The C-C motif chemokine receptor 8 (CCR8) belongs to the class A G protein-coupled receptor (GPCR). CCR8 interacts with the human-specific chemokine ligand CCL1/I-309, which is produced by a variety of cells, including tumor-associated macrophages and regulatory T cells (Tregs). CCR8 is highly expressed in Tregs and T helper 2 (TH2) cells recruited to sites of inflammation and has been implicated in allergies, asthma, and cancer progression. CCR8+ Tregs are considered important regulators of the immunosuppressive tumor microenvironment (TME); therefore, the development of sensitive monoclonal antibodies (mAbs) targeting CCR8 has been highly anticipated. Here, researchers developed a specific mAb against human CCR8 (hCCR8) for flow cytometric detection in cell-based immunity and screening (CBIS) approaches. Flow cytometry revealed that the established anti-human CCR8 monoclonal antibody, C8Mab-21 (murine IgM, κ type), reacted with hCCR8-overexpressing Chinese Hamster Ovary-K1 cells (CHO/hCCR8), TALL-1 cells (acute T-lymphoblastic leukemia), CCRF-HSB2 cells (human T-lymphoblastic leukemia), and natural killer cells, which express endogenous hCCR8 by flow cytometry. Furthermore, C8Mab-21 exhibited moderate affinity for both CHO/hCCR8 and TALL-1 cells, with dissociation constants of 6.5×10⁻⁸ M and 2.0×10⁻⁸ M, respectively. C8Mab-21, constructed using the CBIS method, can serve as an effective tool for flow cytometric analysis of hCCR8-related biological responses.
Flow cytometric analysis of CHO-K1, hCCR8-overexpressing CHO-K1 cells, TALL-1, CCRF-HSB2, and NK cells was performed using C8Mab-21 and commercially available anti-human CD198 (CCR8) mAbs (clones S19017D and L263G8). C8Mab-21, S19017D, and L263G8 recognized hCCR8-overexpressing CHO-K1 cells in a dose-dependent manner (Figure 1A). Even at a mAb concentration of 20 μg/mL, C8Mab-21 and L263G8 did not react with parental CHOK1 cells. S19017D showed a slight reaction with CHO-K1 cells at 20 μg/mL and 2 μg/mL (Figure 1B). Regarding endogenously hCCR8-expressing cells, C8Mab-21 recognized TALL-1, CCRF-HSB2, and NK cells at a concentration of 2 μg/mL or 20 μg/mL. S19017D also responded to TALL-1 and CCRF-HSB2 in a dose-dependent manner even at a concentration of 0.02 μg/mL. However, S19017D failed to recognize NK cells even at a concentration of 20 μg/mL. L263G8 also responded to TALL-1 and CCRF-HSB2 even at a concentration of 0.02 μg/mL and also to NK cells at mAb concentrations of 2 μg/mL or higher. C8Mab-21 detected both exogenously and endogenously expressed naïve hCCR8 by flow cytometry.
Figure 1. Flow cytometric analysis of anti-hCCR8 mAbs against CHO/hCCR8 and CHO-K1. (Tanaka T, et al. 2024)