FOXN2, identified as a novel biomarker in serum, modulates the transforming growth factor-beta signaling pathway through its interaction with partitioning defective 6 homolog alpha, contributing to the pathogenesis of gastric cancer.

BACKGROUND AND OBJECTIVE: Dysregulated expression of Forkhead Box N2 (FOXN2) has been detected in various cancer types. However, the underlying mechanisms by which FOXN2 contributes to the onset and progression of gastric cancer (GC) remain largely unexplored. This study aimed to elucidate the potential role of FOXN2 within GC, its downstream molecular mechanisms, and its feasibility as a novel serum biomarker for GC. METHODS: Tissue samples from GC patients and corresponding non-cancerous tissues were collected. Peripheral blood samples were obtained from GC patients and healthy controls. The expression of FOXN2 was determined using quantitative real-time PCR, western blotting, and immunohistochemistry. The expression of FOXN2 in GC cells was modulated by transfection with small interfering RNA (siRNA) or the pcDNA 3.1 expression vector. Cell proliferation was assessed using the Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine incorporation assays. The migratory and invasive capacities of cells were evaluated by Transwell assays, apoptosis rates were measured by flow cytometry, and the expression of proliferative, apoptotic, and epithelial-mesenchymal transition (EMT) markers were assessed by western blot analysis. RESULTS: FOXN2 was found to be overexpressed in the serum, tissues, and cells of GC, correlating with distant metastasis and TNM staging. FOXN2 demonstrated diagnostic value in differentiating GC patients from healthy individuals, with higher levels of FOXN2 being indicative of poorer survival rates. Silencing FOXN2 in vitro inhibited the proliferation, invasion, migration, and EMT of GC cells, while promoting apoptosis. FOXN2 was shown to regulate the transforming growth factor-beta (TGFß) receptor signaling pathway in GC cells via its interaction with Partitioning Defective 6 Homolog Alpha (PARD6A). CONCLUSION: In summary, our data suggest that FOXN2 acts as an oncogenic factor in GC, modulating the TGFß pathway by binding to PARD6A, thereby influencing gastric carcinogenesis. This study underscores the functional significance of FOXN2 as a potential serum biomarker and therapeutic target in GC.

Persistent vocal learning in an aging open-ended learner reflected in neural FoxP2 expression.

BACKGROUND: Most vocal learning species exhibit an early critical period during which their vocal control neural circuitry facilitates the acquisition of new vocalizations. Some taxa, most notably humans and parrots, retain some degree of neurobehavioral plasticity throughout adulthood, but both the extent of this plasticity and the neurogenetic mechanisms underlying it remain unclear. Differential expression of the transcription factor FoxP2 in both songbird and parrot vocal control nuclei has been identified previously as a key pattern facilitating vocal learning. We hypothesize that the resilience of vocal learning to cognitive decline in open-ended learners will be reflected in an absence of age-related changes in neural FoxP2 expression. We tested this hypothesis in the budgerigar (Melopsittacus undulatus), a small gregarious parrot in which adults converge on shared call types in response to shifts in group membership. We formed novel flocks of 4 previously unfamiliar males belonging to the same age class, either "young adult" (6 mo - 1 year) or "older adult" (≥ 3 year), and then collected audio-recordings over a 20-day learning period to assess vocal learning ability. Following behavioral recording, immunohistochemistry was performed on collected neural tissue to measure FoxP2 protein expression in a parrot vocal learning center, the magnocellular nucleus of the medial striatum (MMSt), and its adjacent striatum. RESULTS: Although older adults show lower vocal diversity (i.e. repertoire size) and higher absolute levels of FoxP2 in the MMSt than young adults, we find similarly persistent downregulation of FoxP2 and equivalent vocal plasticity and vocal convergence in the two age cohorts. No relationship between individual variation in vocal learning measures and FoxP2 expression was detected. CONCLUSIONS: We find neural evidence to support persistent vocal learning in the budgerigar, suggesting resilience to aging in the open-ended learning program of this species. The lack of a significant relationship between FoxP2 expression and individual variability in vocal learning performance suggests that other neurogenetic mechanisms could also regulate this complex behavior.

MiR-135b-5p promotes cetuximab resistance in colorectal cancer by regulating FOXN3.

Despite advances in targeted therapies, primary and acquired resistance make the treatment of colorectal cancer (CRC) a pressing issue to be resolved. According to reports, the development of CRC is linked to miRNA dysregulation. Multiple studies have demonstrated that miR-135b-5p has an aberrant expression level between CRC tissues and adjacent tissues. However, it is unclear whether there is a correlation between miR-135b-5p and cetuximab (CTx) resistance in CRC. Use the GEO database to measure miR-135b-5p expression in CRC. Additionally, RT-qPCR was applied to ascertain the production level of miR-135b-5p in three human CRC cells and NCM460 cells. The capacity of cells to migrate and invade was examined utilizing the wound-healing and transwell assays, while the CCK-8 assay served for evaluating cell viability, as well as colony formation assays for proliferation. The expected target protein of miR-135b-5p in CRC cell cetuximab resistance has been investigated using western blot. Suppression of miR-135b-5p could increase the CTx sensitivity of CTx-resistant CRC cells, as manifested by the attenuation of proliferation, migration, and invasion ability. Mechanistic studies revealed miR-135b-5p regulates the epithelial-to-mesenchymal transition (EMT) process and Wnt/ß-catenin signaling pathway through downgulating FOXN3. In short, knockdowning miR-135b-5p could increase FOXN3 expression in CRC cells, promote the EMT process, and simultaneously activate the Wnt/ß-catenin signaling pathway to elevate CTx resistance in CRC cells.

MEK-inhibitor treatment reduces the induction of regulatory T cells in mice after influenza A virus infection.

Regulatory T cells (Tregs) play a crucial and complex role in balancing the immune response to viral infection. Primarily, they serve to regulate the immune response by limiting the expression of proinflammatory cytokines, reducing inflammation in infected tissue, and limiting virus-specific T cell responses. But excessive activity of Tregs can also be detrimental and hinder the ability to effectively clear viral infection, leading to prolonged disease and potential worsening of disease severity. Not much is known about the impact of Tregs during severe influenza. In the present study, we show that CD4+/CD25+FoxP3+ Tregs are strongly involved in disease progression during influenza A virus (IAV) infection in mice. By comparing sublethal with lethal dose infection in vivo, we found that not the viral load but an increased number of CD4+/CD25+FoxP3+ Tregs may impair the immune response by suppressing virus specific CD8+ T cells and favors disease progression. Moreover, the transfer of induced Tregs into mice with mild disease symptoms had a negative and prolonged effect on disease outcome, emphasizing their importance for pathogenesis. Furthermore, treatment with MEK-inhibitors resulted in a significant reduction of induced Tregs in vitro and in vivo and positively influenced the progression of the disease. Our results demonstrate that CD4+/CD25+FoxP3+ Tregs are involved in the pathogenesis of severe influenza and indicate the potential of the MEK-inhibitor zapnometinib to modulate CD4+/CD25+FoxP3+ Tregs. Thus, making MEK-inhibitors even more promising for the treatment of severe influenza virus infections.

FOXK2 facilitates the airway remodeling during chronic asthma by promoting glycolysis in a SIRT2-dependent manner.

Asthma is a chronic pulmonary disease with the worldwide prevalence. The structural alterations of airway walls, termed as "airway remodeling", are documented as the core contributor to the airway dysfunction during chronic asthma. Forkhead box transcription factor FOXK2 is a critical regulator of glycolysis, a metabolic reprogramming pathway linked to pulmonary fibrosis. However, the role of FOXK2 in asthma waits further explored. In this study, the chronic asthmatic mice were induced via ovalbumin (OVA) sensitization and repetitive OVA challenge. FOXK2 was upregulated in the lungs of OVA mice and downregulated after adenovirus-mediated FOXK2 silencing. The lung inflammation, peribronchial collagen deposition, and glycolysis in OVA mice were obviously attenuated after FOXK2 knockdown. Besides, the expressions of FOXK2 and SIRT2 in human bronchial epithelial cells (BEAS-2B) were increasingly upregulated upon TGF-ß1 stimulation and downregulated after FOXK2 knockdown. Moreover, the functional loss of FOXK2 remarkably suppressed TGF-ß1-induced epithelial-mesenchymal transition (EMT) and glycolysis in BEAS-2B cells, as manifested by the altered expressions of EMT markers and glycolysis enzymes. The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) inhibited the EMT in TGF-ß1-induced cells, making glycolysis a driver of EMT. The binding of FOXK2 to SIRT2 was validated, and SIRT2 overexpression blocked the FOXK2 knockdown-mediated inhibition of EMT and glycolysis in TGF-ß1-treated cells, which suggests that FOXK2 regulates EMT and glycolysis in TGF-ß1-treated cells in a SIRT2-dependnet manner. Collectively, this study highlights the protective effect of FOXK2 knockdown on airway remodeling during chronic asthma.

FOXP3 snatches transcription factors depending on the context.

FOXP3 hijacks DNA-binding proteins to regulate gene expression. In this issue of JEM, He et al. (https://doi.org/10.1084/jem.20232068) propose a dynamic model in which FOXP3 associates with DNA-binding proteins to regulate Treg cell function in response to environmental cues.

Hypermethylation of the FOXP3 gene regulates Tregs immunodysregulation in chronic idiopathic thrombocytopenic purpura.

BACKGROUND: Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by a breakdown of immune tolerance; in ITP, the body's immune system mistakenly attacks and destroys platelets. This study aims to investigate the role and underlying mechanisms of FOXP3 in chronic ITP. METHODS: Flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in CD4+CD25+ T lymphocytes from 20 patients with chronic ITP (CITP), 20 acute ITP (AITP) controls, and 20 healthy individuals.CD4+CD25+ Treg cells were isolated from peripheral blood of patients with CITP using magnetic beads and then treated with phosphate-buffered saline solution or decitabine (a methylation inhibitor) for 48 h. The levels of interleukin-2 (IL-2), IL-10, and transforming growth factor-beta1 (TGF-ß1) in the plasma and CD4+CD25+ Treg cells were assessed by Enzyme-linked-immunosorbent serologic assay and quantitative real-time polymerase chain reaction (qRT-PCR). FOXP3 level was measured by qRT-PCR and Western blot analysis. Methylation-specific PCR (MS-PCR) was adopted to detect the status of FOXP3 methylation. RESULTS: The number of Treg cells and the contents of IL-2, IL-10, and TGF-ß1 decreased in patients with CITP, compared to the AITP control group and normal group. FOXP3 expression was reduced and FOXP3 methylation increased in patients with CITP, compared to the AITP control group and normal group. Hypermethylation of FOXP3 promoter led to decrease in FOXP3 level in Treg cells. Inhibition of FOXP3 promoter hypermethylation promoted the secretion of IL-2, IL-10, and TGF-ß1 in Treg cells. CONCLUSION: The number of Treg cells in CITP patients decreased, and the hypermethylation of FOXP3 promoter led to reduction of its expression in Treg cells, thus affecting the immune functioning of Treg cells.

Peripheral T Cell Development and Immunophenotyping of Twins with Heterozygous FOXN1 Mutations.

The transcription factor FOXN1 plays an established role in thymic epithelial development to mediate selection of maturing thymocytes. Patients with heterozygous loss-of-function FOXN1 variants are associated with T cell lymphopenia at birth and low TCR excision circles that can ultimately recover. Although CD4+ T cell reconstitution in these patients is not completely understood, a lower proportion of naive T cells in adults has suggested a role for homeostatic proliferation. In this study, we present an immunophenotyping study of fraternal twins with low TCR excision circles at birth. Targeted primary immunodeficiency testing revealed a heterozygous variant of uncertain significance in FOXN1 (c.1205del, p.Pro402Leufs*148). We present the immune phenotypes of these two patients, as well as their father who carries the same FOXN1 variant, to demonstrate an evolving immune environment over time. While FOXN1 haploinsufficiency may contribute to thymic defects and T cell lymphopenia, we characterized the transcriptional activity and DNA binding of the heterozygous FOXN1 variant in 293T cells and found the FOXN1 variant to have different effects across several target genes. These data suggest multiple mechanisms for similar FOXN1 variants pathogenicity that may be mutation specific. Increased understanding of how these variants drive transcriptional regulation to impact immune cell populations will guide the potential need for therapeutics, risk for infection or autoimmunity over time, and help inform clinical decisions for other variants that might arise.

KK-LC-1, a biomarker for prognosis of immunotherapy for primary liver cancer.

PURPOSE: There is mounting evidence that patients with liver cancer can benefit from Immune checkpoint inhibitors. However, due to the high cost and low efficacy, we aimed to explore new biomarkers for predicting the efficacy of immunotherapy. METHODS: Specimens and medical records of liver cancer patients treated at Drum Tower Hospital of Nanjing University were collected, and the expression of Kita-Kyushu lung cancer antigen-1 (KK-LC-1) in tissues as well as the corresponding antibodies in serum were examined to find biomarkers related to the prognosis of immunotherapy and to explore its mechanism in the development of liver cancer. RESULTS: KK-LC-1 expression was found to be 34.4% in histopathological specimens from 131 patients and was significantly correlated with Foxp3 expression (P = 0.0356). The expression of Foxp3 in the tissues of 24 patients who received immunotherapy was significantly correlated with overall survival (OS) (P = 0.0247), and there was also a tendency for prolonged OS in patients with high expression of KK-LC-1. In addition, the expression of KK-LC-1 antibody in the serum of patients who received immunotherapy with a first efficacy evaluation of stable disease (SD) was significantly higher than those with partial response (PR) (P = 0.0413). CONCLUSIONS: Expression of KK-LC-1 in both tissues and serum has been shown to correlate with the prognosis of patients treated with immunotherapy, and KK-LC-1 is a potential therapeutic target for oncological immunotherapy.

Regulatory mechanism of cold-inducible diapause in Caenorhabditis elegans.

Temperature is a critical environmental cue that controls the development and lifespan of many animal species; however, mechanisms underlying low-temperature adaptation are poorly understood. Here, we describe cold-inducible diapause (CID), another type of diapause induced by low temperatures in Caenorhabditis elegans. A premature stop codon in heat shock factor 1 (hsf-1) triggers entry into CID at 9 °C, whereas wild-type animals enter CID at 4 °C. Furthermore, both wild-type and hsf-1(sy441) mutant animals undergoing CID can survive for weeks, and resume growth at 20 °C. Using epistasis analysis, we demonstrate that neural signalling pathways, namely tyraminergic and neuromedin U signalling, regulate entry into CID of the hsf-1 mutant. Overexpression of anti-ageing genes, such as hsf-1, XBP1/xbp-1, FOXO/daf-16, Nrf2/skn-1, and TFEB/hlh-30, also inhibits CID entry of the hsf-1 mutant. Based on these findings, we hypothesise that regulators of the hsf-1 mutant CID may impact longevity, and successfully isolate 16 long-lived mutants among 49 non-CID mutants via genetic screening. Furthermore, we demonstrate that the nonsense mutation of MED23/sur-2 prevents CID entry of the hsf-1(sy441) mutant and extends lifespan. Thus, CID is a powerful model to investigate neural networks involving cold acclimation and to explore new ageing mechanisms.

Epidermal factor Foxn1 as a regulator of antioxidant defense in the skin / Naskórkowy czynnik Foxn1 regulatorem obrony antyoksydacyjnej skóry.

The skin, as the largest organ of the body, is constantly exposed to environmental threats, including: injuries and oxidative stress. The thioredoxin system is one of the skin antioxidant systems , which protects cells against oxidative stress, regulates cell migration, proliferation and apoptosis, and also participates in signal transmission by regulating the activity of transcription factors. Recent studies have shown a correlation between the epidermal transcription factor Foxn1 and the thioredoxin system in mouse skin. Mass spectrometry analysis, followed by in vitro and in vivo experiments, showed that Foxn1 in keratinocytes regulates elements of the electron transport chain as well as the thioredoxin system (Txn2, Txnrd3), especially under hypoxic condition. High levels of Txnrd3 mRNA were detected for the first time in the injured skin of Foxn1+/+ mice compared to Foxn1-/- mice, and also showed that Foxn1 in keratinocytes upregulates Txnrd3 protein expression. Moreover, in silico analyzes indicated possible binding sites of the transcription factor Foxn1 in the Txn system. In conclusion, the data presented in this review identify Foxn1 as a novel component of the skin antioxidant system.

Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma.

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.

Prognostic Role of Tumor-Infiltrating Lymphocytes in Oral Squamous Cell Carcinoma.

BACKGROUND: In oral squamous cell carcinoma (OSCC), the tumor-node-metastasis (TNM) staging system is a significant factor that influences prognosis and treatment decisions for OSCC patients. Unfortunately, TNM staging does not consistently predict patient prognosis and patients with identical clinicopathological characteristics may have vastly different survival outcomes. Host immunity plays an important role in tumor progression but is not included in the TNM staging system. Tumor-infiltrating lymphocytes (TILs) are part of the host immune response that recognizes tumor cells; and the presence of TILs has emerged as potential candidates for prognostic markers for many types of cancers. The present study aims to determine the association of T cell-specific markers (CD3, CD4, CD8, and FOXP3) with clinicopathological characteristics and survival outcomes in OSCC patients. The prognostic value of CD3, CD4, and CD8 will also be evaluated based on tumor stage. METHODS: Tissue microarrays were constructed containing 231 OSCC cases and analyzed by immunohistochemical staining for the expression of CD3, CD4, CD8, and FOXP3. The expression scores for each marker were correlated with clinicopathological parameters and survival outcomes. The prognostic impact of CD3, CD4 and CD8 were further analyzed based on tumor stage (early or advanced). RESULTS: CD3, CD4, and CD8 were found to be significantly associated with both overall survival and progression-free survival using univariate analysis. However, none of these markers were found to independently predict the survival outcomes of OSCC using multivariate analysis. Only conventional factors such as nodal status, tumor differentiation and perineural invasion (PNI) were independent predictors of survival outcomes, with nodal status being the strongest independent predictor. Additionally, low CD4 (but not CD3 or CD8) expression was found to identify early-stage OSCC patients with exceptionally poor prognosis which was similar to that of advanced staged OSCC patients. CONCLUSIONS: TIL markers such as CD3, CD4, CD8, and FOXP3 can predict the survival outcomes of OSCC patients, but do not serve as independent prognostic markers as found with conventional factors (i.e. nodal status, tumor differentiation and PNI). CD4 expression may assist with risk stratification in early-stage OSCC patients which may influence treatment planning and decision making for early-stage OSCC patients.

Jujubae Fructus extract prolongs lifespan and improves stress tolerance in Caenorhabditis elegans dependent on DAF-16/SOD-3.

Jujubae Fructus, the fruit of Ziziphus jujuba Mill has been used as one of the medicine food homology species for thousands of years in China. Studies have shown that the active ingredients of Jujubae Fructus have a variety of biological effects, but its role in the aging process still lacks knowledge. Here, we investigated the effect of Jujubae Fructus extract (JE) on Caenorhabditis elegans lifespan and its potential mechanism. The lifespan of C. elegans treated with JE was signifificantly increased in a dose-dependent manner. In addition, JE treatment prolonged the reproductive period and increased normal activity during aging in C. elegans. Similarly, JE supplementation also enhanced the resistance to heat and oxidative stress in C. elegans. Furthermore, the mutant worms' lifespan assays demonstrated that JE requires daf-16 to prolong lifespan. DAF-16::GFP analysis of TJ356 showed that JE treatment translocates DAF-16::GFP to nucleus in transgenic worms. By analyzing the downstream of daf-16, we identify that JE may regulate sod3 downstream of daf-16. Mutant worms' lifespan and transgenic reporter gene expression assays revealed that increasing SOD-3 expression was critical for extending longevity in C. elegans with JE therapy. Collectively, these data indicate that JE may have an important role in C. elegans longevity that is dependent on DAF-16 and SOD-3.

DOS-3 mediates cell-non-autonomous DAF-16/FOXO activity in antagonizing age-related loss of C. elegans germline stem/progenitor cells.

Age-related depletion of stem cells causes tissue degeneration and failure to tissue regeneration, driving aging at the organismal level. Previously we reported a cell-non-autonomous DAF-16/FOXO activity in antagonizing the age-related loss of germline stem/progenitor cells (GSPCs) in C. elegans, indicating that regulation of stem cell aging occurs at the organ system level. Here we discover the molecular effector that links the cell-non-autonomous DAF-16/FOXO activity to GSPC maintenance over time by performing a tissue-specific DAF-16/FOXO transcriptome analysis. Our data show that dos-3, which encodes a non-canonical Notch ligand, is a direct transcriptional target of DAF-16/FOXO and mediates the effect of the cell-non-autonomous DAF-16/FOXO activity on GSPC maintenance through activating Notch signaling in the germ line. Importantly, expression of a human homologous protein can functionally substitute for DOS-3 in this scenario. As Notch signaling controls the specification of many tissue stem cells, similar mechanisms may exist in other aging stem cell systems.

Pre-Transplant Frequencies of FoxP3+CD25+ in CD3+CD8+ T Cells as Potential Predictors for CMV in CMV-Intermediate Risk Kidney Transplant Recipients.

Cytomegalovirus (CMV) infection detrimentally influences graft survival in kidney transplant recipients, with the risk primarily determined by recipient and donor serostatus. However, recipient CD8+ T cells play a crucial role in CMV control. The optimal preventive strategy (prophylaxis vs. pre-emptive treatment), particularly for seropositive (intermediate risk) recipients, remains uncertain. We investigated CD8+ T cell subpopulation dynamics and CMV occurrence (DNAemia ≥ 100 IU/mL) in 65 kidney transplant recipients, collecting peripheral blood mononuclear cells before (T1) and 1 year after transplantation (T2). Comparing the two timepoints, we found an increase in granulocyte, monocyte and CD3+CD8+ T cells numbers, while FoxP3+CD25+, LAG-3+ and PD-1+ frequencies were reduced at T2. CMV DNAemia occurred in 33 recipients (55.8%) during the first year. Intermediate risk patients were disproportionally affected by posttransplant CMV (N = 29/45, 64.4%). Intermediate risk recipients developing CMV after transplantation exhibited lower leukocyte, monocyte, and granulocyte counts and higher FoxP3+CD25+ frequencies in CD3+CD8+ T cells pre-transplantation compared to patients staying CMV negative. Pre-transplant FoxP3+CD25+ in CD3+CD8+ T cells had the best discriminatory potential for CMV infection prediction within the first year after transplantation (AUC: 0.746). The FoxP3+CD25+ CD3+CD8+ T cell subset may aid in selecting intermediate risk kidney transplant recipients for CMV prophylaxis.

Regulatory and memory T lymphocytes infiltrating prostate tumors predict long term clinical outcomes.

Introduction: The localization, density but mostly the phenotype of tumor infiltrating lymphocytes (TIL) provide important information on the initial interaction between the host immune system and the tumor. Our objective was to assess the prognostic significance of T (CD3+), T regulatory (Treg) (FoxP3+) and T memory (Tmem) (CD45RO+) infiltrating lymphocytes and of genes associated with TIL in prostate cancer (PCa). Methods: Immunohistochemistry (IHC) was used to assess the infiltration of CD3+, FoxP3+ and CD45RO+ cells in the tumor area, tumor margin and adjacent normal-like epithelium of a series of 98 PCa samples with long clinical follow-up. Expression of a panel of 31 TIL-associated genes was analyzed by Taqman Low-Density Array (TLDA) technology in another series of 50 tumors with long clinical follow-up. Kaplan-Meier and Cox proportional hazards regression analyses were performed to determine association of these markers with biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa. Results: TIL subtypes were present at different densities in the tumor, tumor margin and adjacent normal-like epithelium, but their density and phenotype in the tumor area were the most predictive of clinical outcomes. In multivariate analyses, a high density of Treg (high FoxP3+/CD3+ cell ratio) predicted a higher risk for need of definitive ADT (HR=7.69, p=0.001) and lethal PCa (HR=4.37, p=0.04). Conversely, a high density of Tmem (high CD45RO+/CD3+ cell ratio) predicted a reduced risk of lethal PCa (HR=0.06, p=0.04). TLDA analyses showed that a high expression of FoxP3 was associated with a higher risk of lethal PCa (HR=5.26, p=0.02). Expression of CTLA-4, PD-1, TIM-3 and LAG-3 were correlated with that of FoxP3. Amongst these, only a high expression of TIM-3 was associated with a significant higher risk for definitive ADT in univariate Cox regression analysis (HR=3.11, p=0.01). Conclusion: These results show that the proportion of Treg and Tmem found within the tumor area is a strong and independent predictor of late systemic progression of PCa. Our results also suggest that inhibition of TIM-3 might be a potential approach to counter the immunosuppressive functions of Treg in order to improve the anti-tumor immune response against PCa.

Human iPSC-derived CD4+ Treg-like cells engineered with chimeric antigen receptors control GvHD in a xenograft model.

CD4+ T cells induced from human iPSCs (iCD4+ T cells) offer a therapeutic opportunity for overcoming immune pathologies arising from hematopoietic stem cell transplantation. However, most iCD4+ T cells are conventional helper T cells, which secrete inflammatory cytokines. We induced high-level expression of FOXP3, a master transcription factor of regulatory T cells, in iCD4+ T cells. Human iPSC-derived, FOXP3-induced CD4+ T (iCD4+ Treg-like) cells did not secrete inflammatory cytokines upon activation. Moreover, they showed demethylation of the Treg-specific demethylation region, suggesting successful conversion to immunosuppressive iCD4+ Treg-like cells. We further assessed these iCD4+ Treg-like cells for CAR-mediated immunosuppressive ability. HLA-A2 CAR-transduced iCD4+ Treg-like cells inhibited CD8+ cytotoxic T cell (CTL) division in a mixed lymphocyte reaction assay with A2+ allogeneic CTLs and suppressed xenogeneic graft-versus-host disease (GVHD) in NSG mice treated with A2+ human PBMCs. In most cases, these cells suppressed the xenogeneic GvHD progression as much as natural CD25+CD127- Tregs did.

Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

Transcription factor FOXF2 promotes the development and progression of pancreatic cancer by targeting MSI2.

Pancreatic cancer (PC) is a malignant tumor possessing high mortality. The role of transcription factor Forkhead Box F2 (FOXF2) in PC remains unverified. The current study investigated the roles of FOXF2 in developing PC in vitro and in vivo. A xenograft tumor model was constructed with nude mice injected using FOXF2­overexpressing PC cells or FOXF2­silenced PC cells. High FOXF2 expression significantly enhanced the proliferation ability of PC cells in vitro and pancreatic tumor growth in vivo. The cell cycle analysis indicated that transition of G1­S phase was promoted by FOXF2. The cell cycle­associated proteins cyclin D1, CDK2, phosphorylated (p)­CDK2 and p­RB were upregulated in the FOXF2­overexpressing cells and downregulated in the cells with FOXF2 knockdown. Flow cytometric analysis and Hoechst staining showed that the percentage of apoptotic cells was significantly increased after FOXF2 was silenced. FOXF2 knockdown promoted expression of pro­apoptotic proteins (Bad, Bax and cleaved caspase­3) while suppressing the anti­apoptotic proteins (Bcl­2 and Bcl­xl) at the protein level. FOXF2 improved the migration and invasion of PC cells in vitro. Moreover, luciferase and chromatin immunoprecipitation assays revealed that FOXF2 binds to the MSI2 promoter, promoting its transcriptional expression. FOXF2 knockdown inhibited the MSI2 protein translation while enhancing the translation of NUMB protein, suppressing PC development in vivo. MSI2 silencing reversed the promotive effect mediated by FOXF2 on cell proliferation. These results demonstrated that FOXF2 is essential in PC progression, and the potential mechanism includes regulating MSI2 transcription.