About InVivoMAb anti-mouse CXCR6 (CD186) The Cx6Mab-1 monoclonal antibody reacts with the N-terminal extracellular ligand-binding domain of mouse CXC chemokine receptor 6 (mCXCR6) also known as CD186, BONZO, or STRL33. CXCR6 is expressed on naive CD8+ T cells and a subset of memory CD4+ T cells, natural killer T cells (NKTs), dendritic cells (DCs), pulmonary alveolar macrophages, and innate lymphoid cells (ILCs). The ligand for CXCR6 is C-X-C chemokine CXCL16 that is predominantly expressed in DCs, monocytes, and various other tissue cells, primarily epithelial cells. The CXCR6-CL16 interaction guides immune cell homing (T lymphocyte migration to peripheral tissues) and their activation, expansion, and cytotoxic activity. Retroviruses such as simian immunodeficiency viruses (SIVs), some strains of HIV-2, and macrophage-tropic HIV-1 use CXCR6 as a coreceptor in conjunction with CD4 to enter target cells. In solid tumors, CXCR6 mediates CD8+ T-cell homing to tumor stromal perivascular niches and promotes interactions with CXCL16+ DCs and IL-15 expression for facilitating the growth of T-cells inside the tumor microenvironment. CXCR6 expression is also linked to the activation of Akt/mTOR and ERK/MAPK pathways and other downstream targets responsible for cancer growth and metastasis. CXCR6’s prognostic significance varies among malignancies, but it is regarded as a reliable biomarker to predict the response to anti-PD-1 immunotherapy in various cancers. InVivoMAb anti-mouse CXCR6 (CD186) Specifications IsotypeRat IgG1, κ Recommended Isotype Control(s)InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase Recommended Dilution BufferInVivoPure pH 7.0 Dilution Buffer ImmunogenSynthetic peptide corresponding to the N-terminal extracellular region of mouse CXCR6 (AA 1-19) Reported ApplicationsFlow cytometry Western blotting ELISA For details on in vivo applications, please contact technicalservice@bioxcell.com FormulationPBS, 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 Sterility0.2 μm filtered ProductionPurified from cell culture supernatant in an animal-free facility PurificationProtein G Molecular Weight150 kDa StorageThe antibody solution should be stored at the stock concentration at 4°C. Do not freeze. Application ReferencesInVivoMAb anti-mouse CXCR6 (CD186) (CLONE: Cx6Mab-1)Isoda Y, Tanaka T, Suzuki H, Asano T, Yoshikawa T, Kitamura K, Kudo Y, Ejima R, Ozawa K, Kaneko MK, Kato Y (2023). "Epitope Mapping Using the Cell-Based 2 × Alanine Substitution Method About the Anti-mouse CXCR6 Monoclonal Antibody, Cx6Mab-1" Monoclon Antib Immunodiagn Immunother 42(1):22-26. PubMedAn anti-mouse CXC chemokine receptor 6 (mCXCR6) monoclonal antibody (mAb), Cx6Mab-1, was developed recently. Cx6Mab-1 is applicable for flow cytometry, Western blotting, and enzyme-linked immunosorbent assay. The purpose of this study is to determine the binding epitope of Cx6Mab-1 using 2 × alanine mutated mCXCR6. Analysis of flow cytometry revealed that Cx6Mab-1 did not recognize S8A-A9G, L10A-Y11A, D12A-G13A, and H14A-Y15A mutants of mCXCR6. The results clearly indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6. The successful determination of the Cx6Mab-1 epitope might contribute to the pathophysiological investigation of mCXCR6.Kitamura K, Suzuki H, Kaneko MK, Kato Y (2022). "Cx6Mab-1: A Novel Anti-Mouse CXCR6 Monoclonal Antibody Established by N-Terminal Peptide Immunization" Monoclon Antib Immunodiagn Immunother 41(3):133-141. PubMedThe CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, natural killer cells, cytotoxic T lymphocytes, and various type of cells in tumor microenvironment (TME). CXCR6 has been proposed as a therapeutic target against tumors through regulation of the tumor TME. In this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CXCR6 (mCXCR6), which are useful for flow cytometry and Western blotting by N-terminal peptide immunization into rat. The established anti-mCXCR6 mAb, Cx6Mab-1 (rat IgG1, kappa), reacted with not only mCXCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR6) but also mCXCR6-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasm) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx6Mab-1 for CHO/mCXCR6, P388, and J774-1 cells were 1.7 × 10-9 M, 3.4 × 10-7 M, and 3.8 × 10-7 M, respectively. Furthermore, Cx6Mab-1 could detect endogenous mCXCR6 in P388 and J774-1 cells by Western blotting. These results indicated that Cx6Mab-1 is useful for detecting mCXCR6 by flow cytometry and Western blotting, and provides a possibility for targeting CXCR6-expressing cells in vivo experiments.Isoda Y, Tanaka T, Suzuki H, Asano T, Nakamura T, Yanaka M, Handa S, Komatsu Y, Okuno S, Takahashi N, Okada Y, Kobayashi H, Li G, Nanamiya R, Goto N, Tateyama N, Yoshikawa T, Kaneko MK, Kato Y (2022). "Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal Antibody (Cx6Mab-1) Using the 2 × Alanine Scanning Method" Monoclon Antib Immunodiagn Immunother 41(5):275-278. PubMedThe CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.
