InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin

Enterovirus D68 (EV-D68) causes respiratory illness in children. It also causes severe paralysis called acute flaccid myelitis (AFM), which has become a global health threat. Here, we generated an mRNA vaccine expressing virus-like particles (VLPs) of EV-D68. We found that the mRNA vaccine elicited potent neutralizing antibodies against EV-D68 in the blood, and the neutralizing titer was superior to that of the inactivated whole virion (IWV) vaccine. The mRNA vaccine showed protective effects against intranasal challenge with EV-D68, and antisera from the vaccinated mice prevented the paralysis caused by EV-D68 infection in neonatal mice. Moreover, the mRNA vaccine induced neutralizing antibodies in the respiratory tract, which is the entry site for EV-D68. Additionally, it attenuated infection with coxsackievirus B3 (CVB3), which belongs to another enterovirus group, via CD8+ T cell responses. In conclusion, our results suggest that this mRNA vaccine is a promising candidate for EV-D68 prevention.

Intracerebral hemorrhage (ICH) causes hematoma formation, leading to PHE, which is associated with leukocyte mobilization and increased inflammation at the site of brain injury. However, the fate of accumulated leukocytes within the hematoma and their impact on PHE expansion remain unknown. We performed single-cell immune profiling of hematoma cells from patients with acute ICH and reported a distinct phenotypic transformation of CD8+ T cells within the hematoma during the first 24 h after onset. In addition to enhanced IFN-γ production and migration capacity, these CD8+ T cells displayed remarkable glycolytic signatures. The metabolic fitness and functional reprogramming of hematomal CD8+ T cells are associated with the transcription factor FOXO1. Single-cell profiling of brain-infiltrating CD8+ T cells within the perihematomal tissues of ICH patients and cell culture assays revealed their capacity to activate microglia via the production of IFN-γ. Furthermore, the removal of hematomal CD8+ T cells reduced neuroinflammation, PHE expansion and neurological deficits in ICH mice. Thus, CD8+ T cells undergo metabolic and functional reprogramming within the hematoma during the acute phase of ICH, which contributes to PHE formation and neurological deterioration.

KRASG12C inhibitors (G12Ci) have produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment are therefore crucial. To better understand the function of KRASG12C and possible G12Ci bypass mechanisms, we developed an autochthonous KRASG12C-driven PDAC model. Compared with the classical KRASG12D PDAC model, the G12C model exhibits slower tumor growth, yet similar histopathologic and molecular features. Aligned with clinical experience, G12Ci treatment of KRASG12C tumors produced modest impact despite stimulating a "hot" tumor immune microenvironment. Immunoprofiling revealed that CD24, a "do not eat me" signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRASG12D-driven PDAC. Together, this study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC.

Stem-like CD8⁺ exhausted T cells (Tex) sustain antitumor immunity, whereas TGFβ signaling acts as a major immunosuppressive pathway. In patients with colorectal liver metastases, we observe that elevated TβRI expression in peri-metastatic hepatocytes correlates with poor prognosis. We therefore investigate whether disrupting hepatocytic TGFβ signaling can reinvigorate stem-like CD8⁺ Tex cells to restrict liver metastasis. In support of this hypothesis, mice with hepatocyte-specific TβRI depletion exhibit reduced liver metastatic burden across multiple tumor models. Mechanistically, hepatocytic TβRI blockade suppresses Galectin-9 secretion, which reshapes the transcriptional program of intra-tumoral CD8⁺ T cells. This reprogramming promotes a phenotypic transition from terminal exhaustion toward stem-like and effector states, yielding T cell subsets with enhanced metastasis-control capacity. Importantly, this axis functions independently of macrophages and CD4⁺ T cells. Furthermore, therapeutic delivery of Galunisertib using choline-modified lipid nanoparticles synergizes with αPD-1, fostering the conversion of exhausted CD8⁺ T cells into responsive Ly108⁺CX3CR1⁺ subsets and suppressing liver metastases. Collectively, our results identify hepatocyte TGFβ signaling as a targetable checkpoint against liver metastases.

Immune checkpoint inhibitors (ICI) benefit some cancer patients but de novo resistance remains poorly understood. Analyzing transcriptional data from two clinical trial cohorts, GO30140 and IMbrave150, we find B cell lymphoma 9 (BCL9), a Wnt/β-catenin co-factor, associated with resistance. We develop a BCL9-targeting peptide, hsBCL9Z96, which suppresses tumor growth in combination with anti-PD-L1 ab in preclinical hepatocellular carcinoma (HCC) mouse models. Multi-omics analyses implicate targeting BCL9 inhibits BMP4 secretion and downregulates CD24 on tumor cells, reprogramming macrophages toward a tumor-suppressive phenotype and promoting macrophage phagocytosis. This in turn rejuvenates T cell immunity via enhanced macrophage-mediated antigen presentation. Our data extend our understanding of how tumor-derived Wnt/β-catenin signaling impedes the innate and adaptive immune responses in the tumor microenvironment and provide preliminary evidence that targeting BCL9 is a promising preclinical strategy to mitigate ICI resistance in HCC.

Oncolytic virus M1 (OVM), a naturally occurring alphavirus, has demonstrated potent antitumor activity in various solid tumor models by inducing immunogenic cell death and activating CD8+ T cells. However, its in vivo efficacy varies widely, and resistance mechanisms remain poorly understood. Tumor-associated macrophages (TAMs), key immunosuppressive cells within the tumor microenvironment, may limit OVM therapeutic potential.

The skull marrow niche has recently been identified as a reservoir that supplies the brain with monocytes and neutrophils in the context of disease and injury, but its role in brain cancers remains unknown. Here we show that glioblastoma, the most malignant type of brain tumor, induces calvarial bone abnormalities in murine models and patients with glioblastoma, altering osteoclast activities and increasing the number of skull channels in mice. Single-cell RNA sequencing revealed glioblastoma-mediated alterations in the immune landscape of skull marrow and femoral bone marrow, including expansion of neutrophils and deterioration of various B cell subsets. In vivo inhibition of bone resorption reduced bone abnormalities, but promoted tumor progression in mesenchymal subtype tumors. This also abolished the survival benefit of the checkpoint inhibitor anti-PD-L1, by reducing activated T cell and increasing inflammatory neutrophil numbers. Together, these data provide insight into how brain tumors affect skull bone and the immune environment.

Following central nervous system injury, astrocytes form borders that were traditionally regarded as physical barriers. Emerging evidence demonstrates their capacity to regulate inflammation and repair; however, the specific characteristics of these border astrocytes and their interactions with immune cells remain insufficiently characterized. Using single-cell sequencing and spatial transcriptomics, we identified astrocytes expressing the interferon-inducible protein bone marrow stromal cell antigen 2 (BST2) enriched at injury boundaries that promote microglial recruitment via C3/C3aR signaling. Astrocyte-specific Bst2 knockout reduced astrocyte-microglia interactions and attenuated border formation, correlating with early neurological improvement after stroke. Mechanistically, BST2 enhanced C3 expression through protein kinase C-βII (PKCβII) phosphorylation. Moreover, treatment with a BST2 monoclonal antibody diminished astrocyte-microglia interactions and improved neurological function. Together, these findings highlight the pivotal role of astrocyte-microglia interactions in lesion border formation and suggest that BST2 may represent a therapeutic target to modulate these interactions and reduce early brain injury after stroke.

Tumor-derived exosomes carry programmed death-ligand 1 (PD-L1), which binds programmed cell death protein 1 (PD-1) on T cells, suppressing immune responses locally and systemically. However, the mechanisms governing exosomal PD-L1 sorting and secretion remain elusive. Here, we identify Munc13-4 as a crucial regulator of this process. Deletion of Munc13-4 in breast tumors enhances T cell-mediated anti-tumor immunity, suppresses tumor growth, and improves the efficacy of immune checkpoint inhibitors. Mechanistically, Munc13-4 collaborates with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), Rab27, and SNAREs to facilitate PD-L1 sorting and secretion via exosomes. Cryogenic electron microscopy (cryo-EM) analysis of the Munc13-4-Rab27a complex provide structural insights into exosome secretion. Importantly, PD-L1 sorting relies on a ternary complex composed of Munc13-4, PD-L1 and HRS, which is regulated by interferon gamma (IFNγ) signaling. A designed peptide that disrupts Munc13-4-PD-L1 interaction impedes PD-L1 sorting, enhances antitumor immunity, and suppresses tumor growth, highlighting the therapeutic potential of targeting this pathway.

B cells express many protein ligands, yet their regulatory functions are incompletely understood. We profiled ligand expression across murine B sublineage cells, including those activated by defined receptor signals, and assessed their regulatory capacities and specificities through in silico analysis of ligand-receptor interactions. Consequently, we identified a B cell subset that expressed cytokine interleukin-27 (IL-27) and chemokine CXCL10. Through the IL-27-IL-27 receptor interaction, these IL-27/CXCL10-producing B cells targeted CD40-activated B cells in vitro and, upon induction by immunization and viral infection, optimized antibody responses and antiviral immunity in vivo. Also present in breast cancer tumors and retained there through CXCL10-CXCR3 interaction-mediated self-targeting, these cells promoted B cell PD-L1 expression and immune evasion. Mechanistically, Il27 and Cxcl10 transcription was induced by synergizing Toll-like receptor (TLR) and CD40 signals and driven by coinduced transcription factor BATF3, which directly targeted these genes. By applying a discovery framework focusing on regulatory cells, our findings expand the recognized scope of B cell regulatory functions.

B cell-rich tertiary lymphoid structures (TLS) are associated with favorable prognosis and positive response to immunotherapy in cancer. Here we show that simultaneous activation of innate immune effectors, STING and lymphotoxin-β receptor (LTβR), results in CD8+ T cell-dependent tumor suppression while inducing high endothelial venule development and germinal center-like B cell responses in tumors to generate functional TLS in a T cell-dependent manner. In a neoadjuvant setting, activation of STING and LTβR by their agonists effectively immunized mice against tumor recurrence, leading to long-term survival. STING activation alone was insufficient for inducing B cell-containing TLS or eliciting long-term therapeutic effects. However, when combined with LTβR activation, it improved the fitness of TLS with B cell expansion and maturation to IgG-producing long-lived plasma cells and memory cells, increased CD4+ T cell recruitment and memory CD8+ T cell expansion, and shifted the TH2/TH17 balance, resulting in the potentiation of humoral and cellular immunity against tumors. These findings suggest a therapeutic approach of simultaneously activating STING and lymphotoxin pathways.

Type 1 diabetes (T1D) is characterised by the autoimmune-mediated destruction of pancreatic β-cells. Although traditionally viewed as a disease dominated by T cells, recent studies have emphasised the crucial role of B cells in the development of T1D. Genome-wide association studies (GWAS) have revealed that CD226 is related to susceptibility to several autoimmune diseases, including T1D. Our recent work identified a pathogenic role of CD226+ CD8+ T cells in T1D. However, the involvement of CD226+ B cells in T1D development remains unclear.

The lack of a favorable tumor immune microenvironment (TIME) results in limited response rates to immune checkpoint blockade (ICB) across human solid tumors, necessitating the development of novel combination strategies. In this study, we repurposed FK228, an US FDA-approved histone deacetylase inhibitor that is used clinically in non-solid tumor treatment, as a novel ICB sensitizer in solid tumors and revealed the diverse regulatory functions of FK228 in the TIME. FK228 serves as a novel necroptosis inducer in cancer cells by triggering endoplasmic reticulum stress. This in turn enhances the immunogenicity of cancer cells and increases the infiltration of tumor-killing immunocytes, including CD8+ T and natural killer cells, particularly activating tumor-infiltrated CD8+ T cells. Meanwhile, FK228 treatment shifts macrophages toward the pro-inflammatory phenotype. Moreover, the combined use of FK228 and a PD-L1 inhibitor significantly delay tumor growth and extend the survival of tumor bearing mice. Overall, our findings reveal new possibilities for the clinical application of FK228 in solid tumors and underscore the critical role of histone deacetylases in maintaining the immune-unfavorable TIME.

Downregulation of antigen presentation and lack of immune infiltration are defining features of small cell lung cancer (SCLC), limiting response to immune checkpoint blockade (ICB). While a high-MHC class I, immune-inflamed subset benefits from ICB, underlying mechanisms of immune response in SCLC have yet to be elucidated. Here we show that in the IMpower133 clinical trial, high, but not low, NOTCH1 expression was significantly associated with longer survival with the addition of ICB to chemotherapy among approximately 80% of SCLC patients with NE-enriched tumors (ASCL1-enriched, HR 0.39, P = 0.0012; NEUROD1-enriched, HR 0.44, P = 0.024). Overexpression or pharmacologic activation of NOTCH1 in ASCL1 and NEUROD1 SCLC cell lines dramatically upregulated MHC class I through epigenetic reactivation of STING. In syngeneic mouse models, Notch1 activation reprogrammed SCLC tumors from immune-excluded to immune-inflamed, facilitating durable, complete responses with ICB combined with a STING agonist. STING1 expression was significantly enriched in high- compared with low-NOTCH1-expressing tumors in IMpower133, validating our proposed mechanism. Our data reveal a previously undiscovered role for NOTCH1 as a critical driver of SCLC immunogenicity and a potential predictive biomarker for ICB in SCLC. NOTCH1 activation may be a therapeutic strategy to unleash antitumor immune responses in SCLC and other neuroendocrine cancers in which NOTCH1 is typically suppressed.

The detailed mechanisms underlying the regulatory significance of dietary components in modulating anti-tumor immunity remain largely unknown. Here, we apply a co-culture-based screening approach using a blood nutrient compound library and identify zeaxanthin (ZEA), a dietary carotenoid pigment found in many fruits and vegetables and known for its role in eye health, as an immunomodulator that enhances the cytotoxicity of CD8+ T cells against tumor cells. Oral supplementation with ZEA, but not its structural isomer lutein (LUT), enhances anti-tumor immunity in vivo. Integrated multi-omics mechanistic studies reveal that ZEA promotes T cell receptor (TCR) stimulation on the CD8+ T cell surface, leading to improved intracellular TCR signaling for effector T cell function. Hence, ZEA treatment augments the efficacy of anti-PD1 immune checkpoint inhibitor in vivo and the cytotoxicity of human TCR gene-engineered CD8+ T cells in vitro. Our findings uncover a previously unknown immunoregulatory function of ZEA, which has translational potential as a dietary element in bolstering immunotherapy.

The immune ecosystem is central to maintaining effective defensive responses. However, it remains largely understudied how immune cells in the peripheral blood interact with circulating tumor cells (CTCs) in metastasis. Here, blood analysis of patients with advanced breast cancer revealed that over 75% of CTC-positive blood specimens contained heterotypic CTC clusters with CD45+ white blood cells (WBCs), which correlates with breast cancer subtypes, racial groups, and decreased survival. CTC-WBC clusters included overrepresented T cells and underrepresented neutrophils. Specifically, a rare subset of CD4 and CD8 double-positive T (DPT) cells was 140-fold enriched in CTC clusters versus their frequency in WBCs. DPT cells shared properties with CD4+ and CD8+ T cells but exhibited unique features of T cell exhaustion and immune suppression. Mechanistically, the integrin heterodimer α4β1, also named very late antigen 4 (VLA-4), in DPT cells and its ligand, VCAM1, in tumor cells are essential mediators of DPT-CTC clusters. Neoadjuvant administration of anti-VLA-4 neutralizing antibodies markedly blocked CTC-DPT clusters, inhibited metastasis, and extended mouse survival. These findings highlight a pivotal role of rare DPT cells in fostering cancer dissemination through CTC clustering. It lays a foundation for developing innovative biomarker-guided therapeutic strategies to prevent and target cancer metastasis.

T cell dysfunction with age underlies an increased incidence of cancer in elderly individuals; however, how T cell aging is triggered in the tumor microenvironment is unclear. Here, we show that an age-associated reduction in adipocyte-derived leptin contributes to the accumulation of tumor-infiltrating senescent CD8+ T cells. Single-cell profiling of human and mouse cancer tissues reveals that the frequency of intratumoral senescent CD8+ T cells increases with age, leading to a weak antitumor effect. Moreover, decreased levels of adipocyte-derived leptin are an indispensable factor for CD8+ T cell aging. Leptin signaling prevents p38-dependent CD8+ T cell senescence. Furthermore, plasma leptin levels are negatively related to intratumoral CD8+ T cell senescence in cancer patients. Our findings identify an unappreciated interplay between metabolic perturbation and T cell aging and suggest that modulating adipocyte-derived leptin levels may be a promising therapeutic strategy for older cancer patients.

Subunits of the SWI/SNF chromatin remodeling complex are altered in ∼20% of human cancers. Exemplifying the alterations is the ARID1A mutation that occurs in ∼50% of ovarian clear-cell carcinoma (OCCC), a disease with limited therapeutic options. In this study, we showed that ARID1A mutations create a dependence on alanine by regulating alanine transporters to increase intracellular alanine levels. ARID1A directly repressed the alanine importer SLC38A2 and simultaneously promoted the alanine exporter SLC7A8. ARID1A inactivation increased alanine utilization predominantly in protein synthesis and passively through the tricarboxylic acid cycle. Indeed, ARID1A-mutant OCCCs were hypersensitive to the inhibition of SLC38A2. In addition, SLC38A2 inhibition enhanced chimeric antigen receptor T-cell assault in vitro and synergized with immune checkpoint blockade using an anti-PD-L1 antibody in a genetically engineered mouse model of OCCC driven by conditional Arid1a inactivation in a CD8+ T-cell-dependent manner. These findings suggest that targeting alanine transport alone or in combination with immunotherapy may represent an effective therapeutic strategy for ARID1A-mutant cancers.

Improving the efficacy of anti-programmed death 1 (PD-1) monoclonal antibody (mAb) therapy remains a major challenge for cancer immunotherapy in non-small cell lung cancer (NSCLC). Gut microbial metabolites can influence immunotherapy efficacy.