InVivoMAb rat IgG2a isotype control, anti-trinitrophenol

CD8+ T cell exhaustion represents a major obstacle to effective cancer immunotherapy. While stem-like progenitor exhausted T (TPEX) cells can differentiate into intermediate (Int-TEX) and terminally exhausted (TEX) subsets, the epigenetic regulation of this process is unclear. We identify the RNA methyltransferase Mettl8 as a critical regulator, with expression significantly higher in TPEX than in TEX subsets. In anti-PD-1 responding non-small cell lung cancer patients, Mettl8 and the stemness factor TCF7 were downregulated. In murine models, Mettl8 deletion restrained tumor progression by driving TPEX differentiation into effective Int-TEX cells. Mechanistically, Mettl8 stabilizes Tcf7 mRNA via m3C modification and enhances Tcf1 protein expression. Additionally, Mettl8 interacts with Tcf1 to facilitate chromatin looping at the Tox locus, maintaining TPEX stemness. Pharmacological Mettl8 inhibition promoted TPEX-to-Int-TEX differentiation and tumor control. Combining this inhibition with anti-PD-1 therapy yielded synergistic efficacy. Our findings establish Mettl8 as a pivotal regulator of TPEX fate and a promising therapeutic target for enhancing immunotherapy.

CD4 tissue-resident memory T cells (TRM) are crucial adaptive immune components involved in preventing influenza A virus (IAV) infection. Despite their importance, their physiological role in the upper respiratory tract, the first site of contact with IAV, remains unclear. Here, we find that, after IAV infection, antigen-specific CD4 TRM persist in the nasal tissue (NT) compartment after infection and provide protection upon heterosubtypic challenge. Single-cell RNA-sequencing analysis reveals that NT CD4 TRM are heterogeneous and transcriptionally distinct as compared with their lung counterparts. Mechanistically, we demonstrate that the CXCR6-CXCL16 axis promotes CD4 TRM residency in the NT. Furthermore, we show that the NT of mice and humans contains a high frequency of Th17 CD4 TRM that aid in local viral clearance and in reducing tissue damage. Collectively, our results support a robust physiological role for NT CD4 TRM in local protection during heterosubtypic IAV infection.

T cell-based immunotherapies have improved outcomes in lung adenocarcinoma (LUAD), yet many patients develop primary or acquired resistance. Tumor-intrinsic mechanisms that suppress CD8+ T cell function remain incompletely understood.

Sepsis remains a major clinical challenge, with high mortality and long-term disability despite current interventions. Here, we identify the tissue hormone acyl-CoA-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), as a biomarker and driver of poor outcome in sepsis. ACBP/DBI was elevated in the plasma of septic patients and associated with organ dysfunction and increased mortality. In murine models of endotoxemia, Escherichia coli infection, and polymicrobial sepsis, genetic deletion or antibody-mediated neutralization of ACBP/DBI conferred robust protection by dampening cytokine storm and preserving organ function. Across these three models, neutralization of ACBP/DBI with monoclonal antibodies restored thermoregulation and reduced mortality. Mechanistically, ACBP/DBI inhibition enhanced resilience to lipopolysaccharide-induced sterile inflammation and improved bacterial clearance by macrophages and granulocytes in vivo and in vitro. These effects were observed in monomicrobial infection models and confirmed by high-dimensional immunophenotyping in a polymicrobial sepsis model. Notably, ACBP/DBI inhibition could be favorably combined with glucocorticoids, enhancing survival and reversing histopathological, transcriptional or metabolic signatures of septic shock across heart, kidney, liver, lung, spleen and plasma. These findings position ACBP/DBI as a mechanistic amplifier of sepsis pathophysiology and propose its neutralization, alone or in combination with corticosteroids, as a promising therapeutic strategy to interrupt the fatal trajectory of septic shock.

Autologous tumor-infiltrating lymphocyte (TIL) therapy holds transformative potential for solid tumors, yet its efficacy in glioblastoma remains limited by T cell exhaustion and immunosuppression. In the current study, we optimized an effective and reliable method for in vitro expansion of TILs from glioblastoma lesions and assessed their tumor-killing capacity both in vitro and in vivo. Single-cell RNA sequencing (scRNA-seq) of expanded TILs uncovered their heterogeneity and identified a cytotoxic tissue-resident memory (TRM) CD8+ TIL subset with a unique exhaustion signature. Notably, the co-stimulatory factor GITR (encoded by TNFRSF18) is highly expressed not only on immunosuppressive regulatory T (Treg) cells but also on exhausted CD8+ TILs. GITR agonism via αGITR antibody achieved dual effects: it directly enhanced CD8+ TIL activation while simultaneously abrogating Treg-mediated immunosuppression. This dual-action mechanism synergized with αPD-1 therapy to amplify TIL reactivation, significantly enhancing tumor control in vivo. Mechanistically, GITR activation potentiated anti-tumor responses by promoting immunological synapse (IS) formation and function in TILs via the NF-κB/KALRN signaling axis. Our findings established GITR as a crucial regulator of CD8+ TIL anti-tumor immunity, positioning GITR targeting as a novel strategy to improve TIL therapy for glioblastoma, with promising implications for clinical application.

Patients with the microsatellite instable (MSI) subtype of colorectal cancer (CRC) have a better prognosis and immunotherapy response than patients with the chromosomally instable (CIN) subtype due to improved cytotoxic T cell responses. This is in part due to high production of the chemokines CXCL10 and CCL5 from constitutive activation of the cytosolic DNA (cyDNA) sensor cGAS/STING by specific features of MSI cyDNA that lead to more effective cGAS/STING pathway activation. Here, we investigate MSI and CIN cyDNA structure and show that MSI cyDNA is enriched in G-quadruplexes that improve cGAS/STING and CD8+ T cell activation. We also show that micronuclei are less effective at inducing anti-tumor immunity and instead increase Treg activation and IL-10 production. Overall, these data highlight the role of specific cyDNA structures in anti-tumor immunity and provide knowledge for improved design of therapeutic DNA-based cGAS/STING agonists to improve the prognosis of poorly immunogenic tumors like CIN CRCs.

Immunosuppression and metastasis are critical hallmarks of breast cancer, often linked to poor patient outcomes. The secreted cytokine chitinase-3-like 1 (CHI3L1) is frequently overexpressed in breast cancer samples and promotes an immunosuppressed tumor microenvironment. Notably, CHI3L1 expression is elevated in metastatic patient samples when compared with the matched primary breast tumor. To investigate its role in breast cancer metastasis, we generated an inducible genetically engineered mouse model that overexpresses CHI3L1 in the mammary epithelium. Ectopic expression of CHI3L1 in the polyomavirus middle T (PyMT) mouse model of breast cancer suppressed antitumor immune responses, accelerated mammary tumor onset, and enhanced lung metastasis. Mechanistically, elevated CHI3L1 expression in the mammary epithelium enhanced neutrophil recruitment, which subsequently degraded the extracellular matrix and increased the number of circulating tumor cells. These findings reveal a key mechanism driving metastatic dissemination and argue that therapeutically targeting Chi3l1 could enhance antitumor immunity and suppress metastasis.

Background/Objectives: Postoperative pain pharmacology is complex. We investigated the sensitivity of analgesic-like effects induced by morphine, ibuprofen, and their combination to peripheral opioid antagonism in a mouse laparotomy model. Methods: Mechanical hypersensitivity was assessed using von Frey filaments, and ongoing pain (abdominal licking and facial expressions) was evaluated using artificial intelligence algorithms. We tested the sensitivity of the analgesic treatments to the opioid antagonist naloxone or its peripherally restricted analog, naloxone methiodide. We also tested the effects of neutrophil depletion using an anti-Ly6G antibody. Gastrointestinal transit and pupillary diameter were measured to assess non-analgesic opioid effects. Results: Morphine reversed all pain-related behaviors; its effect on mechanical hypersensitivity was reversed by peripheral opioid antagonism, whereas its effects on ongoing pain were not. Ibuprofen reduced mechanical hypersensitivity and facial expressions but failed to alter licking. Interestingly, the ibuprofen effect on mechanical hypersensitivity depended on peripheral opioid receptors and neutrophils at the injury site. The morphine-ibuprofen combination produced synergistic analgesia across all endpoints without enhancing opioid-induced gastrointestinal inhibition or mydriasis. Peripheral opioid antagonism reversed the effect of the combination on mechanical hypersensitivity and facial expressions but not on licking. Conclusions: Our results replicate the key clinical phenomena relevant to the postoperative pain context, including the potentiation of morphine analgesia by ibuprofen without the exacerbation of adverse effects. Our results suggest that drug effects on different postoperative pain measures rely on distinct neurobiological mechanisms and are not interchangeable. Therefore, the use of a battery of complementary pain endpoints in preclinical pharmacology studies is advisable.

Bone marrow adipocytes are known to have a critical role within the bone marrow niche. However, our understanding of bone marrow adipose tissue expansion with obesity and the role it plays in immune cell regulation and osteoclastogenesis is limited. Here, we showed the expansion of bone marrow adipocytes promoted osteoclast differentiation and subsequently led to obesity-related trabecular and cortical bone loss through a stimulatory effect of the PD-1/PD-L1 axis. Bone marrow adipocytes isolated from obese mice had increased Mcp-1 expression, a key regulator of osteoclastogenesis and myeloid cell accumulation. With the increase in bone marrow adipose tissue-derived Mcp-1, we found an increase in the number of PD-L1+ myeloid cells. While these cells inhibited activated T-cells, we found evidence of a stimulatory osteoclastogenic effect of PD-L1+ myeloid cells on PD-1-expressing osteoclast precursors. The inhibition of PD-1/PD-L1 signaling during early osteoclastogenesis prevented myeloid cell commitment and resulted in decreased cell fusion, supporting the role of PD-1/PD-L1 signaling in osteoclastogenesis. Using a bone marrow adipocyte depletion mouse model (BMAd-Pparg KO), we demonstrated that obese BMAd-Pparg KO mice had a reduced number of bone marrow PD-L1+ myeloid cells, accompanied by a decrease in PD-1+ osteoclast precursors. The reduction in these precursors resulted in fewer osteoclasts, subsequently leading to improved trabecular bone volume. Since osteoclasts are myeloid cell-derived, these results suggest that bone marrow adipocytes are critical for the commitment and differentiation of myeloid cells into osteoclasts. Targeting bone marrow adipogenesis could ameliorate enhanced osteoclastogenesis and provide a novel approach to treat obesity-related bone loss. Obesity-induced expansion of BM adipocytes leads to PD-1/PD-L1-driven osteoclastogenesis and subsequent bone loss in obese, HFD-fed (OB-HFD) mice. After 12 weeks on a HFD, OB-HFD mice had a significant increase in BM adiposity and BMAT-derived Mcp-1 expression. The increase in BMAT-specific Mcp-1 expression was coupled with an increase in PD-1+ osteoclast (OC) precursors and PD-L1+ myeloid cells. In the context of obesity, the PD-1/PD-L1 axis has a stimulatory effect that enhances osteoclastogenesis and leads to trabecular and cortical bone loss. By depleting BM adipocytes with obesity, BMAT-derived Mcp-1 expression was decreased, as well as a decrease in PD-1+ OC precursors and PD-L1+ myeloid cells. This prevented obesity-related trabecular bone loss. Overall, this work demonstrated a strong correlation between BMAT expansion and PD-1/PD-L1-driven osteoclastogenesis as a mechanism for obesity-induced bone loss. (This image was created using BioRender).

Mutations in DNA mismatch repair (MMR) pathway genes (MSH2, MSH6, MLH1, and PMS2) are linked to acquired resistance to temozolomide (TMZ) and high tumor mutation burden (TMB) in high-grade gliomas (HGGs), including glioblastomas (GBMs). However, the specific roles of individual MMR genes in the initiation, progression, TMB, microsatellite instability (MSI), and resistance to TMZ in gliomas remain unclear. Here, we developed de novo mouse models of germline and somatic MMR-deficient (MMRd) HGGs. Surprisingly, loss of Msh2 or Msh6 did not lead to high TMB, MSI, nor did it confer a response to anti-programmed cell death 1 (anti-PD-1) in GBM. Similarly, human GBM showed discordance between MMR gene mutations and the TMB and MSI. Germline MMRd promoted the progression from low-grade to HGG and reduced survival compared with MMR-proficient (MMRp) tumor-bearing mice. This effect was not tumor cell intrinsic but was associated with MMRd in the tumor immune microenvironment, driving immunosuppressive myeloid programs, reduced lymphoid infiltration, and CD8+ T cell exhaustion. Both MMR-reduced (MMRr) and MMRd GBM were resistant to TMZ, unlike MMRp tumors. Our study shows that N3-(2-fluoroethyl) imidazotetrazine (KL-50), an imidazotetrazine-based DNA targeting agent that induces MMR-independent cross-link-mediated cytotoxicity, was effective against germline and somatic MMRr and MMRd GBMs, offering a potential therapy for TMZ-resistant HGG with MMR alterations.

Lenvatinib resistance and immune exclusion limit outcomes in HCC. We hypothesized that metabolic rewiring orchestrates resistance to lenvatinib and PD-1 blockade.

Adoptive transfer of T lymphocytes specific for neoantigens can elicit immunity against solid tumors in patients. However, how these antigens affect T-cell function, effector differentiation, and persistence remains unclear. We examined how an identical CD8+ T-cell product was shaped by melanoma expressing either a low-avidity self/tumor-associated antigen or high-avidity neoantigen and kinetically profiled T-cell differentiation in these two contexts across host tissues. High-avidity neoantigen expression was sufficient to activate naïve CD8+ T cells-leading to robust tumor regression and long-term protective immunity upon tumor rechallenge. Mechanistically, transferred naïve CD8+ T cells reacting to high-avidity neoantigen exhibited enhanced cytokine production, heightened effector function, and sustained persistence compared with the low-avidity wild-type tumors. Antitumor activity to these high-avidity tumors was preserved even in the absence of functional host T and B lymphocytes, and early lymph node (LN) trafficking was found to be essential for adoptive T-cell therapy efficacy. Expanded effector or stem memory T cells were compared with the naïve pmel-1 T-cell product. Stem memory (but not effector memory) cells exhibited similar antitumor efficacy and LN trafficking patterns to the naïve cells in mice with high-avidity neoantigen-expressing tumors. These findings highlight how differential tumor antigens shape divergent cellular fate and uncover a critical role of T-cell trafficking in LNs in shaping high-avidity neoantigen-specific responses.

Host immunity status and hypoxia are the hallmarks of radiosensitivity. Induction of anti-PD-1 immunotherapy demonstrates promise in locally advanced tumor radiotherapy, but whether anti-PD-1 immunotherapy improves radiosensitivity is unclear.

Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer-related mortality and there are limited therapies1. Although endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are implicated in HCC, the involvement of the UPR transducer ATF6α remains unclear2. Here we demonstrate the function of ATF6α as an ER-stress-inducing tumour driver and metabolic master regulator restricting cancer immunosurveillance for HCC, in contrast to its well-characterized role as an adaptive response to ER stress3. ATF6α activation in human HCC is significantly correlated with an aggressive tumour phenotype, characterized by reduced patient survival, enhanced tumour progression and local immunosuppression. Hepatocyte-specific ATF6α activation in mice induced progressive hepatitis with ER stress, immunosuppression and hepatocyte proliferation. Concomitantly, activated ATF6α increased glycolysis and directly repressed the gluconeogenic enzyme FBP1 by binding to gene regulatory elements. Restoring FBP1 expression limited ATF6α-activation-related pathologies. Prolonged ATF6α activation in hepatocytes triggered hepatocarcinogenesis, intratumoural T cell infiltration and nutrient-deprived immune exhaustion. Immune checkpoint blockade (ICB)4 restored immunosurveillance and reduced HCC. Consistently, patients with HCC who achieved a complete response to immunotherapy displayed significantly increased ATF6α activation compared with those with a weaker response. Targeting Atf6 through germline ablation, hepatocyte-specific ablation or therapeutic hepatocyte delivery of antisense oligonucleotides dampened HCC in preclinical liver cancer models. Thus, prolonged ATF6α activation drives ER stress, leading to glycolysis-dependent immunosuppression in liver cancer and sensitizing to ICB. Our findings suggest that persistently activated ATF6α is a tumour driver, a potential stratification marker for ICB response and a therapeutic target for HCC.

Prevotella melaninogenica is enriched in the lungs of people with cystic fibrosis (pwCF), yet its functional impact on respiratory tract homeostasis remains incompletely understood. Prior studies identified immune modulatory effects following lung exposure to Prevotella, but the relevance of these findings for CF infections is unknown.

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.

Tumour-induced mechanisms of immune evasion hinder immune response to cancer, particularly in melanoma. mRNA translation, by ensuring accurate protein synthesis, regulates cancer phenotypes and immune response, but the underlying mechanisms remain unclear. Here, we reveal how O-sialoglycoprotein endopeptidase (OSGEP), catalysing the tRNA modification N6-threonylcarbamoyladenosine (t6A), drives protein homeostasis in cancer cells to maintain T-cell exclusion and prevent anti-tumour immune response. t6A-deficient melanoma cells disrupt efficient cytoplasmic translation of ANN codons (trinucleotides with A in the first position and N = any nucleotide), causing specific protein aggregation and the formation of integrated stress response-dependent stress granules. We discovered that OSGEP loss triggers melanoma regression by relocating RIG-I to stress granules, leading to its pathway activation. As a result, T-cells are recruited to the tumour site and orchestrate an anti-tumour immune response. Finally, an OSGEP-driven gene signature in melanoma patients is associated with T-cell infiltration and improved overall survival. Together, our findings position t6A tRNA modification as a promising therapeutic target for melanoma treatment.

Most colorectal cancers (CRCs) are mismatch repair-proficient (pMMR) and microsatellite stable (MSS), and they respond poorly to immune checkpoint inhibitors (ICIs). Radiotherapy (RT) can promote antitumor immunity but may also trigger adaptive immune suppression through checkpoint upregulation, providing a rationale for combination therapies.

Radiation therapy (RT) is recognized for its ability to induce DNA damage within cancer cells, leading to cancer cell death and promotion of anti-tumor immune responses. However, this beneficial effect is often counterbalanced by the presence of suppressive Tregs. Although factors such as RT-induced transforming growth factor β (TGF-β) can contribute to increased Treg accumulation within the tumor, the dynamics of Treg movement, and recruitment in the post-RT tumor microenvironment are not fully understood. Our study examined Treg migration following RT, revealing that RT disrupts Treg migration to the tumor-draining lymph node (TdLN) and alters their phenotype. Combining RT with anti-CCR8 therapy, which selectively depletes Tregs within the tumor, significantly reduced tumor burden, and increased survival in preclinical models. This combination also proved effective against distant and unirradiated tumors. Additionally, efficacy of combination therapy was CD8 T cell dependent. These findings highlight the potential of combining RT with Treg-targeting therapies to enhance anti-tumor immunity.

Glioblastoma (GBM) is the most common primary brain tumor with an overall survival under 21 months. Despite extensive research effort, patient outcomes have improved minimally over the past several decades. The Inhibitor of Apoptosis (IAP) proteins are critical survival factors implicated in both immune regulation and gliomagenesis. Small molecule IAP antagonists called SMAC mimetic compounds (SMCs) are under investigation as cancer therapeutics across multiple malignancies, including GBM. SMCs induce GBM cell death in the presence of inflammatory cytokines, synergize with immune checkpoint inhibitors (ICI), and induce death of microglia and macrophages. Although SMCs show significant efficacy in murine models, complete eradication is not achieved. Here, we aimed to understand the limitations of SMCs in murine GBM and identify strategies to enhance efficacy of combination treatment with ICIs with the goal of informing future translational efforts.