InVivoMAb anti-mouse MHC Class I (H-2Kd, H-2Dd)

Catalog #BE0180
Product Citations:
11
Clone:
34-1-2S
Reactivities:
Mouse

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Product Details

The 34-1-2S monoclonal antibody is reported to react with the mouse H-2Kb and H-2Dd MHC class I alloantigens. MHC class I antigens are heterodimers consisting of one alpha chain (44 kDa) associated with β2 microglobulin (11.5 kDa). The antigen is expressed by all nucleated cells at varying levels. MHC Class I molecules present endogenously synthesized antigenic peptides to CD8 T cells.

Specifications

Isotype Mouse IgG2a, κ
Recommended Isotype Control(s) InVivoMAb mouse IgG2a isotype control, unknown specificity
Recommended Dilution Buffer InVivoPure pH 7.0 Dilution Buffer
Conjugation This product is unconjugated. Conjugation is available via our Antibody Conjugation Services.
Immunogen BDF mouse spleen cells
Reported Applications in vivo activation of APCs
Formulation PBS, pH 7.0
Contains no stabilizers or preservatives
Endotoxin <2EU/mg (<0.002EU/μg)
Determined by LAL gel clotting assay
Purity >95%
Determined by SDS-PAGE
Sterility 0.2 µm filtration
Production Purified from cell culture supernatant in an animal-free facility
Purification Protein G
RRID AB_10950841
Molecular Weight 150 kDa
Storage The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.
in vivo activation of APCs
Cleary, S. J., et al. (2020). "Complement activation on endothelium initiates antibody-mediated acute lung injury" J Clin Invest 130(11): 5909-5923. PubMed

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

in vivo activation of APCs
Kapur, R., et al. (2015). "C-reactive protein (CRP) enhances murine antibody-mediated transfusion-related acute lung injury (TRALI)" Blood. pii : blood-2015-09-672592. PubMed

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress triggered by blood transfusions and is the leading cause of transfusion-related mortality. TRALI has primarily been attributed to passive infusion of human leucocyte antigen (HLA) and/or human neutrophil antigen (HNA) antibodies present in transfused blood products and predisposing factors such as inflammation are known to be important for TRALI-initiation. Since the acute phase protein C-reactive protein (CRP) is highly up-regulated during infections and inflammation and can also enhance antibody-mediated responses such as in vitro phagocytosis, respiratory burst and in vivo thrombocytopenia, we investigated whether CRP affects murine antibody-mediated TRALI induced by the anti-MHC antibody, 34-1-2s. We found that BALB/c mice administered with 34-1-2s or CRP alone were resistant to TRALI, but mice injected with 34-1-2s together with CRP had significantly enhanced lung damage and pulmonary edema. Mechanistically, 34-1-2s injection with CRP resulted in a significant synergistic increase in plasma levels of the neutrophil chemoattractant, macrophage inflammatory protein-2 (MIP-2) and pulmonary neutrophil accumulation. Importantly, murine MIP-2 is the functional homologue of human IL-8, a known risk factor for human TRALI. These results suggest that elevated in vivo CRP levels, like those observed during infections, may significantly predispose recipients to antibody-mediated TRALI reactions and support the notion that modulating CRP levels is an effective therapeutic strategy to reduce TRALI-severity.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors.

    In Nature Communications on 3 November 2023 by Chiaro, J., Antignani, G., et al.

    PubMed

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients' primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors. © 2023. The Author(s).

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    Neutrophil-activating therapy for the treatment of cancer.

    In Cancer Cell on 13 February 2023 by Linde, I. L., Prestwood, T. R., et al.

    PubMed

    Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer. Copyright © 2023 Elsevier Inc. All rights reserved.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    Development of mesothelioma-specific oncolytic vaccine exploiting immunopeptidomic analysis of murine and human tumors

    Preprint on Research Square on 8 November 2022 by Chiaro, J., Antignani, G., et al.

    PubMed

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel treatments and therapeutic targets are urgently needed. It has been observed that MPM is responsive to immunotherapeutic cancer treatments, and given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets to engage cytotoxic (CD8+) T cells. In this study, for the first time, we explored the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line), human cell lines (H28, MSTO-211H, H2452, and JL1), and in patients' primary tumors. Applying state-of-art MHC immuno-affinity purification methodologies, we identified MHC-I-restricted peptides presented on the surface of malignant cells. We characterized some newly discovered patients-derived peptides utilizing in vitro co-culturing techniques, demonstrating that eluted peptides showed promising immunogenicity profiles. Additionally, we provided a proof-of-concept for the application of a whole antigen discovery pipeline using immunopeptidomics for cancer vaccine development in a murine model of MPM. Overall, we investigated the antigen landscape of MPM and showed that the discovered peptides show potential to be used for therapeutic cancer vaccine.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Biochemistry and Molecular biology
    In Vivo Imaging of Circadian NET Formation During Lung Injury by Four-Dimensional Intravital Microscopy.

    In Methods in Molecular Biology (Clifton, N.J.) on 25 May 2022 by Aroca-Crevillén, A., Hidalgo, A., et al.

    PubMed

    Neutrophil extracellular traps (NETs) are toxic extracellular structures deployed by neutrophils in response to pathogens and sterile danger signals. NETs are circadian in nature as mouse and human neutrophils preferentially deploy them at night or early morning. Traditionally, NETs have been quantified using a plethora of methods including immunofluorescence and ELISA-based assays; however few options are available to visualize them in vivo. Here we describe a method to directly visualize and quantify NET formation and release in the microvasculature of the lung using intravital imaging in a model of acute lung injury. The method allows four-dimensional capture and quantification of NET formation dynamics over time and should be a useful resource for those interested in visualizing neutrophil responses in vivo. © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

    • Immu-puri
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines.

    In eLife on 22 March 2022 by Feola, S., Chiaro, J., et al.

    PubMed

    Besides the isolation and identification of major histocompatibility complex I-restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective antitumor CD8+ T-cell-mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-γ enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach. © 2022, Feola et al.

    • COVID-19
    • ,
    • Immunology and Microbiology
    Disulfiram inhibits neutrophil extracellular trap formation and protects rodents from acute lung injury and SARS-CoV-2 infection.

    In JCI Insight on 8 March 2022 by Adrover, J. M., Carrau, L., et al.

    PubMed

    Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram - an FDA-approved drug for alcohol use disorder - dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2-infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and it downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for patients with COVID-19. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in 2 rodent models of lung injury for which treatment options are limited.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Biochemistry and Molecular biology
    • ,
    • Cardiovascular biology
    Pulmonary coagulation and fibrinolysis abnormalities that favor fibrin deposition in the lungs of mouse antibody-mediated transfusion-related acute lung injury.

    In Molecular Medicine Reports on 1 August 2021 by Yu, Y., Jiang, P., et al.

    PubMed

    Transfusion‑related acute lung injury (TRALI) is a life‑threatening disease caused by blood transfusion. However, its pathogenesis is poorly understood and specific therapies are not available. Experimental and clinical studies have indicated that alveolar fibrin deposition serves a pathological role in acute lung injuries. The present study investigated whether pulmonary fibrin deposition occurs in a TRALI mouse model and the possible mechanisms underlying this deposition. The TRALI model was established by priming male Balb/c mice with lipopolysaccharide (LPS) 18 h prior to injection of an anti‑major histocompatibility complex class I (MHC‑I) antibody. Untreated mice and mice administered LPS plus isotype antibody served as controls. At 2 h after TRALI induction, blood and lung tissue were collected. Disease characteristics were assessed based on lung tissue histology, inflammatory responses and alterations in the alveolar‑capillary barrier. Immunofluorescence staining was used to detect pulmonary fibrin deposition, platelets and fibrin‑platelet interactions. Levels of plasminogen activator inhibitor‑1 (PAI‑1), thrombin‑antithrombin complex (TATc), tissue factor pathway inhibitor (TFPI), coagulation factor activity and fibrin degradation product (FDP) in lung tissue homogenates were measured. Severe lung injury, increased inflammatory responses and a damaged alveolar‑capillary barrier in the LPS‑primed, anti‑MHC‑I antibody‑administered mice indicated that the TRALI model was successfully established. Fibrin deposition, fibrin‑platelet interactions and platelets accumulation in the lungs of mouse models were clearly promoted. Additionally, levels of TATc, coagulation factor V (FV), TFPI and PAI‑1 were elevated, whereas FDP level was decreased in TRALI mice. In conclusion, both impaired fibrinolysis and enhanced coagulation, which might be induced by boosted FV activity, increased pulmonary platelets accumulation and enhanced fibrin‑platelet interactions and contributed to pulmonary fibrin deposition in TRALI mice. The results provided a therapeutic rationale to target abnormalities in either coagulation or fibrinolysis pathways for antibody‑mediated TRALI.

    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines

    Preprint on BioRxiv : the Preprint Server for Biology on 9 June 2021 by Feola, S., Chiaro, J., et al.

    PubMed

    h4>ABSTRACT/h4> Beside the isolation and identification of MHC-I restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective anti-tumor CD8+ T cell mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific anti-tumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and the HEX software. The latter is a tool previously developed by Chiaro et al. (1), able to identify tumor antigens similar to pathogen antigens, in order to exploit molecular mimicry and tumor pathogen cross-reactive T-cells in cancer vaccine development. The generated list of candidates (twenty-six in total) was further tested in a functional characterization assay using interferon-γ ELISpot (Enzyme-Linked Immunospot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, non-treated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    Complement activation on endothelium initiates antibody-mediated acute lung injury.

    In The Journal of Clinical Investigation on 2 November 2020 by Cleary, S. J., Kwaan, N., et al.

    PubMed

    Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    Programmed 'disarming' of the neutrophil proteome reduces the magnitude of inflammation.

    In Nature Immunology on 1 February 2020 by Adrover, J. M., Aroca-Crevillén, A., et al.

    PubMed

    The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    Treating murine inflammatory diseases with an anti-erythrocyte antibody.

    In Science Translational Medicine on 21 August 2019 by Crow, A. R., Kapur, R., et al.

    PubMed

    Treatment of autoimmune and inflammatory diseases typically involves immune suppression. In an opposite strategy, we show that administration of the highly inflammatory erythrocyte-specific antibody Ter119 into mice remodels the monocyte cellular landscape, leading to resolution of inflammatory disease. Ter119 with intact Fc function was unexpectedly therapeutic in the K/BxN serum transfer model of arthritis. Similarly, it rapidly reversed clinical disease progression in collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis and completely corrected CAIA-induced increase in monocyte Fcγ receptor II/III expression. Ter119 dose-dependently induced plasma chemokines CCL2, CCL5, CXCL9, CXCL10, and CCL11 with corresponding alterations in monocyte percentages in the blood and liver within 24 hours. Ter119 attenuated chemokine production from the synovial fluid and prevented the accumulation of inflammatory cells and complement components in the synovium. Ter119 could also accelerate the resolution of hypothermia and pulmonary edema in an acute lung injury model. We conclude that this inflammatory anti-erythrocyte antibody simultaneously triggers a highly efficient anti-inflammatory effect with broad therapeutic potential. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

    • FC/FACS
    • ,
    • Mus musculus (House mouse)
    Transfer of multiple loci of donor's genes to induce recipient tolerance in organ transplantation.

    In Experimental and Therapeutic Medicine on 1 June 2018 by Li, T., Zhang, W., et al.

    PubMed

    Donor organ rejection remains a significant problem. The present study aimed to assess whether transferring a donor's major histocompatibility complex (MHC) genes to the recipient could mitigate rejection in organ transplantation. Seven loci of MHC genes from donor mice were amplified and ligated into vectors; the vectors either contained one K locus, seven loci or were empty (control). The vectors were subsequently injected into the thymus of recipients (in heterotransplants, recipient rats received the vector containing one K locus), following which donor mouse hearts were transplanted. Following the transplantation of allograft and heterograft, electrocardiosignals were viable for a significantly longer duration in recipient mice and rats receiving the donor histocompatibility-2 complex (H-2)d genes compared with those in controls, and in mice that received seven vectors compared with those receiving one vector. Mixed lymphocyte cultures containing cells from these recipients proliferated significantly less compared with mixed lymphocyte cultures containing controls. Also, hearts from H-2d genes-treated recipients demonstrated less lymphocyte infiltration and necrosis compared with the control recipient. The present study concluded that allograft and heterograft rejection may be mitigated by introducing the donor's MHC into the recipient; transferring seven loci has been demonstrated to be more effective than transferring one locus.