BL/6 wild-type (WT) and interleukin IL-10 knockout (IL-10-/-) mice were fed during 90 days with a standard diet (control groups), or they were fed/supplemented with 10% of the omega-3 polyunsaturated fatty acid diet (omega-3 groups). Muscle, liver, intestinal, and mesenteric lymph node tissue were collected for analysis. RESULTS: the IL-10-/-+O3 group showed greater weight gain compared to the WT+O3 (p = 0.001) group. The IL-10-/-+O3 group exhibited a higher frequency of regulatory T cells than the IL-10-/- group (p = 0.001). It was found that animals in the IL-10-/-+O3 group had lower levels of steatosis when compared to the IL-10-/- group (p = 0.017). There was even greater vitamin E activity in the WT group compared to the IL-10-/-+O3 group (p = 0.001) and WT+O3 compared to IL-10-/-+O3 (p = 0.002), and when analyzing the marker of oxidative stress, MDA, an increase in lipid peroxidation was found in the IL-10-/-+O3 group when compared to the IL-10-/- group (p = 0.03). Muscle tissue histology showed decreased muscle fibers in the IL-10-/-+O3, IL-10-/-, and WT+O3 groups. CONCLUSION: the findings show a decrease in inflammation, an increase in oxidative stress markers, and a decrease in antioxidant markers in the IL-10-/-+O3 group, suggesting that supplementation with omega-3 fish oil might be a potential intervention for inflammaging that characterizes the aging process and age-related diseases.

MiR-326 sponges TET2 triggering imbalance of Th17/Treg differentiation to exacerbate pyroptosis of hepatocytes in concanavalin A-induced autoimmune hepatitis.

INTRODUCTION AND OBJECTIVES: MicroRNA-326 is abnormally expressed in autoimmune diseases, but its roles in autoimmune hepatitis (AIH) are unknown. In this study, we aimed to investigate the effect of miR-326 on AIH and the underlying mechanism. MATERIALS AND METHODS: Concanavalin A was administrated to induce AIH in mice and the expression levels of miR-326 and TET2 was evaluated by qRT-PCR and western blot, respectively. The percentages of Th17 and Treg cells were evaluated by flow cytometry and their marker proteins were determined by western blot and ELISA. The mitochondrial membrane potential (MMP) and ROS level were tested with the JC-1 kit and DCFH-DA assay. The binding relationships between miR-326 and TET2 were verified by dual-luciferase reporter assay. The liver tissues were stained by the HE staining. In vitro, AML12 cells were cocultured with mouse CD4+T cells. The expression levels of pyroptosis-related proteins were assessed by western blot. RESULTS: Concanavalin A triggered AIH and enhanced the expression level of miR-326 in mice. It increased both Th17/Treg ratio and the levels of their marker proteins. The expression of TET2 was decreased in AIH mice. Knockdown of miR-326 could decrease the levels of pyroptosis-related proteins, the ROS level and increase MMP. In mouse CD4+T cells, miR-326 sponged TET2 to release IL-17A. Coculture of AML12 cells with isolated CD4+T cells from miR-326 knockdown AIH mice could relieve pyroptosis. CONCLUSIONS: Knockdown of miR-326 exerted anti-pyroptosis effects via suppressing TET2 and downstream NF-κB signaling to dampen AIH. We highlighted a therapeutic target in AIH.

CD39+ conventional CD4+ T cells with exhaustion traits and cytotoxic potential infiltrate tumors and expand upon CTLA-4 blockade.

Conventional CD4+ T (Tconv) lymphocytes play important roles in tumor immunity; however, their contribution to tumor elimination remains poorly understood. Here, we describe a subset of tumor-infiltrating Tconv cells characterized by the expression of CD39. In several mouse cancer models, we observed that CD39+ Tconv cells accumulated in tumors but were absent in lymphoid organs. Compared to tumor CD39- counterparts, CD39+ Tconv cells exhibited a cytotoxic and exhausted signature at the transcriptomic level, confirmed by high protein expression of inhibitory receptors and transcription factors related to the exhaustion. Additionally, CD39+ Tconv cells showed increased production of IFNγ, granzyme B, perforin and CD107a expression, but reduced production of TNF. Around 55% of OVA-specific Tconv from B16-OVA tumor-bearing mice, expressed CD39. In vivo CTLA-4 blockade induced the expansion of tumor CD39+ Tconv cells, which maintained their cytotoxic and exhausted features. In breast cancer patients, CD39+ Tconv cells were found in tumors and in metastatic lymph nodes but were less frequent in adjacent non-tumoral mammary tissue and not detected in non-metastatic lymph nodes and blood. Human tumor CD39+ Tconv cells constituted a heterogeneous cell population with features of exhaustion, high expression of inhibitory receptors and CD107a. We found that high CD4 and ENTPD1 (CD39) gene expression in human tumor tissues correlated with a higher overall survival rate in breast cancer patients. Our results identify CD39 as a biomarker of Tconv cells, with characteristics of both exhaustion and cytotoxic potential, and indicate CD39+ Tconv cells as players within the immune response against tumors.

Highly purified and functionally stable in vitro expanded allospecific Tr1 cells expressing immunosuppressive graft-homing receptors as new candidates for cell therapy in solid organ transplantation.

The development of new strategies based on the use of Tr1 cells has taken relevance to induce long-term tolerance, especially in the context of allogeneic stem cell transplantation. Although Tr1 cells are currently identified by the co-expression of CD49b and LAG-3 and high production of interleukin 10 (IL-10), recent studies have shown the need for a more exhaustive characterization, including co-inhibitory and chemokines receptors expression, to ensure bona fide Tr1 cells to be used as cell therapy in solid organ transplantation. Moreover, the proinflammatory environment induced by the allograft could affect the suppressive function of Treg cells, therefore stability of Tr1 cells needs to be further investigated. Here, we establish a new protocol that allows long-term in vitro expansion of highly purified expanded allospecific Tr1 (Exp-allo Tr1). Our expanded Tr1 cell population becomes highly enriched in IL-10 producers (> 90%) and maintains high expression of CD49b and LAG-3, as well as the co-inhibitory receptors PD-1, CTLA-4, TIM-3, TIGIT and CD39. Most importantly, high dimensional analysis of Exp-allo Tr1 demonstrated a specific expression profile that distinguishes them from activated conventional T cells (T conv), showing overexpression of IL-10, CD39, CTLA-4 and LAG-3. On the other hand, Exp-allo Tr1 expressed a chemokine receptor profile relevant for allograft homing and tolerance induction including CCR2, CCR4, CCR5 and CXCR3, but lower levels of CCR7. Interestingly, Exp-allo Tr1 efficiently suppressed allospecific but not third-party T cell responses even after being expanded in the presence of proinflammatory cytokines for two extra weeks, supporting their functional stability. In summary, we demonstrate for the first time that highly purified allospecific Tr1 (Allo Tr1) cells can be efficiently expanded maintaining a stable phenotype and suppressive function with homing potential to the allograft, so they may be considered as promising therapeutic tools for solid organ transplantation.

Hypothyroidism-associated immunosuppression involves induction of galectin-1-producing regulatory T cells.

Hypothyroidism exerts deleterious effects on immunity, but the precise role of the hypothalamic-pituitary-thyroid (HPT) axis in immunoregulatory and tolerogenic programs is barely understood. Here, we investigated the mechanisms underlying hypothyroid-related immunosuppression by examining the regulatory role of components of the HPT axis. We first analyzed lymphocyte activity in mice overexpressing the TRH gene (Tg-Trh). T cells from Tg-Trh showed increased proliferation than wild-type (WT) euthyroid mice in response to polyclonal activation. The release of Th1 pro-inflammatory cytokines was also increased in Tg-Trh and TSH levels correlated with T-cell proliferation. To gain further mechanistic insights into hypothyroidism-related immunosuppression, we evaluated T-cell subpopulations in lymphoid tissues of hypothyroid and control mice. No differences were observed in CD3/CD19 or CD4/CD8 ratios between these strains. However, the frequency of regulatory T cells (Tregs) was significantly increased in hypothyroid mice, and not in Tg-Trh mice. Accordingly, in vitro Tregs differentiation was more pronounced in naïve T cells isolated from hypothyroid mice. Since Tregs overexpress galectin-1 (Gal-1) and mice lacking this lectin (Lgals1-/- ) show reduced Treg function, we investigated the involvement of this immunoregulatory lectin in the control of Tregs in settings of hypothyroidism. Increased T lymphocyte reactivity and reduced frequency of Tregs were found in hypothyroid Lgals1-/- mice when compared to hypothyroid WT animals. This effect was rescued by the addition of recombinant Gal-1. Finally, increased expression of Gal-1 was found in Tregs purified from hypothyroid WT mice compared with their euthyroid counterpart. Thus, a substantial increase in the frequency and activity of Gal-1-expressing Tregs underlies immunosuppression associated with hypothyroid conditions, with critical implications in immunopathology, metabolic disorders, and cancer.

Dysfunctional phenotype of systemic and pulmonary regulatory T cells associate with lethal COVID-19 cases.

Severe cases of COVID-19 present hyperinflammatory condition that can be fatal. Little is known about the role of regulatory responses in SARS-CoV-2 infection. In this study, we evaluated the phenotype of regulatory T cells in the blood (peripheral blood mononuclear cell) and the lungs (broncho-alveolar) of adult patients with severe COVID-19 under invasive mechanical ventilation. Our results show important dynamic variation on Treg cells phenotype during COVID-19 with changes in number and functional parameters from the day of intubation (Day 1 of intensive care unit admission) to Day 7. We observed that compared with surviving patients, non-survivors presented lower numbers of Treg cells in the blood. In addition, lung Tregs of non-survivors also displayed higher PD1 and lower FOXP3 expressions suggesting dysfunctional phenotype. Further signs of Treg dysregulation were observed in non-survivors such as limited production of IL-10 in the lungs and higher production of IL-17A in the blood and in the lungs, which were associated with increased PD1 expression. These findings were also associated with lower pulmonary levels of Treg-stimulating factors like TNF and IL-2. Tregs in the blood and lungs are profoundly dysfunctional in non-surviving COVID-19 patients.

Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-ß in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-ß. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Crosstalk between incretin hormones, Th17 and Treg cells in inflammatory diseases.

Intestinal epithelial cells constantly crosstalk with the gut microbiota and immune cells of the gut lamina propria. Enteroendocrine cells, secrete hormones, such as incretin hormones, which participate in host physiological events, such as stimulating insulin secretion, satiety, and glucose homeostasis. Interestingly, evidence suggests that the incretin pathway may influence immune cell activation. Consequently, drugs targeting the incretin hormone signaling pathway may ameliorate inflammatory diseases such as inflammatory bowel diseases, cancer, and autoimmune diseases. In this review, we discuss how these hormones may modulate two subsets of CD4 + T cells, the regulatory T cells (Treg)/Th17 axis important for gut homeostasis: thus, preventing the development and progression of inflammatory diseases. We also summarize the main experimental and clinical findings using drugs targeting the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP-1) signaling pathways and their great impact on conditions in which the Treg/Th17 axis is disturbed such as inflammatory diseases and cancer. Understanding the role of incretin stimulation in immune cell activation and function, might contribute to new therapeutic designs for the treatment of inflammatory diseases, autoimmunity, and tumors.

The increased ratio of Treg/Th2 in promoting metastasis of hepatocellular carcinoma.

OBJECTIVE: Liver cancer is the fifth most common cancer in the world. Research on the pathogenesis and detailed molecular mechanisms of liver cancer is very important. The immune system plays an important role in regulating the incidence and metastasis of liver cancer. MATERIALS AND METHODS: This work collected 20 blood samples from patients with clinical hepatocellular carcinoma without metastasis, 20 blood samples from patients with metastatic hepatocellular carcinoma, and 20 blood samples from healthy subjects. Flow cytometry was used to analyze the content of Treg and Th2 cells in the three groups of blood samples. Immunofluorescence was applied to analyze the relative expression of CTLA-4 and CD28 in lymphocytes of each group of blood samples. Western blot was used to analyze the T cell surface protein CTLA-4, CD28, GATA3, and FOXP3 expression in each group of blood samples. RESULTS: The expression of CD28 and GATA3 in the blood of patients with hepatocellular carcinoma without metastasis was obviously higher than that of patients with metastasis of hepatocellular carcinoma, which is contrary to the expression trend of CTLA-4 and FOXP3, and corresponds to the content ratio of Treg and Th2 cells, thus verifying the relationship between Treg/Th2 ratio and metastasis of hepatocellular carcinoma. CONCLUSIONS: In the microenvironment of liver cancer, the ratio of Treg/Th2 will increase significantly, thereby promoting the metastasis of hepatocellular carcinoma.

OBJETIVO: El cáncer de hígado es el quinto cáncer más común en el mundo. La investigación sobre la patogenia y los mecanismos moleculares detallados del cáncer de hígado es muy importante. El sistema inmunológico juega un papel importante en la regulación de la incidencia y metástasis del cáncer de hígado. MATERIAL Y MÉTODOS: Este trabajo recogió 20 muestras de sangre de pacientes con carcinoma hepatocelular clínico sin metástasis, 20 muestras de sangre de pacientes con carcinoma hepatocelular metastásico y 20 muestras de sangre de sujetos sanos. Se utilizó citometría de flujo para analizar el contenido de células Treg y Th2 en los tres grupos de muestras de sangre. Se aplicó inmunofluorescencia para analizar la expresión relativa de CTLA-4 y CD28 en linfocitos de cada grupo de muestras de sangre. Se utilizó Western blot para analizar la expresión de la proteína de superficie de células T CTLA-4, CD28, GATA3, FOXP3 en cada grupo de muestras de sangre. RESULTADOS: La expresión de CD28 y GATA3 en la sangre de pacientes con carcinoma hepatocelular sin metástasis fue obviamente mayor que la de pacientes con metástasis de carcinoma hepatocelular, lo cual es contrario a la tendencia de expresión de CTLA-4 y FOXP3, y corresponde al contenido relación de células Treg y Th2, verificando así la relación entre la relación Treg/Th2 y la metástasis del carcinoma hepatocelular. CONCLUSIONES: En el microambiente del cáncer de hígado, la proporción de Treg/Th2 aumentará significativamente, promoviendo así la metástasis del carcinoma hepatocelular.

A specific structure and high richness characterize intestinal microbiota of HIV-exposed seronegative individuals.

Intestinal microbiota facilitates food breakdown for energy metabolism and influences the immune response, maintaining mucosal homeostasis. Overall, HIV infection is associated with intestinal dysbiosis and immune activation, which has been related to seroconversion in HIV-exposed individuals. However, it is unclear whether microbiota dysbiosis is the cause or the effect of immune alterations and disease progression or if it could modulate the risk of acquiring the HIV infection. We characterize the intestinal microbiota and determine its association with immune regulation in HIV-exposed seronegative individuals (HESN), HIV-infected progressors (HIV+), and healthy control (HC) subjects. For this, feces and blood were collected. The microbiota composition of HESN showed a significantly higher alpha (p = 0.040) and beta diversity (p = 0.006) compared to HC, but no differences were found compared to HIV+. A lower Treg percentage was observed in HESN (1.77%) than HC (2.98%) and HIV+ (4.02%), with enrichment of the genus Butyrivibrio (p = 0.029) being characteristic of this profile. Moreover, we found that Megasphaera (p = 0.017) and Victivallis (p = 0.0029) also are enriched in the microbiota composition in HESN compared to HC and HIV+ subjects. Interestingly, an increase in Succinivibrio and Prevotella, and a reduction in Bacteroides genus, which is typical of HIV-infected individuals, were observed in both HESN and HIV+, compared to HC. Thus, HESNs have a microbiota profile, similar to that observed in HIV+, most likely because HESN are cohabiting with their HIV+ partners.

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines.

The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV- Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-ß), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

Artesunate strongly modulates myeloid and regulatory T cells to prevent LPS-induced systemic inflammation.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria and is usually administrated to establish models of inflammation. Artesunate (ART), a water-soluble artemisinin derivative, displays multiple pharmacological actions against tumors, viral infections, and inflammation, and has been used as a therapeutic weapon against malaria. In this study, our aim was to evaluate whether ART pretreatment is capable of preventing inflammation induced by LPS. BALB/c mice were treated with 100 mg/kg of ART i.p. for 7 days followed by a single dose of LPS. ART pretreatment led to an improvement in clinical score, prevented alterations in biochemical markers, and reestablished the platelet counts. Flow cytometry analysis showed that ART protected the inflammation mainly by reducing the percentage of M1 macrophages while increasing M2 macrophages and a reestablishment of classical monocytes in the BM. In the spleen, ART pretreatment increased N2 neutrophils, myeloid-derived suppressor cells (MDSC), and regulatory T cells, the latter was also increased in peripheral blood. In addition, a marked decrease in inflammatory cytokines and chemokines was observed in the ART treated group. Our data suggest that ART prevents inflammation, reducing tissue damage and restoring homeostasis.

Phenotypes of STAT3 gain-of-function variant related to disruptive regulation of CXCL8/STAT3, KIT/STAT3, and IL-2/CD25/Treg axes.

STAT3 is a cytokine-signaling transcription factor critical for gene regulation. Gain-of-function (GOF) mutations in STAT3 are associated with lymphoproliferation, autoimmune cytopenias, increased susceptibility to infection, early-onset solid-organ autoimmunity, short stature, and eczema. We studied the JAK/STAT signaling pathway gene expression and the cytokine profile in two families carrying STAT3-GOF variants to shed light on the STAT3-GOF-associated variable expressivity, including the identification of disease markers. Considering 92 target genes, KIT and IL2RA were downregulated only in patients with high clinical penetrance, while CXCL8 was markedly downregulated for all of them. Unlike previous studies, SOCS3-a STAT3 inhibitor-was not upregulated in patients. In addition, low levels of IL-2 and a reduced numbers of Tregs cells were strikingly prevalent in patients. This study shows a disruptive role of STAT3-GOF variants in the regulatory axis activities CXCL8/STAT3, KIT/STAT3, IL2/CD25/Treg, which, by slightly different mechanisms, underlie the broad clinical spectrum seen in the studied patients. In addition, we suggest the investigation of CXCL8 as a biomarker for identifying STAT3-GOF mutation.

Interleukin-35 is a critical regulator of immunity during helminth infections associated with multiple sclerosis.

Multiple sclerosis (MS) is currently thought to arise by interactions between genetic susceptibility and environmental factors. Infections in general trigger autoimmune responses causing clinical manifestations of disease. However, as a result of regulatory T (Treg)- and regulatory B (Breg)-cell induction, helminth infections tend to dampen disease activity. IL-35, the newest member of the IL-12 family, is an inhibitory cytokine composed of an EBI3ß chain subunit, and an IL-12p35 subunit. The aim of this study was to investigate the role of IL-35 during parasite infections occurring in individuals with MS. Numbers of IL-35-producing Breg cells are higher in CSF from helminth-infected than from uninfected MS subjects, a finding associated with decreased MRI disease activity. Interestingly, stimulation of CD19+ B cells with IL-35 promotes conversion of these cells to Breg cells producing both IL-35 and IL-10. Coculture of B cells from helminth-infected MS patients inhibits proliferation of Th1 and Th17 myelin peptide-specific T cells, as well as production of IFN-γ and IL-17. Following activation, CD4+ CD25+ Treg cells significantly upregulate expression of EBI3 and IL-12p35 mRNA. Furthermore, CD4+ CD25- T cells activated in the presence of IL-35 induce a population of cells with regulatory function, known as iTR35. Finally, B cells from normal individuals cultured in vitro in the presence of the helminth antigen SEA increase expression of the transcription BATF, IRF4 and IRF8, acquiring a pattern similar to that of IL-35 Breg cells. These data highlight the important immunoregulatory effects of IL-35 on both Breg and Treg cells, observed in helminth-infected MS subjects.

Phenotypical characterization of regulatory T cells in acute Zika infection.

Zika virus (ZIKV), alongside Dengue virus (DENV), Chikungunya virus (CHIKV), and Yellow Fever Virus (YFV) are prevalent arboviruses in the Americas. Each of these infections is associated with the development of associated disease immunopathology. Immunopathological processes are an outcome of counter-balancing impacts between effector and regulatory immune mechanisms. In this context, regulatory T cells (Tregs) are key in modulating the immune response and, therefore, in tissue damage control. However, to date, Treg phenotypes and mechanisms during acute infection of the ZIKV in humans have not been fully investigated. The main aim of this work was to characterize Tregs and their immunological profile related to cytokine production and molecules that are capable of controlling the exacerbated inflammatory profile in acute Zika infected patients. Using whole blood analyses of infected patients, an ex vivo phenotypical characterization of Tregs, circulating during acute Zika virus infection, was conducted by flow cytometry. We found that though there are no differences in absolute Treg frequency between infected and healthy control groups. However, pro-inflammatory cytokine up-regulation such as IFN-γ and LAP was observed in the acute disease. Furthermore, acute ZIKV patients expressed increased levels of CD39/CD73, perforin/granzyme B, PD-1, and CTLA-4, all markers involved in mechanisms used by Tregs to attempt to control strong inflammatory responses. Thus, the data indicates a potential contribution of Tregs during the inflammatory ZIKV infection response.

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.