Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

Cistromic Reprogramming of the Diurnal Glucocorticoid Hormone Response by High-Fat Diet.

The glucocorticoid receptor (GR) is a potent metabolic regulator and a major drug target. While GR is known to play integral roles in circadian biology, its rhythmic genomic actions have never been characterized. Here we mapped GR's chromatin occupancy in mouse livers throughout the day and night cycle. We show how GR partitions metabolic processes by time-dependent target gene regulation and controls circulating glucose and triglycerides differentially during feeding and fasting. Highlighting the dominant role GR plays in synchronizing circadian amplitudes, we find that the majority of oscillating genes are bound by and depend on GR. This rhythmic pattern is altered by high-fat diet in a ligand-independent manner. We find that the remodeling of oscillatory gene expression and postprandial GR binding results from a concomitant increase of STAT5 co-occupancy in obese mice. Altogether, our findings highlight GR's fundamental role in the rhythmic orchestration of hepatic metabolism.

A model of Th2 differentiation based on polarizing cytokine repression.

Conventional dendritic cells (cDCs) can integrate multiple stimuli from the environment and provide three separate outputs in terms of antigen presentation, costimulation, and cytokine production; this guides the activation, expansion, and differentiation of distinct functional T helper subsets. Accordingly, the current dogma posits that T helper cell specification requires these three signals in sequence. Data show that T helper 2 (Th2) cell differentiation requires antigen presentation and costimulation from cDCs but does not require polarizing cytokines. In this opinion article, we propose that the 'third signal' driving Th2 cell responses is, in fact, the absence of polarizing cytokines; indeed, the secretion of the latter is actively suppressed in cDCs, concomitant with acquired pro-Th2 functions.

A Tunable Diffusion-Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell-to-Cell Communication in the Immune System.

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 µm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors.

Chemokine CCL19 promotes type 2 T-cell differentiation and allergic airway inflammation.

BACKGROUND: Allergic asthma is driven largely by allergen-specific TH2 cells, which develop in regional lymph nodes on the interaction of naive CD4+ T cells with allergen-bearing dendritic cells that migrate from the lung. This migration event is dependent on CCR7 and its chemokine ligand, CCL21. However, is has been unclear whether the other CCR7 ligand, CCL19, has a role in allergic airway disease. OBJECTIVE: This study sought to define the role of CCL19 in TH2 differentiation and allergic airway disease. METHODS: Ccl19-deficient mice were studied in an animal model of allergic asthma. Dendritic cells or fibroblastic reticular cells from wild-type and Ccl19-deficient mice were cultured with naive CD4+ T cells, and cytokine production was measured by ELISA. Recombinant CCL19 was added to CD4+ T-cell cultures, and gene expression was assessed by RNA-sequencing and quantitative PCR. Transcription factor activation was assessed by flow cytometry. RESULTS: Lungs of Ccl19-deficient mice had less allergic airway inflammation, reduced airway hyperresponsiveness, and less IL-4 and IL-13 production compared with lungs of Ccl19-sufficient animals. Naive CD4+ T cells cocultured with Ccl19-deficient dendritic cells or fibroblastic reticular cells produced lower amounts of type 2 cytokines than did T cells cocultured with their wild-type counterparts. Recombinant CCL19 increased phosphorylation of STAT5 and induced expression of genes associated with TH2 cell and IL-2 signaling pathways. CONCLUSIONS: These results reveal a novel, TH2 cell-inducing function of CCL19 in allergic airway disease and suggest that strategies to block this pathway might help to reduce the incidence or severity of allergic asthma.

The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib in vitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both in vitro and in vivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

A family-wide assessment of latent STAT transcription factor interactions reveals divergent dimer repertoires.

The conversion of signal transducer and activator of transcription (STAT) proteins from latent to active transcription factors is central to cytokine signaling. Triggered by their signal-induced tyrosine phosphorylation, it is the assembly of a range of cytokine-specific STAT homo- and heterodimers that marks a key step in the transition of hitherto latent proteins to transcription activators. In contrast, the constitutive self-assembly of latent STATs and how it relates to the functioning of activated STATs is understood less well. To provide a more complete picture, we developed a co-localization-based assay and tested all 28 possible combinations of the seven unphosphorylated STAT (U-STAT) proteins in living cells. We identified five U-STAT homodimers-STAT1, STAT3, STAT4, STAT5A, and STAT5B-and two heterodimers-STAT1:STAT2 and STAT5A:STAT5B-and performed semi-quantitative assessments of the forces and characterizations of binding interfaces that support them. One STAT protein-STAT6-was found to be monomeric. This comprehensive analysis of latent STAT self-assembly lays bare considerable structural and functional diversity in the ways that link STAT dimerization before and after activation.

PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

STAT5 phosphorylation plus minimal residual disease defines a novel risk classification in adult B-cell acute lymphoblastic leukaemia.

The dysregulation of the Janus family tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) is closely related to acute lymphoblastic leukaemia (ALL), whereas the clinical value of phosphorylated STAT5 (pSTAT5) remains elusive. Herein we performed a prospective study on clinical significance of flow cytometry-based pSTAT5 in adult B-ALL patients. A total of 184 patients were enrolled in the Precision-Classification-Directed-Target-Total-Therapy (PDT)-ALL-2016 cohort between January 2018 and December 2021, and STAT5 phosphorylation was detected by flow cytometry at diagnosis. Based on flow-pSTAT5, the population was classified into pSTAT5low (113/184, 61.1%) and pSTAT5high (71/184, 38.9%). Overall survival (OS) and event-free survival (EFS) were inferior in pSTAT5high patients than in those with pSTAT5low (OS, 44.8% vs. 65.2%, p = 0.004; EFS, 23.5% vs. 52.1%, p < 0.001), which was further confirmed in an external validation cohort. Furthermore, pSTAT5 plus flow-based minimal residual disease (MRD) postinduction defines a novel risk classification as being high risk (HR, pSTAT5high + MRD+), standard risk (SR, pSTAT5low + MRD-) and others as moderate-risk group. Three identified patient subgroups are distinguishable with disparate survival curves (3-year OS rates, 36.5%, 56.7% and 76.3%, p < 0.001), which was confirmed on multivariate analysis (hazard ratio 3.53, p = 0.003). Collectively, our study proposed a novel, simple and flow-based risk classification by integrating pSTAT5 and MRD in favour of risk-guided treatment for B-ALL.

Identification of concurrent STAT3::RARA and RARA::STAT5b fusions in a variant APL case.

Acute promyelocytic leukemia (APL) with typically PML::RARA fusion gene caused by t (15;17) (q22; q12) was distinguished from other types of acute myeloid leukemia. In a subset of patients with APL, t (15;17) (q22;q21) and PML::RARA fusion cannot be detected. In this report, we identified the coexistence of STAT3::RARA and RARA::STAT5b fusions for the first time in a variant APL patient lacking t (15;17)(q22;q21)/PML::RARA fusion. Then, this patient was resistant to all-trans retinoic acid combined arsenic trioxide chemotherapy. Accurate detection of RARA gene partners is crucial for variant APL, and effective therapeutic regime is urgently needed.

Effect of the mRNA decapping enzyme scavenger (DCPS) inhibitor RG3039 on glioblastoma.

BACKGROUND: Patients with glioblastoma (GBM) have a poor prognosis and limited treatment options. The mRNA decapping enzyme scavenger (DCPS) is a cap-hydrolyzing enzyme. The DCPS inhibitor RG3039 exhibited excellent central nervous system bioavailability in vivo and was safe and well tolerated in healthy volunteers in a phase 1 clinical trial. In this study, we investigated the expression of DCPS in GBM and the anti-tumor activity of RG3039 in various preclinical models of GBM. METHODS: DCPS expression was examined in human GBM and paired peritumoral tissues. Its prognostic role was evaluated together with clinicopathological characteristics of patients. The anti-GBM effect of RG3039 was determined using GBM cell lines, patient-derived organoids, and orthotopic mouse models. The therapeutic mechanisms of DCPS inhibition were explored. RESULTS: DCPS is overexpressed in GBM and is associated with poor survival of patients with GBM. The DCPS inhibitor RG3039 exhibited robust anti-GBM activities in GBM cell lines, patient-derived organoids and orthotopic mouse models, with drug exposure achievable in humans. Mechanistically, RG3039 downregulated STAT5B expression, thereby suppressing proliferation, survival and colony formation of GBM cells. CONCLUSIONS: DCPS is a promising target for GBM. Inhibition of DCPS with RG3039 at doses achievable in humans downregulates STAT5B expression and reduces proliferation, survival and colony formation of GBM cells. Given the excellent anti-cancer activity and central nervous system bioavailability in vivo and good tolerance in humans, RG3039 warrants further study as a potential GBM therapy.

STAT5-mediated transcription of miR-33-5p in Mycoplasma gallisepticum-infected DF-1 cells.

RESEARCH HIGHLIGHTS: MG-HS regulates the expression of transcription factor STAT5.Transcription factor STAT5 can target miR-33-5p promoter element.MG-influenced STAT5 regulates miR-33-5p and its target gene expression.

Effects of hydrostatic compression on milk production-related signaling pathways in mouse mammary epithelial cells.

Mammary epithelial cells (MECs) secrete milk into the mammary alveolar lumen during lactation. The secreted milk accumulates in the alveolar lumen until milk ejection occurs, and excess milk accumulation downregulates milk production in alveolar MECs. Intramammary hydrostatic pressure also increases in the alveolar lumen in a manner dependent on milk accumulation. In this study, we investigated whether high hydrostatic compression directly affects lactating MECs, using a commercial compression device and a lactation culture model of MECs, which have milk production ability and less permeable tight junctions. High hydrostatic compression at 100 kPa for 8 h decreased ß-casein and increased claudin-4 levels concurrently with inactivation of STAT5 and glucocorticoid receptor signaling pathways. In addition, high hydrostatic compression for 1 h inactivated STAT5 and activated p38 MAPK signaling. Furthermore, repeated rises and falls of the hourly hydrostatic compression induced activation of positive (Akt, mTOR) and negative (STAT3, NF-κB) signaling pathways for milk production concurrently with stimulation of casein and lactoferrin production in MECs. These results indicate that milk production-related signaling pathways in MECs change in response to hydrostatic compression. Hydrostatic compression of the alveolar lumen may directly regulate milk production in the alveolar MECs of lactating mammary glands.

Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling.

Lymphodepleting pre-conditioning is a nearly universal component of T cell adoptive transfer protocols. The side effects of pre-conditioning regimens used in adoptive cell therapy are clinically significant and include pan-cytopenia, immune suppression, and reactive myelopoiesis. We conducted studies to test the hypothesis that the mechanisms underlying effective engraftment are cell autonomous and not dependent on a lymphodepleted host immune status. These studies leveraged mouse models to examine the role of Stat5 signaling during T cell adoptive transfer. We observed that, by transiently expressing a constitutively active mutamer of Stat5b during the process of adoptive transfer, we could completely obviate the need for lymphodepletion prior to adoptive transfer. Using several functional assays, we benchmark the function of the engrafted T cells against T cells transferred after conventional lymphodepletion. These studies identify a cell-autonomous mechanism driven by transient Stat5b signaling with lasting effects on T cell phenotype and function. Furthermore, the results presented suggest that adoptive T cell therapy could be improved by removing lymphodepletion protocols entirely and replacing them with RNA transfection of T cells with transcripts encoding active Stat5.

Oral administration of Lactobacillus plantarum expressing aCD11c modulates cellular immunity alleviating inflammatory injury due to Klebsiella pneumoniae infection.

BACKGROUND: Klebsiella pneumoniae (KP), responsible for acute lung injury (ALI) and inflammation of the gastrointestinal tract, is a zoonotic pathogen that poses a threat to livestock farming worldwide. Nevertheless, there is currently no validated vaccine to prevent KP infection. The development of mucosal vaccines against KP using Lactobacillus plantarum (L. plantarum) is an effective strategy. RESULTS: Firstly, the L. plantarum strains NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c were constructed via homologous recombination to express the aCD11c protein either inducibly or constitutively. Both NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c strains could enhance the adhesion and invasion of L. plantarum on bone marrow-derived dendritic cells (BMDCs), and stimulate the activation of BMDCs compared to the control strain NC8-pSIP409 in vitro. Following oral immunization of mice with NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c, the cellular, humoral, and mucosal immunity were significantly improved, as evidenced by the increased expression of CD4+ IL-4+ T cells in the spleen, IgG in serum, and secretory IgA (sIgA) in the intestinal lavage fluid (ILF). Furthermore, the protective effects of L. plantarum against inflammatory damage caused by KP infection were confirmed by assessing the bacterial loads in various tissues, lung wet/dry ratio (W/D), levels of inflammatory cytokines, and histological evaluation, which influenced T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood and lung. CONCLUSIONS: Both the inducible and constitutive L. plantarum strains NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c have been found to stimulate cellular and humoral immunity levels and alleviate the inflammatory response caused by KP infection. These findings have provided a basis for the development of a novel vaccine against KP.

A zebrafish model of growth hormone insensitivity syndrome with immune dysregulation 1 (GHISID1).

Signal transducer and activator of transcription (STAT) proteins act downstream of cytokine receptors to facilitate changes in gene expression that impact a range of developmental and homeostatic processes. Patients harbouring loss-of-function (LOF) STAT5B mutations exhibit postnatal growth failure due to lack of responsiveness to growth hormone as well as immune perturbation, a disorder called growth hormone insensitivity syndrome with immune dysregulation 1 (GHISID1). This study aimed to generate a zebrafish model of this disease by targeting the stat5.1 gene using CRISPR/Cas9 and characterising the effects on growth and immunity. The zebrafish Stat5.1 mutants were smaller, but exhibited increased adiposity, with concomitant dysregulation of growth and lipid metabolism genes. The mutants also displayed impaired lymphopoiesis with reduced T cells throughout the lifespan, along with broader disruption of the lymphoid compartment in adulthood, including evidence of T cell activation. Collectively, these findings confirm that zebrafish Stat5.1 mutants mimic the clinical impacts of human STAT5B LOF mutations, establishing them as a model of GHISID1.

Melatonin enhances NK cell function in aged mice by increasing T-bet expression via the JAK3-STAT5 signaling pathway.

Natural killer (NK) cells are crucial innate immune cells that provide defense against viruses and tumors. However, aging is associated with alterations in NK cell composition and compromised cell functions. Melatonin, known for its anti-tumor effects, has been reported to improve NK cell function. However, the molecular mechanism underlying melatonin's effect on senescent NK cells remains unclear. In this study, we aimed to elucidate the mechanism by which melatonin enhances the function of senescent NK cells. Our findings revealed that melatonin significantly increased the number and function of NK cells in aging mice. The results suggest that melatonin enhances NK cell proliferation, degranulation, and IFN-γ secretion. Further investigations demonstrated that melatonin promotes NK cell maturation and activation, mainly via the JAK3/STAT5 signaling pathway, leading to increased expression of T-bet. These discoveries provide a theoretical basis for potential immunotherapy strategies based on melatonin-mediated modulation of NK cell function in aging individuals.

Discovery of novel and potent CDK8 inhibitors for the treatment of acute myeloid leukaemia.

It has been reported that CDK8 plays a key role in acute myeloid leukaemia. Here, a total of 40 compounds were rational designed and synthesised based on the previous SAR. Among them, compound 12 (3-(3-(furan-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide) showed the most potent inhibiting activity against CDK8 with an IC50 value of 39.2 ± 6.3 nM and anti AML cell proliferation activity (molm-13 GC50 = 0.02 ± 0.01 µM, MV4-11 GC50 = 0.03 ± 0.01 µM). Mechanistic studies revealed that this compound 12 could inhibit the phosphorylation of STAT-1 and STAT-5. Importantly, compound 12 showed relative good bioavailability (F = 38.80%) and low toxicity in vivo. This study has great significance for the discovery of more efficient CDK8 inhibitors and the development of drugs for treating AML in the future.

The Mechanism of Leptin Resistance in Obesity and Therapeutic Perspective.

Leptin resistance is induced via leptin signaling blockade by chronic overstimulation of the leptin receptor and intracellular signaling defect or increased hypothalamic inflammation and suppressor of cytokine signaling (SOCS)-3 expression. High-fat diet triggers leptin resistance induced by at least two independent causes: first, the limited ability of peripheral leptin to activate hypothalamic signaling transducers and activators of transcription (STAT) signaling and secondly a signaling defect in leptin-responsive hypothalamic neurons. Central leptin resistance is dependent on decreased leptin transport efficiency across the blood brain barrier (BBB) rather than hypothalamic leptin insensitivity. Since the hypothalamic phosphorylated STAT3 (pSTAT3) represents a sensitive and specific readout of leptin receptor-B signaling, the assessment of pSTAT3 levels is the gold standard. Hypertriglyceridemia is one of important factors to inhibit the transport of leptin across BBB in obesity. Mismatch between high leptin and the amount of leptin receptor expression in obesity triggers brain leptin resistance via increasing hypothalamic inflammation and SOCS-3 expression. Therapeutic strategies that regulate the passage of leptin to the brain include the development of modifications in the structure of leptin analogues as well as the synthesis of new leptin receptor agonists with increased BBB permeability. In the hyperleptinemic state, polyethylene glycol (PEG)-modified leptin is unable to pass through the BBB. Peripheral histone deacetylase (HDAC) 6 inhibitor, tubastatin, and metformin increase central leptin sensitization. While add-on therapy with anagliptin, metformin and miglitol reduce leptin concentrations, the use of long-acting leptin analogs, and exendin-4 lead to the recovery of leptin sensitivity. Contouring surgery with fat removal, and bariatric surgery independently of the type of surgery performed provide significant improvement in leptin concentrations. Although approaches to correcting leptin resistance have shown some success, no clinically effective application has been developed to date. Due to the impairment of central and peripheral leptin signaling, as well as the extensive integration of leptin-sensitive metabolic pathways with other neurons, the effectiveness of methods used to eliminate leptin resistance is extremely limited.

ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury.

In this study we investigate the role of Zipper-interacting protein kinase (ZIPK) in high glucose-induced vascular injury, focusing on its interaction with STAT5A and its effects on p53 and inducible nitric oxide synthase (NOS2) expression. Human umbilical vein endothelial cells (HUVECs) are cultured under normal (5 mM) and high (25 mM) glucose conditions. Protein and gene expression levels are assessed by western blot analysis and qPCR respectively, while ROS levels are measured via flow cytometry. ZIPK expression is manipulated using overexpression plasmids, siRNAs, and shRNAs. The effects of the ZIPK inhibitor TC-DAPK6 are evaluated in a diabetic rat model. Our results show that high glucose significantly upregulates ZIPK, STAT5A, p53, and NOS2 expressions in HUVECs, thus increasing oxidative stress. Silencing of STAT5A reduces p53 and NOS2 expressions and reactive oxygen species (ROS) accumulation. ZIPK is essential for high glucose-induced p53 expression and ROS accumulation, while silencing of ZIPK reverses these effects. Overexpression of ZIPK combined with STAT5A silencing attenuates glucose-induced alterations in p53 and NOS2 expression, thereby preventing cell damage. Coimmunoprecipitation reveals a direct interaction between ZIPK and STAT5A in the nucleus under high-glucose condition. In diabetic rats, TC-DAPK6 treatment significantly decreases ZIPK, p53, and NOS2 expressions. Our findings suggest that ZIPK plays a critical role in high glucose-induced vascular injury via STAT5A-mediated pathways, proposing that ZIPK is a potential therapeutic target for diabetic vascular complications.