Treg Levels Research Articles

Dynamics of Plasmodium berghei NK-65 parasitaemia and CD3+CD4+CD25+Fox-p3+ T-regulatory cells in experimentally induced malaria during early, mid, and late-pregnancy in BALB/c mice.

Malaria in pregnancy causes a dual brunt on the mother as well as the foetus. Upregulation of T-regulatory cells (Tregs) during pregnancy allows tolerance towards the growing foetus, their suppression predisposes the mother to infections. This study analyzed the levels of CD3+CD4+CD25+Fox-p3+ Tregs, parasitaemia, maternal and foetal outcomes in BALB/c mice infected with P. berghei NK65 during early-, mid-, and late-pregnancy. Total of 114 mice, non-pregnant non-infected (n = 6), non-pregnant infected (n = 12), pregnant non-infected (n = 48) and pregnant infected (n = 48) were included in the study. Infected groups were inoculated intra-peritoneally with 1 × 106 P. berghei infected RBCs during early-, mid-, and late- pregnancy (D6, D10, and D14 respectively). Six mice from each stage were sacrificed on the 5th and 7th day post-infection (DPI) to evaluate parasitaemia (staining) and Tregs from splenocytes (by flow cytometry). The parasitaemia was significantly higher among early pregnancy infected mice (≥ 70%) than mid-pregnancy infected (40-70%), late pregnancy infected (50-65%), and non-pregnant infected mice (≤ 50%) (p < 0.05). The level of Tregs was significantly higher among non-pregnant infected mice as compared to non-pregnant non-infected mice (%Tregs 0.86 vs. 0.44). Among pregnant mice, the levels of Tregs in infected mice were lower than in non-infected mice during all stages of pregnancy. None of the mice infected during early- and mid-pregnancy survived at 6DPI and 7DPI, respectively, and those infected during late-pregnancy delivered premature pups. In contrast to non-pregnant mice, the levels of Tregs among pregnant mice decrease when malaria infection is acquired thereby leading to adverse pregnancy outcomes. The online version contains supplementary material available at 10.1007/s12088-023-01089-2.

Intravenous BCG vaccination reduces SARS-CoV-2 severity and promotes extensive reprogramming of lung immune cells

Bacillus Calmette-Guérin (BCG) confers heterologous immune protection against viral infections and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here, we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model. BCG vaccination conferred a modest reduction on lung SCV2 viral load, bronchopneumonia scores, and weight loss, accompanied by a reversal of SCV2-mediated Tcell lymphopenia, and reduced lung granulocytes. BCG uniquely recruited immunoglobulin-producing plasma cells to the lung suggesting accelerated local antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, with a transcriptional shift away from exhaustion markers and toward antigen presentation and repair. Similarly, BCG enhanced recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, that show reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Astragaloside IV ameliorates experimental autoimmune myasthenia gravis by regulating CD4 + T cells and altering gut microbiota

BackgroundMyasthenia gravis (MG) is an antibody-mediated autoimmune disease and its pathogenesis is closely related to CD4 + T cells. In recent years, gut microbiota is considered to play an important role in the pathogenesis of MG. Astragaloside IV (AS-IV) is one of the main active components extracted from Astragalus membranaceus and has immunomodulatory effects. To study the immunomodulatory effect of AS-IV and the changes of gut microbiota on experimental autoimmune myasthenia gravis (EAMG) mice, we explore the possible mechanism of AS-IV in improving MG.MethodsIn this study, network pharmacology was utilized to screen the crucial targets of AS-IV in the treatment of MG. Subsequently, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify potential pathways through which AS-IV acts against MG. Furthermore, experimental investigations were conducted to validate the underlying mechanism of AS-IV in MG treatment. Before modeling, 5 mice were randomly selected as the control group (CFA group), and the other 10 were induced to EAMG model. These mice were randomly divided into EAMG group and EAMG + AS-IV group, n = 5/group. In EAMG + AS-IV group, AS-IV was administered by gavage. CFA and EAMG groups were given the same volume of PBS. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. At the last administration, the feces were collected for 16S RNA microbiota analysis. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected by flow cytometry. The levels of IFN-γ, IL-17 and TGF-β in serum were measured by ELISA. Furthermore, fecal microbial transplantation (FMT) experiments were performed for exploring the influence of changed intestinal flora on EAMG. After EAMG model was induced, the mice were treated with antibiotics daily for 4 weeks to germ-free. Then germ-free EAMG mice were randomly divided into two groups: FMT EAMG group, FMT AS-IV group, n = 3/group. Fecal extractions from EAMG and EAMG + AS-IV groups as gathered above were used to administered daily to the respective groups for 4 weeks. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected at the last administration. The levels of IFN-γ, IL-17 and TGF-β in serum were measured by ELISA.ResultsThe network pharmacology and KEGG pathway analysis revealed that AS-IV regulates T cell pathways, including T cell receptor signaling pathway and Th17 cell differentiation, suggesting its potential in improving MG. Further experimental verification demonstrated that AS-IV administration improved muscle strength and body weight, reduced the level of Th1 and Th17 cells, enhanced the level of Treg cells, and resulted in alterations of the gut microbiota, including changes in beta diversity, the Firmicutes/Bacteroidetes (F/B) ratio, and the abundance of Clostridia in EAMG mice. We further conducted FMT tests and demonstrated that the EAMG Abx-treated mice which were transplanted the feces of mice treated with AS-IV significantly alleviated myasthenia symptoms, reduced Th1 and Th17 cells levels, and increased Treg cell levels.ConclusionThis study speculated that AS-IV ameliorates EAMG by regulating CD4 + T cells and altering the structure and species of gut microbiota of EAMG.

Could the Crosstalk Between Myeloid-Derived-Suppressor Cells and Regulatory T Cells Have a Role in Beta-Thalassemia?

Secondary iron overload, alloimmunization, and increased risk of infection are common complications in patients with transfusion-dependent thalassemia (TDT). Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) play an essential role in preventing excessive immune response. This research aimed to study the interaction between Tregs and MDSCs in TDT patients and to evaluate the association of these cell types with disease severity. This case-control study included 26 patients with TDT and 23 healthy, age- and sex-matched controls. All patients were investigated for complete blood count (CBC), serum ferritin, and flow cytometric analysis of peripheral blood to detect Tregs, MDSCs, and MDSC subsets. A significant increase was observed in the frequencies of Tregs and MDSCs, particularly monocytic MDSCs (MO-MDSCs), in TDT patients compared with controls. The frequencies of these cells showed a direct association with ferritin level and total leukocyte count and an inverse association with hemoglobin level. Furthermore, a positive correlation was observed between Tregs and each of the total MDSCs and MO-MDSCs. Levels of Tregs and MDSCs increased in TDT and may probably have a role in suppressing the active immune systems of TDT patients.

Anti-PD-L1 antibody reverses the immune tolerance induced by multiple MUC1-MBP vaccine immunizations by increasing the CD80/PD-L1 ratio, resulting in DC maturation, and decreasing Treg activity in B16-MUC1 melanoma-bearing mice.

In this study, we explored the possible mechanism of tumor tolerance induced by multiple repeated immunizations with a tumor vaccine (MUC1-MBP fusion protein plus CpG2006). We first analyzed the mechanism of tolerance by immunizing tumor-bearing mice 2, 5, or 8 times and found that compared with five immunizations with the M-M vaccine, eight immunizations increased tumor volume and weight and Treg levels, while the proportions of Th1 and Tc1 cells in the spleen and lymph nodes were decreased. In particular, the M-M vaccine induced PD-L1 expression in CD11c+DCs and decreased their CD80/PD-L1 ratio. Therefore, the mechanism of tolerance induction by multiple immunizations with the M-M vaccine was investigated by focusing on the CD80/PD-L1 ratio, and an anti-PD-L1 antibody (αPD-L1) and the M-M vaccine were used in combination to treat melanoma. The results showed that αPD-L1 increased the CD80/PD-L1 ratio and enhanced the maturation of cDC1s by blocking PD-L1 on DCs, which potentially increased the activity of Th1 and Tc1 cells. Furthermore, the combination of the M-M vaccine with αPD-L1 decreased the activity and proportion of Tregs, which reversed the immune tolerance induced by eight immunizations with the vaccine. This study reveals the mechanism of the combination of M-M and αPD-L1 and provides a new combination strategy for improving the therapeutic effect of the M-M vaccine, laying a theoretical basis for the clinical application of the vaccine.

14:30-14:40 Modification of peripheric Treg and CD56 levels in RIF women after egg-donation treated with GM-CSF and controls

14:30-14:40 Modification of peripheric Treg and CD56 levels in RIF women after egg-donation treated with GM-CSF and controls

Tuberculosis and COVID-19 Dually Affect Human Th17 Cell Immune Response.

We analyzed the features of immune response in tuberculosis and COVID-19 patients. For this, we analyzed publications released from December 2019 to March 2023; those which were published in accessible international databases ("Medline", "PubMed", "Scopus") and with keywords such as "COVID-19", "SARS-CoV-2", "tuberculosis", "pulmonary tuberculosis", "latent tuberculosis infection", "Treg", "follicular Treg", and "Treg subsets", we considered. Through our analysis, we found that tuberculosis patients who had been infected with COVID-19 previously and elevated Th1 and Th2 cell levels. High levels of Th1 and Th2 cells may serve as a positive marker, characterizing activated immune response during TB infection. COVID-19 or post-COVID-19 subjects showed decreased Th17 levels, indicating a lack of tuberculosis development. Moreover, the typical course of tuberculosis is associated with an increase in Treg level, but COVID-19 contributes to a hyperinflammatory response. According to the data obtained, the course of tuberculosis proceeds in a dissimilar way due to the distinct immune response, elicited by SARS-CoV-2. Importantly, the development of active tuberculosis with a severe course is associated with a decline in Treg levels. Both pathogens lead to disturbed immune responses, increasing the risk of developing severe TB. The insights and findings of this paper may be used to improve the future management of individuals with latent and active tuberculosis.

Chlorella vulgaris Modulates Gut Microbiota and Induces Regulatory T Cells to Alleviate Colitis in Mice.

Chlorella vulgaris (C. vulgaris) is unicellular green algae consumed worldwide as a functional food. The immune stimulatory function of C. vulgaris is known; however, no study has elucidated its immune regulatory potential and associated microbiome modulation. In the current study, we aimed to validate the immune regulatory role of C. vulgaris mediated through two mechanisms. Initially, we assessed its ability to promote the expansion of the regulatory T cell (Treg) population. Subsequently, we investigated its impact on gut microbiota composition and associated metabolites. The supplementation of C. vulgaris altered the gut microbiota composition, accompanied by increased short-chain fatty acid (SCFAs) production in mice at homeostasis. We later used C. vulgaris in the treatment of a DSS-induced colitis model. C. vulgaris intervention alleviated the pathological symptom of colitis in mice, with a corresponding increase in Treg levels. As C. vulgaris is a safe and widely used food supplement, it can be a feasible strategy to instigate cross-talk between the host immune system and the intestinal flora for the effective management of inflammatory bowel disease (IBD).

Long-term changes of Th17 and regulatory T cells in peripheral blood of dogs with spinal cord injury after intervertebral disc herniation

BackgroundIntervertebral disc herniation (IVDH) is one of the most common causes of spinal cord injury (SCI) in dogs. As a result of acute SCI, a complex inflammatory response occurs in the spinal cord. Th17 cells (Th17) produce pro-inflammatory cytokines, while regulatory T cells (Treg) have opposite effects producing anti-inflammatory cytokines. Therefore, the aim of this study was to determine whether Th17- and Treg cells are involved in the pathogenesis of SCI or whether cellular changes occur due to coexisting inflammatory diseases. We hypothesized that chronic alterations in the Th17/Treg ratio are associated with a worse outcome after SCI.MethodsTwenty-six paretic or plegic dogs with IVDH with and without coexisting inflammatory disease were investigated in the acute stage of the disease and after recovery of SCI. In addition, a healthy control group was included (n = 14). Quantification of Th17 and Treg cells, from peripheral blood samples, was performed by multicolor flow cytometry and IL17 was measured using an enzyme-linked immunosorbent assay (ELISA).ResultsAfter recovery significantly higher levels of Th17 (p = 0.0265) and Treg cells (p = 0.00025) were detected compared to acute IVDH but Th17/Treg ratio did not differ significantly. Recovered dogs and the control group did not differ significantly from each other. No association between an imbalance in the ratio and neurologic severity or underlying inflammatory diseases was found.ConclusionThis study demonstrated that altered Th17 and Treg levels in peripheral blood are altered in the acute stage of IVDH, preexisting inflammatory diseases seem not to influence these cell populations. Th17 and Treg cells could be considered when evaluating new treatment strategies for SCI.

Yin Yang 1 impacts upon preeclampsia by regulating Treg/TH17 cells and PI3K/AKT pathway

Preeclampsia (PE) is a common obstetric syndrome with an unclear etiology and pathogenesis. The study here aimed to investigate the role of Yin Yang 1 (YY1) in PE, and to reveal any YY1-regulated mechanisms in PE. Peripheral blood, placenta, and endometrial tissues of PE patients, healthy volunteers, and patients who had undergone an elective Cesarean section and had a scarred uterus (control group) were collected for analyses. Rat PE models were established by lipopolysaccharide induction. Subsets of these rats were then made to over-express YY1. At 18 d after the PE was established, urine, blood, and placental tissues from all rats were collected. Levels of regulatory-T (Treg) and helper T-type 17 (TH17) cells in both human and rat blood were measured by flow cytometry. ELISA kits were used to evaluate blood levels of inflammatory factors (i.e. IL-6, IL-10, and IL-17) as well. RT-qPCR and Western blot assays were performed to quantify levels of forkhead box P3 (Foxp3), retinoic acid-related orphan receptor C (RORc), and YY1 in the human and rat placenta and endometrial tissues. Expressions of PI3K/AKT pathway-related proteins were also evaluated by Western blots. The results indicated that the PE patients, relative to levels in control group and the healthy control subjects, had decreased circulating levels of Treg cells/increased TH17 cells; tissues from these patients also had relatively-decreased FoxP3 mRNA and protein expressions and elevated RORc mRNA and protein expressions. YY1 was expressed only at low levels in the PE patient placenta and endometrial tissues. In rats, PE rats treated with over-expressed YY1 had (relative to in PE rats without over-induced YY1) increased circulating levels of Treg cells/decreased TH17 cells; tissues from these rats had elevated FoxP3 mRNA and protein expressions and reduced mRNA and protein RORc expressions, as well as indications of alleviated inflammation. In the rat placenta samples, YY1 was also determined to activate the PI3K/AKT pathway. In summary, YY1 regulates the balance among Treg/TH17 cells and so affect the PE process in part through activation of the PI3K/AKT pathway.

Immunomodulatory effects of canine mesenchymal stem cells in an experimental atopic dermatitis model.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multi-lineage cells, suggesting their future applicability in regenerative medicine and biotechnology. The immunomodulatory properties of MSCs make them a promising replacement therapy in various fields of animal research including in canine atopic dermatitis (AD), a skin disease with 10-15% prevalence. We investigated the immunomodulatory effects of MSCs in an experimental canine AD model induced by Dermatophagoides farinae extract ointment. Canine adipose tissue-derived MSCs (cAT-MSCs) were differentiated into mesodermal cell lineages at the third passage. Alterations in immunomodulatory factors in control, AD, and MSC-treated AD groups were evaluated using flow cytometric analysis, enzyme-linked immunosorbent assay, and quantitative reverse transcription PCR. In the MSC-treated AD group, the number of eosinophils decreased, and the number of regulatory T cells (Tregs) increased compared to those in the AD group. In addition, the immunoglobulin E (IgE) and prostaglandin E2 levels were reduced in the MSC-treated AD group compared to those in the AD group. Furthermore, the filaggrin, vascular endothelial growth factor, and interleukin-5 gene expression levels were relatively higher in the MSC-treated AD group than in the AD group, however, not significantly. cAT-MSCs exerted immunomodulatory effects in an AD canine model via a rebalancing of type-1 and -2 T helper cells that correlated with increased levels of Tregs, IgE, and various cytokines.

Combination of pembrolizumab and pelareorep promotes anti-tumour immunity in advanced pancreatic adenocarcinoma (PDAC).

We previously reported activity of pelareorep, pembrolizumab and chemotherapy. Patients developed new T-cell clones and increased peripheral T-cell clonality, leading to an inflamed tumour. To evaluate a chemotherapy-free regimen, this study assesses if pelareorep and pembrolizumab has efficacy by inducing anti-tumour immunological changes (NCT03723915). PDAC patients who progressed after first-line therapy, received iv pelareorep induction with pembrolizumab every 21-days. Primary objective is overall response rate. Secondary objectives included evaluation of immunological changes within tumour and blood. Clinical benefit rate (CBR) was 42% amongst 12 patients. One patient achieved partial response (PR) and four stable disease (SD). Seven progressed, deemed non-responders (NR). VDAC1 expression in peripheral CD8+T cells was higher at baseline in CBR than NR but decreased in CBR upon treatment.On-treatment peripheral CD4+Treg levels decreased in CBR but not in NR. Analysis of tumour demonstrated PD-L1+cells touchingCD8+T cells, and NK cellswere more abundant post-treatment vs. baseline. A higher intensity of PD-L1 intumour infiltrates at baseline, particularly in CBR vs. NR. Finally, higher levels of soluble (s)IDO, sLag3, sPD-1 observed at baseline among NR vs. CBR. Pelareorep and pembrolizumab showed modest efficacy in unselected patients, although potential immune and metabolic biomarkers were identified to warrant further evaluation.

The features of regulatory T-blood cells subpopulation and the level of CD25 expression in patients with Graves’ disease in dynamics after radioactive iodine therapy

BACKGROUND: The content of regulatory T cells (Treg) at different stages in formation of effector subpopulations and the level of CD25 expression on the membrane of their various fractions in Graves’ disease can determine the long-term autoimmune process persistence and be the target of immunotropic therapy of the disease.AIM: To study the features of regulatory T-blood cells subpopulation and the level of CD25 expression in patients with Graves’ disease in dynamics after radioactive iodine therapy (RIT) to identify the specific Treg subpopulations for potential immunotropic therapy targets of the disease.MATERIALS AND METHODS: A single-center, prospective, cohort, open, controlled study was conducted with the participation of women with laboratory-confirmed Graves’ disease. The features of regulatory T-blood cells subpopulation and the level of expression (MFI) CD25 surface receptor were studied by flow cytometry using direct immunofluorescence using monoclonal antibodies.RESULTS: The study included 36 women with recurrent Graves’ disease, middle age 46.34±14.32 years. In patients with Graves’ disease before and during the entire period after RIT a low percentage of naive (CD45R0-CD62L+) and terminally differentiated (CD45R0-CD62L-) Treg was established relative to the control, and on 3 and 6 months after RIT a significant decrease of cells with this phenotype was revealed relative to the values detected in patients before and 1 month after RIT (p&lt;0.001). Against the background of compensated hypothyroidism the most significant changes of expression CD25 receptor in patients with Graves’ disease were found on 3 and 6 months after RIT: reduced levels of MFI CD25 on surface of naive and terminally differentiated Treg.CONCLUSION: A decrease in the level of naive Treg was found (apparently due to a violation of differentiation processes in thymus) and terminally differentiated Tregs (due to maturation and survival processes), which are supplemented by a reduced expression of the CD25 receptor on the surface of these cells and do not depend on hyperthyroidism compensation, the titer of TSH receptor antibodies, previous conservative therapy with thiamazole and RIT. The obtained new data reveal the role of naive and terminally differentiated Treg subpopulations in immunopathogenesis and help to outline further ways to develop approaches for immunotropic therapy.

Modification of peripheric Treg and CD56brightNK levels in RIF women after egg donation, treated with GM-CSF or placebo

Recurrent implantation failure (RIF) is defined as when implantation repeatedly failed to reach a stage recognizable by pelvic ultrasound in IVF cycle and it may be due to several causes. The GM-CSF is a cytokine promoting leukocyte growth and trophoblast development: we tested it to treat these patients in a pilot-controlled trial evaluating the modification of peripheric Treg and CD56brightNK levels after the treatment with this cytokine and in control patients affected by RIF after egg donation cycles. This study was performed on 24 RIF women after egg donation cycles. Single good quality blastocyst transfer was performed in the cycle object of this study. Patients were randomly assigned to two groups: 12 women treated with subcutaneous GM-CSF 0.3 mg/kg/daily from the day before embryo transfer to the β-hCG day, and 12 women treated with subcutaneous saline solution infusion as control. All patients were tested for Treg and CD56brightNK cell levels in blood circulation before and after treatment using flow-cytometry with specific antibodies. The two groups of patients were similar for epidemiologic characteristics, the ongoing pregnancy rate in the GM-CSF group was 83.3% whereas in the control group was 25.0% (P = 0.0123). In the study group there was a significative increase of Treg cells (P < 0.001) with respect to the levels before treatment and to control group. Instead, the levels of CD56brightNK did not show any significative variation. Our study showed that the treatment with GM-CSF increases the Treg cells in the peripheric blood.

Intensity- and time-matched acute interval and continuous endurance exercise similarly induce an anti-inflammatory environment in recreationally active runners: focus on PD-1 expression in Tregs and the IL-6/IL-10 axis.

Acute exercise elicits a transient anti-inflammatory state during the early recovery period. Since recent studies reported on regimen-specific effects on immune-related humoral factors and cellular subsets, this study compared the effects of intensity- and time-matched acute interval and continuous exercise on peripheral anti-inflammatory cellular and humoral immune parameters with a particular focus on the PD-1 expression in CD4+ regulatory T cells (Tregs). Twenty-four recreationally active runners (age: 29.7 ± 4.3years, BMI: 22.2 ± 2.4, VO2peak: 56.6 ± 6.4ml×kg-1×min-1) participated in this crossover RCT. Each subject conducted a moderate continuous (MCE) and a high-intensity interval exercise (HIIE) session in a counterbalanced design. Blood was drawn before, immediately after, and 1 h after exercise. Treg subsets and levels of PD-1 and Foxp3 were assessed by flow cytometry. Serum levels of IL-10 and IL-6 were quantified by ELISA. PD-1 levels on Tregs increased within the recovery period after HIIE (p < .001) and MCE (p < 0.001). Total counts of Tregs (HIIE: p = 0.044; MCE: p = .021), naïve Tregs (HIIE: p <0.001; MCE: p <0.001), and PD-1+ effector Tregs (eTregs) (HIIE: p = .002) decreased 1 h after exercise. IL-10 increased 1 h after HIIE (p < 0.001) and MCE (p = 0.018), while IL-6 increased immediately after both HIIE (p = 0.031) and MCE (p = 0.021). Correlations between changes in IL-6 and IL-10 (p = 0.017, r = 0.379) and baseline VO2peak and Treg frequency (p = 0.002, r = 0.660) were identified. This is the first study that investigates PD-1 expression in circulating Tregs after acute exercise, revealing an increase in PD-1 levels on eTregs during the early recovery period after intensity- and time-matched HIIE and MCE. Future studies are needed to investigate the PD-1 signalosome in eTregs, together with the expression of key effector molecules (i.e., IL-10, TGF-β, IL-35, CTLA-4) to elucidate PD-1-dependent changes in cellular function. Based on changes in serum cytokines, this study further reveals a regimen-independent establishment of an anti-inflammatory milieu and underpins the role of the IL-6/IL-10 axis.

Effect and Mechanism of Sodium Butyrate on Neuronal Recovery and Prognosis in Diabetic Stroke.

Ischemic stroke is a cerebrovascular lesion caused by local ischemia and hypoxia. Diabetes mellitus (DM) is a chronic inflammatory disease that disturbs immune homeostasis and predisposes patients to ischemic stroke. The mechanism by which DM exacerbates stroke remains unclear, although it may involve disturbances in immune homeostasis. Regulatory T cells (Tregs) play a regulatory role in many diseases, but the mechanism of Tregs in diabetes complicated by stroke remains unclear. Sodium butyrate is a short-chain fatty acid that increases Treg levels. This study examined the role of sodium butyrate in the prognosis of neurological function in diabetic stroke and the mechanism by which Tregs are amplified in the bilateral cerebral hemispheres. We evaluated the brain infarct volume, observed 48-h neuronal injury and 28-day behavioral changes, and calculated the 28-day survival rate in mice. We also measured Treg levels in peripheral blood and brain tissue, recorded changes in the blood‒brain barrier and water channel proteins and neurotrophic changes in mice, measured cytokine levels and peripheral B-cell distribution in bilateral hemispheres and peripheral blood, and examined the polarization of microglia and the distribution of peripheral T-cell subpopulations in bilateral hemispheres. Diabetes significantly exacerbated the poor prognosis and neurological deficits in mice with stroke, and sodium butyrate significantly improved infarct volume, prognosis, and neurological function and showed different mechanisms in brain tissue and peripheral blood. The potential regulatory mechanism in brain tissue involved modulating Tregs/TGF-β/microglia to suppress neuroinflammation, while that in peripheral blood involved improving the systemic inflammatory response through Tregs/TGF-β/T cells.

#5000 DIFFERENCES IN CIRCULATING IMMUNE CELLS PROFILE BETWEEN MALES WITH CARDIORENAL SYNDROME TYPE II AND CKD PATIENTS WITHOUT CARDIOVASCULAR DISEASE

Abstract Background and Aims Maladaptive activation of the immune system plays an important role in the pathogenesis of cardiovascular disease (CVD) and chronic kidney disease (CKD). The immune system components can act as mediators of organ cross-talk and may be involved in the reciprocal dysfunction that occurs in cardiorenal syndromes (CRS). The aim of our study was to investigate potential differences in blood levels of specific immune cells subsets between a cohort of type II CRS patients and CKD patients without established CVD. Method 40 stable male patients with CRS type II and 30 male CKD patients without CVD, matched for eGFR (CKD-EPI) were enrolled in this cross-sectional study. Exclusion criteria were history of malignancy, autoimmunity and active or chronic infections. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16+ and CD14+CD16++ absolute values and percentages out of total monocytes and NK cells (CD3+CD16+56+), CD3-CD19+ B lymphocytes, CD3+ CD4+ T cells, CD3+CD8+ T cells and Tregs (CD4+CD25+FoxP3+) absolute values and percentages out of total lymphocytes were measured by flow cytometry. Simultaneously, clinical, laboratory and echocardiographic data were assessed. Multiple regression analyses to assess independent relationships were performed by building separate models with each of the immune cells as the dependent variable and respective univariate correlates including patient group. Results Mean age of patients with CRS and CKD was 72±10 versus 66±10 years respectively (p = 0.01), mean eGFR was 37±14 and 33±16ml/min/1.73 m2 respectively (p = 0.28) and median urinary protein to creatine ratio (UPCR) was 0.19 (IQR, 0.10-0.52) versus 1.03 (IQR, 0.17-2.09) gr protein/gr creatinine (p = 0.02) respectively. CRS patients displayed increased levels of pro-inflammatory, intermediate CD14++CD16+ monocytes [41 (IQR, 24-78)/µL] compared to their CKD counterparts [35 (IQR, 18-43)/µL] (p = 0.04). A higher Tregs percentage was found in CRS patients [2.7% (IQR, 2.0%-3.9%)] compared to CKD patients [2.0% (IQR, 1.6%-2.6%)] (p = 0.03). Lower mean levels of lymphocytes were observed in CRS patients (1557±691/µL) compared to the CKD cohort (1920±545/µL) (p = 0.04). Finally, CRS patients displayed lower NK cell counts [148 (IQR, 103-258)/µL] compared to CKD patients [324 (IQR, 179-368)/µL] (p = 0.001). Age and UPCR did not correlate with immune cells subsets in the whole cohort, neither in the two groups separately. At multivariate regression analysis, the differences in immune cells between the two groups remained statistically significant. Specifically, in patients with CRS the intermediate CD14++CD16+ monocytes counts correlated positively with CRP (p = 0.002) and ESR (p = 0.02). A positive correlation was found between eGFR and total lymphocytes (p = 0.009), T cells (p = 0.005) and CD4+ T cells (p = 0.005) counts. The number and percentage of nonclassical CD14+CD16++ monocytes were higher in CRS patients with left ventricular ejection fraction (LVEF) less than 30% compared to patients with LVEF above 30% [33 (IQR, 18-37)/µL versus 13 (IQR, 10-29)/µL (p = 0.02) and 4.5% (IQR, 3.4%-7.2%) versus 2.7% (IQR, 1.9%-5.4%) (p = 0.03) respectively]. With regard to etiology, patients with dilated cardiomyopathy compared to patients with ischemic CVD displayed increased counts of intermediate CD14++CD16+ monocytes [75 (IQR, 41-104)/µL versus 36 (IQR, 22-61)/µL (p = 0.01)] and non-classical CD14+CD16++ monocytes [37 (IQR, 35-49)/µL versus 21 (IQR, 12-32)/µL (p = 0.02)]. Finally, NK cells and Tregs levels were lower in patients with atrial fibrillation compared to those without [133 (IQR, 79-173)/µL versus 260 (IQR, 151-314)/µL (p = 0.01)] and [32 (IQR, 21-43)/µL vs 47 (IQR, 34-85)/µL (p = 0.006)] respectively. Conclusion Patients with CRS type II exhibit alterations of the immune cells subsets profile in the peripheral blood compared to CKD patients of similar kidney function but without CVD. Our findings suggest that distinct immune mechanisms might be involved in the pathogenesis or the chronic course of CRS type II as compared to CKD. Future research is required to evaluate their pathophysiological or prognostic significance.

Combinatorial analysis of CD4+Tregs and CD8+Teff to predict response to ICI in patients with ES-SCLC.

e20633 Background: Recently, the antiPD-L1 agent, durvalumab, was approved in first line treatment in combination with chemo. However, there is not a clear benefit from this regimen for the majority of patients. In this study, we explored the predictive role of regulatory T-cells in response of patients with ES-SCLC treated with the aforementioned regimen. Moreover, we evaluated the potential role of inflammatory hematological indices such as Neutrophil to Lymphocyte ratio (NLR) and CRP levels to predict responders. Methods: Blood was collected before initiation of immunotherapy from 24 patients with ES-SCLC. PBMCs were isolated with Ficoll density gradient centrifugation from patients and 1O healthy donors (HD). Immunophenotyping was performed by multi-color flow cytometry using antibodies against CD3,CD8,CD45RA,CD45RO,CCR7,PD-1, for CD8 T-cells and CD3,CD4,FoxP3,CD25,CD127,CTLA-4,CD39 for Tregs. Results: High percentages of CD39+ (p = 0.0061) and CTLA-4+ (p = 0.012) Tregs were detected in circulation of ES-SCLC compared to healthy individuals. Additionally, higher percentages of CD8+ Teff were detected in SCLC patients (p = 0.027). Patients with high numbers of Teff had significant higher PFS (p = 0.021, median 183 and 97 days) and better OS (p = 0.075, median 318 and 202 days) in response to immunotherapy compared to those with low numbers. Spearman analysis revealed a significant negative correlation between Tregs and Teff (Spearman r2 = -0.39,p = 0.043) that was accompanied with significant higher PFS (p = 0.041, median 207 and 130 days ) and OS (p = 0.029, median 318 and 202 days ) in patients with high levels of Teff and low Tregs comparing to the rest. Further analysis on inflammatory clinical signatures demonstrated that patients with better clinical response to immunotherapy have significantly lower NLR (p = 0.034) while no significant difference was revealed for CRP (p = 0.401) levels before therapy initiation. Conclusions: Combinatorial analysis of Tregs and Teff cells may be used as a predictive tool for response to immunotherapy in ES-SCLC patients.

Dynamics of T helper subpopulations in the critical period of severe injury in children

Severe mechanical injury is one of the main reasons behind children’s disability and mortality. Severe injury induces a complex host immune response to tissue injury, a parallel pro- and anti-inflammatory state, bearing an elevated risk for infectious complications (IC) and/or multiple organ failure (MOF). This study aimed to determine the informative immunological criteria of traumatic injury severity and prognosis outcome in children (severe injury group (SInj, ISS ≥ 16), n = 87; mild/moderate injury group (MInj, ISS &lt; 16), n = 34) based on the assessment of absolute cell count (abs) and percentage of such T helper subpopulations as regulatory T lymphocytes – CD4+CD127lowCD25high(Treg), Th17 lymphocytes – CD4+CD161+ and CD4+CD127higtCD25high T cells(T127hi) in severe injury cases grouped by the outcome (favorable, n = 47; unfavorable, n = 40) and depending on IC (n = 16) and the development of MOF (n = 11) on the 1st, 3d , 5th, 7th, 14th day after injury. The control group was comprised of 80 apparently healthy children comparable in age and sex. An inverse relationship between severity of injury, degree of blood loss and outcome of injury was revealed with the abs of all Th populations, but for Th abs and Treg abs the most significant correlation was found (Spearman’s R ≤ -0,70, p &lt; 0.00001). For SInj group, a pronounced decrease of Th abs, Treg abs, T127hi abs and Th17 abs, in the acute post-traumatic period with an increase to 14 days was revealed. The values of in the first day for indicators of patients with MInj group correspond to the values of control group and significantly differ from SInj group. There are different kinetics of percentage Th subpopulations in peripheral blood of children with severe injuries. The Th17%CD4+ and T127hi%CD4+ significant increase in 1st-3d and 3d -7th days after injury respectively in comparation with сontrol and MInj groups. There were no differences between groups in terms of Treg%CD4+. The lower-level Treg abs in trauma patients admitted to the ICU is significantly associated with develop the infectious complications and outcome of trauma. The Th17 abs is significantly reduced in 3-7th days after the injury in the SInj group with MOF. The results of the study indicate that in children levels of Treg, T127hi and Th17 is significantly associated with severity of injury and may be used to predict outcome of trauma and assess the risk of IC and MOF.

Effects of rIL2/anti-IL2 antibody complex on chikungunya virus-induced chronic arthritis in a mouse model

Chikungunya virus (CHIKV) is characterized by disabling joint pain that can cause persistent arthritis in approximately one-fourth of patients. Currently, no standard treatments are available for chronic CHIKV arthritis. Our preliminary data suggest that decreases in interleukin-2 (IL2) levels and regulatory T cell (Treg) function may play a role in CHIKV arthritis pathogenesis. Low-dose IL2-based therapies for autoimmune diseases have been shown to up-regulate Tregs, and complexing IL2 with anti-IL2 antibodies can prolong the half-life of IL2. A mouse model for post-CHIKV arthritis was used to test the effects of recombinant IL2 (rIL2), an anti-IL2 monoclonal antibody (mAb), and the complex on tarsal joint inflammation, peripheral IL2 levels, Tregs, CD4 + effector T cells (Teff), and histological disease scoring. The complex treatment resulted in the highest levels of IL2 and Tregs, but also increased Teffs, and therefore did not significantly reduce inflammation or disease scores. However, the antibody group, which had moderately increased levels of IL2 and activated Tregs, resulted in a decreased average disease score. These results suggest the rIL2/anti-IL2 complex stimulates both Tregs and Teffs in post-CHIKV arthritis, while the anti-IL2 mAb increases IL2 availability enough to shift the immune environment towards a tolerogenic one.