InVivoMAb anti-mouse RANKL (CD254)
Product Details
The IK22/5 monoclonal antibody reacts with mouse RANKL (receptor activator of nuclear factor kappa-B ligand) also known as CD254 and TRANCE (TNF-related activation-induced cytokine). RANKL is a 35 kDa type II membrane protein that belongs to the TNF superfamily. RANKL is expressed on activated T lymphocytes in the lung, thymus, and lymph nodes and on osteoclasts. RANKL has been implicated in the regulation of T cell and dendritic cell interactions as well as osteoclast differentiation. Additionally, RANKL was found to be critical for osteoclast differentiation.Specifications
| Isotype | Rat IgG2a,Ā Īŗ |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb rat IgG2a isotype control, anti-trinitrophenol |
| Recommended Dilution Buffer | InVivoPure pH 6.5 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | NSO-derived recombinant mouse RANKL |
| Reported Applications | in vivo RANKL blockade |
| 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_10949003 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb rat IgG2a isotype control, anti-trinitrophenol
-
Recommended Dilution Buffer
InVivoPure pH 6.5 Dilution Buffer
in vivo RANKL blockade
Wang, W., et al. (2020). "The Interaction between Lymphoid Tissue Inducer-Like Cells and T Cells in the Mesenteric Lymph Node Restrains Intestinal Humoral Immunity" Cell Rep 32(3): 107936. PubMed
Lymphoid tissue inducer (LTi)/LTi-like cells are critical for lymphoid organogenesis and regulation of adaptive immunity in various tissues. However, the maintenance and regulation mechanisms of LTi-like cells among different tissues are not clear yet. Here, we find that LTi-like cells from different tissues display heterogeneity. The maintenance of LTi-like cells in the mesenteric lymph node (mLN), but not the gut, requires RANKL signaling from CD4(+) T cells. LTi-like cells from the mLN, but not the gut, could in turn inhibit the development of T follicular helper cells and subsequent humoral responses during intestinal immunization in an ID2- and PD-L1-dependent manner. Together, our findings implicate that the interaction between LTi-like cells and T cells in the mLN could precisely control the intestinal mucosal adaptive immune response.
in vivo RANKL blockade
Khan, I. S., et al. (2014). "Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance" J Exp Med 211(5): 761-768. PubMed
Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.
in vivo RANKL blockade
Metzger, T. C., et al. (2013). "Lineage tracing and cell ablation identify a post-Aire-expressing thymic epithelial cell population" Cell Rep 5(1): 166-179. PubMed
Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and major histocompatibility complex II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire(+) mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire(-) mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates toward the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.
in vivo RANKL blockade
Sherlock, J. P., et al. (2012). "IL-23 induces spondyloarthropathy by acting on ROR-gammat+ CD3+CD4-CD8- entheseal resident T cells" Nat Med 18(7): 1069-1076. PubMed
The spondyloarthropathies are a group of rheumatic diseases that are associated with inflammation at anatomically distal sites, particularly the tendon-bone attachments (entheses) and the aortic root. Serum concentrations of interleukin-23 (IL-23) are elevated and polymorphisms in the IL-23 receptor are associated with ankyosing spondylitis, however, it remains unclear whether IL-23 acts locally at the enthesis or distally on circulating cell populations. We show here that IL-23 is essential in enthesitis and acts on previously unidentified IL-23 receptor (IL-23R)(+), RAR-related orphan receptor gammat (ROR-gammat)(+)CD3(+)CD4(-)CD8(-), stem cell antigen 1 (Sca1)(+) entheseal resident T cells. These cells allow entheses to respond to IL-23 in vitro-in the absence of further cellular recruitmentāand to elaborate inflammatory mediators including IL-6, IL-17, IL-22 and chemokine (C-X-C motif) ligand 1 (CXCL1). Notably, the in vivo expression of IL-23 is sufficient to phenocopy the human disease, with the specific and characteristic development of enthesitis and entheseal new bone formation in the initial complete absence of synovitis. As in the human condition, inflammation also develops in vivo at the aortic root and valve, which are structurally similar to entheses. The presence of these entheseal resident cells and their production of IL-22, which activates signal transducer and activator of transcription 3 (STAT3)-dependent osteoblast-mediated bone remodeling, explains why dysregulation of IL-23 results in inflammation at this precise anatomical site.
RANKL inhibition reduces lesional cellularity, GĪ±s variant expression and enables osteogenic maturation in fibrous dysplasia
Preprint on Research Square on 30 May 2023 by de Castro, L., Whitlock, J., et al.
PubMed
Fibrous dysplasia (FD) is a rare, disabling skeletal disease with no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear Kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated mechanisms underlying RANKL neutralization with the monoclonal antibody denosumab on FD osteoprogenitors, by evaluating human FD tissue pre- and post-treatment, and in murine in vivo and ex vivo pre-clinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic GĪ± s variant in FD lesions after RANKL neutralization. RNA sequencing of human and mouse tissue supported these findings. Interplay between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, indicating that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclastogenesis. Results from this study demonstrate that, beyond its expected anti-osteoclastic effects, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased lesional bone formation. These findings highlight an unappreciated role for cellular crosstalk between osteoclasts and pre-osteoblast/osteoblasts as a driver of FD pathology, and demonstrate a novel mechanism of denosumab action in the treatment of bone disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT03571191
- In Vivo,
- Mus musculus (House mouse),
- Cancer Research
RANKL Blockade Reduces Cachexia and Bone Loss Induced by Non-Metastatic Ovarian Cancer in Mice.
In Journal of Bone and Mineral Research on 1 March 2022 by Pin, F., Jones, A. J., et al.
PubMed
Tumor- and bone-derived soluble factors have been proposed to participate in the alterations of skeletal muscle size and function in cachexia. We previously showed that mice bearing ovarian cancer (OvCa) exhibit cachexia associated with marked bone loss, whereas bone-targeting agents, such as bisphosphonates, are able to preserve muscle mass in animals exposed to anticancer drugs. De-identified CT images and plasma samples from female patients affected with OvCa were used for body composition assessment and quantification of circulating cross-linked C-telopeptide type I (CTX-I) and receptor activator of NF-kB ligand (RANKL), respectively. Female mice bearing ES-2 tumors were used to characterize cancer- and RANKL-associated effects on muscle and bone. Murine C2C12 and human HSMM myotube cultures were used to determine the OvCa- and RANKL-dependent effects on myofiber size. To the extent of isolating new regulators of bone and muscle in cachexia, here we demonstrate that subjects affected with OvCa display evidence of cachexia and increased bone turnover. Similarly, mice carrying OvCa present high RANKL levels. By using in vitro and in vivo experimental models, we found that elevated circulating RANKL is sufficient to cause skeletal muscle atrophy and bone resorption, whereas bone preservation by means of antiresorptive and anti-RANKL treatments concurrently benefit muscle mass and function in cancer cachexia. Altogether, our data contribute to identifying RANKL as a novel therapeutic target for the treatment of musculoskeletal complications associated with RANKL-expressing non-metastatic cancers. Ā© 2021 American Society for Bone and Mineral Research (ASBMR). Ā© 2021 American Society for Bone and Mineral Research (ASBMR).
Single-Cell Multiomics Defines Tolerogenic Extrathymic Aire-Expressing Populations with Unique Homology to Thymic Epithelium
Preprint on BioRxiv : the Preprint Server for Biology on 6 November 2021 by Wang, J., Lareau, C. A., et al.
PubMed
The Autoimmune Regulator ( Aire ) gene, well defined for its role in medullary thymic epithelial cells (mTECs) and immune self-tolerance, is also expressed in extrathymic Aire -expressing cells (eTACs) in the secondary lymphoid organs. eTACs have been shown to be hematopoietic antigen presenting cells (APCs) and potent inducers of immune tolerance (1ā3). However, the precise identity and function of these cells remain unclear. Here, we use high-dimensional single-cell multiomics and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and a unique Aire-hi population co-expressing Aire and RAR-related orphan receptor gamma-t (RORĪ³t). The latter, which have significant transcriptional and genomic homology to migratory dendritic cells (migDCs) and mTECs, we term Janus cells (JCs). All eTACs, and JCs in particular, have a highly accessible chromatin structure and high levels of broad gene expression, including tissue-specific antigens, as well as remarkable transcriptional and genomic homology to thymic medullary epithelium. As in the thymus, Aire expression in eTACs is also dependent on RANK-RANK-ligand interactions. Furthermore, lineage-tracing shows that JCs are not precursors to the majority of AmDCs. Finally, self-antigen expression by eTACs is sufficient to mediate negative selection of T cells escaping thymic selection and can prevent autoimmune diabetes in non-obese diabetic mice. This transcriptional, genomic, and functional symmetry between a hematopoietic Aire-expressing population in the periphery and an epithelial Aire-expressing population in the thymus suggests that a core biological program may influence self-tolerance and self-representation across the spectrum of immune development.
- Immunology and Microbiology
An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion.
In Bone Research on 1 November 2021 by Zhen, G., Dan, Y., et al.
PubMed
Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP+ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP+ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture. Ā© 2021. The Author(s).
- In Vivo,
- Block,
- Mus musculus (House mouse),
- Immunology and Microbiology
The Interaction between Lymphoid Tissue Inducer-Like Cells and T Cells in the Mesenteric Lymph Node Restrains Intestinal Humoral Immunity.
In Cell Reports on 21 July 2020 by Wang, W., Li, Y., et al.
PubMed
Lymphoid tissue inducer (LTi)/LTi-like cells are critical for lymphoid organogenesis and regulation of adaptive immunity in various tissues. However, the maintenance and regulation mechanisms of LTi-like cells among different tissues are not clear yet. Here, we find that LTi-like cells from different tissues display heterogeneity. The maintenance of LTi-like cells in the mesenteric lymph node (mLN), but not the gut, requires RANKL signaling from CD4+ TĀ cells. LTi-like cells from the mLN, but not the gut, could in turn inhibit the development of T follicular helper cells and subsequent humoral responses during intestinal immunization in an ID2- and PD-L1-dependent manner. Together, our findings implicate that the interaction between LTi-like cells and TĀ cells in the mLN could precisely control the intestinal mucosal adaptive immune response.Copyright Ā© 2020 The Author(s). Published by Elsevier Inc. All rights reserved.
- In Vivo,
- Immu-depl,
- Mus musculus (House mouse),
- Neuroscience
Gut-Innervating Nociceptor Neurons Regulate Peyer's Patch Microfold Cells and SFB Levels to Mediate Salmonella Host Defense.
In Cell on 9 January 2020 by Lai, N. Y., Musser, M. A., et al.
PubMed
Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.Copyright Ā© 2019 Elsevier Inc. All rights reserved.
