InVivoMAb anti-mouse CD28

Sphingosine-1-phosphate receptor 1 (S1PR1) signaling has been linked to the regulation of immunosuppressive cell populations within the tumor microenvironment (TME); however, its role in shaping anti-tumor CD8⁺ T cell responses remains poorly defined. Herein, we demonstrate that intratumoral CD8⁺ T cells express S1PR1, with expression predominantly enriched in the terminally exhausted subset. Transcriptomic profiling, combined with pharmacological inhibition and genetic knockdown, reveals that S1PR1-S1P signaling activates the PERK (protein kinase R (PKR)-like endoplasmic reticulum kinase)-CHOP (C/EBP homologous protein) axis of the endoplasmic reticulum stress response. CHOP, in turn, upregulates transcription of Map3k13 and Map3k15, triggering downstream MAPK signaling and culminating in activation of p38MAPK. Activation of this pathway impairs CD8⁺ T cell metabolism and effector function while increasing apoptotic susceptibility. This ultimately limits the persistence and accumulation of functional CD8⁺ T cells within the TME, thereby compromising their responsiveness to anti-PD-1 therapy. Targeting the S1PR1-S1P axis or its downstream effectors offers a promising strategy to improve cancer immunotherapy outcomes.

CD4+ regulatory T cells (Treg cells) are essential for immune tolerance1. Peripherally induced Treg cells (pTreg cells) complement thymic Treg cells by broadening Treg cell reactivity in response to a changing antigenic landscape2. Although both TGFβ and IL-2 synergistically promote functional pTreg cell development in vitro3-6, their combined roles in inducing pTreg cell generation in vivo have not been exploited for tolerizing immunotherapy. Here we designed an IL-2-TGFβ 'surrogate' co-agonist by creating a single-chain fusion protein between IL-2 and a low-affinity TGFβ mimic agonist derived from a helminth parasite7. This IL-2-TGFβ surrogate functions as an AND-gated co-agonist and enabled simultaneous cis-activation of IL-2-STAT5 and TGFβ-SMAD2/3 signalling specifically in T cells that express IL-2 receptors. The IL-2-TGFβ surrogate agonist robustly induced antigen-specific, functional and stable pTreg cells in vivo within peripheral lymphoid organs in mice immunized with ovalbumin (OVA) and myelin oligodendrocyte glycoprotein (MOG)35-55. The induced pTreg cells display an effector-like, actively expanding state with high RORγt expression, enabling efficient migration and suppression of intestinal inflammation. Treatment with this agonist effectively quelled immune activation in mouse models of allergen-induced allergic inflammation and self-antigen-driven autoimmune neuroinflammation, suggesting a strategy for the induction of antigen-specific pTreg cells in vivo to establish immune tolerance in inflammatory, allergic and autoimmune diseases.

Immune checkpoint blockade (ICB) faces limitations owing to high cost and restricted efficacy. This study identifies SNX17 as a key mediator of ICB resistance. Elevated SNX17 correlates with poor anti-PD-1 response in humans and mice. SNX17 deletion in tumor cells inhibits tumor growth via CD8+ T cell-dependent mechanisms. SNX17 reduces uridine in the tumor microenvironment (TME), suppressing IFN-γ and upregulating PD1 in CD8+ T cells. Exogenous uridine shows antitumor efficacy comparable to anti-PD-1/PD-L1 in low-SNX17 tumors and overcomes resistance in high-SNX17 models. Uridine enhances CD8+ T cell function by promoting CD45 N-glycosylation and LCK phosphorylation. Mechanistically, SNX17 stabilizes RUNX2, promoting UPP1 transcription and uridine degradation in the TME. These findings position SNX17 as an ICB response biomarker and nominate uridine as a cost-effective immunotherapeutic strategy.

Interferon-induced transmembrane protein 1 (IFITM1) restricts virus infection. IFITM proteins are involved in Th2 cell differentiation in allergic asthma. The epithelial‒mesenchymal transition (EMT) regulates allergic airway remodeling. We sought to explore the functional contributions and underlying mechanisms of IFITM1 in the EMT associated with allergic asthma.

Background: Immune checkpoint inhibitors are ineffective in the majority of colorectal cancers (CRCs) that are microsatellite stable. However, the underlying reasons for their unresponsiveness and mechanisms of immune evasion are poorly understood. In the present study, we aimed to elucidate the mechanisms underlying the immune evasion driven by CRC cells. Methods: We performed single-cell RNA sequencing of tumor tissues from 30 CRC patients and syngeneic mice implanted with transformation-related protein 53 (Trp53) knockout CT26 cells. Gene expression and correlations of individual tumor microenvironment (TME) components were analyzed, and their functional significance was investigated using syngeneic mouse models and cell line co-culture experiments. Results: CCAAT enhancer-binding protein beta (CEBPB) expression was increased in tumor protein 53 (TP53)-mutated CRCs. We confirmed that wild-type TP53 negatively regulated CEBPB expression in CRC cell lines. CEBPB expression was associated with decreased intratumoral T cell infiltration and negatively impacted survival in CRC patients. In the intercellular correlation analysis of gene expression, tumor epithelial cell CEBPB expression was significantly correlated with cytotoxic T-lymphocyte associated protein 4 (CTLA4) expression in T cells, especially in regulatory and exhausted T cells. Cebpb overexpression promoted tumor growth in the immunocompetent syngeneic mouse models, which was accompanied by increased CTLA-4 expression in tumor-infiltrating CD4+ T cells. In vitro co-culture experiments also showed that tumor cell CEBPB overexpression increased CTLA4 in T cells. Conclusions: Tumor cell CEBPB expression, up-regulated by TP53 mutation, can increase CTLA4 expression in T cells and negatively affect patient outcomes. These findings suggested a central role of tumor cell CEBPB in shaping an immunosuppressive TME.

Immune cell infiltration into the central nervous system is increasingly recognized as a driver of neurodegeneration, yet its role in Huntington's disease remains unresolved. Addressing this question requires models that replicate the selective vulnerability of striatal neurons observed in patients, a challenge unmet by rodent systems. Here we use a previously established and genetically engineered pig model carrying the human huntingtin mutation with an expanded cytosine-adenine-guanine repeat, enabling investigation of immune-neural interactions in a physiologically relevant context. Using single-nucleus and spatial transcriptomics, integrated with immunohistochemistry and T cell receptor sequencing, we constructed a cellular map of the striatum. We identified an interferon-responsive microglial state that secretes chemokine ligand eight, recruiting cytotoxic CD8-positive T cells that release perforin and granzyme, thereby accelerating neuronal loss. Functional experiments confirmed the pathogenic role of chemokine ligand eight and demonstrated that its neutralization mitigates neurodegeneration. These findings uncover a species-dependent immune mechanism in Huntington's disease and nominate chemokine ligand eight-mediated T cell infiltration as a therapeutic target.

Chronic low-grade inflammation is a hallmark of atherosclerosis and cardiovascular diseases, with monocytes playing a central role in sustaining this pathological state. In this study, we demonstrate that prolonged exposure to oxidized low-density lipoprotein (oxLDL) or cholesterol reprograms murine bone marrow-derived monocytes into a persistent pro-inflammatory phenotype. This is characterized by elevated surface markers (CD49d, CD74, CD38, CD86), enhanced endothelial and T cell interactions, and sustained activation of the Src-SYK-mTORC1-STAT3/5 signaling axis. Notably, the inflammatory state persisted even after stimulus withdrawal, suggesting the establishment of an immune memory-like phenotype. Mechanistically, we defined the membrane clustering of Src is responsible for the generation of intra-cellular stress signaling and sustained monocyte activation, which can be alleviated by the administration of fumagillin, a selective inhibitor of protein myristoylation and Src membrane clustering. Our findings uncover mechanistic insights into the generation of sustained monocyte low-grade inflammatory memory and pinpoint potential therapeutic strategies in erasing low-grade inflammation related to chronic diseases.

Adoptive cell therapy (ACT) relies on durable and functional T cells to mediate tumor clearance. Th9 cells are a metabolically fit CD4+ T cell subset with strong persistence but limited cytotoxicity. Here, we identified endomelipeptide A (EpA), a cyclic peptide isolated from Ganoderma lucidum-associated endophytic fungi, as a potent enhancer of Th9 cell differentiation. EpA promoted a cytotoxic Th9 phenotype with enhanced mitochondrial function and metabolic fitness. Mechanistically, EpA dually targeted ZAP70 and SREBP1, coupling T cell receptor signaling activation with lipid metabolism suppression. EpA-treated Th9 cells mediated robust, CD8+ T cell-dependent tumor control and enhanced the efficacy of human Th9 CAR T cell therapy in vivo. These findings establish EpA as a distinct cyclic peptide that reprograms Th9 cells and provides a potential approach to boost ACT efficacy.

Metformin shows clinical promise beyond diabetes, yet its immunological safety in non-diabetic contexts remains uncertain. We found that metformin induces apoptosis in double-positive thymocytes across various mouse models and, importantly, creates a "selection trap" by promoting phenotypic maturation (TCRβ+CD69+) while simultaneously triggering their elimination. Mechanistically, this trap is sprung via mitochondrial dysfunction initiated by complex I inhibition, which causes ATP depletion and elevated mitochondrial reactive oxygen species. This metabolic stress drives sustained AMP-activated protein kinase (AMPK) activation, repurposing extracellular signal-regulated kinase 1/2 signaling to expose the BH3 domain of B cell lymphoma-2 (Bcl-2), thereby neutralizing its anti-apoptotic function. Transcriptomics further reveal that AMPK remodels metabolic pathways to augment oxidative injury and energy crisis, facilitating apoptosis. Notably, thymotoxicity persists even at subtherapeutic doses (25 mg/kg), challenging metformin's indiscriminate use in non-diabetic populations due to risks to central immune homeostasis.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease primarily mediated by T helper 17 (TH17) cells. We previously showed that Itch/WWP2 double knockout (DKO) T cells produce high levels of type 2 cytokines, driving spontaneous autoinflammation. Here, we report that DKO TH2-high carrying autoantigen-specific TCR (2D2) develop atypical spontaneous experimental autoimmune encephalomyelitis (EAE), with CD4+ T cells simultaneously producing IL-4 and GM-CSF, directly causing neuroinflammation. Unexpectedly, IL-4 deletion in DKO TH2-high 2D2 mice exacerbates TH17-driven classical EAE, indicating a TH2 to TH17 conversion. Furthermore, we show that the JAK3/STAT5 signaling pathway is critical for maintaining TH2 lineage stability by modulating Blimp1 and c-Maf thereby suppressing TH17 differentiation. Importantly, we find that this phenomenon can also be observed in dupilumab-treated patients with atopic dermatitis who develop psoriasis. Thus, our findings uncover the molecular antagonism and plasticity in the TH2 and TH17 cell programs and identify potential therapeutic targets for modulating TH2 and TH17 cell responses in autoimmune diseases.

Antibiotics influence both gut microbial composition and immune regulation, but the detailed mechanisms are still undefined. Shifts in the microbiome caused by antibiotic exposure can modulate immune activity through various pathways. Therefore, we aimed to explore how antibiotics affect immune-inflammation by regulating Th17 cells through the gut microbiota of mice with experimental autoimmune prostatitis (EAP). Antibiotic-driven shifts in gut microbial communities and metabolite profiling in EAP mice were performed by integrating 16S rRNA sequencing with mass spectrometry-driven metabolomic analysis. Antibiotic cocktail (ABX) therapy mitigated EAP, modified the gut microbiome composition, and influenced bile acid metabolism. Fecal microbiota transplantation (FMT) using microbiota from ABX-treated feces into EAP mice effectively altered gut microbiome composition and alleviated disease symptoms, indicating that microbiome intervention reduces autoimmune inflammation and decreases deoxycholic acid (DCA) in mice. Subsequent experiments demonstrated that DCA suppresses farnesol X receptor (FXR) expression which can inhibit the NLRP3‒ IL17A axis, thus promoting Th17 cell development and exacerbating inflammatory cell infiltration of the prostate. Our initial clinical examination of patients with prostatitis and antibiotic treatment indicated that bile acid metabolism and Th17 cell development are affected by antibiotic therapy. This work revealed that antibiotic-induced gut microbiota dysbiosis decreases the bile acid metabolite DCA, further restraining Th17 cell differentiation via the FXR‒NLRP3 axis to alleviate autoimmune prostatitis. Our results reveal new perspectives regarding the interconnected dynamics of antibiotics, gut microbiota, bile acid metabolism, and immune regulation, with potential relevance for therapies targeting immune-mediated diseases.

Monocyte exhaustion is a dysfunctional state characterized by prolonged pathogenic inflammation and immune suppression, commonly observed in chronic infections and sepsis. However, the mechanisms underlying the generation and propagation of exhausted monocytes remain poorly understood. In this study, we investigate the impacts of exhausted monocytes on neighboring naïve monocytes, endothelial cells, and T cell function. Using an in vitro co-culture system, we demonstrate that exhausted monocytes induced by prolonged LPS stimulation propagate the exhaustion phenotype to neighboring naïve monocytes. Meanwhile these exhausted monocytes can promote endothelial apoptosis, upregulate adhesion molecules ICAM-1 and VCAM-1, and enhance monocyte transmigration, contributing to endothelial dysfunction. Pharmacological inhibition of CD38, a key marker of monocyte exhaustion, significantly mitigates these effects, highlighting its critical role in monocyte-driven endothelial alterations. Furthermore, we show that exhausted monocytes suppress T cell proliferation and activation, a process reversed by CD38 inhibition. We also identify mTOR signaling as a key regulator of monocyte exhaustion and its propagation, with mTOR inhibition partially restoring monocyte functionality by downregulating exhaustion markers and STAT1/STAT3/S6K signaling. Collectively, our findings highlight the CD38-mTOR axis as a central driver of monocyte exhaustion and its pathological consequences, offering potential therapeutic targets for reversing immune dysfunction in inflammatory diseases.

Osteoarthritis (OA) pathogenesis is profoundly influenced by dysregulated immune dynamics, where persistent interleukin-17 (IL-17)/T helper 17 (Th17) cell mediated inflammation coordinates with failed regenerative processes to perpetuate joint destruction. Here, we unveil the role of myeloid-derived suppressor cells (MDSCs) as dual-phase regulators that paradoxically orchestrate both inflammatory escalation and tissue repair in OA progression. Intra-articular administration of MDSCs in OA mice amplified IL-17 dependent inflammatory cascades and chemokine-driven leukocyte recruitment, revealing a context-dependent pro-inflammatory phenotype. Unexpectedly, MDSC depletion failed to attenuate joint damage, implying their indispensable yet multifaceted role in OA pathogenesis. Mechanistically, MDSCs exhibited functional plasticity by upregulating arginase-1 to polarize M2 macrophages, fostering a regenerative niche alongside their inflammatory activity. To resolve this duality, we developed a bio-responsive hydrogel-microsphere system integrating transforming growth factor β1 (TGF-β1) and interleukin-1 β1 antibody (anti-IL-1β) loaded mesoporous silica nanoparticles (MSNs). This spatiotemporally controlled platform selectively suppressed MDSC-mediated Th17 cell expansion while harnessing their intrinsic capacity to drive M2 macrophage polarization and chondrogenesis. The resultant shift from a pro-inflammatory to pro-regenerative microenvironment significantly attenuated cartilage erosion and restored joint integrity in OA models. Our findings redefine MDSCs as bifunctional immune orchestrators in OA and establish precision biomaterial guided immune decoding as a paradigm-shifting therapeutic strategy. By engineering MDSCs plasticity through antagonistic cytokine delivery, this work provides a blueprint for microenvironment remodeling in degenerative joint diseases.

β-adrenergic signaling has been suggested to promote tumor growth, and β-blockers are being evaluated for repurposing for cancer treatment. Here, we identify a β-adrenergic signaling axis involved in metastasis formation. We show that the β-blocker propranolol has strong anti-metastatic activity in multiple murine models, with this effect being completely dependent on CD4 + T cells and independent of NK or CD8 + T cells. We also observe that CD4 + T cells are required for the anti-tumor effect of propranolol in a syngeneic subcutaneous model of colon cancer. Mechanistically, propranolol induces a Th1-polarized and cytotoxic CD4 + T cell response, which requires MHC class II expression by cancer cells for full efficacy. We also report propanolol-driven systemic changes in the monocyte compartment, and upon depletion of monocytes, propranolol loses its anti-tumor effects. Finally, we show that propranolol treatment synergizes with anti-CTLA-4 therapy to further enhance CD4 + T cell infiltration and control metastasis. Thus, we show that β-adrenergic signaling limits CD4 T cell-mediated anti-tumor immunity, highlighting the potential of repurposing β-blockers for cancer treatment.

Tumor necrosis factor (TNF)-induced RIPK1-mediated cell death is implicated in various human diseases. However, the mechanisms RIPK1-mediated cell death is regulated by metabolic processes remain unclear. Here, we identify hexokinase 2 (HK2), a critical regulator of glycolysis, as a suppressor of TNF-induced RIPK1 kinase-dependent cell death through its non-metabolic function. HK2 inhibits RIPK1 kinase activity through constitutively phosphorylation at serine 32 of RIPK1. Inhibition of RIPK1 S32-phosphorylation results in RIPK1 kinase activation and subsequent cell death in response to TNFα stimulation. We further show that HK2 is elevated under pathological conditions including liver ischemia-reperfusion (IR) injury and hepatocellular carcinoma (HCC) via the transcriptional factor HMGA1. Moreover, the upregulation of HK2 in the liver confers protection against liver IR injury mediated by RIPK1 kinase, while depleting HK2 in HCC cells enhances TNFα-induced cell death and synergistically improves the efficacy of anti-PD1 therapy in an HCC model. Thus, the findings reveal a potential therapeutic avenue for RIPK1-related diseases through manipulating HK2 non-metabolic function.

Sepsis is a life-threatening disease caused by a dysfunctional host response to infection. During sepsis, inflammation-related immunosuppression is the critical factor causing secondary infection and multiple organ dysfunction syndrome. The regulatory mechanisms underlying Treg differentiation and function, which significantly contribute to septic immunosuppression, require further clarification. In this study, we found that neutrophil extracellular traps (NETs) participated in the development of sepsis-induced immunosuppression by enhancing Treg differentiation and function via direct interaction with CD4+ T cells. Briefly, NETs anchored enolase 1 (ENO1) on the membrane of CD4+ T cells through its key protein myeloperoxidase (MPO) and subsequently recruited interferon-induced transmembrane protein 2 (IFITM2). IFITM2 acted as a DNA receptor that sensed NET-DNA and activated intracellular RAS-associated protein 1B (RAP1B) and its downstream ERK signaling pathway to promote Treg differentiation and function. ENO1 inhibition significantly attenuated NET-induced Treg differentiation and alleviated sepsis in mice. Overall, we demonstrated the role of NETs in sepsis-induced immunosuppression by enhancing Treg differentiation, identified ENO1 as an anchor of NET-MPO, and elucidated the downstream molecular mechanism by which IFITM2-RAP1B-ERK regulates Treg differentiation. These findings improve our understanding of the immunopathogenesis of sepsis and provide potential therapeutic targets for sepsis-induced immunosuppression.

Regulatory T (Treg) cells, expressing the transcription factor Foxp3, are obligatory gatekeepers of immune responsiveness, yet the mechanisms by which Foxp3 governs the Treg transcriptional network remain incompletely understood. Using a novel chemogenetic system of inducible Foxp3 protein degradation in vivo, we found that while Foxp3 was indispensable for the establishment of transcriptional and functional programs of newly generated Treg cells, Foxp3 loss in mature Treg cells resulted in minimal functional and transcriptional changes under steady state. This resilience of the Foxp3-dependent program in mature Treg cells was acquired over an unexpectedly long timescale; however, in settings of severe inflammation, Foxp3 loss led to a pronounced perturbation of Treg cell transcriptome and fitness. Furthermore, tumoral Treg cells were uniquely sensitive to Foxp3 degradation, which led to impairment in their suppressive function and tumor shrinkage in the absence of pronounced adverse effects. These studies demonstrate a context-dependent differential requirement for Foxp3 for Treg transcriptional and functional programs.

Mechanisms of T cell aging involve cell-intrinsic alterations and interactions with immune and stromal cells. Here we found that splenic T cells exhibit greater functional decline than lymph node T cells within the same aged mouse, prompting investigation into how the aged spleen contributes to T cell aging. Proteomic analysis revealed increased expression of heme detoxification in aged spleen-derived lymphocytes. Exposure to the heme- and iron-rich aged splenic microenvironment induced aging phenotypes in young T cells, including reduced proliferation and CD39 upregulation. T cells survived this hostile niche by maintaining a low labile iron pool, at least in part, via IRP2 downregulation to resist ferroptosis but failed to induce sufficient iron uptake for activation. Iron supplementation enhanced antigen-specific T cell responses in aged mice. This study identifies the aged spleen as a source of hemolytic signals that systemically impair T cell function, underscoring a trade-off between T cell survival and function and implicating iron metabolism in immune aging.

The heart is one of the least regenerative organs in humans, and ischemic heart disease is the leading cause of death worldwide. Understanding the cellular and molecular processes that occur during cardiac wound healing is an essential prerequisite to reducing health burden and improving cardiac function after myocardial tissue damage. Here, by integrating single-cell RNA sequencing with high-resolution spatial transcriptomics, we reconstruct the spatiotemporal dynamics of the fibrotic niches after cardiac injury in adult mice. We reveal a complex multicellular network that regulates cardiac repair, including fibroblast proliferation silencing by Trem2high macrophages to prevent excessive fibrosis. We further discovered a rare population of progenitor-like cardiomyocytes after lesion, promoted by myeloid and lymphoid niche signals. Culturing non-regenerative mouse cardiomyocytes or human heart tissue with these niche factors reactivated progenitor gene expression and cell cycle activity. In summary, this spatiotemporal atlas provides valuable insights into the heterocellular interactions that control cardiac repair.