About InVivoMAb anti-mouse CCR8 (CDw198) The C8Mab-2 monoclonal antibody reacts with an epitope within the N-terminal region of mouse C-C chemokine receptor type 8 (CCR8), also known as CKR-8, CDw198, CMKBRL2, CMKBR8, and GPRCY6. CCR8 is a seven-pass transmembrane chemokine receptor and a member of the G protein-coupled receptor (GPCR) family. CCR8 ligands include CCL1, CCL16, and CCL8 (mCCL8) or CCL18 (hCCL18, a functional analog of mouse CCL8). Human and mouse CCR8 as well as its primary ligand CCL1 are structurally related, and this ligand is critical for skin homing of T cells and the survival of the regulatory T cells (Tregs) as well as their chemotaxis into tumors. Murine CCR8 is primarily expressed by the Tregs, and it is critical for their function. A subset of Th2 cells, skin-resident memory T cells, monocytic dendritic cells, and NK cells also express CCR8, but this receptor is not expressed by Th1 cells. Recent in vivo studies have documented the involvement of CCR8 in type 2 inflammatory diseases, including atopic dermatitis (AD) and allergic enteritis (AE). In the tumor microenvironment, CCR8+ Treg numbers directly correlate with an advanced state of cancer, and therapeutic depletion of CCR8+ tumor-infiltrating Tregs (ti-Tregs) is shown to exert antitumor immunity and synergism with anti-PD-1 therapy. InVivoMAb anti-mouse CCR8 (CDw198) Specifications IsotypeRat IgG2b, κ Recommended Isotype Control(s)InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin Recommended Dilution BufferInVivoPure pH 7.0 Dilution Buffer ImmunogenmCCR8 expressing CHO cells Reported ApplicationsFlow cytometry Western blotting Immunofluorescence 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 CCR8 (CDw198) (CLONE: C8Mab-2)Kobayashi H, Suzuki H, Tanaka T, Kaneko MK, Kato Y (2024). "Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C8Mab-2 Using Flow Cytometry" Monoclon Antib Immunodiagn Immunother 43(4):101-107. PubMedThe C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2.Saito M, Tanaka T, Asano T, Nakamura T, Yanaka M, Handa S, Komatsu Y, Harigae Y, Tateyama N, Nanamiya R, Li G, Suzuki H, Kaneko MK, Kato Y (2022). "C8Mab-2: An Anti-Mouse C-C Motif Chemokine Receptor 8 Monoclonal Antibody for Immunocytochemistry" Monoclon Antib Immunodiagn Immunother 41(2):115-119. PubMedC-C motif chemokine receptor 8 (CCR8) is a G protein-coupled receptor predominantly expressed in regulatory T (Treg) and T helper 2 cells. The evidence that CCR8 expression in Treg is increased in cancers, CCR8 increases migration activity of Treg, and CCR8 induces the anti-apoptotic activity in T cell leukemia and lymphoma suggests that CCR8 is associated with cancer development. Thus, developing a specific monoclonal antibody (mAb) for CCR8 is useful for diagnostic and therapeutic purposes and the anti-CCR8 mAb becomes a remarkable experimental tool for basic research. We previously developed an anti-mouse CCR8 (mCCR8) mAb called C8Mab-2 (rat IgG2b, kappa) that was applicable to flow cytometric analysis for both endogenous and exogenous mCCR8. This study showed that C8Mab-2 and recombinant C8Mab-2 (recC8Mab-2) were specifically bound to exogenously expressed mCCR8 in mCCR8-overexpressed Chinese hamster ovary-K1 cells. In addition, we found that C8Mab-2 and recC8Mab-2 recognized endogenous mCCR8 in P388 (a mouse lymphocyte-like cell line) and J774-1 cells (a mouse macrophage-like cell line). These data demonstrate that C8Mab-2 and recC8Mab-2 are useful for immunocytochemical analysis.Tanaka T, Nanamiya R, Takei J, Nakamura T, Yanaka M, Hosono H, Sano M, Asano T, Kaneko MK, Kato Y (2021). "Development of Anti-Mouse CC Chemokine Receptor 8 Monoclonal Antibodies for Flow Cytometry" Monoclon Antib Immunodiagn Immunother 40(2):65-70. PubMedCC chemokine receptor 8 (CCR8) belongs to the class A of G protein-coupled receptor. It is highly expressed on Treg and T helper 2 (TH2) cells recruited to the inflammation site and is implicated in allergy and asthma. Recently, CCR8+Treg cells have been suggested to be a master regulator in the immunosuppressive tumor microenvironment; therefore, developing sensitive monoclonal antibodies (mAbs) for CCR8 has been desired. This study established a specific and sensitive mAb for mouse CCR8 (mCCR8), which is useful for flow cytometry by using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCCR8 mAb, C8Mab-2 (rat IgG2b, kappa), reacted with mCCR8-overexpressed Chinese hamster ovary-K1 (CHO/mCCR8) cells and P388 (mouse lymphoid neoplasma) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR8 by flow cytometry. C8Mab-2, which was established by the CBIS method, could be useful for elucidating the mCCR8-related biological response by flow cytometry.
