InVivoPlus rat IgG2a isotype control, anti-trinitrophenol

Ischemic stroke results in blood-brain barrier (BBB) disruption, during which the reciprocal interaction between ischemic neurons and components of the BBB appears to play a critical role. However, the underlying mechanisms for BBB protection remain largely unknown. In this study, we found that Serpina3n, a serine protease inhibitor, was significantly upregulated in the ischemic brain, predominantly in ischemic neurons from 6 hours to 3 days after stroke. Using neuron-specific adeno-associated virus (AAV), intranasal delivery of recombinant protein, and immune-deficient Rag1-/- mice, we demonstrated that Serpina3n attenuated BBB disruption and immune cell infiltration following stroke by inhibiting the activity of granzyme B (GZMB) and neutrophil elastase (NE) secreted by T cells and neutrophils. Furthermore, we found that intranasal delivery of rSerpina3n significantly attenuated the neurologic deficits after stroke. In conclusion, Serpina3n is a novel ischemic neuron-derived proteinase inhibitor that counterbalances BBB disruption induced by peripheral T cell and neutrophil infiltration after ischemic stroke. These findings reveal a novel endogenous protective mechanism against BBB damage with Serpina3n being a potential therapeutic target in ischemic stroke.

Secondary bacterial infection, often caused by Streptococcus pneumoniae (Spn), is one of the most frequent and severe complications of influenza A virus (IAV)-induced pneumonia. Phenotyping of the pulmonary immune cell landscape after IAV infection revealed a substantial depletion of the tissue-resident alveolar macrophage (TR-AM) population at day 7, which was associated with increased susceptibility to Spn outgrowth. To elucidate the molecular mechanisms underlying TR-AM depletion, and to define putative targets for treatment, we combined single-cell transcriptomics and cell-specific PCR profiling in an unbiased manner, using in vivo models of IAV infection and IAV/Spn co-infection. The TNF superfamily 14 (TNFSF14) ligand-receptor axis was revealed as the driving force behind post-influenza TR-AM death during the early infection phase, enabling the transition to pneumococcal pneumonia, while intrapulmonary transfer of genetically modified TR-AMs and antibody-mediated neutralization of specific pathway components alleviated disease severity. With a mainly neutrophilic expression and a high abundance in the bronchoalveolar fluid (BALF) of patients with severe virus-induced ARDS, TNFSF14 emerged as a key determinant of virus-driven lung injury. Targeting the TNFSF14-mediated intercellular communication network in the virus-infected lung can, therefore, improve host defense, minimizing the risk of subsequent bacterial pneumonia, and ameliorating disease outcome.

Treatment options for advanced or metastatic intrahepatic cholangiocarcinoma (ICC) are limited. In this single-arm, phase 2 trial (CASTLE-01, NCT05010668 ), 28 participants with advanced or metastatic ICC who have progressed after chemotherapy were treated with cryoablation, followed by anti-PD1 sintilimab (200 mg every 3 weeks) plus lenvatinib (8-12 mg per day) 2 weeks later. The objective response rate assessed by Response Evaluation Criteria in Solid Tumors version 1.1 was 75.0% (95% confidence interval (CI): 59-91%), meeting the prespecified primary endpoint. Secondary endpoints of disease control rate, median progression-free survival and overall survival were respectively 100% (95% CI: 100-100%), 16.8 months (95% CI: 11.5-not reached (NR)) and 25.4 months (95% CI: 13.3-NR). Treatment was well tolerated. Post hoc multiomics analysis of paired pretreatment and on-treatment tumor biopsies suggested that cryoablation increased the tumor immunogenicity and dendritic cell activation, followed by triggering continuous replenishment of intratumoral CD8+PD1hi effectors from peripheral blood. The addition of lenvatinib transitioned endothelial cells into inflamed venules to boost lymphocyte influx and targeted tumor stroma to promote CD8+PD1hi effectors penetrating into tumor cell nests. Therefore, cryoablation combined with sintilimab plus lenvatinib represents a promising approach for the treatment of advanced or metastatic ICC. These findings also support the notion that cryoablation may trigger abscopal antitumor immunity in ICC when combined with lenvatinib and PD1 blockade. ClinicalTrials.gov registration: NCT05010668 .

Immune checkpoint inhibitors (ICIs), particularly programmed cell death protein 1 (PD-1) blockades, have redefined oncology in the last decade. Previous studies on PD-1 blockades mostly concentrate on their interactions with immune cells. This study aims to investigate how PD-1 blockades affect endothelial cell (EC) heterogeneity in the tumor microenvironment (TME) and to explore potential targets for enhancing the anti-tumor effects of PD-1 blockades. Here, we established a pan-cancer EC atlas from the public database and revealed that PD-1 blockades repress the angiogenic population in ECs and inhibit the CXCL12-CXCR4 signaling derived from ECs. Using a murine tumor model built with Lewis Lung Carcinoma cell line, we further validated our findings that a PD-1 blockade, as well as a CXCR4 antagonist AMD3100, inhibited EC population in tumors and their CXCL12 expression. In addition, the combo therapy of the PD-1 blockade and AMD3100 showed superior anti-tumor effects to monotherapy. Moreover, we predicted MYC to be the potential regulator through which PD-1 blockades affect ECs. Together, our results suggest that PD-1 blockades have an anti-angiogenic effect besides boosting T cell immunity, and the CXCL12/CXCR4 pathway is a potential target for enhancing the effectiveness of PD-1 blockades.

Perineural invasion (PNI) is a well-established factor of poor prognosis in multiple cancer types1, yet its mechanism remains unclear. Here we provide clinical and mechanistic insights into the role of PNI and cancer-induced nerve injury (CINI) in resistance to anti-PD-1 therapy. Our study demonstrates that PNI and CINI of tumour-associated nerves are associated with poor response to anti-PD-1 therapy among patients with cutaneous squamous cell carcinoma, melanoma and gastric cancer. Electron microscopy and electrical conduction analyses reveal that cancer cells degrade the nerve fibre myelin sheets. The injured neurons respond by autonomously initiating IL-6- and type I interferon-mediated inflammation to promote nerve healing and regeneration. As the tumour grows, the CINI burden increases, and its associated inflammation becomes chronic and skews the general immune tone within the tumour microenvironment into a suppressive and exhaustive state. The CINI-driven anti-PD-1 resistance can be reversed by targeting multiple steps in the CINI signalling process: denervating the tumour, conditional knockout of the transcription factor mediating the injury signal within neurons (Atf3), knockout of interferon-α receptor signalling (Ifnar1-/-) or by combining anti-PD-1 and anti-IL-6-receptor blockade. Our findings demonstrate the direct immunoregulatory roles of CINI and its therapeutic potential.

Fibrous dysplasia (FD) is a rare disorder caused by somatic activating mutations in GNAS, encoding the alpha subunit of the Gs protein. Activating GNAS mutations result in focal expansile bone lesions, which cause pain, deformity, and increased risk of fracture. Somatic mosaicism in FD leads to both GNAS mutant and genetically WT osteoprogenitor cells, which jointly contribute to the formation of fibrotic lesions within the bone. Additionally, these lesions contain numerous osteoclasts formed in response to robust lesional expression of RANKL. Neutralizing antibody to RANKL is effective in reducing lesion growth in patients with FD and in preclinical models. To determine the effect of RANKL neutralization specifically on mutant cells early after onset of FD, we used a murine model of C57BL/6 Sox9CreERT;Gnas(R201H)fl/+;Rosa26LSL-tdTomato mice, which recapitulates the somatic mosaicism of FD bone lesions and in which mutant cells are lineage traced. Analysis of Gnas(R201H)fl/+ mice showed a diffuse accumulation of SMA+ early osteoblastic cells, with contribution from both tdTomato+ mutant and tdTomato- WT populations. Anti-RANKL treatment of Gnas(R201H)fl/+ mice inhibited osteoclast formation and substantially reduced fibrosis, detected by Masson's trichrome staining within the proximal metaphysis of the femur and the femoral head. Treatment with anti-RANKL decreased the accumulation of both mutant and WT SMA+ cells, accompanied by an increased number of mutant cells expressing the mature osteoblast marker osteocalcin, and an increase in overall osteoblast density. To elucidate the role of RANKL expression by mutant cells in the formation of FD lesions, we generated Sox9CreERT;Gnas(R201H)fl/+;Rosa26LSL-tdTomato;Ranklfl/fl mice. Deletion of Rankl in Gnas(R201H)fl/+ mutant cells did not prevent fibrosis in this model. The results suggest that while anti-RANKL treatment promotes osteoprogenitor differentiation to reduce fibrosis, the loss of RANKL expression from GNAS mutant cells alone is not sufficient to reverse the pathology of FD bone lesions.

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.

Radiotherapy (RT)-elicited antitumor immunity serves as a pivotal mechanism in RT-mediated tumor control. The strategic integration of RT with immunotherapies, particularly immune checkpoint blockade (ICB), is revolutionizing cancer therapeutics, demonstrating remarkable clinical potential. In this context, identifying molecular targets to potentiate radioimmunotherapy (RIT) efficacy represents a critical research priority. Emerging as a central immunomodulatory axis, the cGAS/STING signaling pathway bridges DNA damage response with antitumor immunity, positioning itself as a prime therapeutic target for radiation sensitization. Our study unveils caspase-activated DNase (CAD) as a previously unrecognized suppressor of cGAS/STING signaling that governs radiosensitivity in colorectal cancer (CRC). CAD physically blocks STING dimerization and cGAMP binding through a nuclease-independent function, thereby compromising RT-induced STING activation and subsequent type I interferon (IFN-I) production. Functional analyses demonstrated that CAD ablation potentiates CD8+ T cell infiltration/activation within the tumor microenvironment and synergizes with anti-PD-1 immunotherapy upon radiation. Translational validation revealed clinical correlations between CAD overexpression in CRC specimens and suboptimal radiotherapy responses coupled with diminished intratumoral CD8+ T cell infiltration. Collectively, our findings establish CAD as a novel rheostat of cGAS-STING signaling and propose CAD inhibition as a promising combinatorial strategy to enhance RT and RIT efficacy in CRC.

In metastasis, the dynamics of tumor-immune interactions during micrometastasis remain unclear. Identifying the vulnerabilities of micrometastases before outbreaking into macrometastases can reveal therapeutic opportunities for metastasis. Here, we report a function of T cell immunoglobulin and mucin domain 3 (TIM3) in tumor cells during micrometastasis using breast cancer (BC) metastasis mouse models. TIM3 is highly upregulated in micrometastases, promoting survival, stemness, and immune escape. TIM3+ tumor cells are specifically selected during early seeding of micrometastasis. Mechanistically, TIM3 increases β-catenin/interleukin-1β (IL-1β) signaling, leading to stemness and immune-evasion by inducing immunosuppressive γδ T cells and reducing CD8 T cells during micrometastasis. Clinical data confirm increased TIM3+ tumor cells in BC metastasis and TIM3+ tumor cells as a biomarker of poor outcome in BC patients. (Neo)adjuvant TIM3 blockade reduces the metastatic seeding and incidence in preclinical models. These findings unveil a specific mechanism of micrometastasis immune-evasion and the potential use of TIM3 blockade for subclinical metastasis.

Immune checkpoint blockade (ICB) immunotherapies are a powerful tool in the clinical management of cancer, but response rates to ICB remain limited, and treatment-related toxicities can be significant. Therapeutic efficacy of ICB can be enhanced by delivering synergistic immunomodulators to tumor-draining lymph nodes (TdLNs). However, achieving sustained release of small molecule immunomodulators into the lymphatics and TdLNs remains challenging. To address this limitation, a sustained release system for delivering an oligonucleotide adjuvant to lymph nodes (LNs) was developed. CpG oligonucleotide was complexed with a redox-responsive cationic polymer and mixed with F127-g-Gelatin to generate a thermosensitive hydrogel that releases lymph-draining polyplex micelles in situ. This CpG/BPEI-SS-/F127-g-Gelatin (CpG-HG) system enhanced the quantity and duration of CpG delivery to TdLNs following locoregional administration compared with free drug and enabled targeted, potent, and prolonged immunomodulation within TdLNs from a single administration. This augmented, localized immune response synergized with systemic ICB treatment, both markedly amplifying the systemic circulating CD8+ T cell response and improving antitumor therapeutic efficacy while enabling ICB dose reduction. These results highlight the potential for this drug delivery system as an adjunct to existing clinical ICB protocols to improve patient outcomes.

Type I interferons (IFNs-I), a group of pleiotropic cytokines, critically modulate host response in various inflammatory diseases. However, the role of the IFN-I pathway in periodontitis remains largely unknown. In this report, we describe that the IFN-β levels in the gingival crevicular fluid of human subjects were negatively associated with periodontitis and clinical gingival inflammation. Disruption of IFN-I signaling worsened alveolar bone resorption in a ligature-induced periodontitis murine model. Deficiency of the IFN-I pathway resulted in an exaggerated inflammatory response in myeloid cells and drastically increased the interleukin-17 (IL-17)-mediated neutrophil recruitment in the gingiva. We further identified that the myeloid lineage-specific IFN-I response was essential in safeguarding against periodontal inflammation by suppressing the IL-17-producing γδ T cells in gingiva. IFN-I signaling also directly repressed osteoclastogenesis in monocytes, which are precursor cells for osteoclasts. Therefore, our findings demonstrate that an integral myeloid-specific IFN-I pathway protects against bone loss by keeping the IL-17-neutrophil axis in check and directly inhibiting osteoclast formation in periodontitis.

Mice lacking apolipoprotein E (APOE, Apoe-/- mice) on a high cholesterol (HC) diet are highly susceptible to infection with Mycobacterium tuberculosis (Mtb) but the underlying immune dysregulation has been unclear. While neutrophils are often the predominant cell type in the lungs of humans with severe tuberculosis (TB), they are relatively scarce in the lungs of some strains of mice that are used to study the disease. The neutrophil levels in the lungs of Mtb-infected Apoe-/- HC mice are very high, and thus studies in this model offer the opportunity to examine the role of specific neutrophil functions in the pathology of severe TB. We determined that depleting neutrophils, depleting plasmacytoid dendritic cells (pDCs), or blocking type I interferon signaling improved the outcome of TB in Apoe-/- HC mice. We also demonstrated that blocking the activation of peptidylarginine deiminase 4 (PAD4), an enzyme critical to NET formation, leads to fewer NETs in the lungs and dramatically improves the outcome of TB in Apoe-/- HC mice without affecting the number of neutrophils in the lung. We found that the transcriptional profile of neutrophils in Mtb-infected Apoe-/- HC mice is biased towards a state that resembles the "N2" phenotype that has been defined in cancer models and has been implicated in matrix degradation and tissue destruction. Our observations strongly suggest that the state of the neutrophil when it encounters the Mtb-infected lung is one of the main drivers of severe disease and implies that targeted interventions that alter specific states or functions, such as the production of NETs, may improve outcome while preserving sufficient capacity for host-defense.

Fetal hematopoiesis takes place in the liver before colonizing the bone marrow where it will persist for life. This colonization is thought to be mediated by specification of a microenvironment that selectively recruits hematopoietic cells to the nascent bone marrow. The identity and mechanisms regulating the specification of this colonization niche are unclear. Here we identify a VCAM1+ sinusoidal colonization niche in the diaphysis that regulates neutrophil and hematopoietic stem cell colonization of the bone marrow. Using confocal microscopy, we find that colonizing hematopoietic stem and progenitor cells (HSPC) and myeloid cells selectively localize to a subset of VCAM1+ sinusoids in the center of the diaphysis. Vcam1 deletion in endothelial cells impairs hematopoietic colonization while depletion of yolk-sac-derived osteoclasts disrupts VCAM1+ expression, and impairs neutrophil and HSPC colonization to the bone marrow. Depletion of yolk-sac-derived myeloid cells increases fetal liver hematopoietic stem cell numbers, function and erythropoiesis independent of osteoclast activity. Thus, the yolk sac produces myeloid cells that have opposite roles in fetal hematopoiesis: while yolk-sac derived myeloid cells in the bone marrow promote hematopoietic colonization by specifying a VCAM1+ colonization niche, a different subset of yolk-sac-derived myeloid cells inhibits HSC in the fetal liver.

Immune checkpoint therapy for colorectal cancer (CRC) has been found to be unsatisfactory for clinical treatment. Fecal microbiota transplantation (FMT) has been shown to remodel the intestinal flora, which may improve the therapeutic effect of αPD-1. Further exploration of key genera that can sensitize cells to αPD-1 for CRC treatment and preliminary exploration of immunological mechanisms may provide effective guidance for the clinical treatment of CRC.

FoxP3+ regulatory T cells (Tregs) are responsible for immune homeostasis by suppressing excessive anti-self-immunity. Tregs facilitate tumor growth by inhibiting anti-tumor immunity. Here, we explored the targeting of FoxP3 as a basis for new immunotherapies. In a high-throughput phenotypic screening of a drug repurposing library using human primary T cells, we identified quinacrine as a FoxP3 downregulator. In silico searches based on the structure of quinacrine, testing of sub-libraries of analogs in vitro, and validation identified a subset of 9-amino-acridines that selectively abrogated Treg suppressive functions. Mechanistically, these acridines interfered with the DNA-binding activity of FoxP3 and inhibited FoxP3-regulated downstream gene regulation. Release from Treg suppression by 9-amino-acridines increased anti-tumor immune responses both in cancer patient samples and in mice in a syngeneic tumor model. Our study highlights the feasibility of screening for small molecular inhibitors of FoxP3 as an approach to pursuing Treg-based immunotherapy.

The limited infiltration of CD8+ T cells in tumors hampers the effectiveness of T cell-based immunotherapy, yet the mechanisms that limit tumor infiltration by CD8+ T cells remain unclear. Through bulk RNA sequencing of human tumors, we identified a strong correlation between WNT7A expression and reduced CD8+ T-cell infiltration. Further investigation demonstrated that inhibiting WNT7A substantially enhanced MHC-I expression on tumor cells. Mechanistically, WNT7A inhibition inactivated the Wnt/β-catenin signaling pathway and thus resulted in reduced physical interaction between β-catenin and p65 in the cytoplasm, which increased the nuclear translocation of p65 and activated the NF-κB pathway, ultimately promoting the transcription of genes encoding MHC-I molecules. We found that our lead compound, 1365-0109, disrupted the protein-protein interaction between WNT7A and its receptor FZD5, resulting in the upregulation of MHC-I expression. In murine tumor models, both genetic and pharmaceutical suppression of WNT7A led to increased MHC-I levels on tumor cells, and consequently enhanced the infiltration and functionality of CD8+ T cells, which bolstered antitumor immunity and improved the effectiveness of immune checkpoint blockade therapy. These findings have elucidated the intrinsic mechanisms of WNT7A-induced immune suppression, suggesting that therapeutic interventions targeting WNT7A hold promise for enhancing the efficacy of immunotherapy.

Steatotic liver enhances liver metastasis of colorectal cancer (CRC), but this process is not fully understood. Steatotic liver induced by a high-fat diet increases cancer-associated fibroblast (CAF) infiltration and collagen and hyaluronic acid (HA) production. We investigated the role of HA synthase 2 (HAS2) in the fibrotic tumor microenvironment in steatotic liver using Has2ΔHSC mice, in which Has2 is deleted from hepatic stellate cells. Has2ΔHSC mice had reduced steatotic liver-associated metastatic tumor growth of MC38 CRC cells, collagen and HA deposition, and CAF and M2 macrophage infiltration. We found that low-molecular weight HA activates Yes-associated protein (YAP) in cancer cells, which then releases connective tissue growth factor to further activate CAFs for HAS2 expression. Single-cell analyses revealed a link between CAF-derived HAS2 and M2 macrophages and CRC cells through CD44; these cells were associated with exhausted CD8+ T cells via programmed death-ligand 1 and programmed cell death protein 1 (PD-1). HA synthesis inhibitors reduced steatotic liver-associated metastasis of CRC, YAP expression, and CAF and M2 macrophage infiltration, and improved response to anti-PD-1 antibody. In conclusion, steatotic liver modulates a fibrotic tumor microenvironment to enhance metastatic cancer activity through a bidirectional regulation between CAFs and metastatic tumors, enhancing the metastatic potential of CRC in the liver.

Treatment of solid tumors remains challenging and therapeutic strategies require continuous development. Tumor-infiltrating macrophages play a pivotal role in tumor dynamics. Here, we present a Macrophage-Drug Conjugate (MDC) platform technology that enables loading macrophages with ferritin-drug complexes. We first show that macrophages actively take up human heavy chain ferritin (HFt) in vitro via macrophage scavenger receptor 1 (MSR1). We further manifest that drug-loaded macrophages transfer ferritin to adjacent cancer cells through a process termed 'TRAnsfer of Iron-binding protein' (TRAIN). The TRAIN process requires direct cell-to-cell contact and an immune synapse-like structure. At last, MDCs with various anti-cancer drugs are formulated with their safety and anti-tumor efficacy validated in multiple syngeneic mice and orthotopic human tumor models via different routes of administration. Importantly, MDCs can be prepared in advance and used as thawed products, supporting their clinical applicability. This MDC approach thus represents a promising advancement in the therapeutic landscape for solid tumors.

Previously, we demonstrated that natural host-defence peptide caerin 1.1/caerin 1.9 (F1/F3) increases the efficacy of anti-PD-1 and therapeutic vaccine, in a HPV16 + TC-1 tumour model, but the anti-tumor mechanism of F1/F3 is still unclear. In this study, we explored the impact of F1/F3 on the tumor microenvironment in a transplanted B16 melanoma model, and further investigated the mechanism of action of F1/F3 using monoclonal antibodies to deplete relevant cells, gene knockout mice and flow cytometry. We show that F1/F3 is able to inhibit the growth of melanoma B16 tumour cells both in vitro and in vivo. Depletion of macrophages, blockade of IFNα receptor, and Stat1 inhibition each abolishes F1/F3-mediated antitumor responses. Subsequent analysis reveals that F1/F3 increases the tumour infiltration of inflammatory macrophages, upregulates the level of IFNα receptor, and promotes the secretion of IFNα by macrophages. Interestingly, F1/F3 upregulates CD47 level on tumour cells; and blocking CD47 increases F1/F3-mediated antitumor responses. Furthermore, F1/F3 intratumor injection, CD47 blockade, and therapeutic vaccination significantly increases the survival time of B16 tumour-bearing mice. These results indicate that F1/F3 may be effective to improve the efficacy of ICB and therapeutic vaccine-based immunotherapy for human epithelial cancers and warrants consideration for clinical trials.