C 3 Mab-3: A Monoclonal Antibody for Mouse CC Chemokine Receptor 3 for Flow Cytometry

Cell lines

Chinese hamster ovary (CHO)-K1, LN229, and P3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (Manassas, VA). P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University (Miyagi, Japan). Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding mCCR3 (Accession No. NM_009914.4) was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). mCCR3 plasmid was transfected into CHO-K1 and LN229 cells using the Neon transfection system (Thermo Fisher Scientific Inc., Waltham, MA). Stable transfectants were established through cell sorting using a cell sorter (SH800; Sony Corp., Tokyo, Japan) and cultivation in a medium containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA).

CHO-K1, P3U1, mCCR3-overexpressed CHO-K1 (CHO/mCCR3), P388, and J774-1 were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan). LN229 and mCCR3-overexpressed LN229 (LN229/mCCR3) were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.). RPMI 1640 and DMEM were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). Cells were grown in a humidified incubator at 37°C with an atmosphere of 5% CO₂ and 95% air.

Antibodies

An anti-mCCR3 mAb (clone J073E5) was bought from BioLegend (San Diego, CA). Secondary Alexa Fluor 488-conjugated anti-rat IgG was bought from Cell Signaling Technology, Inc. (Danvers, MA).

Production of hybridomas

Sprague Dawley (SD) rats (6 weeks old) were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions. All animal experiments were conducted according to relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit No. 2019NiA-001). A rat was monitored daily for health during the full 4-week duration of the experiment. A reduction of more than 25% of total body weight was defined as a humane endpoint. A rat was euthanized through cervical dislocation, and death was verified through respiratory and cardiac arrest.

To develop mAbs against mCCR3, the CBIS method was employed. In brief, one SD rat was immunized with LN229/mCCR3 cells (1 × 10⁹) by the intraperitoneal route together with the Inject Alum (Thermo Fisher Scientific Inc.). The procedure included three additional immunization, followed by a final booster intraperitoneal injection administered 2 days before the harvest of spleen cells. Harvested spleen cells were subsequently fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in an RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific Inc.). Flow cytometry was used to screen the supernatants with CHO/mCCR3, CHO-K1, P388, and J774-1 cells.

Flow cytometry

Cells were harvested after a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). They were washed with 0.1% bovine serum albumin in phosphate-buffered saline and treated with primary mAbs for 30 minutes at 4°C. The cells were then treated with Alexa Fluor 488-conjugated anti-rat IgG (1:2000; Cell Signaling Technology, Inc.). Fluorescence data were obtained using BD FACSLyric (BD Biosciences, Franklin Lakes, NJ).

Determination of dissociation constants (K_Ds) by flow cytometry

CHO/mCCR3, P388, and J774-1 were suspended in 100 μL of serially diluted anti-mCCR3 mAbs, and then 50 μL of Alexa Fluor 488-conjugated anti-rat IgG (1:200; Cell Signaling Technology, Inc.) was added. Fluorescence data were obtained using the BD FACSLyric. The K_Ds were calculated by fitting saturation binding curves to built-in one-site binding models in GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).

The development of anti-mCCR3 mAbs using the CBIS method

To develop anti-mCCR3 mAbs, we employed CBIS method. CBIS method includes the immunization of mCCR3-overexpressed cells and the high-throughput hybridoma screening using flow cytometry (Fig. 1). In this study, one rat was immunized with LN229/mCCR3 cells (Fig. 1A). The spleen was excised from the rat, and splenocytes were fused with P3U1 cells (Fig. 1B). Developed hybridomas were seeded into 96-well plates and cultivated for 6 days. Culture supernatants positive for CHO/mCCR3 were screened using flow cytometry (Fig. 1C).

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FIG. 1.

Schematic illustration of the production of anti-mCCR3 mAbs. (A) LN229/mCCR3 cells were immunized into an SD rat using an intraperitoneal injection. (B) Spleen cells were fused with P3U1 cells. (C) Culture supernatants were screened with CHO/mCCR3 using flow cytometry to select hybridomas that are producing anti-mCCR3 mAb. (D) After limiting dilution and many additional screenings, an anti-mCCR3 mAb was finally established. CCR3, CC chemokine receptor 3; CHO, Chinese hamster ovary; mAb, monoclonal antibody; mCCR3, mouse CCR3; SD, Sprague Dawley.

The first screening approach identified strong signals from CHO/mCCR3 cells and weak or no signals from CHO-K1 cells in 30 of 956 wells (3.14%). The second screening procedure identified strong signals from P388 and J774-1 cells in 26 of the 30 hybridoma supernatants identified in the previous step (86.7%). After limiting dilution and several additional screenings, an anti-mCCR3 mAb, C₃Mab-3 (rat IgG_2a, kappa) was finally established (Fig. 1D).

Flow cytometry analysis

We performed flow cytometry using C₃Mab-3 against CHO/mCCR3, CHO-K1, P388, and J774-1. C₃Mab-3 dose-dependently recognized CHO/mCCR3 cells at 10, 1, and 0.1 μg/mL (Fig. 2A, left panel), but not parental CHO-K1 cells (Fig. 2B, left panel). Another anti-mCCR3 mAb (clone J073E5 from BioLegend: positive control) also recognized CHO/mCCR3 in a dose-dependent manner (Fig. 2A, right panel), but not CHO-K1 (Fig. 2B, right panel). Although C₃Mab-3 did not react with CHO/mCCR3 at 0.01 μg/mL, J073E5 reacted with it even at 0.01 μg/mL (Fig. 2A). C₃Mab-3 and J073E5 also recognized P388 cells (Fig. 2C) and J774-1 cells (Fig. 2D) expressing endogenous mCCR3 in a dose-dependent manner. These results suggested that C₃Mab-3 is specific for mCCR3 and is useful for detecting endogenous mCCR3 using flow cytometry.

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FIG. 2.

Flow cytometry using anti-mCCR3 mAbs. CHO/mCCR3 (A), CHO-K1 (B), P388 (C), and J774-1 (D) cells were treated with 0.01 μg/mL (dashed-dotted line), 0.1 μg/mL (dotted line), 1 μg/mL (dashed line), and 10 μg/mL (solid line) of C₃Mab-3 (left panels) or J073E5 (right panels), followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG. Filled, without 1st Ab as negative controls.

Determination of the K_Ds using C₃Mab-3 against mCCR3-expressing cells by flow cytometry

To assess the K_D of C₃Mab-3 with mCCR3-expressed cells, we performed kinetic analysis of the interaction of C₃Mab-3 with CHO/mCCR3, P388, and J774-1 using flow cytometry. The geometric mean of fluorescence intensity was plotted versus the concentration of C₃Mab-3 and fitted by one-site binding models in GraphPad PRISM 8. The K_Ds of C₃Mab-3 for CHO/mCCR3, P388, and J774-1 cells were determined to be 4.3 × 10⁻⁸ M (Fig. 3A), 2.6 × 10⁻⁷ M (Fig. 3B), and 2.4 × 10⁻⁷ M (Fig. 3C), respectively. These results show that C₃Mab-3 possesses moderate affinity for CHO/mCCR3 cells and low affinity for P388 and J774-1.

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FIG. 3.

Determination of the K_Ds of C₃Mab-3 against mCCR3-expressing cells. CHO/mCCR3 (A) was suspended in 100 μL serially diluted C₃Mab-3 (0.006–100 μg/mL). P388 (B) and J774-1 (C) were suspended in 100 μL serially diluted C₃Mab-3 (0.06–1000 μg/mL). Alexa Fluor 488-conjugated anti-rat IgG was then added. Fluorescence data were obtained using BD FACSLyric.