Costimulation molecules differentially regulate the ERK-Zfp831 axis to shape T follicular helper cell differentiation.

T follicular helper (Tfh) cells play essential roles in regulating humoral immunity, especially germinal center reactions. However, how CD4+ T cells integrate the antigenic and costimulatory signals in Tfh cell development is still poorly understood. Here, we found that phorbol 12-myristate 13-acetate (PMA) + ionomycin (P+I) stimulation, together with interleukin-6 (IL-6), potently induce Tfh cell-like transcriptomic programs in vitro. The ERK kinase pathway was attenuated under P+I stimulation; ERK2 inhibition enhanced Tfh cell development in vitro and in vivo. We observed that inducible T cell costimulator (ICOS), but not CD28, lacked the ability to activate ERK, which was important in sustaining Tfh cell development. The transcription factor Zfp831, whose expression was repressed by ERK, promoted Tfh cell differentiation by directly upregulating the expression of the transcription factors Bcl6 and Tcf7. We have hence identified an ERK-Zfp831 axis, regulated by costimulation signaling, in critical regulation of Tfh cell development.

The Transcription Factor Tox2 Drives T Follicular Helper Cell Development via Regulating Chromatin Accessibility.

T follicular helper (Tfh) cells provide essential help to B cells in germinal center (GC) reactions. Bcl6 is the obligatory lineage transcription factor in Tfh cells. Here, we examined the molecular pathways that induce Bcl6 gene expression and underscore Bcl6-dependent function during Tfh cell commitment. Integration of genome-wide Bcl6 occupancy in Tfh cells and differential gene expression analyses suggested an important role for the transcription factor Tox2 in Tfh cell differentiation. Ectopic expression of Tox2 was sufficient to drive Bcl6 expression and Tfh development. In genome-wide ChIP-seq analyses, Tox2-bound loci associated with Tfh cell differentiation and function, including Bcl6. Tox2 binding was associated with increased chromatin accessibility at these sites, as measured by ATAC-seq. Tox2-/- mice exhibited defective Tfh differentiation, and inhibition of both Tox2 and the related transcription factor Tox abolished Tfh differentiation. Thus, a Tox2-Bcl6 axis establishes a transcriptional feed-forward loop that promotes the Tfh program.

Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction.

T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment1. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.

Negative regulation of IL-17-mediated signaling and inflammation by the ubiquitin-specific protease USP25.

Interleukin 17 (IL-17) is important in infection and autoimmunity; how it signals remains poorly understood. In this study, we identified the ubiquitin-specific protease USP25 as a negative regulator of IL-17-mediated signaling and inflammation. Overexpression of USP25 inhibited IL-17-triggered signaling, whereas USP25 deficiency resulted in more phosphorylation of the inhibitor IκBα and kinase Jnk and higher expression of chemokines and cytokines, as well as a prolonged half-life for chemokine CXCL1-encoding mRNA after treatment with IL-17. Consistent with that, Usp25(-/-) mice showed greater sensitivity to IL-17-dependent inflammation and autoimmunity in vivo. Mechanistically, stimulation with IL-17 induced the association of USP25 with the adaptors TRAF5 and TRAF6, and USP25 induced removal of Lys63-linked ubiquitination in TRAF5 and TRAF6 mediated by the adaptor Act1. Thus, our results demonstrate that USP25 is a deubiquitinating enzyme (DUB) that negatively regulates IL-17-triggered signaling.

Cutting Edge: Transcription Factor BCL6 Is Required for the Generation, but Not Maintenance, of Memory CD8+ T Cells in Acute Viral Infection.

The differentiation of memory CD8+ T cells is critical to the long-term cellular immunity. The transcription factor BCL6 has been reportedly important for the generation and maintenance of memory CD8+ T cells; however, using the newly established BCL6 conditional knockout mouse model, we demonstrate that BCL6 is dispensable for the maintenance of established memory CD8+ T cell pool, although BCL6 is still required for the generation of CD8+ memory precursors upon acute viral infection. In addition, BCL6 promotes the expression of TCF-1 via directly binding to the Tcf7 (gene symbol for TCF-1) allele in CD8+ memory precursors and forced expression of TCF-1 restores the generation of BCL6-deficient memory precursors. Thus, our findings clarify that BCL6 is dispensable for the maintenance of memory CD8+ T cells, but functions as an important upstream of TCF-1 to regulate the generation of memory precursors in acute viral infection.

Association Between Plasma Total Homocysteine Levels and Risk of Early Hemorrhagic Transformation in Patients with Acute Ischemic Stroke: A Hospital-Based Study.

OBJECTS: In this study, we investigated the association between plasma total homocysteine(tHcy) levels and the risk of early hemorrhagic transformation(HT) in patients with acute ischemic stroke(AIS). METHODS: Consecutive hospitalized participants who met the inclusion criteria were enrolled and grouped according to plasma tHcy levels. Participants were divided into a low homocysteine level(L-tHcy) group (<12 µmol/L) and a high homocysteine level group(H-tHcy) (≥ 12 µmol/L). Baseline computed tomography (CT) examination was performed. HT was determined via CT or magnetic resonance imaging within 1 to 3 days after admission. RESULTS: A total of 1858 patients were screened and 1378 patients completed the this study(797 patients in the H-tHcy group and 581 patients in the L-tHcy group). HT incidence was 5.2% (30/581,) in the L-tHcy group and 11.2% (90/797) in the H-tHcy group(P<0.05). Binary logistic regression analysis showed that initial NIHSS score, tHcy levels, treatment with recombinant tissue plasminogen activator thrombolysis, systolic blood pressure on admission, glucose level on admission, smoking status and estimated glomerular filtration rate were independent risk factors for HT. Receiver operating characteristic analysis showed that tHcy level was a moderately sensitive and specific index to predict the incidence of HT, and the optimal cutoff was 16.56 µmol/L (sensitivity 63.3%, specificity 41.3%). CONCLUSION: Our study findings reveal that high plasma tHcy level is one independent risk factor associated with increased risk of early HT in patients with AIS.

CCL8 secreted by tumor-associated macrophages promotes invasion and stemness of glioblastoma cells via ERK1/2 signaling.

Tumor-associated macrophages (TAMs) constitute a large population of glioblastoma and facilitate tumor growth and invasion of tumor cells, but the underlying mechanism remains undefined. In this study, we demonstrate that chemokine (C-C motif) ligand 8 (CCL8) is highly expressed by TAMs and contributes to pseudopodia formation by GBM cells. The presence of CCL8 in the glioma microenvironment promotes progression of tumor cells. Moreover, CCL8 induces invasion and stem-like traits of GBM cells, and CCR1 and CCR5 are the main receptors that mediate CCL8-induced biological behavior. Finally, CCL8 dramatically activates ERK1/2 phosphorylation in GBM cells, and blocking TAM-secreted CCL8 by neutralized antibody significantly decreases invasion of glioma cells. Taken together, our data reveal that CCL8 is a TAM-associated factor to mediate invasion and stemness of GBM, and targeting CCL8 may provide an insight strategy for GBM treatment.

Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development.

In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4(+) T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4(+) T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.

Integrated analysis identified core signal pathways and hypoxic characteristics of human glioblastoma.

As a hallmark for glioblastoma (GBM), high heterogeneity causes a variety of phenotypes and therapeutic responses among GBM patients, and it contributes to treatment failure. Moreover, hypoxia is a predominant feature of GBM and contributes greatly to its phenotype. To analyse the landscape of gene expression and hypoxic characteristics of GBM cells and their clinical significance in GBM patients, we performed transcriptome analysis of the GBM cell line U87-MG and the normal glial cell line HEB under normoxia and hypoxia conditions, with the results of which were analysed using established gene ontology databases as well as The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia. We revealed core signal pathways, including inflammation, angiogenesis and migration, and for the first time mapped the components of the toll-like receptor 6 pathway in GBM cells. Moreover, by investigating the signal pathways involved in homoeostasis, proliferation and adenosine triphosphate metabolism, the critical response of GBM to hypoxia was clarified. Experiments with cell lines, patient serum and tissue identified IL1B, CSF3 and TIMP1 as potential plasma markers and VIM, STC1, TGFB1 and HMOX1 as potential biopsy markers for GBM. In conclusion, our study provided a comprehensive understanding for signal pathways and hypoxic characteristics of GBM and identified new biomarkers for GBM patients.

Ubc9 Is Required for Positive Selection and Late-Stage Maturation of Thymocytes.

SUMOylation is an important posttranslational modification that regulates protein function in diverse biological processes. However, its role in early T cell development has not been genetically studied. UBC9 is the only E2 enzyme for all SUMOylation. In this study, by selectively deleting Ubc9 gene in T cells, we have investigated the functional roles of SUMOylation in T cell development. Loss of Ubc9 results in a significant reduction of CD4 and CD8 single-positive lymphocytes in both thymus and periphery. Ubc9-deficient cells exhibit defective late-stage maturation post the initial positive selection with increased apoptosis and impaired proliferation, among which attenuated IL-7 signaling was correlated with the decreased survival of Ubc9-deficent CD8 single-positive cells. Furthermore, NFAT nuclear retention induced by TCR signals was regulated by SUMOylation during thymocytes development. Our study thus reveals a novel posttranslational mechanism underlying T cell development.

The ANRIL Genetic Variants and Their Interactions with Environmental Risk Factors on Atherothrombotic Stroke in a Han Chinese Population.

BACKGROUND: Ischemic stroke (IS) is considered to be a heterogeneous, multifactorial disease with a strong genetic background. This study aims to determine whether variants in the antisense noncoding RNA in the INK4 locus (ANRIL) gene are associated with IS in Han Chinese, as well as whether there is evidence of a gene-environment interactions. MATERIALS AND METHODS: A case-controlled association study was conducted in which only patients with atherothrombotic stroke (ATS) were enrolled. Multifactor dimensionality reduction model was employed to screen the best interaction combinations among gene and environmental risk factors; RESULTS: A total of 405 subjects (200 cases and 205 controls) and 16 single nucleotide polymorphisms (SNPs) in ANRIL gene were included in this study. The 4 SNPs (rs1537378, rs2184061, rs7044859, and rs7865618) were found to be significantly related to ATS in Chinese Han nationality. In overall people or subjects aged 45 years or older, the GG genotype and G allele of rs1537378, the AA genotype and A allele of rs2184061 and rs7865618, and the AA genotype of rs7044859 increased the risk of ATS. In males, the GG genotype and G allele of rs1537378, the AA genotype and A allele of rs7865618, and the A allele of rs2184061 conferred a susceptibility to ATS. Additionally, the AAAGAGCAAAAAATAG haplotype exhibited an elevated risk of ATS, and a significant interaction was found in ATS susceptibility between ANRIL gene and dyslipidemia; CONCLUSIONS: The ANRIL gene was related to ATS susceptibility in a Han Chinese. Future studies should be performed with larger samples and among different ethnic populations.

Phosphorylated mTOR and YAP serve as prognostic markers and therapeutic targets in gliomas.

Glioma is the most prevalent type of tumor in the brain and is comprised of grades I-IV, according to the WHO classification system. Grade IV glioma is also known as glioblastoma multiforme (GBM), the most malignant type of glioma. Glioma is characterized by a complex molecular background, and gene profiling studies have disclosed critical genetic events in human gliomas, which make targeted therapies the most promising therapeutic strategy. However, crosstalk between the targeted signaling pathways may hinder the efficacy of targeted therapies in gliomas. Therefore, it is necessary to identify effective markers to stratify patients for specific therapeutic procedures. Although several mechanisms have been proposed based on the crosstalk between PI3K/AKT/mTORC1 and Hippo/YAP pathways, the clinical significance of the two pathways has not yet been assessed in a combinatorial manner. In this study, we evaluated the two pathways in human glioma specimens and observed the positive correlation between protein levels of p-mTORS2448 and YAP in gliomas. The findings indicated that high expression of p-mTORS2448 and YAP correlated with poor overall survival of glioma patients. As p-mTORS2448 is a specific marker of mTORC1 activation, our results reveal a potential interaction between mTORC1 and YAP, which might functionally participate in the development and progression of gliomas. In support of this hypothesis, a combination of inhibitors targeting mTORC1 and YAP showed a better inhibitory effect on growth of glioma cell lines. Altogether, our work, for the first time, reveals that p-mTORS2448 and YAP can be used as markers of PI3K/AKT/mTORC1 and Hippo/YAP pathway activity to predict prognosis and are target candidates for personalized medicine.

Basal NF-κB controls IL-7 responsiveness of quiescent naïve T cells.

T cells are essential for immune defenses against pathogens, such that viability of naïve T cells before antigen encounter is critical to preserve a polyclonal repertoire and prevent immunodeficiencies. The viability of naïve T cells before antigen recognition is ensured by IL-7, which drives expression of the prosurvival factor Bcl-2. Quiescent naïve T cells have low basal activity of the transcription factor NF-κB, which was assumed to have no functional consequences. In contrast to this postulate, our data show that basal nuclear NF-κB activity plays an important role in the transcription of IL-7 receptor α-subunit (CD127), enabling responsiveness of naïve T cells to the prosurvival effects of IL-7 and allowing T-cell persistence in vivo. Moreover, we show that this property of basal NF-κB activity is shared by mouse and human naïve T cells. Thus, NF-κB drives a distinct transcriptional program in T cells before antigen encounter by controlling susceptibility to IL-7. Our results reveal an evolutionarily conserved role of NF-κB in T cells before antigenic stimulation and identify a novel molecular pathway that controls T-cell homeostasis.

Transcriptional regulation of follicular T-helper (Tfh) cells.

T-follicular helper (Tfh) cells are a new subset of effector CD4(+) T cells that are specialized in helping B cells in the germinal center reaction. Tfh cells are distinct from other established CD4(+) T-cell lineages, Th1, Th2, Th17, and T-regulatory cells, in their gene expression profiles. Tfh cell differentiation results from a network of transcriptional regulation by a master transcriptional factor Bcl6 as well as IRF4, c-Maf, Batf, and STAT3/5. During Tfh cell ontogeny, increased CXCR5 expression directs activated T-cell migration to the follicles, and their interaction with B cells leads to Bcl6 upregulation, which helps establish effector and memory Tfh cell program. This review summarizes the recent progress in molecular mechanisms underlying Tfh differentiation and discusses the future perspectives for this important area of research.

SEMA3F prevents metastasis of colorectal cancer by PI3K-AKT-dependent down-regulation of the ASCL2-CXCR4 axis.

Semaphorin-3F (SEMA3F), an axonal repulsant in nerve development, has been shown to inhibit the progression of human colorectal cancer (CRC); however, the underlying mechanism remains elusive. In this study we found a negative correlation between the levels of SEMA3F and CXCR4 in CRC specimens from 85 patients, confirmed by bioinformatics analysis of gene expression in 229 CRC samples from the Cancer Genome Atlas. SEMA3F(high) /CXCR4(low) patients showed the lowest frequency of lymph node and distant metastasis and the longest survival. Mechanistically, SEMA3F inhibited the invasion and metastasis of CRC cells through PI3K-AKT-dependent down-regulation of the ASCL2-CXCR4 axis. Specifically, ASCL2 enhanced the invasion and metastasis of CRC cells in vitro and expression of ASCL2 correlated with distant metastasis, tumour size and poor overall survival in CRC patients. Treatment of CRC cells with the CXCR4 antagonist AMD3100 attenuated SEMA3F knockdown-induced invasion and metastasis of CRC cells in vitro and in vivo. Our study thus demonstrates that SEMA3F functions as a suppressor of CRC metastasis via down-regulating the ASCL2-CXCR4 axis.

Molecular mechanisms of T-cell tolerance.

CD4(+) T cells are the master regulators of adaptive immune responses, and many autoimmune diseases arise due to a breakdown of self-tolerance in CD4(+) T cells. Activation of CD4(+) T cells is regulated by not only the binding of peptide-major histocompatibility complexes to T-cell receptor but also costimulatory signals from antigen-presenting cells. Recently, there has been progress in understanding the extracellular and intracellular mechanisms that are required for implementation and maintenance of T-cell tolerance. Understanding of the molecular mechanisms underlying T-cell tolerance will lead to development of pharmacological approaches either to promote the tolerance state in terms of autoimmunity or to break tolerance in cancer.

Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization.

Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.

The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells.

The FcγRs found on macrophages (Ms) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Ms and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8(+) T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4(+) T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Ms but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4(+) T cells in the endosomal versus phagosomal compartments of Ms versus DCs.

The effects of Sodium-glucose cotransporter 2 inhibitors on adipose tissue in patients with type 2 diabetes: A meta-analysis of randomized controlled trials.

Purpose: To systematically evaluate the effect of Sodium-glucose cotransporter 2 (SGLT2) inhibitors on adipose tissue in patients with type 2 diabetes. Methods: We searched PubMed, Cochrane Library, EMBASE, and Web of science databases for literature pertaining to Randomized controlled trials (RCTs) of SGLT2 inhibitors in treating type 2 diabetes patients. The retrieval time was from the date of establishment of the databases to September 1, 2022. Meta-analysis was performed using RevMan5.4 software. Results: Totally 551 patients were included in 10 articles. Meta-analysis results showed that compared with the control group, the visceral adipose tissue (WMD = -16.29 cm2, 95% CI: -25.07 ~ -7.50, P<0.00001), subcutaneous adipose tissue (WMD = -19.34 cm2, 95% CI: -36.27 ~ -2.41, P<0.00001), body weight (WMD = -2.36 kg, 95% CI: -2.89 ~ -1.83, P<0.00001) and triglyceride (WMD = -24.41 mg/dl, 95% CI: -45.79 ~ -3.03, P = 0.03) of the trial group significantly reduced. Conclusion: SGLT2 inhibitors cause significant reductions in visceral adipose tissue, subcutaneous adipose tissue, body weight and triglycerides in type 2 diabetes patients, which may be attributed to the protective effect of the inhibitors on cardiovascular system.

LAPTM4B promotes AML progression through regulating RPS9/STAT3 axis.

Acute myeloid leukemia (AML) is a heterogeneous disorder with high morbidity and mortality under the existing treatment strategy. Here, we found that lysosome-associated protein transmembrane 4 beta (LAPTM4B) was frequently upregulated in AML, and high LAPTM4B was associated with poor outcome. Moreover, LAPTM4B promoted leukemia progression in vitro and in vivo. Mechanically, LAPTM4B interacted with RPS9, and positively regulated RPS9 protein stability, which enhanced leukemia cell progression via activating STAT3. Our findings indicate for the first time that LAPTM4B contributes to leukemia progression in a RPS9/STAT3-dependent manner, suggesting that LAPTM4B may serve as a promising target for treatment of AML.