Conceptualization, M.K.K. and Y.K.; Funding acquisition, Y.K.; Investigation, N.T., T.A., G.L., T.Y., T.T. and M.K.K.; Methodology, M.K.K.; Supervision, Y.K.; Writing—original draft, N.T., T.A. and H.S.; Writing—review & editing, H.S. and Y.K. All authors agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript.

This research was supported in part by Japan Agency for Medical Research and Development (AMED), under Grant Numbers JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 21K20789 (to T.T.), 22K06995 (to H.S.), 22K15523 (to T.A.), 22K07168 (to M.K.K.), and 22K07224 (to Y.K.).

Figure 1. Schematic illustration of mCCR8 and mCCR3 chimeric proteins. The four extracellular regions of mCCR3, namely, (A) the N-terminal region (aa 1–38), (B) ECL1 (aa 96–111), (C) ECL2 (aa 176–207), and (D) ECL3 (aa 269–285), were substituted into the corresponding regions of mCCR8. ECL, extracellular loop, aa; amino acids.

Figure 1. Schematic illustration of mCCR8 and mCCR3 chimeric proteins. The four extracellular regions of mCCR3, namely, (A) the N-terminal region (aa 1–38), (B) ECL1 (aa 96–111), (C) ECL2 (aa 176–207), and (D) ECL3 (aa 269–285), were substituted into the corresponding regions of mCCR8. ECL, extracellular loop, aa; amino acids.

Figure 2. Determination of the epitope of anti-mCCR3 mAbs using flow cytometry and chimeric proteins. C3Mab-3 (1 µg/mL) (A), C3Mab-4 (1 µg/mL) (B), and J073E5 (1 µg/mL) (C) were treated with CHO-K1 cells that transiently expressed chimeric proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with anti-mCCR3 mAbs treatment, and black lines show cells without anti-mCCR3 mAbs treatment as a negative control.

Figure 2. Determination of the epitope of anti-mCCR3 mAbs using flow cytometry and chimeric proteins. C3Mab-3 (1 µg/mL) (A), C3Mab-4 (1 µg/mL) (B), and J073E5 (1 µg/mL) (C) were treated with CHO-K1 cells that transiently expressed chimeric proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with anti-mCCR3 mAbs treatment, and black lines show cells without anti-mCCR3 mAbs treatment as a negative control.

Figure 3. Determination of the C3Mab-3 epitope using flow cytometry and alanine scanning. (A) C3Mab-3 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with C3Mab-3 treatment, and black lines show cells without Ab treatment as a negative control. (B) The C3Mab-3 epitope for mCCR3 involves Ala2, Phe3, Asn4, and Thr5 of mCCR3.

Figure 3. Determination of the C3Mab-3 epitope using flow cytometry and alanine scanning. (A) C3Mab-3 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with C3Mab-3 treatment, and black lines show cells without Ab treatment as a negative control. (B) The C3Mab-3 epitope for mCCR3 involves Ala2, Phe3, Asn4, and Thr5 of mCCR3.

Figure 4. Determination of the C3Mab-4 epitope using flow cytometry and alanine scanning. (A) C3Mab-4 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with C3Mab-4 treatment, and black lines show cells without Ab treatment as a negative control. (B) The C3Mab-4 epitope for mCCR3 involves Ala2, Phe3, and Thr5 of mCCR3.

Figure 4. Determination of the C3Mab-4 epitope using flow cytometry and alanine scanning. (A) C3Mab-4 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with C3Mab-4 treatment, and black lines show cells without Ab treatment as a negative control. (B) The C3Mab-4 epitope for mCCR3 involves Ala2, Phe3, and Thr5 of mCCR3.

Figure 5. Determination of the J073E5 epitope using flow cytometry and alanine scanning. (A) J073E5 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with J073E5 treatment, and black lines show cells without Ab treatment as a negative control. (B) The J073E5 epitope for mCCR3 involves Ala2 and Phe3 of mCCR3.

Figure 5. Determination of the J073E5 epitope using flow cytometry and alanine scanning. (A) J073E5 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with J073E5 treatment, and black lines show cells without Ab treatment as a negative control. (B) The J073E5 epitope for mCCR3 involves Ala2 and Phe3 of mCCR3.

Figure 6. Cell surface expression of mCCR3 mutants on CHO-K1 cells using flow cytometry. C3Mab-7 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with C3Mab-7 treatment, and black lines show cells without Ab treatment as a negative control.

Figure 6. Cell surface expression of mCCR3 mutants on CHO-K1 cells using flow cytometry. C3Mab-7 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at 4 °C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with C3Mab-7 treatment, and black lines show cells without Ab treatment as a negative control.