Expression Levels Of Foxp3 mRNA Research Articles

CD4+ T-cell Subsets and Cytokine Signature in Pemphigus Foliaceus Clinical Stratification beyond the th1/Th2 Paradigm.

T helper interplay and cytokines monitoring in auto-immune skin disorders such as Pemphigus Foliaceus [PF] may play a central role in predicting the clinical stratification of the pathology. In order to assess the CD4+ T cell imbalance, [i] this study aims to assess the related immune cells [Th1, Th2, Th17, and Treg cells] as well as the related cytokines [IL-1β, IFNγ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-17F, IL- 22, TNF-β, and TNFα] in peripheral blood, and [ii] their respective transcription factors in the lesioned skin of PF endemic patients during the clinical course. Peripheral blood of 22 PF patients was analyzed by flow cytometry to assess the functional associations of Th cell subpopulations and their characteristic cytokines by multiplex bead assay of 14-plex cytokines. Skin mRNA expression of their associated transcription factors was analyzed using the TaqMan detection system. Our findings revealed that the CD4+ T cell subtypes in PF patients compared to Healthy Controls [HC] were characterized by [i] a similar Th1/Th2 ratio and increased Th17/Treg ratio and [ii] significantly higher plasma levels of Th-17 specific cytokines; IL- 6, IL-8, IL-17A. Higher percentages in Th17 and Treg subtypes and a significant increase in plasma IL-17F levels were maintained in relapsing PF patients, arguing the pivotal role of Th17 cells in PF pathogenesis. Furthermore, our findings pointed out the major contribution of the pro-inflammatory cytokine IL-6. Indeed, in addition to being involved in the initial stages of disease development, IL-6 seems to also be involved in the maintenance of the pathophysiological process, probably through its effect on Th17 differentiation. The skin-relative mRNA expression levels of FOXP3 and TBET were significantly higher in relapsing PF patients compared to de novo PF patients. Our results highlight the central role played by Th17 lymphocytes and their related pro-inflammatory cytokines during the clinical course of the disease, reversing the Th1/Th2 dichotomy in PF.

Bifidobacterium breve CCFM1310 enhances immunity in immunosuppressed mice via modulating immune response and gut microbiota

There has been a rising interest in studying how Bifidobacterium breve can affect the immune system over the years. The aim of this study was to evaluate the effect of B. breve on the immune system and to explore the mechanism in a mouse model of cyclophosphamide (CTX)-induced immunosuppression. The results of the study showed that B. breve CCFM1310 significantly increased the thymus index (p < 0.05), promoting the secretion of immunoglobulins and the number of immune cells in the blood. Moreover, B. breve CCFM1310 could repair the spleen injury. It increased the mRNA expression level of T-bet (p < 0.05) and decreased the mRNA expression level of TGF-β and FOXP3 (p < 0.05). B. breve CCFM1310 alleviated oxidative stress by increasing enzymatic activities of SOD, CAT and T-AOC and reducing the concentration of MDA. Moreover, it repaired intestinal damage by increasing the number of goblet cells, upregulating mRNA expression of tight junction proteins, and promoting the production of short-chain fatty acids (SCFA). B. breve CCFM1310 could regulate the gut microbiota by increasing the relative abundance of Lactobacillus (p < 0.05) and decreasing the relative abudance of LachnospiraceaeNK4A136 group. Overall, our study suggests that B. breve CCFM1310 could enhance immunity in CTX-induced immunosuppressed mice by affecting immune responses, oxidative stress and gut microbiota.

MicroRNA‑155‑5p affects regulatory T cell activation and immunosuppressive function by targeting BCL10 in myasthenia gravis.

The imbalance in immune homeostasis plays a crucial role in the pathogenesis of myasthenia gravis (MG). MicroRNAs (miRs) have been identified as key regulators of immune homeostasis. B-cell lymphoma/leukemia 10 (BCL10) has been implicated in the activation and suppressive function of regulatory T cells (Tregs). This study aimed to investigate the potential role of miR-155-5p in modulating the activation and function of Tregs in MG. To achieve this objective, blood samples were collected from MG patients to assess the expression levels of miR-155-5p and BCL10, as well as the proportion of circulating Tregs, in comparison to healthy controls. The correlation between miR-155-5p and BCL10 levels was evaluated in human samples. The expression levels of miR-155-5p and the numbers of circulating Tregs were also examined in an animal model of experimental autoimmune MG (EAMG). A dual-luciferase reporter assay was used to verify whether miR-155-5p can target BCL10. To determine the regulatory function of BCL10 in Tregs, CD4+ CD25+ Tregs were transfected with either small interfering-BCL10 or miR-155-5p inhibitor, and the expression levels of the anti-inflammatory cytokine IL-10 and transcription factors Foxp3, TGF-β1, CTLA4, and ICOS were measured. The results demonstrated that the expression level of miR-155-5p was significantly higher in patients with MG compared with that in healthy controls, whereas the expression level of BCL10 was significantly decreased in patients with MG. Furthermore, there was a significant negative correlation between the expression levels of miR-155-5p and BCL10. The number of circulating Tregs was significantly reduced in patients with MG and in the spleen of rats with EAMG compared with that in the corresponding control groups. The dual-luciferase reporter assay demonstrated that miR-155-5p could target BCL10. The Tregs transfected with si-BCL10 demonstrated significant decreases in the protein levels of TGF-β1 and IL-10, as well as in the mRNA expression levels of Foxp3, TGF-β1, CTLA-4 and ICOS. Conversely, the Tregs transfected with the miR-155-5p inhibitor exhibited a substantial increase in these protein and mRNA expression levels compared with their respective control groups. Furthermore, the knockdown of BCL10 exhibited a decline in the suppressive efficacy of Tregs on the proliferation of CD4+ T cells. Conversely, the suppression of miR-155-5p expression attenuated the inhibition of the BCL10 gene, potentially causing an indirect influence on the suppressive capability of Tregs on the proliferation of CD4+ T cells. BCL10 was thus found to contribute to the activation and immunosuppressive function of Tregs. In summary, the present study demonstrated that miR-155-5p inhibited the activation and immunosuppressive function of Tregs by targeting BCL10, which may be used as a future potential target for the treatment of MG.

Histamine H4 Receptor Antagonist Ameliorates the Progression of Experimental Autoimmune Encephalomyelitis via Regulation of T-Cell Imbalance.

Multiple sclerosis (MS) is a degenerative condition characterized by immune-mediated attacks on the central nervous system (CNS), resulting in demyelination and recurring T-cell responses. The histamine H4 receptor (H4R) is mainly expressed in cellular populations and plays a vital role in inflammation and immunological responses. The role of H4R in neurons of the CNS has recently been revealed. However, the precise role of H4R in neuronal function remains inadequately understood. The objective of this work was to investigate the impact of JNJ 10191584 (JNJ), a highly effective and specific H4R antagonist, on the development of experimental autoimmune encephalomyelitis (EAE) and to gain insight into the underlying mechanism involved. In this study, we examined the potential impact of JNJ therapy on the course of EAE in SJL/J mice. EAE mice were administered an oral dose of JNJ at a concentration of 6 mg/kg once a day, starting from day 10 and continuing until day 42. Afterward, the mice's clinical scores were assessed. In this study, we conducted additional research to examine the impact of JNJ on several types of immune cells, specifically Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγt), and regulatory T (Tregs; Foxp3 and TGF-β1) cells in the spleen. In this study, we further investigated the impact of JNJ on the mRNA expression levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγt, Foxp3, and TGF-β1 in the brain. Daily treatment of JNJ effectively reduced the development of EAE in mice. The percentages of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, and CD4+RORγt+ cells were shown to decrease, whereas the percentages of CD4+TGF-β1+ and CD4+Foxp3+ cells were observed to increase in EAE mice treated with JNJ. Therefore, the HR4 antagonist positively affected the course of EAE by modulating the signaling of transcription factors. The identified results include possible ramifications in the context of MS treatment.

Analysis the effect of cytokine-induced killer cells combined with mFOLFOX6 regimen in chemotherapy for advanced colorectal cancer

Objective: To investigate the effect of cytokine-induced killer cells (CIK) combined with mFOLFOX6 regimen (fluorouracil+oxaliplatin+calcium folinate) on advanced colorectal cancer. Methods: 161 patients with advanced colon cancer admitted to the Fourth Clinical College of Xinxiang Medical College, Xinxiang Central Hospital of Henan Province from 2019 to 2021 were selected and divided into the study group (mFOLFOX6 regimen chemotherapy+CIK therapy) with 80 cases and the control group (mFOLFOX6 regimen chemotherapy) with 81 cases. The KPS score, median survival time, progression-free survival time, the proportion of Treg in peripheral blood mononuclear cells, the levels of CD4+, CD4+/CD8+, the expression levels of Foxp3 and CD127 mRNA, the quality-of-life score and the occurrence of toxic and side effects were compared between the two groups before and after chemotherapy. Results: The ages of patients in the study group and the control group were (64.8±7.5) and (66.1±7.0) years old, respectively, and the proportions of males were 62.5% (50 cases) and 50.6% (41 cases), respectively (both P values>0.05). After 2 cycles of chemotherapy and at the end of chemotherapy, the proportions of Treg, Foxp3 and CD127 mRNA in the study group were lower than those in the control group [2 cycles of chemotherapy: (4.33±0.86)% vs (4.89±1.20)%, (0.61±0.22) vs (0.73±0.20), (0.58±0.13) vs (0.70±0.15); after chemotherapy: (2.66±0.70)% vs (3.31±0.84)%, (0.43±0.18) vs (0.51±0.16), (0.41±0.10) vs (0.50±0.13)] (all P values<0.05). The KPS scores of the study group were higher than those of the control group [2 cycles of chemotherapy: (68.41±5.41) points vs (65.38±5.11) points; after chemotherapy: (72.08±5.94) points vs (67.95±5.51) points] (all P values<0.05). The median survival time of the study group was (16.8±2.2) months, which was higher than that of the control group [(11.2±1.8) months]. The progression-free survival time of the study group was also higher than that of the control group [(9.5±1.1) months vs (6.4±1.2) months], and the mortality rate was lower than that of the control group [11.3% (9 cases) vs 31.3% (25 cases)] (all P values<0.001). After chemotherapy, the scores of physical function, emotional function and role function in the study group were higher than those in the control group, and the scores of pains, fatigue and insomnia in the study group were lower than those in the control group (all P values<0.05). Conclusions: CIK combined with mFOLFOX6 regimen for advanced colorectal cancer can improve and regulate the immune function of patients, prolong the survival time of patients, and enhance the quality of life of patients.

Treg Cells, FoxP3 and TGF-β Expression and Significance in Chronic Myeloid Leukemia

To investigate the expression and significance of regulatory T cells (Tregs), FoxP3 and transforming growth factor-β (TGF-β) in different phase of chronic myeloid leukemia (CML). Peripheral blood of 73 CML patients in Department of Hematology, Heze Municipal Hospital from March 2018 to March 2021 were collected. According to patient's period in CML, they were divided into ND CML group (newly diagnosed), CP CML group (chronic period), and BP CML group (blast phase). The percentage of Tregs, expression level of FoxP3 mRNA and TGF-β were detected by flow cytometry, RT-qPCR, and ELISA, respecitively. The roles of above indices in clinical pathogenesis of patients with CML were analyzed. The proportion of Treg in the ND CML group was slightly higher than the CP CML group, but the difference was not statistically significant (P =0.695), while the BP CML group was significantly higher than the other two groups (P =0.008, P <0.001). The expression levels of FoxP3 mRNA in ND CML group, CP CML group and BP CML group were 11.61±2.21, 6.46±1.35 and 8.54±2.13, respectively. Significant difference in FoxP3 mRNA levels was observed among patients in different phases of CML (F =55.199, P <0.001). The expression levels of FoxP3 mRNA both in ND CML group and BP CML group were significantly higher than that in CP CML group (P <0.001), and the ND CML group was the highest (P <0.001). However, the expression levels of TGF-β in different phases of CML showed no statistical differences (H =0.634, P =0.728). The abnormal distribution of Treg subset in different phases of CML and the significant increase of the expression level of FoxP3 mRNA in the new onset and blast phase of CML suggest that Tregs may promote the occurrence and progression of CML through immune regulation.

Imbalance of follicular regulatory T (Tfr) cells/follicular helper T (Tfh) cells in adult patients with primary immune thrombocytopenia.

This study is to investigate the role of follicular regulatory T (Tfr) cells/follicular helper T (Tfh) cells imbalance in adult patients with primary immune thrombocytopenia (ITP). Totally, 40 cases of primary ITP patients and 30 healthy controls were enrolled. Blood samples were collected from ITP patients (pre- and post-therapy) and controls. Flow cytometry was used to detect the proportion of Tfr and Tfh cells in peripheral blood. Real-time quantitative polymerase chain reaction (PCR) was performed to detect the mRNA expression levels of FOXP3, BCL-6, and BLIMP-1. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect interleukin (IL)-10 and IL-21 levels. Spearman's correlation was used for correlation analysis. Compared with control, Tfr cell proportion, FOXP3 mRNA, and IL-10 were significantly decreased in the pre-therapy ITP group, but were significantly increased post-therapy. Tfh cell proportion, BCL-6 mRNA, and IL-21 were increased, while BLIMP-1 mRNA was decreased, in the pre-therapy ITP group than the control group. These effects were reversed in the post-therapy ITP group. Moreover, the Tfr/Tfh ratio was decreased in the pre-therapy ITP group than control group, whereas was increased in the post-therapy ITP group than the pre-therapy ITP group. Furthermore, Tfr cell proportion, FOXP3 mRNA, IL-10, and Tfr/Tfh ratio were positively correlated with the platelet count (PLT) in the ITP pre-therapy group. In addition, Tfh cell proportion, BCL-6 mRNA, and IL-21 were negatively correlated with the PLT, while BLIMP-1 mRNA was positively correlated with the PLT. Conclusively, Tfr cell proportion in peripheral blood is decreased and Tfh cell proportion is increased, leading to unbalanced Tfr/Tfh ratio in ITP patients pre-therapy. The imbalance of Tfr/Tfh is recovered post-therapy, suggesting that the Tfr and Tfh cells may be involved in ITP pathogenesis. The abnormal expression of FOXP3, BCL-6, and BLIMP-1 mRNA and the changes in IL-10 and IL-21 levels may be related to the imbalance of Tfr/Tfh.

Profiling of Immunomodulatory Genes and Quantification of CD25+ Cells in Different Types of Early Pregnancy Loss.

Maternal regulatory T (Treg) cells play a pivotal role in establishing general immune homeostasis in the decidua for maintenance of pregnancy. We aimed in this study to investigate the relationship between mRNA expression of immunomodulatory genes and CD25+ Treg cells with early pregnancy losses. Our study included 3 groups of early pregnancy losses including sporadic spontaneous abortions, recurrent spontaneous abortions, sporadic spontaneous abortions post IVF treatment and the control group. We performed RT-PCR for analyzing mRNA expression levels of 6 immunomodulatory genes and CD25 immunohistochemistry for quantification of Treg cells. Only FOXP3, CD274 (PDL1), and TGFβ1 mRNA expression levels were significantly decreased in the miscarriage groups in comparison to the control group, whereas there was no significant mRNA expression change of CD4, IL2RA, and IL10. We also found significantly lower number of CD25+ cells in the miscarriages. We conclude that decreased expression of FOXP3 and PD-L1 may play a significant role in the pathogenesis of spontaneous abortion cases whereas decreased expression of TGFβ1 gene may be associated with the occurrence of early loss in IVF-treated pregnancies. Additional immunoprofiling of Treg cell population is needed to quantify Treg cells in early pregnancy losses.

Upregulation of FOXP3 may act on immunological misbalance in Turner syndrome

AbstractTurner syndrome (TS) presents a characteristic immune imbalance among patients. As the X chromosome‐linked FOXP3 gene is a powerful immune regulator, in this work we investigated a possible relationship between FOXP3 and TS. We evaluated 117 TS patients to investigate whether genetic associations within polymorphisms in FOXP3 (rs3761548, rs3761549) were associated with immune conditions. TaqMan Assays genotyping probes were employed in a real‐time PCR‐based method. Subsequently, we compared FOXP3 mRNA expression levels in TS patients and healthy females (control group), using fluorogenic probes through qPCR assay. We detected that the T allele from rs3761549 FOXP3 SNP was significantly associated with chronic inflammatory diseases in TS patients (P = 0.008, OR = 0.17, CI = 0.02‐0.71). Further, we observed an upregulation of FOXP3 mRNA levels in the TS group compared to the HC group (+9.21 FC; P = 2.541 × 10−7). In conclusion, our results demonstrated a differential distribution of the T rs3761549 allele in chronic inflammatory diseases among TS patients, as well as that FOXP3 upregulation may influence appropriate immune or inflammatory regulation in these patients.

404.7: Study on the Regulatory Effect of iPS-Derived Exosomes on the Local Immune Microenvironment in DCD Kidney Transplanted Rats

Introduction: Ischemia reperfusion injury (IRI) of donation After Cardiac death (DCD) transplanted kidney is the key factor of acute renal injury, acute and chronic rejection of renal transplantation and transplanted kidney disease.The latest researches show that mesenchymal stem cells (MSCs) have the function of protecting damaged tissues and organs and strong immune regulation. Induced pluripotent stem (iPS) cells have higher proliferative capacity and exosome secretion potential than MSCs. In addition to the functions of stem cells, exosomes have the unique ability to cross the blood-brain barrier and deliver remotely, which means that exosomes have better advantages in clinical disease treatment compared with the therapeutic potential of stem cells. Therefore, the main purpose of this study is to clarify the role of IPS derived exosomes in protecting the IRI of DCD transplanted kidney and regulating its local immune microenvironment. Methods: The iPS cells were passaged to the third passage and massively expanded. After 48 hours of culture, 100 mL of cell culture supernatant was collected and exosomes were extracted from the supernatant by the kit method, and the morphology and surface membrane marker proteins of the exosomes were identified. The SD rats were established with DCD renal transplantation model and 10, 50, and 100 μg of exosomes were infused into that SD rats respectively, and a PBS control group was set at the same time. On the 3rd, 5th, 7th, 14th and 28th days after transplantation, the content of cytokines in the serum of the recipient rats, the proportion of lymphocyte subsets CD4+CD25+Foxp3+Tregs in the spleen and the relative expression of Foxp3 mRNA were detected. The proliferation of renal tubular cells was detected by PCNA immunofluorescence. The pathological changes of renal tissue were determined by HE staining, and the apoptosis of renal tubular epithelial cells was detected by Tunel staining. The infiltration of inflammatory cells was detected by immunohistochemistry. The expression changes of regulatory T cells in renal tissue and macrophage phenotypic characteristics were analyzed by flow cytometry. Results: The experimental amount of exosomes was successfully obtained, and the morphology was observed by transmission electron microscope. The exosomes were round or oval membranous vesicles with uniform size, and the edges were clearly visible. The TSG101, CD63, and CD9 proteins were highly expressed on the exosome membrane surface by western-blot. A DCD rat kidney transplantation model was successfully established. The expression levels of IL-12, IL-2, IFN-γ, and TNF-α in the serum of each group were increased to varying degrees from the 3rd day after operation. On the 3rd and 7th days, the levels of IL-12, IL-2, IFN-γ, and TNF-α in the 100 μg exosome infusion group were significantly lower than those in the 10 μg, 50 μg and PBS groups (P<0.05), and the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than 10μg, 50μg and PBS groups (P<0.05). On the 28th day, the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than other groups (P<0.01). Compared with other groups, the CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs accounted for the highest proportion of CD4+T cells in the spleen of the 100μg infusion group (P<0.05). On the 28th day, the expression level of Foxp3 mRNA in the 100 μg infusion group was significantly higher than that in the 50 μg group (P<0.05). The stainings of PCNA, HE and Tunel showed that SD rats infused with 100μg exosomes could significantly reduce the pathological damage, inflammatory response and apoptosis of renal tubular epithelial cells in the transplanted kidney, promote the proliferation of renal tubular epithelial cells, and have a protective effect on IRI kidney. On the 28th day, the expression of CD4+CD25+Foxp3+Tregs cells in the transplanted kidney tissue was significantly increased, which further promoted the differentiation of macrophages into the immunomodulatory M2 subset. Conclusions: The infusion of iPS-derived high-dose exosomes is involved in the protection of the DCD kidney transplant during IRI process, and alleviates the acute and chronic injury of the DCD kidney transplant, and promotes the formation of the local immunosuppressive microenvironment in the DCD kidney transplantation. The National Natural Science Foundation of China (No.81900686).

Prognostic value of regulatory T cells and T helper 17 cells in high grade serous ovarian carcinoma

PurposeIn recent years the tumor microenvironment and its interaction with the tumor has emerged into research focus with increased attention to the composition of Tumor-infiltrating lymphocytes. We wanted to quantify the composition of Regulatory T cells (Tregs) and T helper 17 cells (Th17 cells) and their prognostic impact in high-grade serous tubo-ovarian carcinoma.MethodsTregs and Th17 cells were determined by immunohistochemical analysis of CD25 FoxP3 and RORγt, respectively on tissue microarrays of a cohort of 222 patients with reviewed histology and available clinical data. Expression was analyzed with Qupath for quantification and integration with clinical data enabled calculation of prognostic impact. For validation FOXP3 and RORC mRNA expression levels from 502 patients with HGSC in publicly available datasets were evaluated.ResultsAn average percentage of 0.93 Tregs and of 0.06 Th17 cells was detected per cells in overall tissue. Optimal cut-offs were determined and higher Tregs were associated with a better overall survival in stroma (p = 0.006), tumor area (p = 0.0012) and overall tissue (p = 0.02). After accounting for well-known prognostic factors age at diagnosis, residual tumor and FIGO stage, this association remained significant for stromal Tregs with overall survival (p = 0.02). Survival analysis for Th17 cells revealed no significant association with survival rates. Moreover, lower Th17/Treg ratios had a positive impact on patient overall survival (p = 0.025 tumor, p = 0.049 stroma and p = 0.016 overall tissue).ConclusionOur results outline a positive prognostic effect for higher Tregs but not for Th17 in high grade serous tubo-ovarian carcinoma.

Rationale and design of phase 1 FTIH study of FOXP3 antisense oligonucleotide AZD8701 in patients with selected advanced solid tumors.

TPS3166 Background: The forkhead box family transcription factor FOXP3 is essential for T regulatory cells (Tregs) development and immune suppressive function. Tregs are an integral component of the adaptive immune system and contribute to maintaining tolerance to self-antigens and preventing autoimmune diseases. In the context of cancer, however, Tregs contribute to tumor progression by suppressing antitumor immunity. To date inhibition of Treg-mediated immunosuppression tested in the clinic has lacked specificity. Targeting FOXP3 provides a selective approach to impair the immunosuppressive function of Tregs but targeting transcription factors has been a challenge using conventional drug modalities. AZD8701 employs next-generation antisense oligonucleotide (ASO) technology (Ionis Pharmaceuticals) to bind mRNA with high affinity and selectively reduce human Foxp3 mRNA expression levels. Foxp3-specific ASOs promote potent dose-dependent reductions in Foxp3 mRNA and protein in vitro. In preclinical models, AZD8701 induced Foxp3 knockdown results in Tregs with a reduced immunosuppressive capacity, loss of immunosuppressive markers, and increased markers of activation on CD8+ T-cells. AZD8701 reduces tumor growth as monotherapy in preclinical models and increased tumor inhibition is obtained by combining AZD8701 with a PD-L1 inhibitor. Methods: This is a Phase I multicenter study of AZD8701 alone or in combination with durvalumab in participants with selected advanced solid tumors. Eligible patients must have ECOG performance status 0 or 1, measurable target lesion per RECIST v1.1 and be diagnosed with selected tumor types as described below. Monotherapy and combination dose escalation phase is open for participants with head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), non-small-cell lung cancer (NSCLC), clear cell renal cell carcinoma (ccRCC), gastroesophageal cancer, melanoma, cervical cancer, small-cell lung cancer (SCLC), and/or solid tumors that have demonstrated a response to prior programmed death-ligand-1 (PD-[L]1) treatment (as defined by duration of response &gt; 18 weeks). Participants with NSCLC, HNSCC, TNBC, and ccRCC will be included in the pharmacodynamic cohort at the selected monotherapy dose and/or disease expansion cohorts. The primary objectives are to assess safety and tolerability and to determine the preliminary antitumor activity of AZD8701 (objective response rate) when administered as monotherapy or in combination with durvalumab. Secondary endpoints include, disease control rate, duration of response, progression free survival and overall survival, pharmacokinetics and pharmacodynamics (including changes in Foxp3 mRNA in paired tumor samples). The trial is currently recruiting. Clinical trial information: NCT04504669.

CXCR2 antagonist SB332235 mitigates deficits in social behavior and dysregulation of Th1/Th22 and T regulatory cell-related transcription factor signaling in male BTBR T+ Itpr3tf/J mouse model of autism

Autism spectrum disorders is a complex neurodevelopmental disorder characterized by abnormal social interaction, defective communication, repetitive and stereotyped patterns of behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) inbred mice are generally used as a model for ASD, display a range of autistic phenotypes. Recent studies suggest that the CXCR2 antagonist is crucial for targets in the treatment of inflammatory and neurodegenerative diseases. In this study, we investigated the potential effects of the CXCR2 antagonist SB332235 on sociability behaviors, marble burying, and self-grooming, we also explored the treatment of SB332235 on Th1 (IFN-γ, Stat1, and T-bet), Th22 (IL-22, TNF-α, and AhR), and T regulatory (Treg, IL-10, Helios and Foxp3) production in CD4+ T cells in male BTBR and C57BL/6 (C57) mice in spleen. We also investigated the effects of SB332235 on IFN-γ, IL-10, IL-22, T-bet, AhR, and Foxp3 mRNA expression levels in the brain tissues. The SB332235-treated mice significantly improve behavioral abnormalities in BTBR mice. In addition, SB332235 administration causes a significantly decreases in IFN-γ, Stat1, T-bet, IL-22, TNF-α, and AhR, and increases in IL-10, Foxp3 and Helios production CD4+ T cells in BTBR mice. We further observed that SB332235 downregulated IFN-γ, IL-10, IL-22, T-bet, and AhR, and upregulated IL-10 and Foxp3 mRNA expression in the brain tissues. Our findings demonstrated that SB332235 treatment attenuated behavior deficits, through inhibiting Th1/Th22 and upregulating Treg cell-related transcription factors signaling pathway. Therefore, CXCR2 antagonist administration may be a promising therapeutic agent to attenuate behavior deficits via its anti-inflammatory effect.

Mesenchymal Stem Cell-Secreted TGF-β1 Restores Treg/Th17 Skewing Induced by Lipopolysaccharide and Hypoxia Challenge via miR-155 Suppression

Background Regulatory T cell (Treg)/T helper (Th) 17 skewing is important in the development of acute respiratory distress syndrome (ARDS). Immunomodulatory effects of mesenchymal stem cell- (MSC-) secreted transforming growth factor- (TGF-) β1 on CD4+ T cells are environment-sensitive and lack discussion in hypoxic and inflammatory conditions. Methods Mouse splenic CD4+ T cells were precoated with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) overnight. RAW264.7 cells were added as antigen-presenting cells (APCs). T cells with and without RAW264.7 cells were treated with various LPS concentrations of 0, 10, 100, and 1000 ng/ml or/and at hypoxia condition of 5% O2. Based on LPS (100 ng/ml) and hypoxia conditions (5% O2) as stimuli, MSCs were set as direct coculture or indirect coculture by transwell system. Anti-TGF-β1 neutralization antibody was added to explore the role of TGF-β1 among the soluble factors secreted by MSCs; miR-155 overexpression of CD4+ T cells was performed by transfection, and then, cells were added to the MSC-CD4+ T cell coculture system in hypoxic- and LPS-stimulated condition. After 48 hours, cells or supernatants were collected for detection of frequency of Treg and Th17 subsets, CD4+ T cell apoptosis and proliferation capacity assay by flow cytometry, secretion of INF-γ, IL-17A, IL-21, TGF-β1, and IL-10 by ELISA, and levels of miR-155, Rorc, Foxp3, and Ptpn2 mRNA expression of CD4+ T cells by RT-PCR. Results MSCs could restore skewed Treg/Th17 induced by LPS and hypoxia compared to groups without MSCs with increased secretion of TGF-β1, IL-10, and IL-17A (P < 0.05) and attenuate the increased expression of miR-155 in CD4+ T cells via cell-to-cell contact mechanism while TGF-β1 neutralization significantly inhibited the effects of MSCs restoring skewed Treg/Th17 and abolished its effect on miR-155 expression in CD4+ T cells. Conclusions These findings suggested miR-155 suppression of CD4+ T cells mediated MSC-secreted TGF-β1 modulating skewed Treg/Th17 induced by LPS-hypoxia challenge, providing evidence when proposing future T lymphocyte-targeted cell therapy in a specific condition.

Expression and Significance of BLIMP-1 in Regulatory T Cells of Children with Aplastic Anemia

To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β in plasma. The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-β in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-β in plasma. Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-β were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-β in plasma (r=0.500, P=0.029; r=0.486, P=0.030). The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-β expressions.

Metformin improves FOXP3 mRNA expression through suppression of interferon gamma levels in pristane-induced murine models of lupus

Background: A recent study has indicated the potential of metformin therapy for lupus in animal models, but there has been no study evaluating the effect on pristane-induced lupus. This study aims to evaluate the effect of intraperitoneal versus oral metformin on interferon (IFN)-γ levels and FOXP3 mRNA expression on pristane-induced female BALB/c mice. Methods: In total, 31 female BALB/c mice, aged 6 weeks, were intraperitoneally induced with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane). After 120 days, the mice were grouped and treated with various treatments: normal saline 100 MCL, oral metformin 100mg/kg-BW, or intraperitoneal metformin 100mg/kg-BW. After 60 days of treatment, all treatment groups were sacrificed, and kidney specimens prepared and stained using hematoxylin and eosin. Results: IFNγ levels of saline controls vs. oral metformin group was 309.39 vs. 292.83 pg/mL (mean difference 16.56 pg/mL; 95% CI 0.74-32.37; p=0.042), and saline control vs. intraperitoneal metformin group was 309.39 vs. 266.90 pg/mL (mean difference 42.49 pg/mL; 95% CI 29.24-55.73 pg/mL; p&lt;0.001). FOXP3 mRNA expression changes in saline controls vs. oral metformin group was 6.90 vs. 7.79-fold change (mean difference -0.89-fold change; 95% CI -1.68-(-0.11); p=0.03) and in saline controls vs. intraperitoneal metformin group was 6.90 vs. 9.02-fold change (mean difference -2.12-fold change; 95% CI -2.99-(-1.25); p=&lt;0.001). Correlation analysis of FOXP3 mRNA expression and IFNγ level changes revealed a Pearson correlation of -0.785 (p=0.001) and R2 value of 0.616 (p=0.001). Conclusion: Metformin is a potential new therapy to reduce the levels of IFNγ and increase FOXP3 mRNA expression in mice models of systemic lupus erythematosus.

Metformin improves FOXP3 mRNA expression through suppression of interferon gamma levels in pristane-induced murine models of lupus

Background: A recent study has indicated the potential of metformin therapy for lupus in animal models, but there has been no study evaluating the effect on pristane-induced lupus. This study aims to evaluate the effect of intraperitoneal versus oral metformin on interferon (IFN)-γ levels and FOXP3 mRNA expression on pristane-induced female BALB/c mice. Methods: In total, 31 female BALB/c mice, aged 6 weeks, were intraperitoneally induced with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane). After 120 days, the mice were grouped and treated with various treatments: normal saline 100 MCL, oral metformin 100mg/kg-BW, or intraperitoneal metformin 100mg/kg-BW. After 60 days of treatment, all treatment groups were sacrificed, and kidney specimens prepared and stained using hematoxylin and eosin. Results: IFNγ levels of saline controls vs. oral metformin group was 309.39 vs. 292.83 pg/mL (mean difference 16.56 pg/mL; 95% CI 0.74-32.37; p=0.042), and saline control vs. intraperitoneal metformin group was 309.39 vs. 266.90 pg/mL (mean difference 42.49 pg/mL; 95% CI 29.24-55.73 pg/mL; p<0.001). FOXP3 mRNA expression changes in saline controls vs. oral metformin group was 6.90 vs. 7.79-fold change (mean difference -0.89-fold change; 95% CI -1.68-(-0.11); p=0.03) and in saline controls vs. intraperitoneal metformin group was 6.90 vs. 9.02-fold change (mean difference -2.12-fold change; 95% CI -2.99-(-1.25); p=<0.001). Correlation analysis of FOXP3 mRNA expression and IFNγ level changes revealed a Pearson correlation of -0.785 (p=0.001) and R2 value of 0.616 (p=0.001). Conclusion: Metformin is a potential new therapy to reduce the levels of IFNγ and increase FOXP3 mRNA expression in mice models of systemic lupus erythematosus.

In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer

The Laminated layer of Echinococcus granulosus (LL) is the outer layer of the hydatic cyst. It plays a pivotal role in protecting the metacestode from host immunity. In our current study, we investigated the immunomodulatory effect of the LL on mouse spleen cells in presence of Lipopolysaccharide (LPS). Mouse spleen cells were cultured with or without LL in presence of LPS. After 24 h, the nitrites level representative of Nitric oxide (NO) production was measured in the culture supernatant by Griess-modified method. In addition, the mRNA expression levels of cytokines (IFN-γ, IL-1β, TGF-β, IL-10), Foxp3, and CTLA-4 were measured by quantitative Real-Time Polymerase chain reaction (qRT-PCR). Interestingly, our results showed a significant decrease (p< 0.01) in NO production and IFN-γ mRNA level (p< 0.001) from LPS- induced spleen cells in response to LL after 24h of culture. Moreover, LPS induced high level of IL-1β that was significantly (p<0.05) down regulated by LL. Importantly, mRNA levels of TGF-β (p< 0.01), Foxp3 and IL-10 (p< 0.05) were significantly upregulated by LL. In conclusion, our data indicated the in vitro immuno-regulatory and anti-inflammatory effects of the hydatic Laminated Layer on mouse spleen cells. These effects are related to an innate response implicating up-regulation of Foxp3, IL-10 and TGF-β expression and down-regulation of IFN-γ and IL-1β expression. LL could constitute a potential candidate for controlling inflammation during inflammatory disease.

5-Aminoisoquinolinone, a PARP-1 Inhibitor, Ameliorates Immune Abnormalities through Upregulation of Anti-Inflammatory and Downregulation of Inflammatory Parameters in T Cells of BTBR Mouse Model of Autism.

Autism spectrum disorder (ASD) covers a range of neurodevelopmental disorders involving impairments in communication and repetitive and stereotyped patterns of behavior and reciprocal social interaction. 5-Aminoisoquinolinone (5-AIQ), a PARP-1 inhibitor, has neuroprotective and anti-inflammatory effects. We investigated the influence of 5-AIQ-treatment in BTBR T+ Itpr3tf/J (BTBR) mice as an autism model and used flow cytometry to assess the effect of 5-AIQ on FOXP3, Helios, GATA3, IL-9, IL-10 and IL-17A production by CXCR6+ and CD4+ T cells in the spleen. We also confirmed the effect of 5-AIQ treatment on expression of FOXP3, Helios, GATA3, IL-17A, IL-10, and IL-9 mRNA and protein expression levels in the brain tissue by quantitative PCR and western blotting. Our results demonstrated that 5-AIQ-treated BTBR mice had significantly increased numbers of CXCR6+FOXP3+, CXCR6+IL-10+, and CXCR6+Helios+ cells and decreased numbers of CD4+GATA3+, CD4+IL-9+, and CD4+IL-17A+ cells as compared with those in untreated BTBR mice. Our results further demonstrated that treatment with 5-AIQ in BTBR mice increased expression for FOXP3, IL-10, and Helios, and decreased expression for GATA3, IL-17A, and IL-9 mRNA. Our findings support the hypotheses that 5-AIQ has promising novel therapeutic effects on neuroimmune dysfunction in autism and is associated with modulation of Treg and Th17 cells.

Correlation Analysis of PTPN22 Gene Polymorphism, Th17 / Treg Cells and Their Cytokines in GD with Different Iodine Nutritional Status

Background: PTPN22 is an important candidate gene for autoimmune diseases and its SNP is involved in the pathogenesis of a variety of autoimmune diseases. Objective: To investigate the relationship between the PTPN22 gene polymorphism (rs2488457) and the Th17 and Treg cells and factors in the peripheral blood mononuclear cells (PBMC) of Graves' disease (GD) patients with different iodine nutrition conditions. Methods: We selected 100 GD patients and 60 healthy people in Cangzhou Central Hospital and People's Hospital from September 2019 to August 2020 and used SASP-PCR technology to calculate the PTPN22-1123 genotype and allele frequency. Flow cytometrywere used to detect the ratio and ratio of Th17 and Treg cells in PBMC and real-time PCR were used to detect the mRNA expression levels of ROR-γt and Foxp3. ELISA method was used to detect serum IL-17 and TGF-β levels. Results: The PTPN22 gene -1123G>C (rs2488457) of GD patients has a certain correlation with Th17 and Treg cells and their factors in PBMC. The distribution frequencies of various genotypes and alleles were statistically different between GD patients and the control group (P<0.05), but they had nothing to do with iodine nutrition status. The C/G and C/C genotypes at the PTPN22 gene (1123 G>C) of may increase the risk of GD, while the G/G genotype may reduce the risk of GD. Conclusion: PTPN22 gene polymorphism (rs2488457) in GD patients and Th17 and Treg cells and their factors in PBMC are related to GD susceptibility.