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Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103- counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.

Background: Semaphorins are axonal guidance molecules involved in neural development and contribute to the regulation of various phases of the immune response. This study aimed to investigate the plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and the regulatory T (Treg) cell-related cytokine interleukin-10 (IL-10), as well as the gene expression levels of forkhead box P3 (FoxP3), Semaphorin-3A (Sema-3A), Neuropilin-1 (Nrp-1), Semaphorin-4A (Sema-4A), and Plexin-D1 (Plxn-D1), in the peripheral blood of newly diagnosed rheumatoid arthritis (RA) patients treated with conventional disease-modifying antirheumatic drugs (DMARDs) for 6 months compared with healthy controls. Methods: Peripheral blood samples were obtained from 40 newly diagnosed RA patients (before and after treatment) and 40 age- and sex-matched healthy subjects. The plasma concentrations of IL-6 and IL-10 were quantified via enzyme-linked immunosorbent assay (ELISA), and the mRNA expression levels of FoxP3, Sema-3A, Nrp-1, Sema-4A, and Plxn-D1 were assessed via quantitative real-time PCR. Results: Compared with those in the controls, the plasma IL-6 levels in the RA patients (both pre- and post-treatment) were significantly greater (P < 0.001). Compared with the pre-treatment levels, the plasma IL-6 levels decreased significantly after DMARD therapy (P < 0.05). Moreover, plasma IL-10 levels were significantly greater in post-treatment RA patients than in controls (P < 0.05). The gene expression of FoxP3, Sema-3A, and Nrp-1 was significantly lower in pre-treated RA patients than in controls (P < 0.001). Compared with that in pre-treatment RA patients, the gene expression of FoxP3, Sema-3A, and Nrp-1 in DMARDs-treated RA patients was strongly increased (P < 0.05, P < 0.01, and P < 0.01, respectively). There was a positive correlation between Sema-3A gene expression and the gene expression of FoxP3 (r = 0.292, P < 0.01) and Nrp-1 (r = 0.569, P < 0.0001). Conclusion: Conventional DMARDs therapy effectively reduces disease activity and inflammation in newly diagnosed RA patients by increasing FoxP3, Sema-3A, and Nrp-1 gene expression. [ABSTRACT FROM AUTHOR]

Background: FOXP3 is a transcription factor that regulates the development and function of Treg, playing an essential role in preventing autoimmune diseases. Variation in FOXP3 can impair the function of Treg cells, thus destroying their inhibitory capacity and leading to autoimmune diseases. This paper investigated whether the three SNPs in the FOXP3 gene (-3279 C/A, -924 A/G and -6054 del/ATT) are associated with systemic lupus erythematosus (SLE) susceptibility in the Han Chinese population. Materials and methods: The study cohort comprised 122 SLE patients and 268 healthy controls. Genotyping was performed by polymerase chain reaction sequence-specific primer (PCR-SSP). Furthermore, we examined the potential clinical manifestations associated with FOXP3 polymorphisms in SLE patients. Results: The results showed that the -3279 (C > A) was significantly associated with the SLE risk in a homozygote (OR = 3.24, 95% CI = 1.23-8.52, p = .013, AA vs. CC), dominant (OR = 1.68, 95% CI = 1.07-2.65, p = .025, AC + AA vs. CC), recessive (OR = 2.90, 95% CI = 1.12-7.55, p = .023, AA vs. AC + CC) and allelic (OR = 1.72, 95% CI = 1.18-2.53, p = .005, A vs. C) models. In addition, -924 (A >G) was positively associated with SLE risk in the heterozygote (OR = 1.66, 95% CI = 1.04-2.66, p = .033, AG vs. AA) and dominant (OR = 1.59, 95% CI = 1.01-2.49, p = .042, AG + GG vs. AA) models, whereas -6054 (del> ATT) was not associated with SLE. Moreover, the immunological index analysis suggested that decreased complement C4 occurred more frequently in SLE patients carrying the minor allele (A) -3279 (C > A) than those not (p = .005). Conclusions: We demonstrated that -3279 (C > A) and -924 (A >G) were associated with an increased risk of SLE and the immunological index, indicating that the FOXP3 variation is potentially related to the occurrence and development of SLE. [ABSTRACT FROM AUTHOR]

Due to its capacity to drive osteoclast differentiation, the receptor activator of nuclear factor kappa-β ligand (RANKL) is believed to exert a pathological influence in periodontitis. However, RANKL was initially identified as an activator of dendritic cells (DCs), expressed by T cells, and exhibits diverse effects on the immune system. Hence, it is probable that RANKL, acting as a bridge between the bone and immune systems, plays a more intricate role in periodontitis. Using ligature-induced periodontitis (LIP), rapid alveolar bone loss was detected that was later halted even though the ligature was still present. This late phase of LIP was also linked with immunosuppressive conditions in the gingiva. Further investigation revealed that the ligature prompted an immediate migration of RANK-expressing Langerhans cells (LCs) and EpCAM+ DCs, the antigen-presenting cells (APCs) of the gingival epithelium, to the lymph nodes, followed by an expansion of T regulatory (Treg) cells in the gingiva. Subsequently, the ligatured gingiva was repopulated by monocyte-derived RANK-expressing EpCAM+ DCs, while gingival epithelial cells upregulated RANKL expression. Blocking RANKL signaling with monoclonal antibodies significantly reduced the frequencies of Treg cells in the gingiva and prevented gingival immunosuppression. In addition, RANKL signaling facilitated the differentiation of LCs from bone marrow precursors. To further investigate the role of RANKL, we used K14-RANKL mice, in which RANKL is overexpressed by gingival epithelial cells. The elevated RANKL expression shifted the steady-state frequencies of LCs and EpCAM+ DCs within the epithelium, favoring LCs over EpCAM+ DCs. Following ligature placement, heightened levels of Treg cells were observed in the gingiva of K14-RANKL mice, and alveolar bone loss was significantly reduced. These findings suggest that RANKL-RANK interactions between gingival epithelial cells and APCs are crucial for suppressing gingival inflammation, highlighting a protective immunological role for RANKL in periodontitis that was overlooked due to its osteoclastogenic activity. [ABSTRACT FROM AUTHOR]

Purpose: Resistance to Trastuzumab is a significant challenge in the management of HER2-positive Metastatic Breast cancer (HER2-MBC), and a better understanding of the molecular causes of resistance is required to develop more effective treatment plans. While elevated plasma levels of miR-200 and FOXP3 have been linked to breast cancer progression and treatment response, no clinical studies have confirmed these results. Methods: The study involved 40 patients with HER2-positive metastatic breast cancer (HER2-MBC). The expression levels of miR-200c-3p and the FOXP3 gene were assessed in plasma samples at two time points: baseline (BL) and after the consent completion of one cycle of Trastuzumab, utilizing quantitative polymerase chain reaction (qPCR). Clinical response to Trastuzumab was evaluated 12 months post-therapy and correlated with the time to progression (TTP) through Kaplan-Meier analysis. Results: Low plasma expression level of miR-200c-3p was detected before therapy in HER2-MBC, compared to healthy controls, and decreased dramatically in the follow-up sample at disease progression, while increased after one cycle of Trastuzumab therapy in patients who were sensitive to Trastuzumab. At baseline, a low expression level of miR-200c was significantly associated with overexpression of FOXP3, poor prognosis, and shorter time to progression. Conclusions: The findings suggest that miR-200c-3p may be a promising biomarker for predicting the response to Trastuzumab in HER2-MBC patients. [ABSTRACT FROM AUTHOR]

Tregs have the potential to be utilized as a novel therapeutic agent for the treatment of various chronic diseases, including diabetes, Alzheimer's disease, asthma, and rheumatoid arthritis. One of the challenges associated with developing a therapeutic product based on Tregs is the non-selectivity of polyclonal cells. A potential solution to this issue is a generation of antigen-specific CAR-Tregs. Other challenges associated with developing a therapeutic product based on Tregs include the phenotypic instability of these cells in an inflammatory microenvironment, discrepancies between engineered Treg-like cells and natural Tregs, and the expression of dysfunctional isoforms of Treg marker genes. This review presents a summary of proposed strategies for addressing these challenges. [ABSTRACT FROM AUTHOR]

AbstractBackgroundMethodsResultsConclusionsThe abnormality of the immune system caused by infection is a contributor to the organ dysfunctions associated with sepsis. The balance between Th17/Treg cells is essential for maintaining immune homeostasis. Bergapten is a natural furocoumarin and has been reported to alleviate the Th17/Treg imbalance. Here, we explored the effects of bergapten on the inflammation and immune state in mouse models of sepsis.The model was established using the cecal ligation and puncture method. Mice were administered 30 mg/kg bergapten. Histological examination, RT-qPCR, enzyme-linked immunosorbent assay, immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry were used to evaluate the effects of bergapten in vivo.Bergapten ameliorated lung damage, reduced lung wet/dry weight ratio, inhibited myeloperoxidase activity, and reduced inflammatory cell infiltration. Bergapten also restrained sepsis-induced inflammation via inhibition of inflammatory cytokines and NF-κB signaling. These effects were accompanied by the restored Th17/Treg balance induced by bergapten. Bergapten decreased the number of Th17 cells and elevated the number of Tregs, and this effect was mediated by the signal transducer and activator of transcription 5 (STAT5)/Forkhead box P3 (Foxp3) and STAT3/retinoid-related orphan receptor-γt (RORγt) pathways.Bergapten exerted anti-inflammatory effects in acute lung injury by improving the Th17/Treg balance, which suggested a potential of bergapten as an immunomodulatory drug treating sepsis-associated diseases. [ABSTRACT FROM AUTHOR]

Pro-inflammatory autoantigen-specific CD4+ T helper (auto-Th) cells are central orchestrators of autoimmune diseases (AIDs). We aimed to characterize these cells in human AIDs with defined autoantigens by combining human leukocyte antigen (HLA)-tetramer-based and activation-based multidimensional ex vivo analyses. In aquaporin4-antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) patients, auto-Th cells expressed CD154, but proliferative capacity and pro-inflammatory cytokines were strongly reduced. Instead, exhaustion-associated co-inhibitory receptors were expressed together with FOXP3, the canonical regulatory T cell (Treg) transcription factor. Auto-Th cells responded in vitro to checkpoint inhibition and provided potent B cell help. Cells with the same exhaustion-like (ThEx) phenotype were identified in soluble liver antigen (SLA)-antibody-autoimmune hepatitis and BP180-antibody-positive bullous pemphigoid, AIDs of the liver and skin, respectively. While originally described in cancer and chronic infection, our data point to T cell exhaustion as a common mechanism of adaptation to chronic (self-)stimulation across AID types and link exhausted CD4+ T cells to humoral autoimmune responses, with implications for therapeutic targeting. [Display omitted] • Auto-Th cells from AID patients have an exhausted-like (ThEx) phenotype • Auto-ThEx cell frequencies are stable for years despite successful therapy • ThEx cells respond to checkpoint inhibition and provide B cell help • ThEx phenotype development during chronic stimulation in vitro is regulated by FOXP3 CD4+ T cells are important contributors to autoimmune disease pathology. Saggau, Bacher et al. characterize autoantigen-specific CD4+ T helper cells in various human autoimmune diseases, revealing features of exhaustion. These findings provide insight into CD4+ T cell exhaustion in the context of chronic stimulation and suggest a role for exhausted autoantigen-specific CD4+ Th cells in promoting humoral autoimmunity. [ABSTRACT FROM AUTHOR]

Purpose: To examine the mitigating influence of curcumin on asthma in children, and the involvement of Foxp3 in the process. Methods: A rat model of asthma was successfully induced using ovalbumin (OVA). Twenty-four (24) rats were assigned to 3 cohorts: model, curcumin and control groups. At the cellular level, sorted CD4+ T cells were differentiated into regulatory T (Treg) cells and divided into 4 groups: Treg, Treg + curcumin, Treg + STAT5-IN-1 (STAT5 inhibitor) and Treg + curcumin + STAT5-IN-1 groups. Results: Curcumin significantly inhibited OVA-induced increases in eosinophils and lymphocytes and reduced airway pathological changes, relative to model rats (p < 0.05). There were marked increases in TGF-ß and IL-10 contents in bronchial alveolar lavage fluid (BALF), relative to model rats. Furthermore, curcumin up-regulated p-STAT5 and Foxp3 in the lung tissues of rats, when compared with model group (p < 0.05). In cell experiments, curcumin significantly increased p-STAT5, Foxp3 and IL-10 levels, and enhanced the transformation of CD4+ T cells to more Treg cells (p < 0.05). Conclusion: Curcumin may affect the differentiation of Treg cells, as well as alleviate asthma in rats by targeting Foxp3 through STAT5 regulation. This research has identified the likely beneficial role of curcumin as therapeutic agent for pediatric asthma, but this has to be clinically validated. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGFβ) and FoxP3 regulatory T cells (Treg) are involved in human breast carcinogenesis. This topic is not well documented in canine mammary tumors (CMT). In this work, the tumoral TGFβ expression was assessed by immunohistochemistry in 67 malignant CMT and its correlation to previously determined FoxP3, VEGF, and CD31 markers and other clinicopathologic parameters was evaluated. The high levels of TGFβ were statistically significantly associated with skin ulceration, tumor necrosis, high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastases. The observed levels of TGFβ were positively correlated with intratumoral FoxP3 (strong correlation), VEGF (weak correlation) and CD31 (moderate correlation). Tumors that presented a concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31 markers were statistically significantly associated with parameters of tumor malignancy (high HGM, presence of vascular emboli and nodal metastasis). Additionally, shorter overall survival (OS) time was statistically significantly associated with tumors with an abundant TGFβ expression and with concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31. The presence of lymph node metastasis increased 11 times the risk of disease-related death, arising as an independent predictor of poor prognosis in the multivariable analysis. In conclusion, TGFβ and Treg cells seem involved in tumor progression emerging as potential therapeutic targets for future immunotherapy studies.

Antonia Becker,1 Karoline Röhrich,1 Amanda Leske,1 Ulrike Heinicke,1 Tilo Knape,2 Aimo Kannt,2,3 Verena Trümper,4 Kai Sohn,5 Annett Wilken-Schmitz,1 Holger Neb,1 Elisabeth H Adam,1 Volker Laux,2 Michael J Parnham,2 Valerie Onasch,4 Andreas Weigert,4 Kai Zacharowski,1,2 Andreas von Knethen1 1Goethe University Frankfurt, Department of Anaesthesiology, Intensive Care Medicine, and Pain Therapy, University Hospital Frankfurt, Frankfurt, 60590, Germany; 2Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Frankfurt, 60596, Germany; 3Institute of Clinical Pharmacology, Goethe University, Frankfurt, 60590, Germany; 4Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Frankfurt, 60590, Germany; 5Innovation Field in-vitro Diagnostics, Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, 70569, GermanyCorrespondence: Andreas von Knethen, Email andreas.vonknethen@ukffm.deBackground: COVID-19 is a serious viral infection, which is often associated with a lethal outcome. Therefore, understanding mechanisms, which affect the immune response during SARS-CoV2 infection, are important.Methods: To address this, we determined the number of T cells in peripheral blood derived from intensive care COVID-19 patients. Based on our previous studies, evaluating PPARγ-dependent T cell apoptosis in sepsis patients, we monitored PPARγ expression. We performed a next generation sequencing approach to identify putative PPARγ-target genes in Jurkat T cells and used a PPARγ transactivation assay in HEK293T cells. Finally, we translated these data to primary T cells derived from healthy donors.Results: A significantly reduced count of total CD3+ T lymphocytes and the CD4+ and CD8+ subpopulations was observed. Also, the numbers of anti-inflammatory, resolutive Th 2 cells and FoxP3-positive regulatory T cells (Treg) were decreased. We observed an augmented PPARγ expression in CD4+ T cells of intensive care COVID-19 patients. Adapted from a next generation sequencing approach in Jurkat T cells, we found the chemoattractant receptor‐homologous molecule expressed on T helper type 2 cells (CRTH2) as one gene regulated by PPARγ in T cells. This Th 2 marker is a receptor for prostaglandin D and its metabolic degradation product 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), an established endogenous PPARγ agonist. In line, we observed an increased PPARγ transactivation in response to 15d-PGJ2 treatment in HEK293T cells overexpressing CRTH2. Translating these data to primary T cells, we found that Th 2 differentiation was associated with an increased expression of CRTH2. Interestingly, these CRTH2+ T cells were prone to apoptosis.Conclusion: These mechanistic data suggest an involvement of PPARγ in Th 2 differentiation and T cell depletion in COVID-19 patients.Keywords: Treg, PPARγ, Th 2, COVID-19, NGS, IL-4, CRTH2, FoxP3

The impaired function of regulatory T (Treg) cells and the imbalance of Treg/Th17 cells play a central role in developing autoimmune diseases such as systemic lupus erythematosus (SLE). Treg cells are crucial for maintaining immune homeostasis and tolerance to self-antigens. One of the most important transcription factors that regulate the differentiation and function of Treg cells is the FOXP3 protein. Aberrant epigenetic modifications affecting FOXP3 gene expression and consequently dysregulated function of Treg cells have been implicated in the pathogenesis of SLE. Therefore, understanding the intricate interplay between FOXP3 expression pattern in Treg cells and epigenetic regulatory mechanisms (e.g., DNA methylation, histone modifications and non-coding RNAs such as microRNAs and long non-coding RNAs) is crucial for unravelling the underlying mechanisms of SLE. Moreover, targeting these epigenetic pathways may offer novel therapeutic strategies for restoring immune balance and ameliorating autoimmune pathology. This review report aimed to provide an update on the epigenetic controlling of FOXP3 gene expression in SLE disease.

Chunlei Tan,1 Jinling Xu,2 Shiyuan Zhang,1 Shuqiang Liu,1 Xiaotian Yang,1 Danping Wu,1 Boqian Yu,1 Yuanxi Huang1 1Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150081, People’s Republic of China; 2Endoscope Department, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150081, People’s Republic of ChinaCorrespondence: Yuanxi Huang, Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150081, People’s Republic of China, Tel/Fax +86-0451-86298053, Email rxwk@163.comPurpose: The current investigation is to assess FOXP3 expression in breast cancer patients and evaluate the predictive significance of FOXP3.Patients and Methods: A cohort of 313 cases between January 2015 and November 2015 were enrolled this research. Immunohistochemistry (IHC) assay was utilized to detect the expression levels of FOXP3 in primary breast carcinoma specimens. These patients were separated into two groups by semiquantitative scoring approach. Chi-square test and Fisher’s exact test were conducted to investigate the correlations between FOXP3 expression in tumors and clinicopathological variables. Kaplan–Meier method and Log rank test were utilized to generate survival curves for disease-free survival (DFS) and overall survival (OS). The independent factors were examined using Cox regression analysis. Nomogram models were created for assessing DFS and OS rates.Results: Depending on the levels of FOXP3 expression in tumors, these patients were categorized into two groups: low FOXP3 expression (174 cases) and high FOXP3 expression (139 cases). The patients exhibiting low levels of FOXP3 expression in tumors demonstrated a longer survival duration contrasted with those with high expression (DFS: 88.75 vs 65.87 months, χ2=36.1100, P< 0.0001; OS: 89.70 vs 78.37 months, χ2=32.4900, P< 0.0001). Multivariate analysis revealed that FOXP3 was a significant prognostic factor [DFS: hazard ratio (HR): 2.822, 95% CI: 1.595– 4.992, P< 0.0001; OS: HR: 3.232, 95% CI: 1.812– 5.763, P< 0.0001]. The good predictive clinical utility of FOXP3-based nomograms within the threshold probability range for different survival rates was demonstrated by calibration curve and decision curve analyses.Conclusion: FOXP3 expression serves as a crucial prognostic indicator in breast cancer patients, and may aid preoperative evaluation in clinical practice.Keywords: breast cancer, FOXP3, chemotherapy, radiotherapy, survival

A 3-year-old boy initially presented with purpura-like rashes and nephrotic syndrome, suspected to be IgA vasculitis nephritis (IgAVN). The suggestion of kidney biopsy was rejected. Although the patient responded well to glucocorticoids, they later developed recurrent proteinuria, refractory diarrhea, and subsequent metabolic acidosis. Kidney biopsy showed membranous nephropathy with positive semaphorin 3B expression, indicative of other kidney diseases rather than IgAVN. Although his kidney responded well to glucocorticoid combined with cyclosporine A treatment regimen, enteropathy and severe food allergy still progressed afterwards as evidenced by villous atrophy on gastrointestinal endoscopy examination. Whole exome sequencing identified a heterozygous missense variant in exon 11 of FOXP3: c.1121 T > G, confirming the diagnosis of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. The case expanded the phenotypic spectrum of IPEX syndrome, suggesting high phenotypic heterogeneity despite similar genotypes. It also put emphasis on the significance of kidney biopsy to differentiate IgA vasculitis nephropathy from other immune disorders. [ABSTRACT FROM AUTHOR]

Abstract Background Regulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma. Methods We performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings. Results High CD25+, FOXP3+, and CD25+FOXP3+CD45RA− stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA− stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA− : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021). Conclusion The current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA− cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA− effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.

ObjectiveTo investigate the potential significance of FOXP3 expression in BRCA1/2-mutant breast cancer. MethodsA total of 48 BRCA mutation carriers (16 with BRCA1 and 32 with BRCA2) and 78 age-matched non-carriers were included in this study. Immunohistochemistry was used to detect the expression of FOXP3 in breast cancer tissues. The FOXP3 RNA expression in 39 BRCA1, 36 BRCA2, and 948 non-carrier breast cancer patients from TCGA-BRCA and the correlation with homologous recombination deficiency scores were evaluated to validate the immunohistochemistry results. ResultsThe FOXP3 positive rate was 43.8% (7/16) in BRCA1 mutation carriers, 59.4% (19/32) in BRCA2 mutation carriers, and 9.0% (7/78) in non-carriers. The FOXP3 positive rates in patients with BRCA1/2 mutant breast cancer were significantly higher than those in non-carriers (P=0.002; P

Background: Regulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma. Methods: We performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings. Results: High CD25+, FOXP3+, and CD25+FOXP3+CD45RA− stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA− stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA− : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021). Conclusion: The current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA− cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA− effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas. [ABSTRACT FROM AUTHOR]

Simple Summary: T regulatory cells are specialized lymphocytes that prevent excessive immune responses, yet they can suppress beneficial anti-tumor immunity when infiltrating malignant tumors. Osteopontin, a versatile protein produced by various cells, including T lymphocytes, influences both inflammation-related diseases and cancer immunity. Its role in T regulatory cells, however, remains less explored. Our study examined the impact of osteopontin deficiency on T regulatory cells. We generated mice lacking osteopontin in these cells and observed impaired immune suppression capabilities, which resulted in increased tumor-fighting activity against melanoma. These findings emphasize osteopontin's crucial role in T regulatory cell stability and functionality within the tumor microenvironment, providing valuable insights for developing new immunotherapeutic strategies. Foxp3-expressing regulatory T (Treg) cells represent the most highly immunosuppressive cell in the tumor microenvironment (TME) that halts effective anti-tumor immunity. Osteopontin (Opn), an extracellular matrix (ECM) glycophosphoprotein, plays key roles in many types of immune-related diseases and is associated with cancer aggressiveness when expressed by tumor cells. However, its role in Foxp3Treg heterogeneity, function, and stability in the TME is poorly defined. We generated mice with a Foxp3-specific deletion of Opn and assessed the ability of Opn-deficient Tregs to suppress inflammation. As these mice aged, they developed a scurfy-like syndrome characterized by aberrant and excessive activation of effector T cells. We evaluated and further confirmed the reduced suppressive capacity of Opn-deficient Tregs in an in vivo suppression assay of colitis. We also found that mice with Opn-deficient Foxp3+ Tregs have enhanced anti-tumor immunity and reduced tumor burden, associated with an unstable Treg phenotype, paralleled by reduced Foxp3 expression in tumor-infiltrating lymphocytes. Finally, we observed reduced Foxp3 and Helios expression in Opn-deficient Tregs compared to wild-type controls after in vitro activation. Our findings indicate that targeting Opn in Tregs reveals vigorous and effective ways of promoting Treg instability and dysfunction in the TME, facilitating anti-tumor immunity. [ABSTRACT FROM AUTHOR]

Our goal is to improve the outcomes of cancer immunotherapy by targeting FOXP3+ T-regulatory (Treg) cells with a next generation of antisense oligonucleotides (ASO), termed FOXP3 AUMsilence ASO. We performed in vitro experiments with human healthy donor PBMC and clinical samples from patients with lung cancer, mesothelioma and melanoma, and tested our approach in vivo using ASO FOXP3 in syngeneic murine cancer models and in humanized mice. ASO FOXP3 had no effects on cell viability or cell division, did not affect expression of other FOXP members, but decreased expression of FOXP3 mRNA in PBMC by 54.9% and in cancer samples by 64.7%, with corresponding 41.0% (PBMC) and 60.0% (cancer) decreases of Treg numbers (all p<0.0001). Hence, intratumoral Treg were more sensitive to the effects of ASO FOXP3 than peripheral blood Tregs. Isolated human Treg, incubated with ASO FOXP3 for 3.5 hours, had significantly impaired suppressive function (66.4%) versus Scramble control. In murine studies, we observed a significant inhibition of tumor growth, while 13.6% (MC38) to 22% (TC1) of tumors were completely resorbed, in conjunction with ~50% decrease of Foxp3 mRNA by qPCR and decreased numbers of intratumoral Tregs. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, confirming that intratumoral Treg had enhanced sensitivity to ASO FOXP3 in vivo compared to other Treg populations. ASO FOXP3 Treg targeting in vivo and in vitro was accompanied by significant downregulation of multiple exhaustion markers, and by increased expression of perforin and granzyme-B by intratumoral T cells. To conclude, we report that targeting the key Treg transcription factor FOXP3, with ASO FOXP3, has a powerful anti-tumoral effect and enhances T cell response in vitro and in vivo. [ABSTRACT FROM AUTHOR]

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Conventional CD4pos regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αβpos CD4negCD8αneg double-negative (dn) T cells of dogs, a novel FoxP3pos Treglike subset was described that, similar to conventional CD4pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβpos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4). Here, to verify their regulatory function, the dnCD25pos (enriched for FoxP3pos Treg-like cells) and the dnCD25neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4posCD25pos Treg cells (positive control) and CD4posCD25neg T cells (negative control). Canine dnCD25pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4posCD25pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4posCD25pos populations, even dnCD25neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25neg T cells have a FoxP3neg phenotype comparable to non-suppressive CD4posCD25neg T cells. Both, CD25pos and CD25neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4posCD25neg T cells to secrete inhibitory factors in contrast to CD4posCD25pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25pos and CD4posCD25pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25neg T cells. Taken together, unique canine nonconventional dnCD25pos FoxP3pos Treg-like cells are potent suppressor cells in vitro. Moreover, inhibition of proliferation of responder T cells by the dnCD25neg fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory nonconventional dn T cell subpopulations of the dog in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs), characterized by the expression of Forkhead Box P3 (FOXP3), constitute a distinct subset of T cells crucial for immune regulation. Tregs can exert direct and indirect control over immune homeostasis by releasing inhibitory factors or differentiating into Th-like Treg (Th-Treg), thereby actively contributing to the prevention and treatment of autoimmune diseases. The epigenetic regulation of FOXP3, encompassing DNA methylation, histone modifications, and post-translational modifications, governs the development and optimal suppressive function of Tregs. In addition, Tregs can also possess the ability to maintain homeostasis in diverse microenvironments through non-suppressive mechanisms. In this review, we primarily focus on elucidating the epigenetic regulation of Tregs as well as their multifaceted roles within diverse physiological contexts while looking forward to potential strategies involving augmentation or suppression of Tregs activity for disease management, particularly in light of the ongoing global COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

The prevalence of HCV infection in Egypt has decreased following the introduction of direct-acting antiviral therapy. However, treatment response is influenced by various factors, particularly host immunogenetics such as IL-28B and FOXP3 polymorphisms. The current study examined the impact of SNPs in the FOXP3 gene promoter region on HCV-infected Egyptian patients, along with SNPs in the IL28B gene.This study involved 99 HCV patients who achieved SVR12 after a 12 week DAA treatment while 63 HCV patients experienced treatment failure. IL28B rs12979860 SNP was identified using real-time PCR, while IL28B rs8099917, FOXP3 rs3761548, and rs2232365 SNPs were analyzed using RFLP-PCR. Serum levels of IL28B and FOXP3 were quantified using ELISA technique in representative samples from both groups. The IL28B rs12979860 T > C (P = 0.013) and FOXP3 rs2232365 A > G polymorphisms (P = 0.008) were found to significantly increase the risk of non-response. Responders had higher IL28B serum levels (P = 0.046) and lower FOXP3 levels (P < 0.001) compared to non-responders. Regression analysis showed an association between IL28B rs12979860 and FOXP3 rs2232365 with treatment response, independent of age and gender. A predictive model was developed with 76.2% sensitivity and 91.9% specificity for estimating DAAs response in HCV patients.Our findings confirmed the IL28B rs12979860 T > C and FOXP3 rs2232365 A > G polymorphisms significantly affect DAA treatment response in HCV Egyptian patients. Lower levels of IL-28B along with higher levels of FOXP3 are linked to poor response. Our results may lead to new insights into DAA responsiveness contributing to personalized medicine and improving therapeutic decision-making for HCV patients. [ABSTRACT FROM AUTHOR]

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1β. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1β, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus. [ABSTRACT FROM AUTHOR]

Autoimmune neutropenia (AIN) in early childhood is characterized by chronic neutropenia and positivity for human neutrophil antibodies (HNA), resulting in the excessive destruction of neutrophils. The association between regulatory T cells (Tregs) and AIN has been described, and in this study, we investigated three Treg‐associated genes, IL‐2, IL‐10 and FOXP3. The frequencies of three single nucleotide polymorphisms (SNPs) in IL‐2 −330T>G (rs2069762), +114G>T (rs2069763) and IVS3‐116 A>G (rs2069772), four SNPs in IL‐10 −3575T>A (rs1800890), −1082G>A (rs1800896), −819 C>T (rs1800871) and −592 C>A (rs1800872) and three SNPs in FOXP3 –3499 A>G (rs3761547), −3279 C>A (rs3761548) and −924 A>G (rs2232365) were compared between 166 Danish AIN patients and 358 healthy controls. Disease association was observed for IL‐2 IVS3‐116 GG (p = 0.0081, OR = 0.35 [0.15–0.80]), IL‐10 −3575 TT (p = 0.0078, OR = 1.71 [1.16–2.54]) and IL‐10 −1082 AA (p = 0.014, OR = 1.76 [1.14–2.72]) in all patients and FOXP3 –924 (p = 0.0005, A OR = 0.41 [0.25–0.68] and G OR = 2.42 [1.46–4.01]) in male patients. None of the associations were linked to antibody specificity. Disease‐associated haplotypes were observed in IL‐2 and FOXP3. IL‐2 −330T/+114 T/IVS3‐116A was associated with anti‐FcγRIIIb‐positive patients (p = 0.012, OR = 2.07 [1.18–3.62]). FOXP3 –3499A/–3279C/−924A was associated with anti‐HNA‐1a‐positive male patients (p = 0.016, OR = 0.41 [0.20–0.83]), and ACG was associated with female patients, both in the combined group (p = 0.006, OR = NA) and the anti‐FcγRIIIb‐positive group (p = 0.002, OR = NA). We conclude that our findings reveal a correlation between SNP in Treg‐associated genes and AIN, indicating that AIN could be driven by dysfunction of immune homeostatic‐evolving Tregs. [ABSTRACT FROM AUTHOR]

Introduction: Young onset colorectal cancer (YOCRC) is a heterogenous CRC phenotype with an increasing trend globally. This study aims to determine FOXP3+ Treg cells, Mismatch Repair (MMR) proteins, and proto-oncogene B-Raf (BRAF) V600E status among YOCRC patients at Hospital Universiti Sains Malaysia. Materials and Methods: This was a retrospective study of YOCRC (<50 years) over 8 years (January 2013 to December 2021). Immunohistochemistry staining of FOXP3, BRAFV600E, and MMR protein expression was performed using monoclonal antibodies. The staining intensity and percentage of positive cells were used to evaluate the staining using immunoreactive scoring. All data were analysed using descriptive and correlation statistics. A p-value of ≤ 0.05 was taken as statistically significant. Results: A total of 65 YOCRC patients were diagnosed, out of which 53.8% had proficient MMR (pMMR) with a mean age of 41, while 46.2% had deficient MMR (dMMR) with a mean age of 35.5. The pMMR with the BRAFV600E+ group expressed higher FOXP3+Tregs (54.2%) than the dMMR with the BRAFV600E+ group (22.9%). Patients with lower FOXP3+Tregs were observed more in dMMR with BRAFV600E- (47%) than in pMMR with BRAFV600E- (5.9%). There was a statistically significant association between the density of expressed FOXP3+Tregs with MMR and BRAFV600E status (p=0.002). Conclusion: While most of the YOCRC had pMMR, others exhibited dMMR with loss of one or more MMR proteins. The presence of BRAFV600E demonstrated the YOCRC's sporadic nature. A high FOXP3+Treg expression was significantly associated with MMR and BRAFV600E status. Future research must be expanded to cover other hospitals to increase the sample size and include MLH1 hypermethylation testing. [ABSTRACT FROM AUTHOR]

Forkhead box P3 (FOXP3) has been identified as a novel molecular marker in various types of cancer. The present study assessed the expression of FOXP3 in patients with head and neck squamous cell carcinoma (HNSCC) and its potential as a clinical prognostic indicator, and developed a radiomics model based on enhanced computed tomography (CT) imaging. Data from 483 patients with HNSCC were downloaded from the Cancer Genome Atlas for FOXP3 prognostic analysis and enhanced CT images from 139 patients included in the Cancer Imaging Archives, which were subjected to the maximum relevance and minimum redundancy and recursive feature elimination algorithms for radiomics feature extraction and processing. Logistic regression was used to build a model for predicting FOXP3 expression. A prognostic scoring system for radiomics score (RS), FOXP3, and patient clinicopathological factors was established to predict patient survival. The area under the receiver operating characteristic (ROC) curve (AUC) and calibration curve and decision curve analysis (DCA) were used to evaluate model performance. Furthermore, the relationship between FOXP3 and the immune microenvironment, as well as the association between RS and immune checkpoint-related genes, was analyzed. Results of analysis revealed that patients with HNSCC and high FOXP3 mRNA expression exhibited better overall survival. Immune infiltration analysis revealed that FOXP3 had a positive correlation with CD4 + and CD8 + T cells and other immune cells. The 8 best radiomics features were selected to construct the radiomics model. In the FOXP3 expression prediction model, the AUC values were 0.707 and 0.702 for the training and validation sets, respectively. Additionally, the calibration curve and DCA demonstrated the positive diagnostic utility of the model. RS was correlated with immune checkpoint-related genes such as ICOS, CTLA4, and PDCD1. A predictive nomogram was established, the AUCs were 0.87, 0.787, and 0.801 at 12, 24, and 36 months, respectively, and DCA demonstrated the high clinical applicability of the nomogram. The enhanced CT radiomics model can predict expression of FOXP3 and prognosis in patients with HNSCC. As such, FOXP3 may be used as a novel prognostic marker to improve individualized clinical diagnosis and treatment decisions. [ABSTRACT FROM AUTHOR]

Massive evidence shows that intestinal tryptophan metabolites affected by intestinal flora can modulate the progression of rheumatoid arthritis (RA). However, the effects and mechanisms of intestinal tryptophan metabolites on RA are not yet detailed. Herein, we investigated the protective effects of intestinal tryptophan metabolites on RA and its detailed mechanisms. In this study, the collagen-induced arthritis (CIA) rat model was established. Based on metabolomics analysis, the contents of β-indole-3-acetic acid (IAA), indolylpropionic acid, and indole-3-β-acrylic acid in the sera of CIA rats were significantly less compared with those of the normal rats. Under the condition of Treg or Th17 cell differentiation, IAA significantly promoted the differentiation and activation of Treg cells instead of Th17 cells. Intestinal tryptophan metabolites are well-known endogenic ligands of aryl hydrocarbon receptor (AhR). Not surprisingly, IAA increased the level of Foxp3 through activating the AhR pathway. Interestingly, IAA had little impact on the level of Foxp3 mRNA, but reducing the ubiquitination and degradation of Foxp3. Mechanically, IAA reduced the expression of the transcriptional coactivator TAZ, which was almost completely reversed by either AhR antagonist CH223191 or siRNA. In vitro, IAA decreased the combination of TAZ and the histone acetyltransferase Tip60, while it increased the combination of Tip60 and Foxp3. In CIA rats, oral administration of IAA increased the number of Treg cells and relieved the inflammation. A combined use with CH223191 almost abolished the effect of IAA. Taken together, IAA attenuated CIA by promoting the differentiation of Treg cells through reducing the ubiquitination of Foxp3 via the AhR-TAZ-Tip60 pathway. [ABSTRACT FROM AUTHOR]

Thymic regulatory lymphocytes (Tregs) are rare in the normal periphery where they mediate immune tolerance but accumulate in the tumor immune microenvironment (TIM), reducing the antitumor response. Subtypes of classical Hodgkin lymphoma (CHL) are characterized by a minority of malignant Hodgkin and Reed–Sternberg cells (HRS) and an abundant TIM that plays a key role in modulating the disease. CHL is related to the Epstein–Barr virus (EBV), whose oncogenes influence the growth of HRS. We analyzed the number of T lymphocytes expressing the regulatory marker FOXP3 in CHL with regard to EBV status. The tumor tissue of 182 patients was stained by double immunohistochemistry for FOXP3, CD4, and CD8, and the number of different phenotypes was analyzed microscopically. EBV status was determined by EBER in situ hybridization. EBV-positive CHL was confirmed in 28% of patients and was associated with mixed cellularity (MC) (p < 0.001), older age (p < 0.001), and unfavorable outcomes (p = 0.038). The number of CD8+ T lymphocytes differed according to the EBV status of MC and nodular sclerosis (NS), and was the lowest in EBV-negative NS (p = 0.001). Likewise, the numbers for FOXP3 and FOXP3/CD4 were different, and were the lowest in EBV-negative MC (p = 0.035 and p = 0.041, respectively). Values above a median of FOXP3 and CD4 are associated with longer progression-free survival (p = 0.039 and p < 0.001, respectively). EBV impacts the composition of T cell phenotypes in TIM, among which the amount of CD4 and FOXP3 is prognostically valuable. [ABSTRACT FROM AUTHOR]

Purpose: Tumor initiating cells (TICs) or cancer stem cells (CSCs) are considered to be the main culprit of hepatocellular carcinoma (HCC) initiation and progression, nevertheless the mechanism by which tumor microenvironment maintains the HCC ‘stemness’ is not fully understood. This study aims to investigate the effect of regulatory T cells (Tregs) on the TICs characteristics of HCC. Methods: Immunocytochemistry, flow cytometry, real-time PCR, western blot, in vitro sphere-formation, and in vivo tumorigenesis assay were used to detect HCC ‘stemness’. Additionally, after forced expression or inhibition of FoxP3, β-catenin expression and HCC ‘stemness’ were investigated. Results: Tregs enhanced the ‘stemness’ of HCC cells by upregulating TIC-related markers CD133, Oct3/4, Sox2, c-Myc, Klf4, Nanog, CD13, EpCAM, and inducting epithelial to mesenchymal transition (EMT), increasing TICs ratio, as well as promoting tumorigenic ability. Moreover, β-catenin and c-Myc were upregulated in HCC cells after co-cultured with Tregs. HCC ‘stemness’ was inhibited after treatment with Wnt/β-catenin pathway inhibitor. Furthermore, forced expression of FoxP3 resulted in increased GSK3β, decreased β-catenin and TIC ratio in HCC. In contrast, FoxP3 interference reduced GSK3β, enhanced β-catenin and TIC ratio of HCC. Conclusion: This study, for the first time, demonstrated that Tregs increased the population of TICs in HCC by inhibiting FoxP3 as well as promoting β-catenin expression. [ABSTRACT FROM AUTHOR]

Objective To investigate the potential significance of FOXP3 expression in BRCA1/2-mutant breast cancer. Methods A total of 48 BRCA mutation carriers (16 with BRCA1 and 32 with BRCA2) and 78 age-matched non-carriers were included in this study. Immunohistochemistry was used to detect the expression of FOXP3 in breast cancer tissues. The FOXP3 RNA expression in 39 BRCA1, 36 BRCA2, and 948 non-carrier breast cancer patients from TCGA-BRCA and the correlation with homologous recombination deficiency scores were evaluated to validate the immunohistochemistry results. Results The FOXP3 positive rate was 43.8% (7/16) in BRCA1 mutation carriers, 59.4% (19/32) in BRCA2 mutation carriers, and 9.0% (7/78) in non-carriers. The FOXP3 positive rates in patients with BRCA1/2 mutant breast cancer were significantly higher than those in non-carriers (P=0.002; P<0.001). TCGA-BRCA results showed that the FOXP3 RNA level in BRCA1/2 mutant breast cancer was significantly higher than that in non-carriers (P=0.02, P=0.004). The FOXP3 RNA level was positively correlated with the homologous recombination deficiency score (Spearman R=0.30, P<2.2e-16). Conclusion Patients with BRCA1/2 mutant breast cancers have higher FOXP3 expression than non-carriers, and may be more sensitive to immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: Although developing a better understanding of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) might provide essential knowledge to predict response to immunotherapy and prognosis, our current knowledge about Foxp3 + TILs is inadequate. This study investigated the prognostic significance of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) in squamous cell lung cancer (SQ-LC) objectively. Methods: Among patients with SQ-LC surgically resected in our institution between 2011 and 2017, those with pathological stage IA3-IIIA were immunohistochemically studied to evaluate Foxp3 + TILs in their tumor stroma. The impact of Foxp3 + TILs on relapse-free survival (RFS) was analyzed with Kaplan–Meier survival analysis and multivariate analysis using a Cox proportional hazards model/Fine-Gray model. Results: This study analyzed 100 patients. Multivariate analysis showed that a large number of Foxp3 + TILs in the stroma does not associate with a poor prognosis, rather that a large number of Foxp3 + TILs (≥ 64 cells) tend to be associated with a more favorable prognosis than a small number of Foxp3 + TILs (< 64 cells) (large vs small number: HR, 0.56; 95% CI, 0.17–1.83; P = 0.34). Exploratory analysis also showed that in the two populations divided by a difference in Foxp3 expression levels, similar trends to the main analysis were observed. Conclusion: Our results showed that a large number of Foxp3 + TILs in the stroma may not associate with a poor prognosis in SQ-LC. To use the seemingly complicated information of Foxp3 + TILs as biomarkers, better understanding the diversity and heterogeneity of Foxp3 + TILs and analyzing their subpopulations that increase in the TME may be needed. [ABSTRACT FROM AUTHOR]

ABSTRACTMosquito-borne viral infections are on the rise worldwide and can lead to severe symptoms such as haemorrhage, encephalitis, arthritis or microcephaly. A protective immune response following mosquito-borne viral infections requires the generation of a controlled and balanced immune response leading to viral clearance without immunopathology. Here, regulatory T cells play a central role in restoring immune homeostasis. In current review, we aim to provide an overview and summary of the phenotypes of FOXP3+ Tregs in various mosquito-borne arboviral disease, their association with disease severity and their functional characteristics. Furthermore, we discuss the role of cytokines and Tregs in the immunopathogenesis of mosquito-borne infections. Lastly, we discuss possible novel lines of research which could provide additional insight into the role of Tregs in mosquito-borne viral infections in order to develop novel therapeutic approaches or vaccination strategies.

ABSTRACTActivins, members of the TGF-beta superfamily, have been isolated and identified in the endocrine system, but have not been substantially investigated in the context of the immune system and endocrine-unrelated cancers. Here, we demonstrated that tumor-bearing mice had elevated systemic activin levels, which correlated directly with tumor burden. Likewise, cancer patients have elevated plasma activin levels compared to healthy controls. We observed that both tumor and immune cells could be sources of activins. Importantly, our in vitro studies suggest that activins promote differentiation of naïve CD4+ cells into Foxp3-expressing induced regulatory T cells (Tregs), particularly when TGF-beta was limited in the culture medium. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1c (ActRIC) was uniquely expressed on Tregs and that both ActRIC and ActRIIB (activin receptor 2b) were highly upregulated during iTreg differentiation. ActRIC-deficient naïve CD4+ cells were found to be defective in iTreg generation both in vitro and in vivo. Treg suppression assays were also performed, and ActRIC deficiency did not change the function or stability of iTregs. Mice lacking ActRIC or mice treated with monoclonal anti-ActRIC antibody were more resistant to tumor progression than wild-type controls. This phenotype was correlated with reduced expression of Foxp3 in CD4+ cells in the tumor microenvironment. In light of the information presented above, blocking activin-ActRIC signaling is a promising and disease-specific strategy to impede the accumulation of immunosuppressive iTregs in cancer. Therefore, it is a potential candidate for cancer immunotherapy.

Biomarkers including Forkhead/winged-helix transcription factor box P3 have been proposed in immunohistochemical techniques to diagnose cervical lesions, but can be objectively quantified and measured in blood using methods that can be standardised. In this study we quantified the serum FOXP3 concentrations and assessed their association with cervical lesions at the cervical cancer clinic of Mbarara Regional Hospital (MRRH) Southwestern Uganda.We performed secondary analysis on archived serum samples from a previous unmatched case control study in which we recruited 90 cervical cancer (CC) cases, 90 cervical intraepithelial neoplasia (CIN) cases before any form of treatment and 90 controls. Clinical and demographic data were recorded. We measured FOXP3 concentrations using quantitative ELISA. We performed descriptive statistics and logistic regression in STATA 17 and took P-values of

Except the widely accepted use of immune Checkpoint inhibitors in the treatment of microsatellite instability-high (MSI-H) mismatch repair-deficient (MMRd) CRCs representing about 5% of all metastatic (m)CRC patients, new strategies are applied to cure MMR-proficient (MMRp) mCRC patients. Tumor microenvironment (TME) is decisive for cancer development. The determination of some immunoeffective and immunosuppressive immune cells and some cytokines, chemokines and growth factors in the TME gives Information about the use of immune Checkpoint inhibitors in MMRp CRCs. The increased level of IL-6 in the serum and increased number of IL-6+ immune cells in TME, the increased number of IL-17+ Th17 cells, and of FoxP3+ cells are used to determine the use of anti-IL-6 antibody and of anti-FoxP3 antibody for treatment. The determination of high CD8+, high PD-1 expression and little or no Th17 cells appoint better response to anti-PD-1 therapy. The used combination therapies are: combination of immunotherapy with chemotherapy, with radiation therapy, with targeted therapy, with vaccines, oncolytic viruses and bispeeifie antibodies. Classical treatment of CRC patients has included chemotherapy, radiotherapy and surgery. Recently, immunotherapy has been added as a mainstay for therapy of CRC. The main checkpoint inhibitors used in CRC immunotherapy are pembrolizumab and nivolumab (anti-PD-1), durvalumab (anti-PD-L1), ipilimumab (anti-CTLA-4), favezelimab (anti-LAG3), etc. They are applied after fluorapyrimidine, oxaliplain, and irinotecan therapy. In conclusion, we may state that the future treatment of MSS CRC is in combination therapies, i.e. conventional and immunotherapies. We consider that immune infiltrate in TME must be assessed in order to determine combination therapies.

A prominent cause of cancer-related fatalities with a poor prognosis is lung adenocarcinoma (LUAD). KIF5A, a crucial member of the kinesin superfamily, is linked to drug resistance in malignancies. This work aims to investigate the mechanism of KIF5A in docetaxel (DTX) resistance in LUAD cells. The results of bioinformatics analysis, qRT-PCR and western blot analysis show that KIF5A, which is involved in the glycolysis pathway, is highly expressed in LUAD and is positively correlated with glycolysis-related genes. We further verify that silencing of KIF5A inhibits DTX resistance, glycolysis, and lactate production in LUAD cells via cell counting kit-8 (CCK-8), flow cytometry, Seahorse XFe 96, lactate, and glucose assays. Mechanistically, KIF5A promotes DTX resistance in LUAD, and this effect is attenuated upon the addition of an LDHA inhibitor. Chromatin immunoprecipitation and dual-luciferase reporter assays reveal that FOXP3 transcriptionally activates KIF5A. Knockdown of FOXP3 reduces lactate production and enhances DTX sensitivity in LUAD, which is restored upon simultaneous overexpression of KIF5A. Our findings reveal that FOXP3 increases DTX resistance in LUAD cells by enhancing lactate production through the upregulation of KIF5A level. In conclusion, our study provides a novel treatment target for improving chemosensitivity in LUAD.

Abstract Gene expression for Th1/Th2 cytokines (IL-4 and IFN-ɣ), regulatory cytokines (TGF-β and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-ɣ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-ɣ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-β in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Foxp3+ regulatory T cells (Foxp3+ Treg) play a role in regulating various types of tumors, but uncertainty still exists regarding the exact mechanism underlying Foxp3+ Treg activation in gastrointestinal malignancies. As of now, research has shown that Foxp3+ Treg expression, altered glucose metabolism, or a hypoxic tumor microenvironment all affect Foxp3+ Treg function in the bodies of tumor patients. Furthermore, it has been demonstrated that post-translational modifications are essential for mature Foxp3 to function properly. Additionally, a considerable number of non-coding RNAs (ncRNAs) have been implicated in the activation of the Foxp3 signaling pathway. These mechanisms regulating Foxp3 may one day serve as potential therapeutic targets for gastrointestinal malignancies. This review primarily focuses on the properties and capabilities of Foxp3 and Foxp3+Treg. It emphasizes the advancement of research on the regulatory mechanisms of Foxp3 in different malignant tumors of the digestive system, providing new insights for the exploration of anticancer treatments. [ABSTRACT FROM AUTHOR]

Simple Summary: Fasciolosis is a parasitic disease of livestock causing important economic losses worldwide, and it is also a zoonosis. The treatment is based on the use of anthelmintic drugs, but the increase in resistance and the risk of drug residues in food make this approach no longer sustainable. Developing protective vaccines for the control of fasciolosis is postulated as an appropriate treatment, but a better knowledge of the host–parasite interaction is needed. The aim of the present study was to evaluate the hepatic lesions in sheep infected and reinfected with Fasciola hepatica during the acute and chronic stages of infection and the characterization of the hepatic inflammatory infiltrates using immunohistochemistry with CD3, Foxp3, iNOS, and CD163 antibodies. The most remarkable histopathological finding was the presence of large necrotic foci and/or hemorrhages adjacent to enlarged bile ducts containing adult parasites, suggesting flukes may have caused these lesions while feeding. In the literature, necrotic foci/hemorrhages are considered a consequence of parasite migration. In both the primoinfected and reinfected groups, and during acute and chronic stages of the infection, an increase in Foxp3 T cells with respect to uninfected controls and a poor expression of iNOS was found accompanied by a strong expression of CD163, suggesting a marked M2 activation of macrophages in the hepatic lesions. Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since the current anthelmintic therapy is no longer sustainable. A better knowledge of the host–parasite interaction is needed to design effective vaccines. To date, few studies have evaluated host–parasite interaction by comparing infected and reinfected animals. The present study evaluates the microscopical hepatic lesions in sheep infected and reinfected with Fasciola hepatica during the acute and chronic stages of infection. The histopathological study revealed the presence of necrotizing foci (NF1) associated with larvae migration during the early stages of infection in the primoinfected (PI) and reinfected (RI) groups. In the late stages of infection of the PI group and at the early and late stages of infection in the RI groups, extensive necrotizing/hemorrhagic foci (NF2) were found in the vicinity of enlarged bile ducts, some containing adult flukes, suggesting parasites may have caused NF2 while feeding. The immunohistochemical study revealed an increase in Foxp3+ T cells in both PI and RI groups with respect to the UC group and in the infiltrates adjacent to NF1 in the RI groups with respect to the PI group, suggesting the F. hepatica induce Foxp3 T cell expansion to facilitate parasite survival. In addition, in both the PI and RI groups, and during acute and chronic stages of the infection, a poor expression of iNOS was found accompanied by a strong expression of CD163, suggesting a marked M2 activation of macrophages in the hepatic lesions, which may be related with healing processes, and it also may facilitate parasite survival. The main differences between PI and RI animals were the more severe infiltration of eosinophils and Foxp3+ T cells, whereas RI did not modify M2 activation of macrophages which occurs since the early stages of primoinfection. [ABSTRACT FROM AUTHOR]

Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we examined immune cell infiltration in 230 breast cancer samples, emphasizing diverse ethnic populations. Leveraging tissue microarrays (TMAs) and core samples, we applied multiplex immunofluorescence (mIF) to dissect immune cell subtypes across TME regions. Our analysis revealed distinct immune cell distribution patterns, particularly enriched in aggressive molecular subtypes triple-negative and HER2-positive tumors. We observed significant correlations between immune cell abundance and key clinicopathological parameters, including tumor size, lymph node involvement, and patient overall survival. Notably, immune cell location within different TME regions showed varying correlations with clinicopathologic parameters. Additionally, ethnicities exhibited diverse distributions of cells, with certain ethnicities showing higher abundance compared to others. In TMA samples, patients of Chinese and Caribbean origin displayed significantly lower numbers of B cells, TAMs, and FOXP3-positive cells. These findings highlight the intricate interplay between immune cells and breast cancer progression, with implications for personalized treatment strategies. Moving forward, integrating advanced imaging techniques, and exploring immune cell heterogeneity in diverse ethnic cohorts can uncover novel immune signatures and guide tailored immunotherapeutic interventions, ultimately improving breast cancer management. [ABSTRACT FROM AUTHOR]

Since transcription factor Forkhead Box P3 (FoxP3) was identified as a specific regulatory T cell (Treg) marker, researchers have scrutinized its value as a potential novel therapeutic target or a prognostic factor in various types of cancer with inconsistent results. The present analysis was performed to assess the influence of Treg FoxP3 expression on the prognosis of primary melanoma and to evaluate the correlations with various clinicopathological prognostic factors. We analyzed all eligible patients with stage pT3 primary malignant melanomas treated in a tertiary cancer center. Immunohistochemical staining for Treg FoxP3 expression was performed on retrospectively identified paraffin blocks and subsequently correlated with the outcomes of the patients. A total of 81% of the patients presented a positive Treg FoxP3 expression, being correlated with a higher risk of lymph node metastasis, tumor relapse, and death. Moreover, positive expression was statistically associated with a shorter OS. The tumor relapse rate was estimated at 36.7%. A positive expression of Treg FoxP3 and lymph node metastasis were associated with a higher risk of death based on multivariate analysis. Treg FoxP3 expression may be used as an independent prognostic factor in patients with malignant melanoma to evaluate tumor progression and survival. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3, CTLA4, and PDCD1), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3), and loss of Treg-mediated suppression. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are essential to maintain immune homeostasis by promoting self-tolerance. Reduced Treg numbers or functionality can lead to a loss of tolerance, increasing the risk of developing autoimmune diseases. An overwhelming variety of human Tregs has been described, based on either specific phenotype, tissue compartment, or pathological condition, yet the bulk of the literature only addresses CD25-positive and CD127-negative cells, coined by naturally occurring Tregs (nTregs), most of which express the transcription factor Forkhead box protein 3 (FOXP3). While the discovery of FOXP3 was seminal to understanding the origin and biology of nTregs, there is evidence in humans that not all T cells expressing FOXP3 are regulatory, and that not all Tregs express FOXP3. Namely, the activation of human T cells induces the transient expression of FOXP3, irrespective of whether they are regulatory or inflammatory effectors, while some induced T cells that may be broadly defined as Tregs (e.g., Tr1 cells) typically lack demethylation and do not express FOXP3. Furthermore, it is unknown whether and how many nTregs exist without FOXP3 expression. Several other candidate regulatory molecules, such as GITR, Lag-3, GARP, GPA33, Helios, and Neuropilin, have been identified but subsequently discarded as Treg-specific markers. Multiparametric analyses have uncovered a plethora of Treg phenotypes, and neither single markers nor combinations thereof can define all and only Tregs. To date, only the functional capacity to inhibit immune responses defines a Treg and distinguishes Tregs from inflammatory T cells (Teffs) in humans. This review revisits current knowledge of the Treg universe with respect to their heterogeneity in phenotype and function. We propose that it is unavoidable to characterize human Tregs by their phenotype in combination with their function, since phenotype alone does not unambiguously define Tregs. There is an unmet need to align the expression of specific markers or combinations thereof with a particular suppressive function to coin functional Treg entities and categorize Treg diversity. [ABSTRACT FROM AUTHOR]

Summary: Objective: Investigating the impact of FOXP3 (transcription factor forkhead box P3) expression on the biological behavior of esophageal squamous cell carcinoma (ESCC) and its influence on the sensitivity of ESCC cells towards cetuximab-targeted (an EGFR monoclonal antibody inhibitor) therapy. Methods: A specifically designed recombinant FOXP3 shRNA plasmid was synthesized to target the human FOXP3 gene, and the plasmid was transfected into TE12 cells using a liposome method. Multiple assays were conducted to evaluate the effect of FOXP3 expression on ESCC cells and their response to cetuximab treatment. Proliferation activity and cetuximab sensitivity of ESCC cells were measured using the CCK‑8 assay. The invasion ability of cells was assessed using an in vitro invasion assay. Furthermore, the efficacy of cetuximab in treating ESCC was analyzed using a tumorigenesis assay in nude mice. Results: Silencing the FOXP3 gene in the TE12 cell line (shFOXP3 group) resulted in a significant reduction in FOXP3 mRNA and protein expression (p = 0.013). The shFOXP3 group exhibited slowed cell growth (p = 0.035), decreased invasion rate (p = 0.031), and increased sensitivity to cetuximab treatment (p = 0.039) compared to the control group (shNC group). In the in vivo tumorigenesis assay, the shFOXP3 group demonstrated a significant reduction in tumor volume and lung metastasis rate following cetuximab treatment (p = 0.028 and 0.007, respectively). Conclusion: High FOXP3 expression promotes the proliferation and migration of ESCC cells, while negatively affecting their sensitivity to cetuximab-targeted therapy. Consequently, targeting FOXP3 shows potential therapeutic implications for enhancing the effectiveness of cetuximab treatment in ESCC patients. [ABSTRACT FROM AUTHOR]

A balance between pro-inflammatory decidual CD4+ T cells and FOXP3+ regulatory T cells (FOXP3+ Tregs) is important for maintaining fetomaternal tolerance. Using single-cell RNA-sequencing and T cell receptor repertoire analysis, we determined that diversity and clonality of decidual CD4+ T cell subsets depend on gestational age. Th1/Th2 intermediate and Th1 subsets of CD4+ T cells were clonally expanded in both early and late gestation, whereas FOXP3+ Tregs were clonally expanded in late gestation. Th1/Th2 intermediate and FOXP3+ Treg subsets showed altered gene expression in preeclampsia (PE) compared to healthy late gestation. The Th1/Th2 intermediate subset exhibited elevated levels of cytotoxicity-related gene expression in PE. Moreover, increased Treg exhaustion was observed in the PE group, and FOXP3+ Treg subcluster analysis revealed that the effector Treg like subset drove the Treg exhaustion signatures in PE. The Th1/Th2 intermediate and effector Treg like subsets are possible inflammation-driving subsets in PE. [ABSTRACT FROM AUTHOR]