InVivoMAb rat IgG2a isotype control, anti-trinitrophenol
Product Details
The 2A3 monoclonal antibody reacts with trinitrophenol. Because trinitrophenol is not expressed by mammals this antibody is ideal for use as an isotype-matched control for rat IgG2a antibodies in mostĀ in vivoĀ andĀ in vitroĀ applications.Specifications
| Isotype | Rat IgG2a,Ā Īŗ |
|---|---|
| Recommended Dilution Buffer | InVivoPure pH 6.5 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Formulation |
PBS, pH 6.5 Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein G |
| RRID | AB_1107769 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Additional Formats
Recommended Products
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Recommended Dilution Buffer
InVivoPure pH 6.5 Dilution Buffer
Bauche, D., et al. (2018). "LAG3(+) Regulatory T Cells Restrain Interleukin-23-Producing CX3CR1(+) Gut-Resident Macrophages during Group 3 Innate Lymphoid Cell-Driven Colitis" Immunity 49(2): 342-352 e345. PubMed
Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintains gut homeostasis but can also promote inflammatory bowel disease (IBD). The regulation of ILC3-dependent colitis remains to be elucidated. Here we show that Foxp3(+) regulatory T cells (Treg cells) prevented ILC3-mediated colitis in an IL-10-independent manner. Treg cells inhibited IL-23 and IL-1beta production from intestinal-resident CX3CR1(+) macrophages but not CD103(+) dendritic cells. Moreover, Treg cells restrained ILC3 production of IL-22 through suppression of CX3CR1(+) macrophage production of IL-23 and IL-1beta. This suppression was contact dependent and was mediated by latent activation gene-3 (LAG-3)-an immune checkpoint receptor-expressed on Treg cells. Engagement of LAG-3 on MHC class II drove profound immunosuppression of CX3CR1(+) tissue-resident macrophages. Our study reveals that the health of the intestinal mucosa is maintained by an axis driven by Treg cells communication with resident macrophages that withhold inflammatory stimuli required for ILC3 function.
Ngiow, S. F., et al. (2015). "A Threshold Level of Intratumor CD8+ T-cell PD1 Expression Dictates Therapeutic Response to Anti-PD1" Cancer Res 75(18): 3800-3811. PubMed
Despite successes, thus far, a significant proportion of the patients treated with anti-PD1 antibodies have failed to respond. We use mouse tumor models of anti-PD1 sensitivity and resistance and flow cytometry to assess tumor-infiltrating immune cells immediately after therapy. We demonstrate that the expression levels of T-cell PD1 (PD1(lo)), myeloid, and T-cell PDL1 (PDL1(hi)) in the tumor microenvironment inversely correlate and dictate the efficacy of anti-PD1 mAb and function of intratumor CD8(+) T cells. In sensitive tumors, we reveal a threshold for PD1 downregulation on tumor-infiltrating CD8(+) T cells below which the release of adaptive immune resistance is achieved. In contrast, PD1(hi) T cells in resistant tumors fail to be rescued by anti-PD1 therapy and remain dysfunctional unless intratumor PDL1(lo) immune cells are targeted. Intratumor Tregs are partly responsible for the development of anti-PD1-resistant tumors and PD1(hi) CD8(+) T cells. Our analyses provide a framework to interrogate intratumor CD8(+) T-cell PD1 and immune PDL1 levels and response in human cancer. Cancer Res; 75(18); 3800-11. (c)2015 AACR.
Dai, M., et al. (2015). "Curing mice with large tumors by locally delivering combinations of immunomodulatory antibodies" Clin Cancer Res 21(5): 1127-1138. PubMed
PURPOSE: Immunomodulatory mAbs can treat cancer, but cures are rare except for small tumors. Our objective was to explore whether the therapeutic window increases by combining mAbs with different modes of action and injecting them into tumors. EXPERIMENTAL DESIGN: Combinations of mAbs to CD137/PD-1/CTLA-4 or CD137/PD-1/CTLA-4/CD19 were administrated intratumorally to mice with syngeneic tumors (B16 and SW1 melanoma, TC1 lung carcinoma), including tumors with a mean surface of approximately 80 mm(2). Survival and tumor growth were assessed. Immunologic responses were evaluated using flow cytometry and qRT-PCR. RESULTS: More than 50% of tumor-bearing mice had complete regression and long-term survival after tumor injection with mAbs recognizing CD137/PD-1/CTLA-4/CD19 with similar responses in three models. Intratumoral injection was more efficacious than intraperitoneal injection in causing rejection also of untreated tumors in the same mice. The three-mAb combination could also induce regression, but was less efficacious. There were few side effects, and therapy-resistant tumors were not observed. Transplanted tumor cells rapidly caused a Th2 response with increased CD19 cells. Successful therapy shifted this response to the Th1 phenotype with decreased CD19 cells and increased numbers of long-term memory CD8 effector cells and T cells making IFNgamma and TNFalpha. CONCLUSIONS: Intratumoral injection of mAbs recognizing CD137/PD-1/CTLA-4/CD19 can eradicate established tumors and reverse a Th2 response with tumor-associated CD19 cells to Th1 immunity, whereas a combination lacking anti-CD19 is less effective. There are several human cancers for which a similar approach may provide clinical benefit.
Kurtulus, S., et al. (2015). "TIGIT predominantly regulates the immune response via regulatory T cells" J Clin Invest. doi : 10.1172/JCI81187. PubMed
Coinhibitory receptors are critical for the maintenance of immune homeostasis. Upregulation of these receptors on effector T cells terminates T cell responses, while their expression on Tregs promotes their suppressor function. Understanding the function of coinhibitory receptors in effector T cells and Tregs is crucial, as therapies that target coinhibitory receptors are currently at the forefront of treatment strategies for cancer and other chronic diseases. T cell Ig and ITIM domain (TIGIT) is a recently identified coinhibitory receptor that is found on the surface of a variety of lymphoid cells, and its role in immune regulation is just beginning to be elucidated. We examined TIGIT-mediated immune regulation in different murine cancer models and determined that TIGIT marks the most dysfunctional subset of CD8+ T cells in tumor tissue as well as tumor-tissue Tregs with a highly active and suppressive phenotype. We demonstrated that TIGIT signaling in Tregs directs their phenotype and that TIGIT primarily suppresses antitumor immunity via Tregs and not CD8+ T cells. Moreover, TIGIT+ Tregs upregulated expression of the coinhibitory receptor TIM-3 in tumor tissue, and TIM-3 and TIGIT synergized to suppress antitumor immune responses. Our findings provide mechanistic insight into how TIGIT regulates immune responses in chronic disease settings.
Ellis, G. T., et al. (2015). "TRAIL+ monocytes and monocyte-related cells cause lung damage and thereby increase susceptibility to influenza-Streptococcus pneumoniae coinfection" EMBO Rep 16(9): 1203-1218. PubMed
Streptococcus pneumoniae coinfection is a major cause of influenza-associated mortality; however, the mechanisms underlying pathogenesis or protection remain unclear. Using a clinically relevant mouse model, we identify immune-mediated damage early during coinfection as a new mechanism causing susceptibility. Coinfected CCR2(-/-) mice lacking monocytes and monocyte-derived cells control bacterial invasion better, show reduced epithelial damage and are overall more resistant than wild-type controls. In influenza-infected wild-type lungs, monocytes and monocyte-derived cells are the major cell populations expressing the apoptosis-inducing ligand TRAIL. Accordingly, anti-TRAIL treatment reduces bacterial load and protects against coinfection if administered during viral infection, but not following bacterial exposure. Post-influenza bacterial outgrowth induces a strong proinflammatory cytokine response and massive inflammatory cell infiltrate. Depletion of neutrophils or blockade of TNF-alpha facilitate bacterial outgrowth, leading to increased mortality, demonstrating that these factors aid bacterial control. We conclude that inflammatory monocytes recruited early, during the viral phase of coinfection, induce TRAIL-mediated lung damage, which facilitates bacterial invasion, while TNF-alpha and neutrophil responses help control subsequent bacterial outgrowth. We thus identify novel determinants of protection versus pathology in influenza-Streptococcus pneumoniae coinfection.
Walsh, K. B., et al. (2014). "Animal model of respiratory syncytial virus: CD8+ T cells cause a cytokine storm that is chemically tractable by sphingosine-1-phosphate 1 receptor agonist therapy" J Virol 88(11): 6281-6293. PubMed
The cytokine storm is an intensified, dysregulated, tissue-injurious inflammatory response driven by cytokine and immune cell components. The cytokine storm during influenza virus infection, whereby the amplified innate immune response is primarily responsible for pulmonary damage, has been well characterized. Now we describe a novel event where virus-specific T cells induce a cytokine storm. The paramyxovirus pneumonia virus of mice (PVM) is a model of human respiratory syncytial virus (hRSV). Unexpectedly, when C57BL/6 mice were infected with PVM, the innate inflammatory response was undetectable until day 5 postinfection, at which time CD8(+) T cells infiltrated into the lung, initiating a cytokine storm by their production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Administration of an immunomodulatory sphingosine-1-phosphate (S1P) receptor 1 (S1P1R) agonist significantly inhibited PVM-elicited cytokine storm by blunting the PVM-specific CD8(+) T cell response, resulting in diminished pulmonary disease and enhanced survival. IMPORTANCE: A dysregulated overly exuberant immune response, termed a ācytokine storm,ā accompanies virus-induced acute respiratory diseases (VARV), is primarily responsible for the accompanying high morbidity and mortality, and can be controlled therapeutically in influenza virus infection of mice and ferrets by administration of sphingosine-1-phosphate 1 receptor (S1P1R) agonists. Here, two novel findings are recorded. First, in contrast to influenza infection, where the cytokine storm is initiated early by the innate immune system, for pneumonia virus of mice (PVM), a model of RSV, the cytokine storm is initiated late in infection by the adaptive immune response: specifically, by virus-specific CD8 T cells via their release of IFN-gamma and TNF-alpha. Blockading these cytokines with neutralizing antibodies blunts the cytokine storm and protects the host. Second, PVM infection is controlled by administration of an S1P1R agonist.
Mittal, D., et al. (2014). "Antimetastatic effects of blocking PD-1 and the adenosine A2A receptor" Cancer Res 74(14): 3652-3658. PubMed
Adenosine targeting is an attractive new approach to cancer treatment, but no clinical study has yet examined adenosine inhibition in oncology despite the safe clinical profile of adenosine A2A receptor inhibitors (A2ARi) in Parkinson disease. Metastasis is the main cause of cancer-related deaths worldwide, and therefore we have studied experimental and spontaneous mouse models of melanoma and breast cancer metastasis to demonstrate the efficacy and mechanism of a combination of A2ARi in combination with anti-PD-1 monoclonal antibody (mAb). This combination significantly reduces metastatic burden and prolongs the life of mice compared with either monotherapy alone. Importantly, the combination was only effective when the tumor expressed high levels of CD73, suggesting a tumor biomarker that at a minimum could be used to stratify patients that might receive this combination. The mechanism of the combination therapy was critically dependent on NK cells and IFNgamma, and to a lesser extent, CD8(+) T cells and the effector molecule, perforin. Overall, these results provide a strong rationale to use A2ARi with anti-PD-1 mAb for the treatment of minimal residual and metastatic disease.
Xiao, N., et al. (2014). "The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells" Nat Immunol 15(7): 657-666. PubMed
Follicular helper T cells (T(FH) cells) are responsible for effective B cell-mediated immunity, and Bcl-6 is a central factor for the differentiation of T(FH) cells. However, the molecular mechanisms that regulate the induction of T(FH) cells remain unclear. Here we found that the E3 ubiquitin ligase Itch was essential for the differentiation of T(FH) cells, germinal center responses and immunoglobulin G (IgG) responses to acute viral infection. Itch acted intrinsically in CD4(+) T cells at early stages of T(FH) cell development. Itch seemed to act upstream of Bcl-6 expression, as Bcl-6 expression was substantially impaired in Itch(-/-) cells, and the differentiation of Itch(-/-) T cells into T(FH) cells was restored by enforced expression of Bcl-6. Itch associated with the transcription factor Foxo1 and promoted its ubiquitination and degradation. The defective T(FH) differentiation of Itch(-/-) T cells was rectified by deletion of Foxo1. Thus, our results indicate that Itch acts as an essential positive regulator in the differentiation of T(FH) cells.
Simons, D. M., et al. (2013). "Autoreactive Th1 cells activate monocytes to support regional Th17 responses in inflammatory arthritis" J Immunol 190(7): 3134-3141. PubMed
We have examined mechanisms underlying the formation of pathologic Th17 cells using a transgenic mouse model in which autoreactive CD4(+) T cells recognize influenza virus hemagglutinin (HA) as a ubiquitously expressed self-Ag and induce inflammatory arthritis. The lymph nodes of arthritic mice contain elevated numbers of inflammatory monocytes (iMO) with an enhanced capacity to promote CD4(+) Th17 cell differentiation, and a regional inflammatory response develops in the paw-draining lymph nodes by an IL-17-dependent mechanism. The activation of these Th17-trophic iMO precedes arthritis development and occurs in the context of an autoreactive CD4(+) Th1 cell response. Adoptive transfer of HA-specific CD4(+) T cells into nonarthritic mice expressing HA as a self-Ag similarly led to the formation of Th1 cells and of iMO that could support Th17 cell formation, and, notably, the accumulation of these iMO in the lymph nodes was blocked by IFN-gamma neutralization. These studies show that autoreactive CD4(+) Th1 cells directed to a systemically distributed self-Ag can promote the development of a regional Th17 cell inflammatory response by driving the recruitment of Th17-trophic iMO to the lymph nodes.
Bamboat, Z. M., et al. (2010). "Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion" J Clin Invest 120(2): 559-569. PubMed
TLRs are recognized as promoters of tissue damage, even in the absence of pathogens. TLR binding to damage-associated molecular patterns (DAMPs) released by injured host cells unleashes an inflammatory cascade that amplifies tissue destruction. However, whether TLRs possess the reciprocal ability to curtail the extent of sterile inflammation is uncertain. Here, we investigated this possibility in mice by studying the role of conventional DCs (cDCs) in liver ischemia/reperfusion (I/R) injury, a model of sterile inflammation. Targeted depletion of mouse cDCs increased liver injury after I/R, as assessed by serum alanine aminotransferase and histologic analysis. In vitro, we identified hepatocyte DNA as an endogenous ligand to TLR9 that promoted cDCs to secrete IL-10. In vivo, cDC production of IL-10 required TLR9 and reduced liver injury. In addition, we found that inflammatory monocytes recruited to the liver via chemokine receptor 2 were downstream targets of cDC IL-10. IL-10 from cDCs reduced production of TNF, IL-6, and ROS by inflammatory monocytes. Our results implicate inflammatory monocytes as mediators of liver I/R injury and reveal that cDCs respond to DAMPS during sterile inflammation, providing the host with protection from progressive tissue damage.
Bamboat, Z. M., et al. (2010). "Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury" Hepatology 51(2): 621-632. PubMed
Endogenous ligands such as high-mobility group box 1 (HMGB1) and nucleic acids are released by dying cells and bind Toll-like receptors (TLRs). Because TLR9 sits at the interface of microbial and sterile inflammation by detecting both bacterial and endogenous DNA, we investigated its role in a model of segmental liver ischemia-reperfusion (I/R) injury. Mice were subjected to 1 hour of ischemia and 12 hours of reperfusion before assessment of liver injury, cytokines, and reactive oxygen species (ROS). Wild-type (WT) mice treated with an inhibitory cytosine-guanosine dinucleotide (iCpG) sequence and TLR9(-/-) mice had markedly reduced serum alanine aminotransferase (ALT) and inflammatory cytokines after liver I/R. Liver damage was mediated by bone marrow-derived cells because WT mice transplanted with TLR9(-/-) bone marrow were protected from hepatic I/R injury. Injury in WT mice partly depended on TLR9 signaling in neutrophils, which enhanced production of ROS, interleukin-6 (IL-6), and tumor necrosis factor (TNF). In vitro, DNA released from necrotic hepatocytes increased liver nonparenchymal cell (NPC) and neutrophil cytokine secretion through a TLR9-dependent mechanism. Inhibition of both TLR9 and HMGB1 caused maximal inflammatory cytokine suppression in neutrophil cultures and conferred even greater protection from I/R injury in vivo. CONCLUSION: TLR9 serves as an endogenous sensor of tissue necrosis that exacerbates the innate immune response during liver I/R. Combined blockade of TLR9 and HMGB1 represents a clinically relevant, novel approach to limiting I/R injury.
AhR Activation Transcriptionally Induces Anti-Microbial Peptide Alpha-Defensin 1 Leading to Reversal of Gut Microbiota Dysbiosis and Colitis.
In Gut Microbes on 1 December 2025 by Palrasu, M., Kakar, K., et al.
Alpha-defensin 1 is a small antimicrobial peptide that acts as the first line of defense against pathogens. It is induced following microbial cues and inflammatory signals in neutrophils and Paneth cells in the small intestine, which suggests that it plays a role in microbial homeostasis in the gut. The gut microbial products also serve as ligands for the aryl hydrocarbon receptor (AhR), an environmental sensor. In the current study, we investigated if there is any crosstalk between AhR and alpha-defensin 1. Interestingly, we found a positive correlation between AhR and alpha-defensin 1 protein levels in ileal tissues from active Crohn's' (CD) patients and epithelial cells (IECs) from multiple models of murine colitis. In vitro downregulation of AhR led to inhibition of Ī±-defensin 1, while activation of AhR induced Ī±-defensin 1 in IECs. AhR directly targeted the dioxin response element 3 (DRE3) region on the Ī±-defensin 1 promoter in IECs. AhR-mediated induction of Ī±-defensin 1 in colitis mice reversed the gut microbial dysbiosis and alleviated colitis. Our data identify a novel signaling pathway in which AhR acts as a transcription factor for Ī±-defensin 1, leading to regulation of homeostasis between gut microbiota, intestinal mucosa, and mucosal immunity.
- Biochemistry and Molecular biology,
- Cancer Research,
- Cell Biology,
- Immunology and Microbiology
Targeting PERP promotes anti-tumor immunity in HNSCC by regulating tumor immune microenvironment and metabolic homeostasis.
In Molecular Cancer on 7 June 2025 by Wang, X., Tian, Y., et al.
PERP may have the potential to function as an oncogene. However, the precise function, prognostic value, and predictive significance remain shrouded in ambiguity. We conducted an in-depth analysis using pan-cancer RNA sequencing data and various online web tools to investigate the correlation between PERP and crucial clinical outcomes such as prognosis, tumor microenvironment, and tumor metabolism. In addition, we explored the tumor-promoting role of PERP and its potential mechanisms through models such as immunofluorescence staining, flow cytometry, cell proliferation assays, wound healing assays, cell migration assays, mass spectrometry analysis and isotope tracing. Further in vivo models confirmed the functional consistency of PERP across pan-cancer. Finally, we analyzed the potential of PERP as a predictive factor for immunotherapy sensitivity in a clinical cohort. PERP exhibits elevated expression in the majority of cancer types and impedes immune cell infiltration as well as immune checkpoint reactivity in pan-cancer. We confirmed that PERP can promote tumor progression by tumor cell proliferation, scratch and transwell experiments. Meanwhile, the absence of PERP restricts the flux of 13C6-glucose into glycolysis and the tricarboxylic acid (TCA) cycle. Importantly, the deficiency of PERP enhances the in vivo anti-tumor efficacy of PD1 monoclonal antibodies. In addition, low PERP expression is highly correlated with the response of head and neck squamous cell carcinoma (HNSCC) patients to immunotherapy. PERP represents a promising predictive/diagnostic biomarker and therapeutic target for HNSCC patients. Ā© 2025. The Author(s).
- Cancer Research,
- Immunology and Microbiology
Glutamate transporter SLC1A6 promotes resistance to immunotherapy in cancer.
In Cancer Immunology, Immunotherapy : CII on 7 June 2025 by Li, C., Lin, Y., et al.
Resistance to immune checkpoint inhibitors remains a significant challenge in the treatment of cancer. Emerging evidence suggests that metabolic reprogramming plays a crucial role in tumor metabolism and progression. Our study strived to investigate the role and underlying mechanisms of the glutamate transporter SLC1A6 in resistance to immunotherapy of cancer. Single-cell RNA sequencing was performed on bladder cancer patients receiving neoadjuvant immunotherapy to identify the expression of SLC1A6 in treatment-resistant cases. The clinical prognostic value of SLC1A6 in cancer was validated using publicly available lung cancer single-cell datasets, as well as transcriptomic data from both bladder and lung cancer cohorts. Flow cytometry was employed to assess the impact of SLC1A6 knockdown on the effector function of CD8āŗ T cell. In vivo tumor models were used to evaluate the role of SLC1A6 in immunotherapy resistance, with immunofluorescence staining performed to examine GZMBāŗ CD8āŗ T cell infiltration. SLC1A6 was highly expressed in bladder cancer patients resistant to neoadjuvant immunotherapy, and its expression was associated with disease progression, poor prognosis, and low immune infiltration. Knockdown of SLC1A6 in tumor cells enhanced CD8āŗ T cell effector function. SLC1A6 knockdown also improved the efficacy of immunotherapy and increased the infiltration of GZMBāŗ CD8āŗ T cells within the tumor microenvironment. SLC1A6 plays a critical role in resistance to immunotherapy in cancer. Targeting SLC1A6 may provide a promising therapeutic strategy for improving responses to neoadjuvant immunotherapy and advancing combination treatment approaches. Ā© 2025. The Author(s).
- Immunology and Microbiology
Characterisation of an autochthonous mouse ccRCC model of immune checkpoint inhibitor therapy resistance.
In Scientific Reports on 5 June 2025 by Peighambari, A., Huang, H., et al.
Many metastatic clear cell renal cell carcinomas (ccRCC) are resistant to immune checkpoint inhibitor therapies, however the mechanisms underlying sensitivity or resistance remain incompletely characterised. We demonstrate that ccRCCs in the Vhl/Trp53/Rb1 mutant mouse model are resistant to combined anti-PD-1/anti-CTLA-4 therapy alone and in combination with additional therapeutic agents that reflect current ccRCC clinical trials. However, in some animals in vivo checkpoint therapy allowed isolated splenic T cells to recognise cultured ccRCC cells from the same animal, implicating the tumour microenvironment in suppression of T cell activation. We identified putative immunosuppressive myeloid cell populations with features similar to myeloid cells in the microenvironment of human ccRCC. The expression patterns of immune checkpoint ligands in both the mouse model and in human ccRCC suggests that several checkpoint systems other than PD-1 and CTLA-4 are likely to represent the dominant T cell suppressive forces in ccRCC. Our findings characterise an autochthonous mouse ccRCC model of immune checkpoint inhibitor therapy resistance and pave the way for a systematic functional dissection of the identified potential molecular barriers to effective immune therapy of ccRCC. Ā© 2025. The Author(s).
- Cancer Research,
- Immunology and Microbiology
B and T lymphocyte attenuator (BTLA) and PD-1 pathway dual blockade promotes antitumor immune responses by reversing CD8+ T-cell exhaustion in non-small cell lung cancer.
In Frontiers in Immunology on 4 June 2025 by Zhang, Y., Yang, Y., et al.
Immunotherapies targeting the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) have shown great promise for a subset of patients with non-small cell lung cancer (NSCLC). However, safe and robust combination therapies are still needed to bring the benefit to broader patient populations. we performed in vivo treatment with PD-L1 antibody in Lewis lung carcinoma (LLC)-derived murine NSCLC model. Expression of B and T lymphocyte attenuator (BTLA) was detected during treatment. We evaluated the effects of the combination of anti-BTLA and anti-PD-L1 mAbs on tumor growth and overall survival of mice. In addition, distribution and function of immune cells were analyzed by flow cytometry. The role of BTLA in human and murine CD8+ T cells and its impact on reversing exhausted phenotype of PD-1+CD8+ T cell by PD-L1 blockade were analyzed. Furthermore, we investigated expression and distribution of BTLA on lymphocytes in tumor microenvironment of different specimens from NSCLC patients. There was no significant difference overall survival between anti-PD-L1 therapy and IgG in LLC-bearing mice, and BTLA expression was increased on CD8+ T cells after PD-L1 antibody treatment. LLC-bearing mice treated with combination of anti-BTLA and anti-PD-L1 therapy had an improved overall survival than anti-BTLA or anti-PD-L1 alone. Compared to monotherapy with anti-BTLA or anti-PD-L1, mice treated with combination therapy demonstrated increased infiltration of CD8+ and CD4+ T cells, as well as increased expression of IFN-Ī³, TNF-Ī± and Ki-67 in CD8+ T cells. In addition, CD8+ T cells co-expressing BTLA and PD-1 exhibited the most exhausted phenotype to resist PD-L1 blockade therapy. Furthermore, BTLA+CD8+ T cells were abnormally increased in different specimens from NSCLC patients, and CD8+ T cells expressing BTLA in NSCLC microenvironment were correlated with clinical response to anti-PD-1 therapy in NSCLC patients. Our results show that BTLA and PD-1 cooperatively inhibit the activity of CD8+ T cells and are associated with resistance to PD-1/PD-L1 pathway blockade in NSCLC patients. Anti-BTLA blockade enhances the antitumor efficacy of anti-PD-L1 therapy. Dual BTLA and PD-1/PD-L1 blockade should be further explored to elicit potent antitumor CD8+ T-cell responses in NSCLC patients. Copyright Ā© 2025 Zhang, Yang, Zeng, Qu, Shen, Mu, Lei, Su, Mao, Gao, Liu, Chen and Huang.
- Cancer Research,
- Cell Biology
Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs.
In The Journal of Clinical Investigation on 2 June 2025 by Liao, Q., Zhou, X., et al.
Colorectal cancer (CRC) is characterized by an immune-suppressive microenvironment that contributes to tumor progression and immunotherapy resistance. The gut microbiome produces diverse metabolites that feature unique mechanisms of interaction with host targets, yet the role of many metabolites in CRC remains poorly understood. In this study, the microbial metabolite 4-hydroxybenzeneacetic acid (4-HPA) promoted the infiltration of PMN myeloid-derived suppressor cells (PMN-MDSCs) in the tumor microenvironment, consequently inhibiting the antitumor response of CD8+ T cells and promoting CRC progression in vivo. Mechanistically, 4-HPA activates the JAK2/STAT3 pathway, which upregulates CXCL3 transcription, thereby recruiting PMN-MDSCs to the CRC microenvironment. Selective knockdown of CXCL3 resensitized tumors to anti-PD-1 immunotherapy in vivo. Chlorogenic acid reduces the production of 4-HPA by microbiota, likewise abolishing 4-HPA-mediated immunosuppression. The 4-HPA content in CRC tissues was notably increased in patients with advanced CRC. Overall, the gut microbiome uses 4-HPA as a messenger to control chemokine-dependent accumulation of PMN-MDSC cells and regulate antitumor immunity in CRC. Our findings provide a scientific basis for establishing clinical intervention strategies to reverse the tumor immune microenvironment and improve the efficacy of immunotherapy by reducing the interaction among intestinal microbiota, tumor cells, and tumor immune cells.
- Cancer Research,
- Immunology and Microbiology
Precision targeting of Ī²-catenin induces tumor reprogramming and immunity in hepatocellular cancers.
In Nature Communications on 30 May 2025 by Lehrich, B. M., Delgado, E. R., et al.
First-line immune checkpoint inhibitor (ICI) combinations show responses in subsets of hepatocellular carcinoma (HCC) patients. Nearly half of HCCs are Wnt-active with mutations in CTNNB1 (encoding for Ī²-catenin), AXIN1/2, or APC, and demonstrate heterogeneous and limited benefit to ICI due to an immune excluded tumor microenvironment. We show significant tumor responses in multiple Ī²-catenin-mutated immunocompetent HCC models to a novel siRNA encapsulated in lipid nanoparticle targeting CTNNB1 (LNP-CTNNB1). Both single-cell and spatial transcriptomics reveal cellular and zonal reprogramming, along with activation of immune regulatory transcription factors IRF2 and POU2F1, re-engaged type I/II interferon signaling, and alterations in both innate and adaptive immunity upon Ī²-catenin suppression with LNP-CTNNB1 at early- and advanced-stage disease. Moreover, ICI enhances response to LNP-CTNNB1 in advanced-stage disease by preventing T cell exhaustion and through formation of lymphoid aggregates (LA). In fact, expression of an LA-like gene signature prognosticates survival for patients receiving atezolizumab plus bevacizumab in the IMbrave150 phase III trial and inversely correlates with CTNNB1-mutatational status in this patient cohort. In conclusion, LNP-CTNNB1 is efficacious as monotherapy and in combination with ICI in CTNNB1-mutated HCCs through impacting tumor cell-intrinsic signaling and remodeling global immune surveillance, providing rationale for clinical investigations. Ā© 2025. The Author(s).
Integrin-mediated mTOR signaling drives TGF-Ī² overactivity and myxomatous mitral valve degeneration in hypomorphic fibrillin-1 mice.
In The Journal of Clinical Investigation on 20 May 2025 by Gao, F., Chen, Q., et al.
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-Ī² 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-Ī² overactivity and leukocytic infiltrates, while long-term inhibition of mTOR or TGF-Ī² 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-Ī² overactivity and myxomatous mitral valve degeneration, and mTOR inhibition may prevent disease progression of mitral valve prolapse.
- Cancer Research
NECTIN4 regulates the cell surface expression of CD155 in non-small cell lung cancer cells and induces tumor resistance to PD-1 inhibitors.
In Cancer Immunology, Immunotherapy : CII on 20 May 2025 by Mizusaki, S., Yoneshima, Y., et al.
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. Ā© 2025. The Author(s).
- Mus musculus (House mouse),
- Biochemistry and Molecular biology,
- Cell Biology,
- Genetics
Histone methyltransferase ASH1L primes metastases and metabolic reprogramming of macrophages in the bone niche.
In Nature Communications on 20 May 2025 by Meng, C., Lin, K., et al.
Bone metastasis is a major cause of cancer death; however, the epigenetic determinants driving this process remain elusive. Here, we report that histone methyltransferase ASH1L is genetically amplified and is required for bone metastasis in men with prostate cancer. ASH1L rewires histone methylations and cooperates with HIF-1Ī± to induce pro-metastatic transcriptome in invading cancer cells, resulting in monocyte differentiation into lipid-associated macrophage (LA-TAM) and enhancing their pro-tumoral phenotype in the metastatic bone niche. We identified IGF-2 as a direct target of ASH1L/HIF-1Ī± and mediates LA-TAMs' differentiation and phenotypic changes by reprogramming oxidative phosphorylation. Pharmacologic inhibition of the ASH1L-HIF-1Ī±-macrophages axis elicits robust anti-metastasis responses in preclinical models. Our study demonstrates epigenetic alterations in cancer cells reprogram metabolism and features of myeloid components, facilitating metastatic outgrowth. It establishes ASH1L as an epigenetic driver priming metastasis and macrophage plasticity in the bone niche, providing a bona fide therapeutic target in metastatic malignancies. Ā© 2025. The Author(s).
- In Vivo,
- Mus musculus (House mouse),
- Cancer Research
Identification of PD-L1-related biomarkers for selecting gastric adenocarcinoma patients for PD-1/PD-L1 inhibitor therapy.
In Discov Oncol on 8 May 2025 by Li, B. Y., Li, H. L., et al.
PD-1/PD-L1 inhibitors have been used to treat gastric cancer, and PD-L1 expression has been identified as a biomarker for predicting the effectiveness of immunotherapy in the treatment of gastric cancer. However, PD-L1 expression prediction for immunotherapy response is inaccurate, and improved response biomarkers are required. Thus, it is important to identify additional biomarkers that can predict the responses to PD-1/PD-L1 monoclonal antibodies in gastric cancer. In this study, GO and KEGG enrichment analysis of 142 DEGs co-expressed with PD-L1 were performed, and 41 genes were identified based on the intersection of the mRNA-significant GO term network and the mRNA-significant signalling pathway network. Further intersection analysis of the 41 candidate genes and 137 positive immunotherapy response genes indicated that BATF2 significantly affects the overall survival of GC patients. The transcription factor prediction for BATF2 identified additional potential predictors and therapeutic targets for GC. STAT and IRF family members were predicted to be transcription factors for BATF2. In addition, BATF2 knockdown significantly promoted GC cell growth, and PD-L1 expression was upregulated in si-BATF2-treated MKN-45 cells. Thus, BATF2 may serve as a biomarker for predicting the efficacy of PD-L1 blockade therapy in GC. BATF2 acts as a tumour suppressor gene during the development of GC. BATF2 is closely related to PD-L1 expression in GC, and high BATF2 expression positively correlates with low PD-L1 expression. BATF2 can be used as a potential biomarker and therapeutic target for responding to anti-PD-1 and anti-PD-L1 immunotherapies in GC. Ā© 2025. The Author(s).
- Cancer Research
Gene Therapy with Enterovirus 3āC Protease: A Promising Strategy for Various Solid Tumors.
In Nature Communications on 8 May 2025 by Yang, X., Li, W., et al.
Current cancer gene therapies rely primarily on antitumor immunity, but the exploration of alternative mRNA cargoes for direct antitumor effects is crucial to expand cancer gene therapies. Here we show that lipid nanoparticles (LNPs) carrying mRNA encoding a viral 3āC protease can efficiently suppress tumors by selectively inducing tumor cell apoptosis. In various solid tumor models, intracranial injection of LNPs carrying mRNA encoding the 3āC protease (3C-LNPs) significantly inhibits tumor growth and prolongs survival in glioblastoma models. Similarly, subcutaneous injection reduces tumor volume and inhibits angiogenesis in a breast cancer model, while intravenous injection inhibits tumor growth and angiogenesis and prolongs survival in hepatocellular carcinoma models. Mass spectrometry and cleavage site prediction assays identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as the main target degraded by the 3āC protease. This study suggests that viral protease mRNA could be a promising broad-spectrum antitumor therapeutic. Ā© 2025. The Author(s).
- Immunology and Microbiology
Colonic inflammation triggers Ī² cell proliferation during obesity development via a liver-to-pancreas interorgan mechanism.
In JCI Insight on 8 May 2025 by Kubo, H., Imai, J., et al.
Under insulin-resistant conditions, such as obesity, pancreatic Ī² cells adaptively proliferate and secrete more insulin to prevent blood glucose elevation. We previously reported hepatic ERK activation during obesity development to stimulate a neuronal relay system, consisting of afferent splanchnic nerves from the liver and efferent vagal nerves to the pancreas, thereby triggering adaptive Ī² cell proliferation. However, the mechanism linking obesity with the interorgan system originating in hepatic ERK activation remains unclear. Herein, we clarified that colonic inflammation promotes Ī² cell proliferation through this interorgan system from the liver to the pancreas. First, dextran sodium sulfate (DSS) treatment induced colonic inflammation and hepatic ERK activation as well as Ī² cell proliferation, all of which were suppressed by blockades of the neuronal relay system by several approaches. In addition, treatment with anti-lymphocyte Peyer's patch adhesion molecule-1 (anti-LPAM1) antibody suppressed Ī² cell proliferation induced by DSS treatment. Importantly, high-fat diet (HFD) feeding also elicited colonic inflammation, and its inhibition by anti-LPAM1 antibody administration suppressed hepatic ERK activation and Ī² cell proliferation induced by HFD. Thus, colonic inflammation triggers adaptive Ī² cell proliferation via the interorgan mechanism originating in hepatic ERK activation. The present study revealed a potentially novel role of the gastrointestinal tract in the maintenance of Ī² cell regulation.
- In Vivo,
- Mus musculus (House mouse),
- Cancer Research
Increased fatty acid delivery by tumor endothelium promotes metastatic outgrowth.
In JCI Insight on 8 May 2025 by Edwards, D. N., Wang, S., et al.
Metastatic outgrowth in distant microscopic niches requires sufficient nutrients, including fatty acids (FAs), to support tumor growth and to generate an immunosuppressive tumor microenvironment (TME). However, despite the important role of FAs in metastasis, the regulation of FA supply in metastatic niches has not been defined. In this report, we show that tumor endothelium actively promotes outgrowth and restricts antitumor cytolysis by transferring FAs into developing metastatic tumors. We describe a process of transendothelial FA delivery via endosomes that requires mTORC1 activity. Thus, endothelial cell-specific targeted deletion of Raptor (RptorECKO), a unique component of the mTORC1 complex, significantly reduced metastatic tumor burden that was associated with improved markers of T cell cytotoxicity. Low-dose everolimus that selectively inhibited endothelial mTORC1 improves immune checkpoint responses in metastatic disease models. This work reveals the importance of transendothelial nutrient delivery to the TME, highlighting a future target for therapeutic development.
- Cancer Research
Tumor suppressors in Sox2-mediated lung cancers promote distinct cell intrinsic and immunologic remodeling.
In JCI Insight on 6 May 2025 by Sengottuvel, N., Whately, K. M., et al.
Non-small cell lung cancer (NSCLC) largely consists of lung squamous (LUSC) and lung adenocarcinoma (LUAD). Alterations in the TRP53 and PTEN tumor suppressors are common in both subtypes, but their relationship with SOX2 is poorly understood. We deleted Trp53 or Pten in a C57BL/6J-Sox2hi;Nkx2-1-/-;Lkb1-/- (SNL) genetic background and generated a highly metastatic LUSC cell line (LN2A; derived from a Sox2hi mouse model, followed by Trp53, Pten, and Cdkn2a deletion). Histologic and single-cell RNAseq analyses corroborated that SNL mice developed mixed tumors with both LUAD and LUSC histopathology while SNL-Trp53 and SNL-Pten mice developed LUAD and LN2A tumors retained LUSC morphology. Compared with SNL mice, additional loss of Trp53 or Pten resulted in significantly reduced survival, increased tumor burden and altered tumor mucin composition. We identified a sub-cluster of CD38+ tumor-associated inflammatory monocytes in the LN2A model that significantly enriched for activation of the classical and alternative complement pathways. Complement Factor B (CFB) is associated with poor survival in LUSC patients, and we observed the LN2A model had significantly improved survival on a Cfb-/- background. Our findings demonstrate a cooperative role of Trp53 and Pten tumor suppressors in Sox2-mediated NSCLC tumor progression, mucin production, and remodeling of the immune tumor microenvironment.
- In Vivo,
- Mus musculus (House mouse),
- Immunology and Microbiology
Caspase-1-licensed pyroptosis drives dsRNA-mediated necroptosis and dampens host defense against bacterial pneumonia.
In PLoS Pathogens on 1 May 2025 by Luo, Q., Shen, L., et al.
Bacterial lung infections cause severe host responses. Here, we showed that global deficiency of caspase-1 can protect against lethal pulmonary Escherichia coli infection by reducing the necroptosis of infiltrated neutrophils, which are key players in immune responses in the lung. Mechanistically, neutrophil necroptosis was not directly triggered in a cell-intrinsic manner by invading bacteria but was triggered by bacteria-stimulated pyroptotic epithelial cell supernatants in vitro. In validation experiments, chimeric mice with nonhematopoietic caspase-1 or GSDMD knockout were protected from lung E. coli infection and exhibited decreased neutrophil death. Nonhematopoietic pyroptosis facilitates the release of dsRNAs and contributes to neutrophil ZBP1-related necroptosis. Moreover, blocking dsRNA or depleting ZBP1 ameliorated the pathophysiological process of pulmonary E. coli infection. Overall, our results demonstrate a paradigm of communication between necroptosis and pyroptosis in different cell types in cooperation with microbes and hosts and suggest that therapeutic targeting of the pyroptosis or necroptosis pathway may prevent pulmonary bacterial infection. Copyright: Ā© 2025 Luo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
- Immunology and Microbiology
Interleukin-35 inhibits NETs to ameliorate Th17/Treg immune imbalance during the exacerbation of cigarette smoke exposed-asthma via gp130/STAT3/ferroptosis axis.
In Redox Biology on 1 May 2025 by Tao, P., Su, B., et al.
Cigarette smoke (CS) exposure amplifies neutrophil accumulation. IL-35, a novel cytokine with anti-inflammatory properties, is involved in protection against asthma. However, the biological roles of neutrophils and the precise molecular mechanisms of IL-35 in CS exposed-asthma remain unclear. We showed that the exacerbation of CS exposed-asthma leads to dramatically increased neutrophil counts and an imbalance in DC-Th17/Treg immune responses. RNA sequencing revealed that NETs, part of a key biological process in neutrophils, were significantly upregulated in the context of CS exposed-asthma exacerbation and that IL-35 treatment downregulated NET-associated gene expression. Targeted degradation of NETs, rather than neutrophil depletion, alleviated the CS exposed-asthma. Mechanistically, STAT3 phosphorylation promoted ferroptosis, exacerbating NET release, which in turn enhanced dendritic cell (DC) antigen presentation, activated T cells, and specifically promoted Th17Ā cell differentiation while inhibiting Treg cells. IL-35 acting on the gp130 receptor alleviated STAT3-mediated ferroptosis-associated NET formation. In summary, our study revealed a novel mechanism by which IL-35 inhibited NET formation, subsequently alleviating neutrophilic inflammation and restoring the DC-Th17/Treg imbalance in CS exposed-asthma, highlighting the potential of IL-35 as a targeted therapeutic strategy. Copyright Ā© 2025 The Authors. Published by Elsevier B.V. All rights reserved.
- Cancer Research,
- Immunology and Microbiology
Development of PVTX-405 as a potent and highly selective molecular glue degrader of IKZF2 for cancer immunotherapy.
In Nature Communications on 1 May 2025 by Chen, Z., Dhruv, H., et al.
IKZF2 (Helios) is a transcription factor that is selectively expressed by Tregs and is essential for preserving the function and stability of Tregs in the tumor microenvironment (TME), where it suppresses the anti-tumor immune response. Targeted IKZF2 degradation by small molecules represents a promising strategy for the development of a new class of cancer immunotherapy. Herein, we describe the discovery of PVTX-405, a potent, effective, highly selective, and orally efficacious IKZF2 molecular glue degrader. PVTX-405 degrades IKZF2 (DC50ā=ā0.7ānM and Dmaxā=ā91%) while sparing other CRBN neo-substrates. Degradation of IKZF2 by PVTX-405 increases production of inflammatory cytokine IL-2 and reduces the suppressive activity of Tregs, leading to an increase in Teff cell proliferation. Once-daily oral administration of PVTX-405 as single agent significantly delays the growth of MC38 tumors in a syngeneic tumor model using humanized CRBN mice. PVTX-405 in combination with anti-PD1 or anti-LAG3 significantly increases animal survival compared to anti-PD1 or anti-LAG3 alone. Together, these results demonstrate that PVTX-405 is a promising IKZF2 degrader for clinical development for the treatment of human cancers. Ā© 2025. The Author(s).
- In Vivo,
- Mus musculus (House mouse),
- Cancer Research
Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils.
In Nature Communications on 28 April 2025 by Wang, X., Cheng, M., et al.
Relatlimab and nivolumab combination therapy shows significant efficacy in treating various types of cancer. Current research on the molecular mechanisms of this treatment is abundant, but in-depth investigations into post-treatment resistance remain notably lacking. In this study, we identify significant enrichment of SRY (sex determining region Y)-box 9 (Sox9)+ tumor cells in resistant samples using single cell RNA sequencing (scRNAseq) in a head and neck squamous cell carcinoma (HNSCC) mouse model. In addition, Sox9 directly regulates the expression of annexin A1 (Anxa1), mediating apoptosis of formyl peptide receptor 1 (Fpr1)+ neutrophils through the Anxa1-Fpr1 axis, which promotes mitochondrial fission, inhibits mitophagy by downregulating BCL2/adenovirus E1B interacting protein 3 (Bnip3) expression and ultimately prevents the accumulation of neutrophils in tumor tissues. The reduction of Fpr1+ neutrophils impairs the infiltration and tumor cell-killing ability of cytotoxic Cd8 T and Ī³Ī“T cells within the tumor microenvironment, thereby leading to the development of resistance to the combination therapy. We further validate these findings using various transgenic mouse models. Overall, this study comprehensively explains the mechanisms underlying resistance to the anti-LAG-3 plus anti-PD-1 combination therapy and identifies potential therapeutic targets to overcome this resistance. Ā© 2025. The Author(s).
- Cardiovascular biology
ApoM-bound S1P acts via endothelial S1PR1 to suppress choroidal neovascularization and vascular leakage.
In Angiogenesis on 23 April 2025 by Jung, B., Yagi, H., et al.
Neovascular age-related macular degeneration (nAMD) is a major cause of vision loss worldwide. Current standard of care is repetitive intraocular injections of vascular endothelial growth factor (VEGF) inhibitors, although responses may be partial and non-durable. We report that circulating sphingosine 1-phosphate (S1P) carried by apolipoprotein M (ApoM) acts through the endothelial S1P receptor 1 (S1PR1) to suppress choroidal neovascularization (CNV) in mouse laser-induced CNV, modeling nAMD. In humans, low plasma ApoM levels were associated with increased choroidal and retinal pathology. Additionally, endothelialĀ S1pr1Ā knockout and overexpressing transgenic mice showed increased and reduced CNV lesion size, respectively. Systemic administration of ApoM-Fc, an engineered S1P chaperone protein, not only attenuated CNV to an equivalent degree as anti-VEGF antibody treatment but also suppressed pathological vascular leakage. We suggest that modulating circulating ApoM-bound S1P action on endothelial S1PR1 provides a novel therapeutic strategy to treat nAMD. Ā© 2025. The Author(s).
