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

Introduction

Diacylglycerol kinases (DGKs) are a family of proteins that phosphorylate the cell membrane lipid diacylglycerol (DG) into phosphatidic acid.^(1–3) DG functions as an important second messenger in T cells.⁽⁴⁾ DG-mediated signal transduction downstream of T cell receptors (TCRs) is terminated by DGKα and DGKζ, 2 of the 10 DGK isoforms.⁽⁵⁾ DGKζ has been shown to be the dominant isoform.⁽⁶⁾ T cells deficient in either DGKα or DGKζ are hyper-responsive, leading to enhanced proliferation and secretion of cytokines 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 DGKζ may increase the efficacy of adoptive T cell and immune checkpoint therapies in the treatment of leukemia.⁽¹¹⁾

In the present study, we selected a commercially available rabbit anti-DGKζ monoclonal antibody (mAb) that is advantageous for immunohistochemical analysis and further characterized its binding epitope using enzyme-linked immunosorbent assay (ELISA).

Materials and Methods

Results

For immunohistochemical analysis, we used formalin-fixed and paraffin-embedded (FFPE) sections, including the tonsils of a patient with oropharyngeal squamous cell carcinoma, because DGKζ is known to be expressed in activated T cells.^(7–9) As shown in Figure 1, the rabbit anti-DGKζ mAb (clone: EPR22040-80) stained the T cells of the tonsils very strongly, indicating that EPR22040-80 is extremely useful for the detection of DGKζ in FFPE tissues.

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FIG. 1.

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

Next, we examined the binding epitope of EPR22040-80. We first produced 92 synthetic peptides of DGKζ, in which all cysteine residues were converted into serine residues to avoid 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 detected by EPR22040-80, except for P790A, Q791A, G792A, and L795A, indicating Pro790, Gln791, Gly792, and Leu795 are included in the critical epitope of EPR22040-80. The epitope of EPR22040-80 is summarized in Figure 2.

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FIG. 2.

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

We further performed an inhibition assay using immunohistochemistry. Although EPR22040-80 reacted with the T cells 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 DGKζ is critical for EPR22040-80 detection.

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FIG. 3.

Inhibition assay. Tissue sections were incubated with 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 the Envision+ Kit for rabbit (Agilent Technologies, Inc.) for 30 minutes. Scale bar = 100 μm. (I, J) Hematoxylin and eosin staining.

Discussion

We have developed anti-DGKα (clone: DaMab-2)⁽¹²⁾ and anti-DGKγ (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 DGKα are important for binding of DaMab-2 to DGKα.⁽¹⁴⁾ These findings could be applied for the production of more functional anti-DGKα mAbs. We have not developed anti-DGKζ mAbs, which are useful for 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-DGKζ mAbs. Among several anti-DGKζ mAbs, a rabbit anti-DGKζ mAb (clone: EPR22040-80 from Abcam) stained the T cells of tonsils very strongly. According to the Abcam product datasheet, EPR22040-80 is produced by immunizing recombinant fragments within human DGKζ (700 aa–1000 aa). However, the critical epitope of EPR22040-80 has not been determined. In this study, ELISA demonstrated that Pro790, Gln791, Gly792, and Leu795 of DGKζ are included in the critical epitope of EPR22040-80. This epitope seems to be included between catalytic domain and ankyrin repeats of DGKζ (Fig. 2). EPR22040-80 could be valuable for immunohistochemical analyses and in clarifying our understanding of the distribution of DGKζ-expressing T cells in a wide range of pathophysiological tissues. These findings can be applied to the production of more functional anti-DGKζ mAbs.