MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0002
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C8Mab-3:
An Anti-Mouse CCR8 Monoclonal Antibody
for Immunocytochemistry

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Hiroyuki Suzuki,1 Masaki Saito,1 Teizo Asano,2 Tomohiro Tanaka,2 Kaishi Kitamura,1Yuma Kudo,1 Mika K. Kaneko,2 and Yukinari Kato1,2

The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also expressedin many cancers and is associated with those progression. The development of monoclonal antibodies (mAbs)
for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer
progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-3, rat IgG1,
kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C8Mab-3 and its
recombinant antibody (recC8Mab-3) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells(CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry. In addition, C8Mab-3 and recC8Mab3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express
endogenous mCCR8. C8Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry.
These results suggest that C8Mab-3, developed using the CBIS method, is useful for immunocytochemistry
against exogenous and endogenous mCCR8.
Keywords: CCR8, monoclonal antibody, CBIS, flow cytometry, immunofluorescence

IntroductionT

he C-C motif chemokine receptor 8 (CCR8) is afamily of the seven transmembrane domain G proteincoupled receptors (GPCRs) mainly expressed by regulatory T
cells (Tregs).(1–5) Human CCR8 has four ligands: C-C motif
chemokine ligand 1 (CCL1), CCL8, CCL16, and CCL18,(6)whereas only three of them, CCL1, CCL8, and CCL16, areexpressed in mouse.(7–9) CCR8 is the only known receptor for
CCL1,(5) whereas the other CCR8 ligands bind several chemokine receptors.(7–9) The CCR8+ Tregs are master drivers of
immune regulation. In the mice model of encephalomyelitis,a T cell-mediated autoimmune disease of the central nervous
system (CNS), the number of CCR8+ Tregs is reduced in the
periphery. Moreover, CNS CCR8+ Tregs are potentiated by
CCL1, which restricts the progression of the disease.(10)Therapeutic monoclonal antibodies (mAbs) targeting
immune checkpoints have changed the therapeutic strategy
of tumors.(11,12) Tumors described as ‘‘hot’’ are signs ofinflammation, particularly massive infiltration and enrichment with CD8+ effector T cells. The immune checkpoint
inhibitors are effective in hot tumors. However, immune
checkpoint inhibitors’ response rate is still low owing to thelack of effector CD8+ T cell infiltration or massive accumulation of Tregs that suppress their activities, which ischaracterized as ‘‘cold tumors.’’(13,14) Anti-mouse CCR8(mCCR8) mAbs could be used to limit cancer growth inseveral cancer models.(15,16) Therefore, anti-CCR8 mAbs,
mostly Tregs depleting antibodies, are currently developed
for clinical use.
We have developed anti-GPCR mAbs including antimouse CCR3 mAb (clone C3Mab-2),(17) anti-human CCR9mAb (clone C9Mab-1),(18) and anti-mCCR8 mAb (clone
C8Mab-2)(19) using the Cell-Based Immunization andScreening (CBIS) method. This study developed anotheranti-mCCR8 mAb (clone C8Mab-3), and showed that
C8Mab-3 recognizes both exogenous and endogenous
mCCR8 in flow cytometry and immunofluorescence.

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

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ANTI-MOUSE CCR8 FOR IMMUNOCYTOCHEMISTRY
Materials and Methods

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Cell lines and animals

P388 and J774-1 were obtained from the Cell Resource
Center for Biomedical Research Institute of Development,
Aging, and Cancer, Tohoku University (Miyagi, Japan). In
addition, Chinese hamster ovary (CHO)-K1 andP3X63Ag8U.1 (P3U1) were obtained from the American
Type Culture Collection (Manassas, VA).
Stable transfectants of CHO/mCCR8 were established in
our previous study.(19) In brief, synthesized DNA (Eurofins
Genomics KK, Tokyo, Japan) encoding mCCR8 (Accession
No.: NM_007720.2) was subcloned into a pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). mCCR8 plasmid was transfected using a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham,MA). CHO/mCCR8 cells were sorted using a cell sorter(SH800; Sony Corp., Tokyo, Japan) and cultivated in a medium containing 0.5 mg/mL of Zeocin (InvivoGen, San
Diego, CA).
P388, J774-1, CHO-K1, P3U1, and CHO/mCCR8 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 of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.). Cells were grown in a humidified incubator at
37C with 5% CO2 and 95% air.
A female Sprague-Dawley (SD) rat (6 weeks old) was
purchased from CLEA Japan (Tokyo, Japan). The animal washoused under specific pathogen-free conditions. All animal
experiments were conducted following the relevant guidelines and regulations to minimize animal suffering and distress in the laboratory.
Animal experiments were approved by the animal care anduse committee of Tohoku University (Permit No.: 2019NiA001). The rat was monitored daily for health during the 4 fullweeks duration of the experiment. A reduction of >25% of thetotal body weight was defined as a humane endpoint. The rat
was euthanized by cervical dislocation, and death was verified by respiratory and cardiac arrest.

111

light chain cDNAs of C8Mab-3 into the pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation) to produce
recombinant C8Mab-3 (recC8Mab-3). C8Mab-3 vectors weretransfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.). The resulting mAb (recC8Mab-3) was purified using Ab-Capcher
ExTra (ProteNova Co., Ltd, Kagawa, Japan).

Hybridoma production

The CBIS method was used to develop mAbs against
mCCR8.(20–26) One SD rat was immunized with
CHO/mCCR8 cells (1 · 109) using intraperitoneal injection
together with the Imject Alum (Thermo Fisher Scientific,Inc.). The procedure included three weekly immunization
followed by a final booster intraperitoneal injection administered 2 days before harvesting spleen cells. Harvested
spleen cells were subsequently fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI 1640 medium supplemented
with hypoxanthine, aminopterin, and thymidine for selection(Thermo Fisher Scientific, Inc.). Culture supernatants werescreened using flow cytometry.
Production of the recombinant antibody

Variable (VH) and constant (CH) regions of heavy chain
cDNAs of C8Mab-3 were subcloned into the pCAG-Neo
vector along with variable (VL) and constant (CL) regions of

FIG. 1. Flow cytometry using anti-mCCR8 mAbs,
C8Mab-3 and recC8Mab-3. CHO-K1, CHO/mCCR8, P388,
and J774-1 were treated with 0.01–10 mg/mL C8Mab-3 orrecC8Mab-3, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG; black line, negative control. CHO,
Chinese hamster ovary; CCR8, CC motif chemokine receptor 8; mAbs, monoclonal antibodies.

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SUZUKI ET AL.

Flow cytometry

We harvested cells after brief exposure to 0.25% trypsin and
1 mM of ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.), washed them with 0.1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS), treated them with primarymAbs (10 lg/mL) for 30 minutes at 4C, and treated with Alexa
Fluor 488-conjugated anti-rat IgG (1:2000; Cell Signaling
Technology, Inc., Danvers, MA). In addition, we collected
fluorescence data using the EC800 Cell Analyzer (Sony Corp.).

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Determination of binding affinity by flow cytometry

We suspended CHO/mCCR8 cells in 100 lL of serially diluted anti-mCCR8 mAbs, and then in 100 lL of Alexa Fluor
488-conjugated anti-rat IgG (1:200; Cell Signaling Technology,Inc.). Fluorescence data were collected using the EC800 Cell
Analyzer. The dissociation constant (KD) was calculated by fitting binding isotherms to built-in one-site binding models in
GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).
Immunocytochemistry

CHO-K1, CHO/mCCR8, P388, and J774-1 cells were cultured on acid-wash coverslips. They were treated with 4%paraformaldehyde (in PBS) for 10 minutes and in the additional
50 mM of NH4Cl (in PBS containing 0.2 mM of Ca2+ and2 mM of Mg2+ [PBSc/m]) for 10 minutes. Subsequently, they
were incubated with a blocking buffer (PBSc/m containing
0.5% BSA) for 30 minutes, C8Mab-3 or recC8Mab-3(10 lg/mL; in a blocking buffer) for 1 hour, and Alexa Fluor
488-conjugated anti-rat IgG (1:400; in a blocking buffer) for 45
minutes. 4¢,6-Diamidino-2-phenylindole (DAPI; Thermo
Fisher Scientific, Inc.) was used for the cell nuclei staining. The
fluorescence images were acquired on a digital microscope BZX800 (Keyence, Osaka, Japan) using a 40 · objective.
Results
Establishment of anti-mCCR8 mAbs

We employed the CBIS method to develop anti-mCCR8
mAbs. In the CBIS method, we used stable transfectant immunization and flow cytometry-mediated hybridoma
screening.(20–26) In this study, a rat was immunized with
CHO/mCCR8 cells. Hybridomas were seeded into 96-wellplates, and CHO/mCCR8-positive and CHO-K1-negative
wells were selected. After limiting dilution, C8Mab-3 (rat
IgG1, kappa) was finally established. We further produced its
recombinant antibody (recC8Mab-3).
Flow cytometry

We conducted flow cytometry using C8Mab-3 andrecC8Mab-3 against CHO/mCCR8 and CHO-K1. Both
C8Mab-3 and recC8Mab-3 reacted with CHO/mCCR8, but‰
FIG. 2. Immunocytochemistry using C8Mab-3 andrecC8Mab-3. CHO-K1, CHO/mCCR8, P388, and J774-1
were treated with buffer control, 10 mg/mL C8Mab-3, or
recC8Mab-3, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG. DAPI was used for nuclear staining.
Scale bars, 20 mm. DAPI, 4¢,6-diamidino-2-phenylindole.

ANTI-MOUSE CCR8 FOR IMMUNOCYTOCHEMISTRY

not with CHO-K1 (Fig. 1). In addition, both C8Mab-3 andrecC8Mab-3 also recognized P388 and J774-1, which express
endogenous mCCR8 in a dose-dependent manner (Fig. 1).
Determination of the binding affinity of C8Mab-3
and recC8Mab-3

The binding affinity of C8Mab-3 and recC8Mab-3 with
CHO/mCCR8 was evaluated using flow cytometry. The KD
of C8Mab-3 and recC8Mab-3 for CHO/mCCR8 was
1.1 · 10-7 and 1.7 · 10-7 M, respectively (Supplementary
Fig. S1), indicating that C8Mab-3 possesses a low affinity for
CHO/mCCR8 cells.

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Immunocytochemical analysis using C8Mab-3
and recC8Mab-3

We explored the availability of C8Mab-3 and recC8Mab-3
in immunocytochemical analysis. First, C8Mab-3 (10 lg/mL)
and recC8Mab-3 (10 lg/mL), but not buffer control, boundto CHO/mCCR8 (Fig. 2), whereas both antibodies did not bindto CHO-K1 (Fig. 2), suggesting that C8Mab-3 and recC8Mab-3
specifically detect exogenous mCCR8. Next, C8Mab-3 andrecC8Mab-3, but not buffer control, are also bound to P388 andJ774-1 cells, suggesting that both antibodies recognize endogenous mCCR8 (Fig. 2). These results show that C8Mab-3
and recC8Mab-3 are available in immunocytochemical analysis
against exogenous and endogenous mCCR8.

113

Since CCR8 expression is increased in tumor-infiltrated
Tregs,(28) CCR8 is one of the promising targets depleting
Tregs selectively in tumors.
In our previous studies, we changed the isotype of mAbs
into mouse IgG2a to retain antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC), which caused high antitumor activities in mouse
xenograft models.(30–32) Since the subclass of C8Mab-3 is rat
IgG1, it does not possess ADCC and CDC. Therefore, infurther studies, the subclass of C8Mab-3 will be converted
into mouse IgG2a to assess the effect of depletion of CCR8+
Treg cells in cancer immunotherapy.
In summary, we developed a novel mAb for mCCR8,
C8Mab-3, in this study. C8Mab-3 and recC8Mab-3 apply toflow cytometry and immunofluorescence. These mAbs
would be valuable for elucidating the roles of CCR8 incancer.
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
Numbers: JP21am0401013 (to Y.K.) and JP21am0101078(to Y.K.).
Supplementary Material

Discussion

We developed a novel mAb for mCCR8, C8Mab-3,
using the CBIS method. Both C8Mab-3 and recC8Mab-3
bind to mCCR8 with low affinity (Supplementary Fig. S1),
but they specifically recognize exogenous and endogenous
mCCR8 in both flow cytometry (Fig. 1) and immunocytochemistry (Fig. 2). The confirmation to other applications, including Western blotting, immunoprecipitation,and immunohistochemistry, is required for basic study inthe future.
The secretion of CCL1, a ligand of CCR8, by
CD11b+CD14+ myeloid cells is involved in Treg cell infiltration.(27) Increased expression of CCR8 is observed in Treg
cells and natural killer T cells, especially in cancer patients.(28) Upon binding of CCL1 to CCR8, the expression of
FoxP3 is elevated through the STAT3 pathway, and the activated CCR8+ Treg cells strongly suppress antitumor immunity by secretion of IL-10 and granzyme B.(10) The overall
survival of breast cancer patients with high infiltration of
CCR8+FoxP3+ Tregs is significantly shorter than that of patients with low infiltration.(27) Therefore, therapies that target
CCL1–CCR8 molecules are desired in the clinic. Currently,
we have developed three mAbs for mCCR8 (C8Mab-1 to 3).
The determination of their epitopes and neutralizing activity for CCL1–CCR8 interaction is required for in vivo
experiments.
Tregs express several cell surface markers, including
CD25, CTLA-4, PD-1, ICOS, GITR, OX40, CD15s, CCR4,
and CCR8, and these markers can be used to deplete Tregs.
Tregs are also indispensable for preventing autoimmunity.
The removal of Tregs improves antitumor immunity and may
also trigger autoimmunity.(29) Therefore, we should specifically deplete Tregs in tumors for cancer immunotherapy.

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: January 11, 2022
Accepted: January 25, 2022