InVivoMAb mouse IgG1 isotype control, unknown specificity

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.

The microenvironment of distant organs affects the colonization and growth of disseminated tumor cells. It remains unclear how tumor-associated neutrophils are influenced by the microenvironment of distant organs. Here, we demonstrate that mature low-density neutrophils in colorectal cancer patients abnormally accumulate neutral lipids and induce the reactivation of dormant tumor cells, a process regulated by hepatic stellate cells. Mechanistically, activated hepatic stellate cells increased DGAT1/2-dependent lipid droplet synthesis in low-density neutrophils through the secretion of IL33, thereby maintaining the survival and immunosuppressive function of these neutrophils. The uptake of lipids from lipid-laden low-density neutrophils drives dormant tumor cell reactivation through the potentiation of β-oxidation and the stimulation of protumorigenic eicosanoid synthesis. In mouse models, targeting IL33 blocked neutrophil lipid synthesis, decreased the colonization of colorectal cancer cells in the liver, and enhanced the efficacy of immunotherapy. Overall, our study revealed that lipid accumulation in mature low-density neutrophils regulates the growth of dormant tumor cells and antitumor immunity to facilitate colorectal cancer liver metastasis. Targeting IL33 could be a promising therapeutic approach for colorectal cancer liver metastases.

Immune checkpoint inhibitors targeting the interaction between PD1 and PDL1 are effective for immunotherapy in non-small cell lung cancer (NSCLC). However, only subgroups of patients respond to therapy, suggesting the existence of resistance mechanisms.

Asthma is a heterogeneous disease characterized by infiltration of immune cells that interact with epithelial cells and release various factors driving chronic inflammation and airway remodeling. Although monoclonal antibody-based biologics alleviate inflammation, their efficacy in suppressing airway remodeling is limited. Interleukin-24 (IL-24) has been implicated in neutrophilic asthma, but its role in eosinophilic asthma remains unclear. Here, we show that IL-24 is mainly secreted by infiltrating eosinophils in mice with OVA- and HDM-induced asthma-like lung disease models. IL-24 knockout mice exhibit reduced inflammatory responses, alleviated pulmonary fibrosis, improved epithelial integrity, and decreased mucus hypersecretion. Mechanistically, IL-24 activates the CXCL5/CXCR1/CXCR2 axis, enhancing eosinophil recruitment to the lungs. IL-24 disrupts epithelial tight junction integrity, contributing to increased permeability. Furthermore, IL-24 targets airway epithelial cells, promoting EMT-like changes and the secretion of profibrotic mediators, which leads to bronchial wall thickening and pulmonary fibrosis. Therefore, targeting IL-24 holds promise for anti-asthmatic interventions by suppressing inflammation and pathological remodeling.

Beyond supporting cancer cell proliferation, tumor growth relies on the ability of cancer cells to evade immune surveillance. Identifying novel molecules that promote tumor immune escape may help develop more effective immunotherapeutic strategies. The histone acetyltransferase E1A-binding protein p300 (EP300) is a key epigenetic regulator that modulates gene transcription through chromatin remodeling and acetylation of histones and transcription factors. However, its role in regulating immune evasion remains incompletely understood. This study investigates the impact of EP300 on tumor immune escape and suggests its potential as an immunotherapeutic target.

Immune checkpoint therapy has changed cancer treatment, including non-small cell lung cancer (NSCLC). The unresponsiveness of PD-L1lo/- tumors to anti-PD-1/PD-L1 immunotherapy is attributed to alternative immune evasion mechanisms that remain elusive. We previously reported that farnesoid X receptor (FXR) was increased in PD-L1lo/- NSCLC. Herein, we found that immune checkpoint HVEM was positively correlated with FXR but inversely correlated with PD-L1 in NSCLC. HVEM was highly expressed in FXRhiPD-L1lo NSCLC. Consistently, clinically relevant FXR antagonist dose-dependently inhibited HVEM expression in NSCLC. FXR inhibited cytokine production and cytotoxicity of cocultured CD8+ T cells in vitro, and it shaped an immunosuppressive tumor microenvironment (TME) in mouse tumors in vivo through the HVEM/BTLA pathway. Clinical investigations show that the FXR/HVEM axis was associated with immunoevasive TME and inferior survival outcomes in patients with NSCLC. Mechanistically, FXR upregulated HVEM via transcriptional activation, intracellular Akt, Erk1/2 and STAT3 signals, and G1/S cycle progression in NSCLC cells. In vivo treatment experiments demonstrated that anti-BTLA immunotherapy reinvigorated antitumor immunity in TME, resulting in enhanced tumor inhibition and survival improvement in FXRhiPD-L1lo mouse Lewis lung carcinomas. In summary, our findings establish the FXR/HVEM axis as an immune evasion mechanism in PD-L1lo/- NSCLC, providing translational implications for future immunotherapy in this subgroup of patients.

As a part of the commensal microbiome, the regulatory role of the intratumoral microbiome in tumor immunity is gradually revealed. However, the relationship between the intratumoral microbiome and the efficacy of immune checkpoint inhibitors (ICIs) clinical treatment remains unclear. Here, we collect RNA sequencing (RNA-seq) data and clinical information from publicly available ICIs therapy cohorts. By developing an improved bioinformatics pipeline to identify the intratumoral microbiome and performing a comprehensive association analysis, we find that the intratumoral microbiome is associated with response to ICIs and characteristics of the tumor microenvironment (TME). In vivo experiments demonstrate that intratumoral injection of Burkholderia cepacia, Priestia megaterium, or Corynebacterium kroppenstedtii, which were selected from our analysis results, would synergize with anti-PD-1 therapy to inhibit tumor growth and enhance antitumor immunity. Our findings highlight the essential role of the intratumoral microbiome in the clinical effectiveness differences of ICIs, suggesting its potential in future ICIs combination therapy.

Maintaining a healthy pool of circulating red blood cells (RBCs) is essential for adequate perfusion, as even minor changes in the population can impair oxygen delivery, resulting in serious health complications including tissue ischemia and organ dysfunction. This responsibility largely falls to specialized macrophages in the spleen, known as red pulp macrophages, which efficiently take up and recycle damaged RBCs. However, questions remain regarding how these macrophages are acutely activated to accommodate increased demand. Proresolving lipid mediators stimulate macrophage phagocytosis and efferocytosis but their role in erythrophagocytosis has only recently been described. To investigate the role of lipid mediators on red pulp macrophage function, we targeted the ALX/FPR2 signaling pathway, as this receptor binds multiple lipid mediator ligands eliciting potent macrophage responses. We found that mice with Fpr2 deletion exhibited disrupted erythrocyte homeostasis resulting in an aged RBC pool, decreased markers of splenic RBC turnover, and altered splenic macrophage phenotype characterized by changes in heme metabolism. Upon activation of ondemand erythrophagocytosis, production of the ALX/FPR2 ligand, lipoxin A4 (LXA4), was induced in the spleen while receptor-deficient animals were unable to efficiently clear damaged RBCs, a defect that was conserved in mice with myeloid-specific FPR2 deletion. Similarly, mice lacking the LXA4 biosynthetic enzyme displayed defective erythrophagocytosis that was rescued with LXA4 administration. These results indicate that the ALX/FPR2 signaling axis is necessary for maintenance of RBC homeostasis and LXA4 activation is a critical aspect of the red pulp macrophage response to acute erythroid stress.

The pathology of severe COVID-19 is due to a hyperinflammatory immune response persisting after viral clearance. To understand how the immune response to SARS-CoV-2 is regulated to avoid severe COVID-19, we tested relevant immunoregulatory cytokines. Transforming growth factor β (TGF-β), interleukin (IL)-10, and IL-4 were neutralized upon infection with mouse-adapted SARS-CoV-2 (CMA3p20), a model of mild disease; lung inflammation was quantified by histology and flow cytometry at early and late time points. Mild weight loss and lung inflammation including consolidation and alveolar thickening were evident 3 d postinfection (dpi), and inflammation persisted to 7 dpi. Coinciding with early monocytic infiltrates, CCL2 and granulocyte colony-stimulating factor were transiently produced 3 dpi, while IL-12 and CCL5 persisted to 7 dpi, modeling viral and inflammatory phases of disease. Neutralization of TGF-β, but not IL-10 or IL-4, significantly increased lung inflammatory monocytes and elevated serum but not lung IL-6. Neutralization of IL-4 prolonged weight loss and increased early perivascular infiltration without changing viral titer. Anti-IL-4 reduced expression of Arg1, a gene associated with alternative activation of macrophages. Neutralizing TGF-β and IL-4 had differential effects on pathology after virus control. Lung perivascular infiltration was reduced 7 dpi by neutralization of IL-4 or TGF-β, and periairway inflammation was affected by anti-TGF-β, while alveolar infiltrates were not affected by either. Anti-IL-4 prolonged IL-12 to 7 dpi along with reduced IL-10 in lungs. Overall, the immunoregulatory cytokines TGF-β and IL-4 dampen initial inflammation in this mouse-adapted SARS-CoV-2 infection, suggesting that promotion of immunoregulation could help patients in early stages of disease.

Cancer immunotherapy has recently achieved a breakthrough status, however, it is not effective in all cancer types. Genetically engineered oncolytic viruses (OVs) with immunomodulators are promising new therapeutic modalities for cancer. CARG-2020 is an engineered trivalent oncolytic viral construct that specifically expresses three immune modulators that inhibit IL-17RA signaling and regulate PD-L1 expression by shRNAs, along with the cytokine IL-12 which activates multiple tumoricidal pathways. Previous work showed that intratumoral (i.t.) injection of CARG-2020 exhibits robust efficacy against established colorectal cancer (CRC). In this study, we report that systemic delivery of CARG-2020 via the intravenous (i.v.) route can successfully control CRC growth. To further expand the scope of CARG-2020 as a pan-cancer candidate, we also show that CARG-2020 works in two additional mouse models of melanoma and triple-negative breast cancer. Administration of CARG-2020 resulted in increased accumulation of CD8+ T lymphocytes in the tumors, and depletion of these T cells results in poor tumor regression mediated by CARG-2020. Overall, our study shows a broad-spectrum efficacy of CARG-2020 in solid tumors and demonstrates the potential of CARG-2020 to be developed as a clinical candidate for the treatment of multiple human cancers that are surgically accessible.

Mitral valve prolapse is often benign, but progression to mitral regurgitation may require invasive intervention and there is no specific medical therapy. An association of mitral valve prolapse with Marfan syndrome resulting from pathogenic FBN1 variants supports the use of hypomorphic fibrillin-1 mgR mice to investigate mechanisms and therapy for mitral valve disease. mgR mice developed severe myxomatous mitral valve degeneration with mitral regurgitation by 12 weeks of age. Persistent activation of TGF-β and mTOR signaling along with macrophage recruitment preceded histological changes at 4 weeks of age. Short-term mTOR inhibition with rapamycin from 4 to 5 weeks of age prevented TGF-β overactivity and leukocytic infiltrates, while long-term inhibition of mTOR or TGF-β signaling from 4 to 12 weeks of age rescued mitral valve leaflet degeneration. Transcriptomic analysis identified integrins as key receptors in signaling interactions, and serologic neutralization of integrin signaling or a chimeric integrin receptor altering signaling prevented mTOR activation. We confirmed increased mTOR signaling and a conserved transcriptome signature in human specimens of sporadic mitral valve prolapse. Thus, mTOR activation from abnormal integrin-dependent cell-matrix interactions drives TGF-β overactivity and myxomatous mitral valve degeneration, and mTOR inhibition may prevent disease progression of mitral valve prolapse.

Accumulating evidence implicates the gut microbiome (GMB) in the pathogenesis and progression of Alzheimer's disease (AD). We recently showed that the GMB regulates reactive astrocytosis and Aβ plaque accumulation in a male APPPS1-21 AD mouse model. Yet, the mechanism(s) by which GMB perturbation alters reactive astrocytosis in a manner that reduces Aβ deposition remain unknown. Here, we performed metabolomics on plasma from mice treated with antibiotics (ABX) and identified a significant increase in plasma propionate, a gut-derived short-chain fatty acid, only in male mice. Administration of sodium propionate reduced reactive astrocytosis and Aβ plaques in APPPS1-21 mice, phenocopying the ABX-induced phenotype. Astrocyte-specific RNA-Seq on ABX- and propionate-treated mice showed reduced expression of proinflammatory and increased expression of neurotrophic genes. Next, we performed flow cytometry experiments, in which we found that ABX and propionate decreased peripheral RAR-related orphan receptor-γ+ (Rorγt+) CD4+ (Th17) cells and IL-17 secretion, which positively correlated with reactive astrocytosis. Last, using an IL-17 mAb to deplete IL-17, we found that propionate reduced reactive astrocytosis and Aβ plaques in an IL-17-dependent manner. Together, these results suggest that gut-derived propionate regulates reactive astrocytosis and Aβ amyloidosis by decreasing peripheral Th17 cells and IL-17 release. Thus, propionate treatment or strategies boosting propionate production may represent novel therapeutic strategies for the treatment of AD.

Psoriasis is an inflammatory disease characterised by chronic recurrent relapses. Previous observational studies have shown that patients with psoriasis are predisposed to cardiovascular comorbidities, but few studies have investigated the impact of psoriasis-related chronic inflammation on cardiac function. In this study, we used imiquimod (IMQ) to establish psoriasis-like mouse models with short-term inflammation (IMQ-ST) or long-term repeated inflammation (IMQ-LT), to mimic acute or chronic recurrent pathophysiology of psoriasis inflammation. The inflammatory pattern in the hearts of IMQ-ST mice and IMQ-LT mice was similar to that in the skin, characterised by increased level of interleukin (IL)-17A and proportion of IL-17A-producing γδT cells. However, only IMQ-LT mice showed declined cardiac function, significant myocardial tissue necrosis, and decreased expression of genes encoding structural and functional proteins in cardiomyocytes. Furthermore, IL-17A neutralisation markedly alleviated myocardial injury and improved cardiac function in IMQ-LT mice. In conclusion, we demonstrated that IL-17A-mediated inflammation was present in the skin and heart of acute and chronic psoriasis-like mouse models. However, only IMQ-LT mice developed myocardial injury and declined cardiac function, which could be prevented by IL-17A neutralisation.

Human metapneumovirus (HMPV) is a leading cause of lower respiratory tract infection in children accounting for 7% of acute care visits and hospitalizations. In particular, neonates and infants have worse outcomes with HMPV infection. The neonatal immune system is regulated to favor anti-inflammatory and tolerogenic responses compared to adults, including prior work demonstrating epigenetic factors in neonatal CD4+ T cells promoting Th2 formation rather than antiviral Th1 differentiation. To interrogate the neonatal immune response to HMPV, 4-to-6 day-old mice or adult 6-to-8 week-old mice were infected with HMPV. Neonates had a decreased Th1 population and increased Th2 and regulatory T-cell (Treg) populations compared to adults. Neonatal Th1 function, but not cell number, was restrained by surface PD-1 expression. To assess if neonatal Th1 formation was intrinsically inhibited after HMPV, neonatal and adult CD4s were transferred into immunocompetent or immunodeficient neonates. Both adult and neonatal CD4s demonstrated reduced Th1 differentiation in the immunocompetent neonates, but robust Th1 differentiation in immunodeficient neonates and immunocompetent adults, suggesting an extrinsic mechanism. Loss of neonatal Tregs led to increased Th1 differentiation after HMPV infection. Neonatal Tregs had increased TGF-β production compared to adult Tregs, and disruption of TGF-β signaling increased Th1 induction. These data demonstrate Tregs provide extrinsic regulation of Th1 formation in the context of respiratory viral infections, rather than an intrinsic limitation of neonatal CD4s. Collectively, these findings identify a nuanced neonatal response to respiratory viruses limiting Th1 formation and function.

Mucosal delivery of vaccine boosters induces robust local protective immune responses even without any adjuvants. Yet, the mechanisms by which antigen alone induces mucosal immunity in the respiratory tract remain unclear. Here we show that an intranasal booster with an unadjuvanted recombinant SARS-CoV-2 spike protein, after intramuscular immunization with 1 μg of mRNA-LNP vaccine encoding the full-length SARS-CoV-2 spike protein (Pfizer/BioNTech BNT162b2), elicits protective mucosal immunity by retooling the lymph node-resident immune cells. On intranasal boosting, peripheral lymph node-primed B cells rapidly migrated to the lung through CXCR3-CXCL9 and CXCR3-CXCL10 signaling and differentiated into antigen-specific IgA-secreting plasma cells. Memory CD4+ T cells in the lung served as a natural adjuvant for developing mucosal IgA by inducing the expression of chemokines CXCL9 and CXCL10 for memory B cell recruitment. Furthermore, CD40 and TGFβ signaling had important roles in mucosal IgA development. Repeated mucosal boosting with an unadjuvanted protein amplified anamnestic IgA responses in both the upper and the lower respiratory tracts. These findings help explain why nasal boosters do not require an adjuvant to induce robust mucosal immunity at the respiratory mucosa and can be used to design safe and effective vaccines against respiratory pathogens.

Despite the therapeutic benefit of immune checkpoint blockade in cancers, there is no consensus on its effect in infectious diseases. Here we investigated whether blocking the immune checkpoint T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) increases T cell immunity in active Mycobacterium tuberculosis infection. TIGIT expression in both peripheral blood and lung lesions in tuberculosis patients was assessed, and the correlation with clinical features analyzed. The functional status of TIGIT+ and TIGIT-CD8+ T cell subsets in tuberculosis patients was analyzed by flow cytometry and transcriptome analysis. To investigate the regulatory effect of TIGIT, the function of CD8+ T cells in tuberculosis patients and bacterial load in a tuberculosis mouse model were assessed after in vitro and in vivo TIGIT blockade. In active tuberculosis patients, TIGIT expression on CD8+ T cells in the peripheral blood was significantly upregulated and positively correlated with disease severity. TIGIT expression in lung lesions was significantly higher in patients with pulmonary tuberculosis than in patients infected with other pathogens. TIGIT+CD8+ T cells exhibited higher activation and differentiation levels, increased expression levels of cytokines and cytotoxic molecules, and showed gene expression features of natural killer-like cytotoxic effector CD8+ T cells. TIGIT blockade increased the ability of human CD8+ T cells to produce effector molecules and kill intracellular bacteria in vitro. Importantly, blocking TIGIT reduced lung bacterial burden in mice infected with M. tuberculosis. The findings reveal that in active tuberculosis patients, activated CD8+ T cells express TIGIT and blocking TIGIT enhances CD8+ T cell function and promotes clearance of M. tuberculosis. The findings also suggest that TIGIT limits T cell immunity in tuberculosis and implicate TIGIT blockade as a novel strategy for tuberculosis therapy.

The development of immune checkpoint inhibitors has changed treatment strategies for some patients with non-small cell lung cancer (NSCLC). However, resistance remains a major problem, requiring the elucidation of resistance mechanisms, which might aid the development of novel therapeutic strategies. The upregulation of CD155, a primary ligand of the immune checkpoint receptor TIGIT, has been implicated in a mechanism of resistance to PD-1/PD-L1 inhibitors, and it is therefore important to characterize the mechanisms underlying the regulation of CD155 expression in tumor cells. The aim of this study was to identify a Nectin that might regulate CD155 expression in NSCLC and affect anti-tumor immune activity. In this study, we demonstrated that NECTIN4 regulated the cell surface expression and stabilization of CD155 by interacting and co-localizing with CD155 on the cell surface. In a syngeneic mouse model, NECTIN4-overexpressing cells exhibited increased cell surface CD155 and resistance to anti-PD-1 antibodies. Of note, combination therapy with anti-PD-1 and anti-TIGIT antibodies significantly suppressed tumor growth. These findings provide new insights into the mechanisms of resistance to anti-PD-1 antibodies and suggest that NECTIN4 could serve as a valuable marker in therapeutic strategies targeting TIGIT.

In anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and systemic lupus erythematosus (SLE), glomerulonephritis is a severe kidney complication driven by immune cells, including T cells. However, the mechanisms underlying T cell activation in these contexts remain elusive. Here we report that in patients with AAV and SLE, type I interferon (IFN-I) induces T cell differentiation into interferon-stimulated genes-expressing T (ISG-T) cells, which are characterized by an elevated IFN-I signature, an immature phenotype, and cytotoxicity in inflamed tissue. Mechanistically, IFN-I stimulates the expression of interferon regulatory factor 7 (IRF7) in T cells, which in turn induces granzyme B production. In mice, blocking IFN-I signaling reduces IRF7 and granzyme B expression in T cells, thus ameliorating glomerulonephritis. In parallel, spatial transcriptomic analyses of kidney biopsies from patients with AAV or SLE reveal an elevated ISG signature and the presence of ISG-T cells in close proximity to plasmacytoid dendritic cells, the primary producers of IFN-I. Our results from both patients and animal models thus suggest that IFN-I production in inflamed tissue may drive ISG-T cell differentiation to expand the pool of cytotoxic T cells in autoimmune diseases.