MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0045

Short Communications
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C8Mab-2: An Anti-Mouse C–C Motif Chemokine Receptor 8
Monoclonal Antibody for Immunocytochemistry

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Masaki Saito,1 Tomohiro Tanaka,2 Teizo Asano,2 Takuro Nakamura,2 Miyuki Yanaka,2Saori Handa,2 Yu Komatsu,2 Yasuhiro Harigae,1 Nami Tateyama,2 Ren Nanamiya,2Guanjie Li,1 Hiroyuki Suzuki,1 Mika K. Kaneko,2 and Yukinari Kato1,2

C–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 andlymphoma 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 aremarkable 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 andexogenous 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.
Keywords: mouse CCR8, C8Mab-2, monoclonal antibody, immunocytochemical analysis

Introduction

C–C motif chemokine receptor 8 (CCR8), a memberof the C–C motif chemokine receptor family, is a G
protein-coupled receptor (GPCR) and is activated using C–C
motif chemokine ligands CCL1/I-309, CCL16, andCCL18.(1,2) CCR8 is primarily expressed in regulatory T(Treg) and T helper 2 (TH2) cells, but little in T helper 1
cells.(3–5) Accordingly, the receptor plays a role in allergy andasthma by recruiting CCR8+ Treg and TH2 cells to the inflammation site.(4–6)
CCR8 is also associated with cancer development. Transcriptome analyses of tumor-infiltrating Tregs revealed that
the CCR8 gene was upregulated in breast cancer, nonsmall
cell lung cancer, colorectal cancer, and oral squamous cell
carcinoma.(7–9) CCL1 increases the migration activity of
CCR8+ Treg and induces the anti-apoptotic activity of T cellleukemia and lymphoma through CCR8.(10–13) The activationof CCR8 through CCL1 promotes suppression of the CCR8+
Treg function through an increase in the expression of CCR8level in Treg.(5)In contrast, CCL18 induces invasion, migration, and epithelial–mesenchymal transition in bladder cancer cells.(14)These reports collectively imply that CCR8 can be a diagnostic and therapeutic target molecule of cancers. Moreover,developing specific antibodies against CCR8 can be thepowerful driving force for basic research to reveal unresolved
CCR8 functions.
We developed monoclonal antibodies (mAbs) for GPCRs,
including anti-mouse CCR3 (clone C3Mab-2) and antihuman CCR9 (clone C9Mab-1) using the Cell-Based Immunization and Screening (CBIS) method.(15,16) We also
developed an anti-mouse CCR8 (mCCR8) clone, C8Mab-2,
and showed that C8Mab-2 was applicable to flow cytometric

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Miyagi, Japan.

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analysis.(17) In this study, we demonstrated that C8Mab-2 wasavailable for immunocytochemical analysis against mCCR8,
which is both endogenously and exogenously expressed incultured mammalian cells.
Materials and Methods

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Cell lines

Chinese hamster ovary (CHO)-K1 cells were provided
from the American Type Culture Collection (Manassas, VA).
The establishment of a mCCR8-overexpressed CHO-K1 cellline (CHO/mCCR8) was described in our previous report.(17)
Mouse lymphocyte-like cell line (P388) and mouse
macrophage-like cell line ( J774-1) were provided from Cell
Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Miyagi,
Japan).
All cells were cultured in Roswell Park Memorial Institute
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.), and were maintained in a humidified atmosphere at 37C under 5% CO2.

SAITO ET AL.
Antibodies

An anti-mCCR8 mAb C8Mab-2 (rat IgG2b, kappa) was
developed as previously described.(17) To generate recombinant C8Mab-2 (recC8Mab-2), we subcloned VH and CH of
cDNAs of C8Mab-2 into the pCAG-Neo vector, along with
VL and CL cDNAs of C8Mab-2 into the pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), respectively. An anti-mCCR8 mAb (clone SA214G2)
was purchased from BioLegend (San Diego, CA).
Immunocytochemical analysis

Immunocytochemical analysis was carried out by a modification of a previously described procedure.(18) In brief,
CHO-K1, CHO/mCCR8, P388, and J774-1 cells were cultured on acid-wash coverslips for 3 days. The cells were fixedin phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 10 minutes at room temperature and quenchedwith PBSc/m (PBS supplemented with 0.2 mM Ca2+ and2 mM Mg2+) containing 50 mM NH4Cl for 10 minutes.
Then, the cells were blocked with PBSc/m supplemented
with 0.5% bovine serum albumin for 30 minutes, and they
were incubated with 1 or 10 lg/mL of primary antibodies for
1 hour followed by Alexa Fluor 488-conjugated anti-rat IgG

FIG. 1. Immunocytochemical analysis of exogenous mCCR8 using 10 mg/mL C8Mab-2 and recC8Mab-2. CHO/mCCR8(A) and CHO-K1 cells (B) were incubated with buffer control, 10 mg/mL SA214G2, 10 mg/mL C8Mab-2, or 10 mg/mL
recC8Mab-2 for 1 hour. They were subsequently incubated with Alexa Fluor 488-conjugated anti-rat IgG and DAPI for 45
minutes. Scale bars, 20 mm. CCR8, C–C motif chemokine receptor 8; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino2-phenylindole; mCCR8, mouse CCR8; recC8Mab-2, recombinant C8Mab-2.

C8MAB-2 FOR IMMUNOCYTOCHEMISTRY(1:400; Cell Signaling Technology, Inc.) for 45 minutes. The
cell nuclei were stained with 4¢,6-diamidino-2-phenylindole(DAPI; Thermo Fisher Scientific Inc.). Fluorescence images
were obtained using a 40 · objective on a fluorescence microscope BZ-X800 (Keyence, Osaka, Japan).

cells.(17) Thus, in this study, we also used 1 lg/mL C8Mab-2,
recC8Mab-2, and SA214G2 for immunocytochemistry. As aresult, 1 lg/mL C8Mab-2 and recC8Mab-2 detected mCCR8
with a similar fluorescent intensity to 10 lg/mL in
CHO/mCCR8 cells (Supplementary Fig. S1).

Results

C8Mab-2 and recC8Mab-2 recognize endogenously
expressed mCCR8 in P388 and J774-1 cells

C8Mab-2 and recC8Mab-2 recognize exogenously
expressed mCCR8 in CHO-K1 cells

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To investigate whether C8Mab-2 and recC8Mab-2 recognize mCCR8 in immunocytochemistry, we used stable
CHO/mCCR8 cells, which highly express mCCR8 protein.(17) It was revealed that both C8Mab-2 (10 lg/mL) andrecC8Mab-2 (10 lg/mL), but not the buffer control, detected
mCCR8 in CHO/mCCR8 cells (Fig. 1A). In contrast, both
antibodies showed no signal from parental CHO-K1 cells
during image acquisition (Fig. 1B). A commercially available anti-mCCR8 antibody (SA214G2) was used as a positive
control and bound to CHO/mCCR8 cells, but backgroundsignals were also obtained from CHO-K1 cells (Fig. 1A, B).
These data suggest that C8Mab-2 and recC8Mab-2 specifically recognize mCCR8.
The result of flow cytometry demonstrated that the fluorescence intensity obtained from 1 and 10 lg/mL of C8Mab-2
labeling was comparable with those of CHO/mCCR8Using immunocytochemistry, we examined whether
C8Mab-2 and recC8Mab-2 recognize endogenous mCCR8 in
P388 and J774-1 cells. As the expression levels of endogenous mCCR8 in both cells are expected to be lower than thelevel of exogenous mCCR8 in CHO/mCCR8 cells, we obtained fluorescence images of endogenous mCCR8 using
longer exposure times. Thus, C8Mab-2, recC8Mab-2, andSA214G2 specifically detected endogenous mCCR8 in P388
and J774-1 cells (Fig. 2A, B). Moreover, the fluorescenceintensity from recC8Mab-2 was slightly stronger than theintensity from C8Mab-2, indicating that the activity ofrecC8Mab-2 was stronger than that of C8Mab-2.
Discussion

Using flow cytometry, we previously evaluated that
C8Mab-2 selectively binds to exogenously expressed
mCCR8 in CHO/mCCR8 cells and endogenous mCCR8 in

FIG. 2. Immunocytochemical analysis of endogenous mCCR8 using 10 mg/mL C8Mab-2 and recC8Mab-2. P388 (A) andJ774-1 cells (B) were incubated with buffer control, 10 mg/mL SA214G2, 10 mg/mL C8Mab-2, or 10 mg/mL recC8Mab-2 for
1 hour. They were subsequently incubated with Alexa Fluor 488-conjugated anti-rat IgG and DAPI for 45 minutes. Scalebars, 20 mm.

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P388 and J774-1 cells.(17) This study demonstrates that using
immunocytochemical analysis, C8Mab-2 recognizes bothexogenous and endogenous mCCR8. In immunocytochemistry, the specificity of C8Mab-2 and recC8Mab-2 was demonstrated by two pieces of evidence: (1) no visible (or veryweak) signal was obtained from control samples (e.g., CHOK1 cells treated with C8Mab-2/recC8Mab-2; CHO/mCCR8,
P388, or J774-1 cells treated with buffer control) and (2)mCCR8 was distributed in the cell membrane in CHO/mCCR8,
P388, and J774-1 cells.
Immunocytochemical analysis of mCCR8 using C8Mab-2
and recC8Mab-2 would provide us valuable information forbasic research. The intracellular distribution of mCCR8
would be identified in detail when the cells are colabeled withany organelle markers, such as plasma membranes. The expression level of the mCCR8 protein in cancer cells would be
detected from fluorescence intensity data. Furthermore, using
image cytometry, T cell types that highly express mCCR8
would be revealed when they are isolated from mice and thecell population is colabeled with surface antigen markers(e.g., CD4, CD25, and CD127).
Some anti-GPCR antibodies have been approved for clinicaluse. Mogamulizumab, an anti-CCR4 mAb, is used for human T
cell lymphoma.(18) Erenumab, an anti-calcitonin gene-related
peptide receptor mAb, is used for migraines.(19) Contrastingly,
the availability of anti-mCCR8 antibodies for cancer therapy
has been investigated in vivo and in vitro. For instance, treatment of colon cancer with a commercially available antimCCR8 mAb suppresses tumor growth.(20) Blockage of human
CCR8 with a commercially available anti-human CCR8 mAb
destabilizes CCR8+ Tregs, resulting in reactivation of antitumor immunity in muscle-invasive bladder cancer.(21)
Van Damme et al.(22) developed a fusion antibody of a
CCR8-specific nanobody and an antibody-dependent cellmediated cytotoxicity (ADCC)-prone Fc region. The antiCCR8 nanobody–Fc fusion significantly reduced the growthof nonsmall cell lung carcinoma.
In general, the development of anti-GPCR antibodies istechnically difficult because of the variable three-dimensional
conformation of GPCR, small extracellular epitopes of GPCR,
and difficult preparation of homogeneous functional antigens.(23) In the CBIS method, mammalian cell lines stably
expressing a target molecule were used as an immunogen,meaning that GPCR-harboring conformational epitopes andphysiological post-translational modification are used as antigens. MAbs developed using the CBIS method are expected tobe highly suitable for detecting GPCR expressed in cells. Thus,in the future, it is necessary to investigate whether C8Mab-2
and recC8Mab-2 possess ADCC activity, complementdependent cytotoxicity, and antitumor activity.
In conclusion, we clarified that C8Mab-2 and recC8Mab-2
could be useful tools for immunocytochemistry in detecting
the expression and intracellular distribution of mCCR8 in
Tregs and cancer cells including T cell lymphoma.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant

SAITO ET AL.

Nos. JP22ama121008 (to Y.K.), JP21am0401013 (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. 21K06059(to M.S.), 21K20789 (to T.T.), 21K15523 (to T.A.),
20K16322 (to M.S.), 21K07168 (to M.K.K.), and19K07705 (to Y.K.).
Supplementary Material

Supplementary Figure S1
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 15, 2021
Accepted: February 14, 2022