MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 5, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0016
Open camera or QR reader andscan code to access this article
and other resources online.

KLMab-1: An Anti-human KLRG1 Monoclonal
Antibody for Immunocytochemistry

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

Masaki Saito,1 Hiroyuki Suzuki,1 Teizo Asano,2 Tomohiro Tanaka,2 Takeo Yoshikawa,3Mika K. Kaneko,2 and Yukinari Kato1–3

Immune checkpoint molecules have received attention as targets of cancer immunotherapy. Killer cell lectinlike receptor subfamily G member 1 (KLRG1) is one of the immune checkpoint molecules expressed in CD4+ T,
CD8+ T, and natural killer (NK) cells. KLRG1 exhibits antiviral and antitumor immunity, and its expression
in T and NK cells is upregulated by viral infectious diseases and some tumors. Thus, monoclonal antibodies(mAbs) for KLRG1 would be useful tools for the diagnosis and immunotherapy against viral infectious diseases
and cancers. We have developed anti-human KLRG1 (hKLRG1) mAb (clone KLMab-1, mouse IgG1, kappa) by
the Cell-Based Immunization and Screening method. We have also demonstrated that KLMab-1 recognizes
both exogenous and endogenous hKLRG1 in flow cytometry. In this study, we first showed that KLMab-1 andits recombinant mAb (recKLMab-1) bound to exogenous hKLRG1 overexpressed in Chinese hamster ovary(CHO)-K1 cells, but not in parental CHO-K1 cells, in immunocytochemistry. We next showed that both mAbs
detected endogenous hKLRG1 expressed in human NK cells. These results demonstrate that KLMab-1 andrecKLMab-1 are available for immunocytochemistry.
Keywords: KLRG1, KLMab-1, monoclonal antibody, immunocytochemistry

IntroductionTcells and natural killer (NK) cells play crucial
roles in antiviral and antitumor immunity.1–4 During the
response to viral infection or cancer development, T cells areactivated, expanded, and differentiated into effector andmemory T cells. Activated NK cells migrate to the infected ortumor site, and the NK cells eliminate the target cells throughthe production of cytokines and exhibition of cytolytic activity. In contrast, the activities of T and NK cells are suppressedby immune checkpoint molecules, including programmed
cell death 1 (PD-1) and cytotoxic T lymphocyte-associatedantigen 4 (CTLA-4).5
Accordingly, immune checkpoint molecules have drawn
attention as targets for cancer immunotherapy. In fact, specific monoclonal antibodies (mAbs) against PD-1 and CTLA-4
have provided great advances in the medical treatment ofcancers.6 However, developing novel mAbs against otherimmune checkpoint molecules has been required because
the number of patients who respond to anti-PD-1 or antiCTLA-4 mAbs is limited.7
Killer cell lectin-like receptor subfamily G member 1(KLRG1), a lectin-like type II transmembrane protein, is an
immune checkpoint molecule expressed in CD4+ T, CD8+ T,
and NK cells.8–10 It harbors an immunoreceptor tyrosinebased inhibitory motif (ITIM) in its cytoplasmic region.
Upon binding to KLRG1 ligands including E-cadherin,
KLRG1 evokes inhibitory signaling through recruitmentof Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) and Src homology region 2
domain-containing phosphatase 2 (SHP2) to ITIM. Then,KLRG1 attenuates interferon g production in T and NK cells,
and suppresses NK cell-mediated cytotoxicity.8,10–16
The mechanism contributes to the progression of viral
infection and tumor by KLRG1. Moreover, the expression of
KLRG1 is increased in NK cells of virus-infected mice andDepartments of 1Molecular Pharmacology, 2Antibody Drug Development, and 3Pharmacology, Tohoku University Graduate Schoolof Medicine, Sendai, Miyagi, Japan.

279

280

T cells of cervical and colorectal cancer patients.8,17,18 These
reports have suggested that KLRG1 can be a target molecule for the diagnosis and immunotherapy of viral infectious
diseases and some cancers.
We have established mAbs against cell surface-expressing
membrane proteins by the Cell-Based Immunization andScreening, including CCR3,19–21 CCR8,22–25 CCR9,26,27
CD10,28,29 CD19,30 CD20,31,32 CD44,33 CD133,34 EpCAM,35,36 HER3,37 PD-L1,38 podoplanin,39–53 TIGIT,54 andTROP2.55,56 We have also established an antihuman KLRG1(hKLRG1) mAb (clone KLMab-1; mouse IgG1, kappa),
which reacts to endogenous and exogenous hKLRG1 inflow cytometry.57 In this study, we showed that KLMab-1
and its recombinant mAb (recKLMab-1) are available for
immunocytochemistry against endogenous and exogenoushKLRG1.

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 cells were obtained
from the American Type Culture Collection (Manassas, VA).
CHO-K1 cells overexpressed with human KLRG1 (CHO/hKLRG1) were established previously.57 CHO-K1 and CHO/hKLRG1 were cultured in Roswell Park Memorial Institute
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mLof penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mLof amphotericin B (Nacalai Tesque, Inc.). The cells weremaintained in a humidified atmosphere at 37C and 5%
carbon dioxide. Human NK cells (donor lot. 4022602, purity>70%) were purchased from Takara Bio (Shiga, Japan).
Antibodies

The development of KLMab-1 was described in ourprevious report.57 To generate a recombinant KLMab-1(recKLMab-1), VH and CH cDNAs of KLMab-1 were subcloned into the pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan), and VL and CL cDNAsof KLMab-1 were subcloned into the pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation). An antihKLRG1 mAb (clone SA231A2) was purchased from BioLegend (San Diego, CA). Alexa Fluor 488-conjugatedanti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).
Immunocytochemistry

For immunocytochemistry of CHO-K1 and CHO/hKLRG1cells, the cells were attached to an acid-wash coverslip andwere fixed with 4% paraformaldehyde (PFA) in phosphatebuffered saline (4% PFA/PBS) for 10 minutes. Subsequently,
the cells were incubated with the blocking buffer (PBS
supplemented with 0.2 mM Ca2+, 2 mM Mg2+, and 0.5%
bovine serum albumin) for 30 minutes, primary antibodies(10 lg/mL in the blocking buffer) for 1 hour, and Alexa Fluor
488-conjugated anti-mouse IgG (1:400 dilution in theblocking buffer) for 45 minutes.
For immunocytochemistry of NK cells, the suspension of
NK cells was centrifuged at 270 · g for 5 minutes at room
temperature, and the cell pellet was suspended in and fixed

SAITO ET AL.

with 4% PFA/PBS for 10 minutes. The cells were further
suspended in the blocking buffer for 30 minutes, primary
antibodies (10 mg/mL in the blocking buffer) for 2 hours, andAlexa Fluor 488-conjugated anti-mouse IgG (1:400 dilution
in the blocking buffer) for 45 minutes. 4’,6-Diamidino-2phenylindole (Thermo Fisher Scientific, Inc.) was used fornuclear staining of CHO-K1, CHO/hKLRG1, and NK cells.
Fluorescence images were acquired using a 40 · objective
on a digital microscope (BZ-X800; Keyence, Osaka, Japan).
Results

Our flow cytometric analysis revealed that CHO/hKLRG1cells highly express hKLRG1 on the cell surface.57 In thisstudy, we applied KLMab-1 and recKLMab-1 in immunocytochemistry using CHO/hKLRG1 cells and found that
KLMab-1 and recKLMab-1, but not buffer control, bound to
CHO/hKLRG1 cells (Fig. 1A). In particular, hKLRG1 wasstrongly detected at the plasma membrane. Both mAbs did
not react to CHO-K1 cells (Fig. 1B). A commercially available anti-hKLRG1 mAb (clone SA231A2) also bound to
CHO/hKLRG1 cells, but not CHO-K1 cells (Fig. 1A, B).
This result shows that KLMab-1 and recKLMab-1 recognize
exogenous hKLRG1 in immunocytochemistry.
We previously showed that KLMab-1 detects endogenously expressing hKLRG1 in human NK cells in flow
cytometry.57 In this study, we incubated NK cells with
KLMab-1 and recKLMab-1 and found that both mAbs, aswell as SA231A2, bound to NK cells (Fig. 1C). This result
demonstrates that KLMab-1 and recKLMab-1 recognize
endogenous hKLRG1 in immunocytochemistry.
Discussion

In this study, we demonstrated that KLMab-1 andrecKLMab-1 were applicable for immunocytochemistry
against endogenous and exogenous hKLRG1. The mAbs
would become useful tools for the detection of hKLRG1positive T and NK cells in viral infectious diseases andcancers.
KLMab-1 and recKLMab-1 provided images with highsignal-to-noise ratios against not only exogenous hKLRG1
overexpressed in CHO-K1 cells but also endogenous hKLRG1
expressed in NK cells. Our previous study showed that
KLMab-1 weakly detected endogenous hKLMab-1 in flow
cytometry.57 We suppose that KLMab-1 and recKLMab-1
are suitable for immunocytochemistry. Moreover, KLMab-1
and recKLMab-1 strongly detected hKLRG1 at the plasma
membrane, which was clearly represented in CHO/hKLRG1cells. The result indicates that the mAbs would be able
to identify the intracellular distribution of hKLRG1, especially by colabeling the cells with any organelle markers. The
information about the intracellular distribution of hKLRG1
would provide advantages in uncovering the unknown functions of KLRG1.
To uncover the KLRG1’s roles and to detect KLRG1positive cells, other applications of KLMab-1 andrecKLMab-1 are also required. In the future, we would liketo test other applications, including immunohistochemistry,
immunoprecipitation, and Western blotting.
Furthermore, confirming the availability of KLMab-1
and recKLMab-1 in viral infectious diseases and cancers
is one of our goals. Some studies have demonstrated that

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

FIG. 1. Immunocytochemistry of hKLRG1 using KLMab-1 and recKLMab-1. (A, B) CHO/hKLRG1 cells (A) or CHO-K1(B) cells were incubated with buffer control, SA231A2 (10 mg/mL), KLMab-1 (10 mg/mL), or recKLMab-1 (10 mg/mL) for 1
hour. Subsequently, the cells were incubated with Alexa 488-conjugated anti-mouse IgG and DAPI for 45 minutes. (C)Immunocytochemistry of endogenously expressing hKLRG1. NK cells were incubated with SA231A2 (10 mg/mL), KLMab-1(10 mg/mL), or recKLMab-1 (10 mg/mL) for 2 hours. Subsequently, NK cells were incubated with Alexa 488-conjugated antimouse IgG and DAPI for 45 minutes. Scale bars, 20 mm. CHO, Chinese hamster ovary; DAPI, 4’,6-diamidino-2-phenylindole;
hKLRG1, human KLRG1; KLRG1, Killer cell lectin-like receptor subfamily G member 1; NK, natural killer.
281

282

anti-KLRG1 mAb reduces cytokine productions in KLRG1overexpressed NK cells8 and KLRG1+ CD4+ T cells.58 An
anti-KLRG1 mAb reduces the progression of breast cancer
cells.59 In addition, blocking of KLRG1 signaling in CD8+
T cells by anti-E-cadherin antibody reduces the proliferation of the cells.60 These studies would promote us toinvestigate the antitumor and/or antiviral effects of KLMab-1
and recKLMab-1.
Author Disclosure Statement

No competing financial interests exist.

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers: JP22ama121008 (to Y.K.), JP22am0401013(to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730(to Y.K.), and JP21am0101078 (to Y.K.).
References

1. Kaech SM, Wherry EJ. Heterogeneity and cell-fate decisions in effector and memory CD8+ T cell differentiation
during viral infection. Immunity 2007;27:393–405; doi:
10.1016/j.immuni.2007.08.007
2. Huntington ND, Cursons J, Rautela J. The cancer-naturalkiller cell immunity cycle. Nat Rev Cancer 2020;20:437–
454; doi: 10.1038/s41568-020-0272-z.
3. Bjorkstrom NK, Strunz B, Ljunggren HG. Natural killer
cells in antiviral immunity. Nat Rev Immunol 2022;22:
112–123; doi: 10.1038/s41577-021-00558-3
4. Thommen DS, Schumacher TN. T cell dysfunction incancer. Cancer Cell 2018;33:547–562; doi: 10.1016/j.ccell.2018.03.012
5. Dyck L, Mills KHG. Immune checkpoints and their inhibition in cancer and infectious diseases. Eur J Immunol
2017;47:765–779; doi: 10.1002/eji.201646875
6. Ribas A, Wolchok JD. Cancer immunotherapy using
checkpoint blockade. Science 2018;359:1350–1355; doi:
10.1126/science.aar4060
7. Kraehenbuehl L, Weng CH, Eghbali S, et al. Enhancing
immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat Rev Clin Oncol 2022;19:37–50;
doi: 10.1038/s41571-021-00552-7
8. Robbins SH, Nguyen KB, Takahashi N, et al. Cutting edge:
Inhibitory functions of the killer cell lectin-like receptor
G1 molecule during the activation of mouse NK cells.
J Immunol 2002;168:2585–2589; doi: 10.4049/jimmunol.168.6.2585
9. Marcolino I, Przybylski GK, Koschella M, et al. Frequent
expression of the natural killer cell receptor KLRG1 inhuman cord blood T cells: Correlation with replicative
history. Eur J Immunol 2004;34:2672–2680; doi: 10.1002/
eji.200425282
10. Guthmann MD, Tal M, Pecht I. A secretion inhibitory
signal transduction molecule on mast cells is anotherC-type lectin. Proc Natl Acad Sci U S A 1995;92:9397–
9401; doi: 10.1073/pnas.92.20.9397
11. Tourdot BE, Brenner MK, Keough KC, et al. Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediatedinhibitory signaling is regulated by sequential phosphory-

SAITO ET AL.

lation mediated by distinct nonreceptor tyrosine kinases:
A case study involving PECAM-1. Biochemistry 2013;52:
2597–2608; doi: 10.1021/bi301461t
12. Tessmer MS, Fugere C, Stevenaert F, et al. KLRG1 binds
cadherins and preferentially associates with SHIP-1. Int
Immunol 2007;19:391–400; doi: 10.1093/intimm/dxm004
13. Herndler-Brandstetter D, Ishigame H, Shinnakasu R, et al.
KLRG1+ effector CD8+ T cells lose KLRG1, differentiate
into all memory T cell lineages, and convey enhanced
protective immunity. Immunity 2018;48:716.e8–729.e8;
doi: 10.1016/j.immuni.2018.03.015
14. Grundemann C, Bauer M, Schweier O, et al. Cutting edge:
Identification of E-cadherin as a ligand for the murine killer
cell lectin-like receptor G1. J Immunol 2006;176:1311–
1315; doi: 10.4049/jimmunol.176.3.1311
15. Ito M, Maruyama T, Saito N, et al. Killer cell lectin-likereceptor G1 binds three members of the classical cadherinfamily to inhibit NK cell cytotoxicity. J Exp Med 2006;
203:289–295; doi: 10.1084/jem.20051986
16. Rosshart S, Hofmann M, Schweier O, et al. Interaction of
KLRG1 with E-cadherin: New functional and structural
insights. Eur J Immunol 2008;38:3354–3364; doi: 10.1002/
eji.200838690
17. Saleh R, Taha RZ, Toor SM, et al. Expression of immune
checkpoints and T cell exhaustion markers in early andadvanced stages of colorectal cancer. Cancer Immunol
Immunother 2020;69:1989–1999; doi: 10.1007/s00262020-02593-w.
18. Guo Y, Feng Y, Fan P, et al. Expression and clinical significance of KLRG1 and 2B4 on T cells in the peripheral
blood and tumour of patients with cervical cancer. Immunol
Invest 2022;51(3):670–687; doi: 10.1080/08820139.2020.1867567
19. Asano T, Nanamiya R, Takei J, et al. Development of antimouse CC chemokine receptor 3 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:107–112; doi: 10.1089/mab.2021.0009
20. Asano T, Suzuki H, Tanaka T, et al. C3Mab-3: A monoclonal antibody for mouse CCR3 for flow cytometry
monoclon. Antib Immunodiagn Immunother 2022;41:74–
79; doi: 10.1089/mab.2021.0062
21. Saito M, Harigae Y, Li G, et al. C3Mab-2: An anti-mouse
CCR3 monoclonal antibody for immunocytochemistry.
Monoclon Antib Immunodiagn Immunother 2022;41:45–
49; doi: 10.1089/mab.2021.0050
22. Tanaka T, Nanamiya R, Takei J, et al. Development ofanti-mouse CC chemokine receptor 8 monoclonal antibodies for flow cytometry. Monoclon Antib Immunodiagn
Immunother 2021;40:65–70; doi: 10.1089/mab.2021.
0005
23. Saito M, Suzuki H, Tanaka T, et al. Development of ananti-mouse CCR8 monoclonal antibody (C8Mab-1) for flow
cytometry and immunocytochemistry monoclon. AntibImmunodiagn. Immunother 2022; [Online ahead of print],
doi: 10.1089/mab.2021.0069
24. Suzuki H, Saito M, Asano T, et al. C8Mab-3: An antimouse CCR8 monoclonal antibody for immunocytochemistry monoclon. Antib Immunodiagn Immunother 2022;41:
110–114; doi: 10.1089/mab.2022.0002
25. Saito M, Tanaka T, Asano T, et al. C8Mab-2: An antimouse C–C motif chemokine receptor 8 monoclonal
antibody for immunocytochemistry. Monoclon AntibImmunodiagn Immunother 2022;41:115–119; doi: 10.1089/
mab.2021.0045

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

ANTI-KLRG1 FOR ICC

26. Nanamiya R, Takei J, Asano T, et al. Development of antihuman CC chemokine receptor 9 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:101–106; doi: 10.1089/mab.2021.0007
27. Saito M, Suzuki H, Harigae Y, et al. C9Mab-1: An antimouse CCR9 monoclonal antibody for immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022;41:
120–124; doi: 10.1089/mab.2021.0052
28. Kawabata H, Ohishi T, Suzuki H, et al. A defucosylatedmouse anti-CD10 monoclonal antibody (31-mG2a-f) exerts
antitumor activity in a mouse xenograft model of renal cell
cancers monoclon. Antib Immunodiagn Immunother 2022;[Online ahead of print], doi: 10.1089/mab.2021.0049
29. Kawabata H, Suzuki H, Ohishi T, et al. A defucosylatedmouse anti-CD10 monoclonal antibody (31-mG2a-f) exerts
antitumor activity in a mouse xenograft model of CD10overexpressed tumors monoclon. Antib Immunodiagn
Immunother 2022;41:59–66; doi: 10.1089/mab.2021.0048
30. Yamada S, Kaneko MK, Sayama Y, et al. Development ofNovel Mouse Monoclonal Antibodies Against Human
CD19. Monoclon Antib Immunodiagn Immunother 2020;
39:45–50; doi: 10.1089/mab.2020.0003
31. Furusawa Y, Kaneko MK, Kato Y. Establishment of
C20Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967;
doi: 10.3892/ol.2020.11753
32. Furusawa Y, Kaneko MK, Kato Y. Establishment of ananti-CD20 monoclonal antibody (C20Mab-60) for immunohistochemical analyses. Monoclon Antib Immunodiagn
Immunother 2020;39:112–116; doi: 10.1089/mab.2020.0015
33. Yamada S, Itai S, Nakamura T, et al. Detection of highCD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5. Biochem Biophys Rep 2018;
14:64–68; doi: 10.1016/j.bbrep.2018.03.007
34. Itai S, Fujii Y, Nakamura T, et al. Establishment ofCMab-43, a sensitive and specific anti-CD133 monoclonal
antibody, for immunohistochemistry. Monoclon AntibImmunodiagn Immunother 2017;36:231–235; doi: 10.1089/
mab.2017.0031
35. Hosono H, Ohishi T, Takei J, et al. The anti-epithelial
cell adhesion molecule (EpCAM) monoclonal antibody
EpMab-16 exerts antitumor activity in a mouse model ofcolorectal adenocarcinoma. Oncol Lett 2020;20:383; doi:
10.3892/ol.2020.12246
36. Kaneko MK, Ohishi T, Takei J, et al. AntiEpCAM monoclonal antibody exerts antitumor activity against oral
squamous cell carcinomas. Oncol Rep 2020;44:2517–2526;
doi: 10.3892/or.2020.7808
37. Asano T, Ohishi T, Takei J, et al. Anti-HER3 monoclonal
antibody exerts antitumor activity in a mouse model ofcolorectal adenocarcinoma. Oncol Rep 2021;46(2):173;
doi: 10.3892/or.2021.8124
38. Yamada S, Itai S, Nakamura T, et al. Monoclonal antibody
L1Mab-13 detected human PD-L1 in lung cancers. Monoclon Antib Immunodiagn Immunother 2018;37:110–115;
doi: 10.1089/mab.2018.0004
39. Furusawa Y, Kaneko MK, Nakamura T, et al. Establishment of a monoclonal antibody PMab-231 for tiger podoplanin. Monoclon Antib Immunodiagn Immunother 2019;
38:89–95; doi: 10.1089/mab.2019.0003
40. Furusawa Y, Takei J, Sayama Y, et al. Development of ananti-bear podoplanin monoclonal antibody PMab-247 forimmunohistochemical analysis. Biochem Biophys Rep
2019;18:100644; doi: 10.1016/j.bbrep.2019.100644

283

41. Furusawa Y, Yamada S, Itai S, et al. Establishment of a
monoclonal antibody PMab-233 for immunohistochemical
analysis against Tasmanian devil podoplanin. Biochem
Biophys Rep 2019;18:100631; doi: 10.1016/j.bbrep.2019.100631
42. Furusawa Y, Yamada S, Itai S, et al. PMab-219: A
monoclonal antibody for the immunohistochemical analysis
of horse podoplanin. Biochem Biophys Rep 2019;18:
100616; doi: 10.1016/j.bbrep.2019.01.009
43. Furusawa Y, Yamada S, Itai S, et al. PMab-210: A
monoclonal antibody against pig podoplanin. Monoclon
Antib Immunodiagn Immunother 2019;38:30–36; doi:
10.1089/mab.2018.0038
44. Furusawa Y, Yamada S, Nakamura T, et al. PMab-235: A
monoclonal antibody for immunohistochemical analysis
against goat podoplanin. Heliyon 2019;5:e02063; doi:
10.1016/j.heliyon.2019.e02063
45. Kaneko MK, Sano M, Takei J, et al. Development andcharacterization of anti-sheep podoplanin monoclonal
antibodies PMab-253 and PMab-260. Monoclon AntibImmunodiagn Immunother 2020;39:144–155; doi: 10.1089/
mab.2020.0018
46. Kato Y, Furusawa Y, Itai S, et al. Establishment of an
anticetacean podoplanin monoclonal antibody PMab-237
for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113; doi: 10.1089/
mab.2019.0013
47. Kato Y, Furusawa Y, Yamada S, et al. Establishment of a
monoclonal antibody PMab-225 against alpaca podoplanin
for immunohistochemical analyses. Biochem Biophys Rep
2019;18:100633; doi: 10.1016/j.bbrep.2019.100633
48. Kato Y, Yamada S, Furusawa Y, et al. PMab-213: A
monoclonal antibody for immunohistochemical analysis
against pig podoplanin. Monoclon Antib Immunodiagn
Immunother 2019;38:18–24; doi: 10.1089/mab.2018.0048
49. Kato Y, Furusawa Y, Sano M, et al. Development of ananti-sheep podoplanin monoclonal antibody PMab-256 forimmunohistochemical analysis of lymphatic endothelial
cells. Monoclon Antib Immunodiagn Immunother 2020;39:
82–90; doi: 10.1089/mab.2020.0005
50. Takei J, Yamada S, Konnai S, et al. PMab-241 specificallydetects bear podoplanin of lymphatic endothelial cells in
the lung of brown bear. Monoclon Antib Immunodiagn
Immunother 2019;38:282–284; doi: 10.1089/mab.2019.0038
51. Tanaka T, Asano T, Sano M, et al. Development of
monoclonal antibody PMab-269 against california sea lion
podoplanin. Monoclon Antib Immunodiagn Immunother2021;40:124–133; doi: 10.1089/mab.2021.0011
52. Goto N, Suzuki H, Tanaka T, et al. Development of a
monoclonal antibody PMab-292 against ferret podoplanin.
Monoclon Antib Immunodiagn Immunother 2022;41:101–
109; doi: 10.1089/mab.2021.0067
53. Yamada S, Itai S, Nakamura T, et al. PMab-52: Specificand sensitive monoclonal antibody against cat podoplanin
for immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230; doi: 10.1089/mab.2017.0027
54. Takei J, Asano T, Nanamiya R, et al. Development ofanti-human T cell immunoreceptor with Ig and ITIM
domains (TIGIT) monoclonal antibodies for flow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:
71–75; doi: 10.1089/mab.2021.0006
55. Sayama Y, Kaneko MK, Kato Y. Development and characterization of TrMab6, a novel anti-TROP2 monoclonal

284

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

antibody for antigen detection in breast cancer. Mol Med
Rep 2021;23(2):92; doi: 10.3892/mmr.2020.11731
56. Sayama Y, Kaneko MK, Takei J, et al. Establishment of
a novel anti-TROP2 monoclonal antibody TrMab-29 for
immunohistochemical analysis. Biochem Biophys Rep
2021;25:100902; doi: 10.1016/j.bbrep.2020.100902
57. Asano T, Nanamiya R, Tanaka T, et al. Developmentof antihuman killer cell lectin-like receptor subfamily G
member 1 monoclonal antibodies for flow cytometry.
Monoclon Antib Immunodiagn Immunother 2021;40:76–
80; doi: 10.1089/mab.2021.0008
58. Hu Z, Zhao HM, Li CL, et al. The role of KLRG1 in humanCD4+ T-cell immunity against tuberculosis. J Infect Dis
2018;217:1491–1503; doi: 10.1093/infdis/jiy046
59. Chan IS, Knutsdottir H, Ramakrishnan G, et al. Cancer
cells educate natural killer cells to a metastasis-promoting
cell state. J Cell Biol 2020;219(9):e202001134; doi:
10.1083/jcb.202001134

SAITO ET AL.

60. Henson SM, Franzese O, Macaulay R, et al. KLRG1 signaling induces defective Akt (ser473) phosphorylationand proliferative dysfunction of highly differentiated CD8+
T cells. Blood 2009;113:6619–6628; doi: 10.1182/blood2009-01-199588

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: yukinari.kato.e6@tohoku.ac.jp
Received: April 6, 2022
Accepted: September 26, 2022