Development of Antihuman Killer Cell Lectin-Like Receptor Subfamily G Member 1 Monoclonal Antibodies for Flow Cytometry

Cell lines

Chinese hamster ovary-K1 (CHO-K1) and P3X63Ag8U.1 (P3U1) were obtained from the American Type Culture Collection (Manassas, VA). For flow cytometry, NK cells (donor lot 4022602 NK1602) were purchased from Takara Bio, Inc. (Shiga, Japan). Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding human KLRG1 (hKLRG1; accession no: NP_001316028.1) plus N-terminal PA tag⁽²⁵⁾ was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The hKLRG1 plasmid was transfected into CHO-K1 cells using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific Inc., Waltham, MA). Stable transfectants were identified using a cell sorter (SH800; Sony Corp., Tokyo, Japan) and cultivated in a medium containing 0.5 mg/mL zeocin (InvivoGen, San Diego, CA) to generate CHO/PA-hKLRG1 cells.

CHO-K1, P3U1, and CHO/PA-hKLRG1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), 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 5% CO₂.

Antibodies

An anti-hKLRG1 mAb (clone 14C2A07; mouse IgG_2a, kappa) was purchased from BioLegend (San Diego, CA). Secondary Alexa Fluor 488-conjugated anti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Hybridoma production

Female BALB/c mice (6 weeks old) were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions. Animal studies were performed according to the relevant guidelines and regulations to minimize animal suffering and distress. Studies were approved by the Animal Care and Use Committee of Tohoku University (permit: 2019NiA-001). Mice were monitored daily during the 4-week study. A reduction of >25% of total body weight was defined as a humane endpoint. Mice were euthanized by cervical dislocation, and death was verified by respiratory and cardiac arrest.

We used CBIS method to develop mAbs against hKLRG1.⁽²⁴⁾ In brief, two BALB/c mice were intraperitoneally immunized with CHO/PA-hKLRG1 cells (1 × 10⁸) together with Imject Alum (Thermo Fisher Scientific Inc.). The procedure required three additional immunizations followed by a final booster intraperitoneal injection administered 2 days before spleen cells were harvested. Once harvested, cells were fused with P3U1 cells using polyethylene glycol 1500 (Roche Diagnostics, Indianapolis, IN). For selection, resultant hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific Inc.). Culture supernatants were screened by flow cytometry.

Flow cytometry

Cells were harvested after a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). They were washed in 0.1% bovine serum albumin in phosphate-buffered saline, treated with primary mAbs for 30 minutes at 4°C, and then treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc.). Fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).

Determination of binding affinity by flow cytometry

CHO/PA-hKLRG1 cells were suspended in 100 μL serially diluted anti-hKLRG1 mAbs, and Alexa Fluor 488-conjugated anti-mouse IgG was added (1:200; Cell Signaling Technology, Inc.). Fluorescence data were collected using the BD FACSLyric instrument (BD Biosciences, Franklin Lakes, NJ). The dissociation constant (K_D) was calculated by fitting binding isotherms to built-in one-site binding models in GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).

Establishment of anti-hKLRG1 mAbs

CBIS method was used to establish anti-hKLRG1 mAbs (Fig. 1). First, two mice were immunized with CHO/PA-hKLRG1 cells, and hybridomas were produced by fusing splenocytes with P3U1 myeloma cells. Next, we selected hybridomas whose supernatants were positive for CHO/PA-hKLRG1 but negative for CHO-K1 cells, using flow cytometry. The first screening identified strong signals from CHO/PA-hKLRG1 cells and weak or no signals from CHO-K1 cells in 22 of 960 wells (2.3%). After limiting dilutions and several additional screenings, KLMab-1 (IgG₁, kappa), which reacted most strongly with CHO/PA-hKLRG1, was established.

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FIG. 1.

Production of anti-hKLRG1 mAbs. Schematic representation of CBIS. (1) CHO/PA-hKLRG1 cells were immunized into BALB/c mice by intraperitoneal injection. (2) Splenocytes were fused with myelomas for production of hybridomas. (3) Screening of anti-hKLRG1 mAb producing hybridomas was performed by flow cytometry using CHO/hKLRG1 and CHO-K1 cells. (4) Anti-hKLRG1 mAb-producing hybridomas were cloned using the limiting dilution method. CBIS, Cell-Based Immunization and Screening; CHO, Chinese hamster ovary-K1; hKLRG1, human KLRG1; KLRG1, Killer cell lectin-like receptor subfamily G member 1; mAbs, monoclonal antibodies.

Flow cytometric analyses

KLMab-1 recognized CHO/PA-hKLRG1 but not CHO-K1 cells (Fig. 2A). Also, KLMab-1 reacted with endogenous KLRG1 in NK cells (Fig. 2A). A 0.01 μg/mL concentration was adequate for KLMab-1 recognition of CHO/PA-hKLRG1, but the KLMab-1 recognition of NK cells was weak (Fig. 2A). As a positive control, clone 14C2A07 (BioLegend) reacted similarly with all cell types (Fig. 2B).

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FIG. 2.

Flow cytometry using anti-hKLRG1 mAbs. (A) CHO-K1, CHO/PA-hKLRG1, and NK cells were treated with 0.01–1 μg/mL KLMab-1, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; negative control (black). (B) CHO-K1, CHO/PA-hKLRG1, and NK cells were treated with 0.01–1 μg/mL of clone 14C2A07, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; negative control (black). (C) Determination of KLMab-1 binding affinity. CHO/PA-hKLRG1 cells were suspended in 100 μL of serially diluted KLMab-1 (0.006–100 μg/mL). Alexa Fluor 488-conjugated anti-mouse IgG was then added. Fluorescence data were collected using the BD FACSLyric instrument. NK, natural killer.

Determination of binding affinity using KLMab-1 in hKLRG1-expressing cells

We conducted a kinetic analysis of the interaction between KLMab-1 and CHO/PA-hKLRG1 cells by flow cytometry to assess the binding affinity of KLMab-1 with CHO/PA-hKLRG1 cells. The geometric mean of fluorescence intensity was plotted against different KLMab-1 concentrations and fitted by one-site binding models. The K_D of KLMab-1 for CHO/PA-hKLRG1 cells was determined to be 2.0 × 10⁻⁸ M (Fig. 2C), suggesting that KLMab-1 possessed a moderate binding affinity for these cells.