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CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T-cell activation, cell adhesion, and cell migration. We have also demonstrated that CD99 and its ligands regulate proinflammatory cytokines in NK cells, monocytes and activated T cells. These data suggest CD99 as a potential therapeutic target in cancer. However, the molecular mechanisms by which CD99 and CD99 counter receptors participate in such processes are unclear. High-quality CD99 recombinant proteins produced in large amounts are essential for biological studies and clinical research. In this study, we optimized the various culture conditions for increasing amounts of recombinant protein production with good biological activity. Intracellular immunofluorescence staining was performed to identify the highly expressing CD99HIgG cells. We further investigated the culture conditions for recombinant protein production. A double antibody sandwich enzyme-linked immunosorbent assay was employed to determine the level of secreted CD99HIgG proteins in the culture supernatant of various culture conditions. Later, affinity chromatography using protein G was used to purify CD99HIgG proteins from the culture supernatant of three proper culture conditions. According to our previous report, which utilized Western blotting, the purified CD99HIgG obtained from all tested culture conditions is composed of the CD99 extracellular part fused with the human IgG Fc part in dimer form. For biological activity, the obtained CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor, resulting in the induction of cytokine production., Competing Interests: Declaration of competing interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: CD99, a leukocyte surface molecule, reportedly plays an important role in several cellular processes. However, the role of CD99 in T cell regulation remains unclear, as the CD99 ligand associated with T-cell regulation has not yet been identified. Our previous study showed that recombinant CD99 bound to CD99 ligands was expressed on monocytes, NK cells and dendritic cells. This interaction regulates the expression of IL-6 and TNF-α in CD3 + T cells following T cell activation. In the present study, we confirmed the presence of CD99 ligands in immune cells., Results: A recombinant CD99-human IgG fusion protein, CD99HIgG, was produced and used to search for CD99 ligand expression in various hematopoietic cell lines. Among several cell lines, THP-1 monocytic cell line showed strong positive reaction for CD99HIgG, and CD99 and CD99 ligand complexes were pulled-down using a DTSSP cross-linker. The study demonstrated the presence of the membrane bound CD99 ligand, and CD99 ligand candidates were identified via LC-MS/MS. These results may be useful to further identify the CD99 ligands, and to fully comprehend the role of CD99 in immunoregulation.

Objective: To examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3., Background: Engagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells., Methods: Total mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA., Results: CD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment., Conclusion: Our study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the nonphagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of nonphagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was downregulated. The proposed method will benefit the study of phagocytic mechanisms in the future.

The leukocyte surface molecule CD99 is an integral membrane glycoprotein encoded by the E2/MIC2 gene. This molecule is broadly expressed on cells of the hematopoietic system and displays two surface forms, a long 32 kDa form and a short 28 kDa form. While the complete function of the CD99 molecule is unclear, it has been reported to be involved in regulation of cell adhesion, migration, and apoptosis. Thus, several CD99 monoclonal antibodies (MAbs) have been generated for biochemical and functional studies of the CD99 molecule. In the present study two CD99 MAbs, MT99/1 and MT99/2, were produced. The MAbs recognized different epitopes of the CD99 molecule. MAb MT99/1, but not MT99/2, was appropriate for biochemical characterization. Binding of MAb MT99/1 with its epitope led to the induction of cell adhesion and apoptosis. The generated MAbs can be used for future study of the function and mechanism of the CD99 molecule, including its role in the immune system, and may have application in tumor diagnosis and treatment.