MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 3, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0005

Epitope Mapping of Anti-Diacylglycerol Kinase z
Monoclonal Antibody for the Detection of T Cells
by Immunohistochemical Analyses

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Yukinari Kato,1,2 Shunsuke Itai,1 Shinji Yamada,1 Hiroyoshi Suzuki,3 and Mika K. Kaneko1

The diacylglycerol kinases (DGKs) are a family of proteins that catalyze the phosphorylation of the cell
membrane lipid diacylglycerol (DG), a cellular component that is important in lipid biochemistry and signaltransduction, into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has
previously been reported to be terminated in most cases by one of 10 DGK isoforms, DGKz. In this study, we
performed immunohistochemical analysis using a rabbit anti-DGKz monoclonal antibody (mAb) (clone
EPR22040-80) against tissues from the tonsils of a patient with oropharyngeal squamous cell carcinoma. We
demonstrated that many DGKz-expressing T cells are localized in the tonsils. We further characterized thebinding epitope using an enzyme-linked immunosorbent assay and found that Pro790, Gln791, Gly792, andLeu795 residues of DGKz are important for facilitating anti-DGKz mAb binding to DGKz. This anti-DGKz
mAb could be valuable in immunohistochemical analyses in determining the distribution of DGKz-expressing
T cells in pathophysiological tissues.
Keywords: diacylglycerol kinase, DGKz, monoclonal antibody, epitope mapping

characterized its binding epitope using enzyme-linked immunosorbent assay (ELISA).

Introduction

Diacylglycerol kinases (DGKs) are a family ofproteins that phosphorylate the cell membrane lipid
diacylglycerol (DG) into phosphatidic acid.(1–3) DG functions as an important second messenger in T cells.(4) DGmediated signal transduction downstream of T cell receptors(TCRs) is terminated by DGKa and DGKz, 2 of the 10 DGK
isoforms.(5) DGKz has been shown to be the dominant isoform.(6) T cells deficient in either DGKa or DGKz are hyperresponsive, leading to enhanced proliferation and secretion ofcytokines in response to TCR activation.(7–9) Riese et al.
demonstrated that CD8+ T cells deficient in DGKs exhibit
enhanced activity against xenografts after adoptive transfer
of T cells when expressing TCRs or chimeric antigen receptors specific for tumor antigens.(10) Jing et al. reported
that targeting DGKz may increase the efficacy of adoptive T
cell and immune checkpoint therapies in the treatment ofleukemia.(11)In the present study, we selected a commercially available
rabbit anti-DGKz monoclonal antibody (mAb) that is advantageous for immunohistochemical analysis and further

1
2
3

Materials and Methods
Immunohistochemical analyses

We used tissues from a patient with oropharyngeal
squamous cell carcinoma who had undergone surgery at
the Sendai Medical Center. Informed consent for sample
procurement and subsequent data analyses was obtained
from the patient or the patient’s guardian. The tissue samples
were processed to produce 4-mm paraffin-embedded tissue
sections that were autoclaved in EnVision FLEX Target
Retrieval Solution High pH (Agilent Technologies, Inc.,
Santa Clara, CA) for 20 minutes and then blocked using
the SuperBlock T20 (PBS) blocking buffer (Thermo
Fisher Scientific, Inc., Waltham, MA). Samples wereincubated with rabbit anti-DGKz mAb (clone: EPR2204080; 1/4000 dilution; Abcam, Cambridge, United Kingdom)for 1 hour at room temperature and then treated using theEnvision+ Kit for rabbit (Agilent Technologies, Inc.) for
30 minutes. The tissue sections were stained using 3,3¢-

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.

124

EPITOPE OF ANTI-DGKZETA mAb

diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 minutes and counterstained using hematoxylin (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan).

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Enzyme-linked immunosorbent assay

The DGKz peptides synthesized using PEPScreen (SigmaAldrich Corp., St. Louis, MO) were immobilized on Nunc
MaxiSorp 96-well immunoplates (Thermo Fisher Scientific,Inc.) at concentrations of 10 mg/mL for 30 minutes at 37C.
Following blocking with SuperBlock T20 (PBS) blocking
buffer, the plates were incubated with 1 mg/mL of rabbit antiDGKz mAb, followed by 1:1000 dilution of peroxidaseconjugated anti-rabbit IgG (Agilent Technologies, Inc.).
The enzymatic reaction was performed using 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific, Inc.). Opticaldensity was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These
reactions were performed at 37C using a total sample
volume of 50–100 mL.

125
Inhibition assay

The 4-mm paraffin-embedded tissue sections were directly
autoclaved in EnVision FLEX Target Retrieval Solution
High pH (Agilent Technologies, Inc.) for 20 minutes andblocked using the SuperBlock T20 (PBS) blocking buffer(Thermo Fisher Scientific, Inc.), incubated with a rabbit antiDGKz mAb (clone: EPR22040-80; 1/4000 dilution; Abcam)
plus peptides (1 mg/mL) for 1 hour at room temperature, andthen treated using the Envision+ Kit for rabbit (Agilent
Technologies, Inc.) for 30 minutes. The tissue sections were
stained using DAB (Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin(FUJIFILM Wako Pure Chemical Corporation).
Results

For immunohistochemical analysis, we used formalinfixed and paraffin-embedded (FFPE) sections, including thetonsils of a patient with oropharyngeal squamous cell carcinoma, because DGKz is known to be expressed in activated T
cells.(7–9) As shown in Figure 1, the rabbit anti-DGKz mAb

FIG. 1. Immunohistochemical analysis of DGKz against oropharyngeal squamous cell carcinomas. Tissue sections wereincubated with EPR22040-80 (1/4000 dilution; A, D, G, J) or blocking buffer (B, E, H, K) for 1 hour at room temperatureand treated using the Envision+ Kit for rabbit (Agilent Technologies, Inc.) for 30 minutes. Scale bar = 100 mm. Hematoxylin
and eosin staining (C, F, I, L). DGK, diacylglycerol kinase.

Table 1. Determination of EPR22040-80 Epitopeby Enzyme-Linked Immunosorbent Assay

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PeptideSequence

1–20
MEPRDGSPEARSSDSESASA
11–30
RSSDSESASASSSGSERDAG
21–40
SSSGSERDAGPEPDKAPRRL
31–50
PEPDKAPRRLNKRRFPGLRL
41–60
NKRRFPGLRLFGHRKAITKS
51–70
FGHRKAITKSGLQHLAPPPP
61–80
GLQHLAPPPPTPGAPSSESE
71–90
TPGAPSSESERQIRSTVDWS
81–100 RQIRSTVDWSESATYGEHIW
91–110 ESATYGEHIWFETNVSGDFS
101–110 FETNVSGDFSYVGEQYSVAR
111–130 YVGEQYSVARMLQKSVSRRK
121–140 MLQKSVSRRKSAASKIVVHT
131–150 SAASKIVVHTPSIEQLEKIN
141–160 PSIEQLEKINFRSKPSFRES
151–170 FRSKPSFRESGSRNVREPTF
161–180 GSRNVREPTFVRHHWVHRRR
171–190 VRHHWVHRRRQDGKSRHSGK
181–200 QDGKSRHSGKGFQQKFTFHS
191–210 GFQQKFTFHSKEIVAISSSW
201–220 KEIVAISSSWSKQAYHSKVS
211–230 SKQAYHSKVSSFMLQQIEEP
221–240 SFMLQQIEEPSSLGVHAAVV
231–250 SSLGVHAAVVIPPTWILRAR
241–260 IPPTWILRARRPQNTLKASK
251–270 RPQNTLKASKKKKRASFKRK
261–280 KKKRASFKRKSSKKGPEEGR
271–290 SSKKGPEEGRWRPFIIRPTP
281–300 WRPFIIRPTPSPLMKPLLVF
291–310 SPLMKPLLVFVNPKSGGNQG
301–320 VNPKSGGNQGAKIIQSFLWY
311–330 AKIIQSFLWYLNPRQVFDLS
321–340 LNPRQVFDLSQGGPKEALEM
331–350 QGGPKEALEMYRKVHNLRIL
341–360 YRKVHNLRILASGGDGTVGW
351–370 ASGGDGTVGWILSTLDQLRL
361–380 ILSTLDQLRLKPPPPVAILP
371–390 KPPPPVAILPLGTGNDLART
381–400 LGTGNDLARTLNWGGGYTDE
391–410 LNWGGGYTDEPVSKILSHVE
401–420 PVSKILSHVEEGNVVQLDRW
411–430 EGNVVQLDRWDLHAEPNPEA
421–440 DLHAEPNPEAGPEDRDEGAT
431–450 GPEDRDEGATDRLPLDVFNN
441–460 DRLPLDVFNNYFSLGFDAHV
451–470 YFSLGFDAHVTLEFHESREA
461–480 TLEFHESREANPEKFNSRFR
471–490 NPEKFNSRFRNKMFYAGTAF
481–500 NKMFYAGTAFSDFLMGSSKD
491–510 SDFLMGSSKDLAKHIRVVSD
501–520 LAKHIRVVSDGMDLTPKIQD
511–530 GMDLTPKIQDLKPQSVVFLN
521–540 LKPQSVVFLNIPRYSAGTMP
531–550 IPRYSAGTMPWGHPGEHHDF
541–560 WGHPGEHHDFEPQRHDDGYL
551–570 EPQRHDDGYLEVIGFTMTSL
561–580 EVIGFTMTSLAALQVGGHGE
571–590 AALQVGGHGERLTQSREVVL
581–600 RLTQSREVVLTTSKAIPVQV
591–610 TTSKAIPVQVDGEPSKLAAS
601–620 DGEPSKLAASRIRIALRNQA
611–630 RIRIALRNQATMVQKAKRRS
621–640 TMVQKAKRRSAAPLHSDQQP
631–650 AAPLHSDQQPVPEQLRIQVS

Table 1. (Continued)

EPR22040-80
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—

PeptideSequence

641–660 VPEQLRIQVSRVSMHDYEAL
651–670 RVSMHDYEALHYDKEQLKEA
661–680 HYDKEQLKEASVPLGTVVVP
671–690 SVPLGTVVVPGDSDLELSRA
681–700 GDSDLELSRAHIERLQQEPD
691–710 HIERLQQEPDGAGAKSPTSQ
701–720 GAGAKSPTSQKLSPKWSFLD
711–730 KLSPKWSFLDATTASRFYRI
721–740 ATTASRFYRIDRAQEHLNYV
731–750 DRAQEHLNYVTEIAQDEIYI
741–760 TEIAQDEIYILDPELLGASA
751–770 LDPELLGASARPDLPTPTSP
761–780 RPDLPTPTSPLPTSPSSPTP
771–790 LPTSPSSPTPRSLQGDAAPP
781–800 RSLQGDAAPPQGEELIEAAK
791–810 QGEELIEAAKRNDFSKLQEL
801–820 RNDFSKLQELHRAGGDLMHR
811–830 HRAGGDLMHRDEQSRTLLHH
821–840 DEQSRTLLHHAVSTGSKDVV
831–850 AVSTGSKDVVRYLLDHAPPE
841–860 RYLLDHAPPEILDAVEENGE
851–870 ILDAVEENGETSLHQAAALG
861–880 TSLHQAAALGQRTISHYIVE
871–890 QRTISHYIVEAGASLMKTDQ
881–900 AGASLMKTDQQGDTPRQRAE
891–910 QGDTPRQRAEKAQDTELAAY
901–920 KAQDTELAAYLENRQHYQMI
911–929 LENRQHYQMIQREDQETAVEPR22040-80
—
—
—
—
—
—
—
—
—
—
—
—
—
—
+++
—
—
—
—
—
—
—
—
—
—
—
—
—

+++, OD655 S 0.6; —, OD655 < 0.2.

Table 2. Determination of EPR22040-80 Epitopeby Enzyme-Linked Immunosorbent Assay
Using Point Mutants(continued)

Mutation

Sequence

EPR22040-80

R781A
S782A
L783A
Q784A
G785A
D786A
A787G
A788G
P789A
P790A
Q791A
G792A
E793A
E794A
L795A
I796A
E797A
A798G
A799G
K800A

ASLQGDAAPPQGEELIEAAKRALQGDAAPPQGEELIEAAKRSAQGDAAPPQGEELIEAAKRSLAGDAAPPQGEELIEAAKRSLQADAAPPQGEELIEAAKRSLQGAAAPPQGEELIEAAKRSLQGDGAPPQGEELIEAAKRSLQGDAGPPQGEELIEAAKRSLQGDAAAPQGEELIEAAKRSLQGDAAPAQGEELIEAAKRSLQGDAAPPAGEELIEAAKRSLQGDAAPPQAEELIEAAKRSLQGDAAPPQGAELIEAAKRSLQGDAAPPQGEALIEAAKRSLQGDAAPPQGEEAIEAAKRSLQGDAAPPQGEELAEAAKRSLQGDAAPPQGEELIAAAKRSLQGDAAPPQGEELIEGAKRSLQGDAAPPQGEELIEAGKRSLQGDAAPPQGEELIEAAA

+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+
—
+++
+++
—
+++
+++
+++
+++
+++

Mutated amino acids (Ala or Gly) are shown in bold letters.
+++, OD655 S 0.6; ++, 0.4 & OD655 < 0.6; +, 0.2 & OD655 < 0.4;—, OD655 < 0.2.

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EPITOPE OF ANTI-DGKZETA mAb

127(clone: EPR22040-80) stained the T cells of the tonsils verystrongly, indicating that EPR22040-80 is extremely usefulfor the detection of DGKz in FFPE tissues.
Next, we examined the binding epitope of EPR22040-80.
We first produced 92 synthetic peptides of DGKz, in which
all cysteine residues were converted into serine residues toavoid self-aggregation (Table 1). Using ELISA, we demonstrated that the 781–800 position peptide was detected by
EPR22040-80. We also synthesized 20 peptides, including a
number of point mutations of the peptide at positions 781–
800 (Table 2). Almost all the point mutations were detectedby EPR22040-80, except for P790A, Q791A, G792A, and
L795A, indicating Pro790, Gln791, Gly792, and Leu795 areincluded in the critical epitope of EPR22040-80. The epitopeof EPR22040-80 is summarized in Figure 2.
We further performed an inhibition assay using immunohistochemistry. Although EPR22040-80 reacted with the Tcells of oropharyngeal squamous cell carcinoma, these reactions were completely neutralized by R781A. L795A did
not block the reactions of EPR22040-80 (Fig. 3). These data
supported the contention that Leu795 of DGKz is critical for
EPR22040-80 detection.
Discussion

We have developed anti-DGKa (clone: DaMab-2)(12)and anti-DGKg (clone: DgMab-6)(13) mAbs, both of which
are extremely useful for immunocytochemical analysis. We
characterized the binding epitope of DaMab-2 using Western blots and revealed that the Cys246, Lys249, Pro252,
and Cys253 residues of DGKa are important for bindingof DaMab-2 to DGKa.(14) These findings could be applied
for the production of more functional anti-DGKa mAbs.
We have not developed anti-DGKz mAbs, which are usefulfor immunocytochemical and immunohistochemical analyses. In particular, it is difficult to develop mAbs for immunohistochemical analyses against FFPE tissue sections.
Therefore, we first characterized commercially available anti-DGKz mAbs. Among several anti-DGKz mAbs, arabbit anti-DGKz mAb (clone: EPR22040-80 from Abcam)
stained the T cells of tonsils very strongly. According to the
Abcam product datasheet, EPR22040-80 is produced byimmunizing recombinant fragments within human DGKz
(700 aa–1000 aa). However, the critical epitope of EPR22040-

FIG. 2. Schematic illustration of EPR22040-80 epitope.
Red amino acids, strong reaction with EPR22040-80.

FIG. 3. Inhibition assay. Tissue sections were incubatedwith EPR22040-80 (A, B), EPR22040-80+R781A (C, D),
EPR22040-80+L795A (E, F), or blocking buffer (G, H)
for 1 hour at room temperature and treated using theEnvision+ Kit for rabbit (Agilent Technologies, Inc.) for
30 minutes. Scale bar = 100 mm. (I, J) Hematoxylin andeosin staining.

80 has not been determined. In this study, ELISA demonstrated that Pro790, Gln791, Gly792, and Leu795 of DGKz
are included in the critical epitope of EPR22040-80. Thisepitope seems to be included between catalytic domain andankyrin repeats of DGKz (Fig. 2). EPR22040-80 could bevaluable for immunohistochemical analyses and in clarifying
our understanding of the distribution of DGKz-expressing
T cells in a wide range of pathophysiological tissues. Thesefindings can be applied to the production of more functionalanti-DGKz mAbs.

128
Acknowledgments

We thank Takuro Nakamura, Miyuki Yanaka, Kayo Hisamatsu, Saori Handa, Yoshimi Nakamura, and Maki Takahashi
for their excellent technical assistance. This research was supported, in part, by AMED under Grant Nos. JP18am0101te078(Y.K.), JP18am0301010 (Y.K.), and JP18ae0101028 (Y.K.),
and by JSPS KAKENHI Grant no. 17K07299 (M.K.K.) and
Grant no. 16K10748 (Y.K.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. The other authors have no conflict of interest.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi
Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: February 1, 2019
Accepted: April 24, 2019