Identification of different functions of CD8+ T cell subpopulations by a novel monoclonal antibody.

The explicit identification of CD8+ T cell subpopulation is important for deciphering the role of CD8+ T cells for protecting our body against invading pathogens and cancer. Our generated monoclonal antibody (mAb), named FE-1H10, recognized two novel subpopulations of peripheral blood CD8+ T cells, FE-1H10+ and FE-1H10- CD8+ T cells. The molecule recognized by mAb FE-1H10 (FE-1H10 molecules) had a higher distribution on effector memory CD8+ T cell subsets. The functions of FE-1H10- and FE-1H10+ CD8+ T cells were investigated. T cell proliferation assays revealed that FE-1H10- CD8+ T cells exhibited a higher proliferation rate than FE-1H10+ CD8+ T cells, whereas FE-1H10+ CD8+ T cells produced higher levels of IFN-γ and TNF-α than FE-1H10- CD8+ T cells. In T cell cytotoxicity assays, FE-1H10+ CD8+ T cells were able to kill target cells better than FE-1H10- CD8+ T cells. RNA-sequencing analysis confirmed that these subpopulations were distinct: FE-1H10+ CD8+ T cells have higher expression of genes involved in effector functions (IFNG, TNF, GZMB, PRF1, GNLY, FASL, CX3CR1) while FE-1H10- CD8+ T cells have greater expression of genes related to memory CD8+ T cell populations (CCR7, SELL, TCF7, CD40LG). The results suggested that mAb FE-1H10 identifies two novel distinctive CD8+ T cell subpopulations. The FE-1H10+ CD8+ T cells carried a superior functionality in response to tumour cells. The uncover of these novel CD8+ T cell subpopulations may be the basis knowledge of an optional immunotherapy for the selection of potential CD8+ T cells in cancer treatment.

Transcriptome Analysis Reveals the Induction of Apoptosis-Related Genes by a Monoclonal Antibody against a New Epitope of CD99 on T-Acute Lymphoblastic Leukemia.

CD99 was demonstrated to be a potential target for antibody therapy on T-acute lymphoblastic leukemia (T-ALL). The ligation of CD99 by certain monoclonal antibodies (mAbs) induced T-ALL apoptosis. However, the molecular basis contributing to the apoptosis of T-ALL upon anti-CD99 mAb engagement remains elusive. In this study, using our generated anti-CD99 mAb clone MT99/3 (mAb MT99/3), mAb MT99/3 engagement strongly induced apoptosis of T-ALL cell lines, but not in non-malignant peripheral blood cells. By transcriptome analysis, upon mAb MT99/3 ligation, 13 apoptosis-related genes, including FOS, TNF, FASLG, BCL2A1, JUNB, SOCS1, IL27RA, PTPN6, PDGFA, NR4A1, SGK1, LPAR5 and LTB, were significantly upregulated. The epitope of CD99 recognized by mAb MT99/3 was then identified as the VDGENDDPRPP at residues 60-70 of CD99, which has never been reported. To the best of our knowledge, this is the first transcriptome data conducted in T-ALL with anti-CD99 mAb engagement. These findings provide new insights into CD99 implicated in the apoptosis of T-ALL. The identification of a new epitope and apoptosis-related genes that relate to the induction of apoptosis by mAb MT99/3 may serve as a new therapeutic target for T-ALL. The anti-CD99 mAb clone MT99/3 might be a candidate for further development of a therapeutic antibody for T-ALL therapy.

Neutralizing antibody and T-cell responses against SARS-CoV-2 variants by heterologous CoronaVac/ChAdOx-1 vaccination in elderly subjects with chronic obstructive pulmonary disease.

BACKGROUND: Data on humoral and cellular immune responses against SARS-CoV-2 after receiving heterologous CoronaVac/ChAdOx-1 (CoVac/ChAd) vaccination in subjects with chronic obstructive pulmonary disease (COPD) are still limited. Therefore, we determined the neutralizing antibody (NAb) and T-cell responses against SARS-CoV-2 wild type (WT) and variants of concern (VOCs) in COPD patients. METHODS: The levels of NAb as well as specific CD4 and CD8 T-cell responses against SARS-CoV-2 WT and VOCs were determined in COPD patients before and after vaccination. RESULTS: Four weeks after vaccinations, the median levels of % inhibition of NAb against SARS-CoV-2 WT, Alpha, Beta, and Delta variants were significantly higher compared to pre-vaccination. The induction of NAb against Omicron was very low compared to other variants. At four weeks after vaccination, in comparison to pre-vaccination, the increasing trend of TNF-α-, IFN-γ-, IL-4-, IL-17-, IL-10-, and FasL-producing CD4 T-cells upon stimulation with WT spike peptides were demonstrated. No difference in T-cell responses to spike peptides of Alpha, Beta, and Delta variants and their WT homologs was observed. CONCLUSION: Heterologous CoVac/ChAd vaccine induced the production of NAb against SARS-CoV-2 WT, Alpha, Beta, and Delta variants, but low for Omicron in COPD patients. Induction of CD4 T-cell subset responses was slightly observed by this vaccine regimen. CLINICAL TRIALS REGISTRY: This study was approved by the Clinical Trials Registry (Study ID: TCTR20210822002).

Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy.

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy.

Neutralizing Antibody and T-Cell Responses against SARS-CoV-2 Wild-Type and Variants of Concern in Chronic Obstructive Pulmonary Disease Subjects after ChAdOx-1/ChAdOx-1 Homologous Vaccination: A Preliminary Study.

Data on immunogenicity of adenovirus-vectored vaccine in chronic obstructive pulmonary disease (COPD) patients is limited. Therefore, we aimed to determine the humoral and cellular immune responses after homologous ChAdOx-1 vaccination in subjects with COPD. COPD subjects and age- and sex-matched healthy elderly receiving ChAdOx-1 homologous vaccination were included. The levels of neutralizing antibodies (NAb) and specific CD4 and CD8 T-cell responses against SARS-CoV-2 wild-type (WT) and variants of concern (VOCs: Alpha, Beta, Delta, and Omicron) were measured. Eight COPD patients were matched with eight control participants. After vaccination for 4 and 12 weeks, % inhibition of NAb against Alpha, Beta, and Delta in both groups were comparable and significantly higher than baseline. The percentage inhibition of NAb at the 12th week was significantly dropped from the 4th week in each group. The NAb against the Omicron variant, however, were much lower than Alpha, Beta, Delta variants. The increasing trend in the number of CD4 T-cells producing TNF-α, IFN-γ, IL-10, and FasL upon stimulation with spike peptides of WT and VOCs was observed in COPD patients compared to the healthy group. These responses were not observed in CD8 T-cells. Homologous ChAdOx-1 vaccination could induce comparable NAb against the SARS-CoV-2 WT, Alpha, Beta, Delta, and Omicron variants between COPD and healthy elderly. The CD4 T-cell responses did not differ between COPD patients and healthy control.

Optimization of culture conditions for stable expression of recombinant fc-fused human extracellular CD99 in HEK293T cells.

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.

Differential CD147 Functional Epitopes on Distinct Leukocyte Subsets.

CD147, a member of the immunoglobulin (Ig) superfamily, is widely expressed in several cell types. CD147 molecules have multiple cellular functions, such as migration, adhesion, invasion, energy metabolism and T cell activation. In particular, recent studies have demonstrated the potential application of CD147 as an effective therapeutic target for cancer, as well as autoimmune and inflammatory diseases. In this study, we elucidated the functional epitopes on CD147 extracellular domains in T cell regulation using specific monoclonal antibodies (mAbs). Upon T cell activation, the anti-CD147 domain 1 mAbs M6-1E9 and M6-1D4 and the anti-CD147 domain 2 mAb MEM-M6/6 significantly reduced surface expression of CD69 and CD25 and T cell proliferation. To investigate whether functional epitopes of CD147 are differentially expressed on distinct leukocyte subsets, PBMCs, monocyte-depleted PBMCs and purified T cells were activated in the presence of anti-CD147 mAbs. The mAb M6-1E9 inhibited T cell functions via activation of CD147 on monocytes with obligatory cell-cell contact. Engagement of the CD147 epitope by the M6-1E9 mAb downregulated CD80 and CD86 expression on monocytes and IL-2, TNF-α, IFN-γ and IL-17 production in T cells. In contrast, the mAb M6-1D4 inhibited T cell function via activation of CD147 on T cells by downregulating IL-2, TNF-α and IFN-γ. Herein, we demonstrated that certain epitopes of CD147, expressed on both monocytes and T cells, are involved in the regulation of T cell activation.

The presence of membrane bound CD99 ligands on leukocyte surface.

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.

Interaction of CD99 and its ligand upregulates IL-6 and TNF-α upon T cell activation.

CD99 has been reported to be involved in T cell regulation. CD99 ligand involvement in the regulation of T cell activation has been postulated. In this study, recombinant CD99 proteins were produced and used as a tool for determining the role of CD99 and its ligand interaction. Recombinant CD99 proteins induced the upregulation of IL-6 and TNF-α expression, but not IFN-γ, in anti-CD3 monoclonal antibody activated T cells. The cytokine alteration was not observed in unstimulated T cells indicating the cytokine upregulation required the signal from T cell activation. The upregulation of IL-6 and TNF-α was, in addition, observed in CD3- mononuclear cell population including monocytes and NK cells. The recombinant CD99 proteins, however, did not affect either CD25, CD69 or MHC class II expression or T cell proliferation, upon T cell activation. The CD99 ligands were demonstrated to be expressed on monocytes, NK cells and dendritic cells, but not on B and T cells. Our results indicated the presence of CD99 ligands on leukocyte surface. Interaction between CD99 and its ligands involves the regulation of cytokine production.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

Triggering of CD99 on monocytes by a specific monoclonal antibody regulates T cell activation.

CD99, a leukocyte surface glycoprotein, has been implicated in many cellular processes including cell adhesion, cell migration and T cell activation. Our previous study demonstrated the anti-CD99 monoclonal antibody (mAb) clone MT99/3 inhibited T cell activation; however, the mechanism is unclear. In this study, we demonstrated that CD99 expressed on monocytes played a role in the inhibition of T cell activation. Anti-CD99 mAb MT99/3 downregulated the expression of costimulatory molecule CD86, but upregulated IL-6, IL-10 and TNF-α production by monocytes. The inhibitory effect of mAb MT99/3 required cell to cell contact between monocytes and lymphocytes. The soluble mediators produced by monocytes alone were insufficient to induce hypo-function of T lymphocytes. In summary, we demonstrated that ligation of CD99 on monocytes by anti-CD99 mAb MT99/3 could mediate T cell hypo-responsiveness. These findings provide the first evidence of the role of CD99 on monocytes that contributes to T cell activation.

Ligation of Na, K ATPase ß3 subunit on monocytes by a specific monoclonal antibody mediates T cell hypofunction.

T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase ß3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase ß3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases.

Overexpression of ADAM9 in oral squamous cell carcinoma.

Overexpression of a disintegrin and metalloproteinase 9 (ADAM9) has been shown in various types of cancer. Some studies have reported inconclusive findings regarding chromosomal aberrations in the ADAM9-containing region and ADAM9 expression in oral cancer. Therefore, in this study, ADAM9 protein expression was determined and compared between oral squamous cell carcinoma (OSCC) and normal oral tissues, and between oral cancer cell lines and human oral keratinocytes (HOKs). In total, 34 OSCC and 10 healthy paraffin-embedded tissue sections were probed with an anti-ADAM9 antibody, and the immunohistochemical score was determined by multiplying the percentage of positively stained cells with the intensity score. Four different oral cancer and eight independent HOK cell lines were cultured, and the expression of membrane ADAM9 and active ADAM9 at 84 kDa in these cell lines was assayed by flow cytometry and western blot hybridization, respectively. The results showed that the median immunohistochemical score of ADAM9 expression in OSCC tissues was significantly greater than that in normal tissues (P<0.001). Furthermore, among OSCC cases, intense staining of ADAM9 expression was detected in well-differentiated and in moderately-differentiated OSCC; ADAM9 expression was also correlated with an increased degree of cell differentiation (r=0.557; P=0.001). Expression of membrane ADAM9 was present in 3/4 cancer cell lines. Expression of active ADAM9 varied among all the tested cell lines, but significantly higher ADAM9 expression was present in certain cancer cell lines than those in HOKs (P<0.05). In summary, ADAM9 expression is enhanced in OSCC and oral cancer cell lines, suggesting its role in the pathogenesis of oral cancer. Similar to the overexpression of ADAM9 in well-differentiated prostate cancer, high degrees of ADAM9 expression have also been observed in well-differentiated OSCC.

Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva.

BACKGROUND: Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva. RESULTS: With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured. CONCLUSIONS: A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

Exploitation of human CD99 expressing mouse myeloma cells as immunogen for production of mouse specific polyclonal antibodies.

In this study, we describe the application of a molecular biology technique for the production of mouse polyclonal antibodies (pAbs) specific to human cell surface molecules. Production of the pAb specific to the human CD99 surface molecule was used as the study model. The retroviral expression system was employed to generate human CD99 expressing mouse myeloma cells. After cell sorting and single cell cloning, a myeloma clone which stably expressed high levels of human CD99 on its surface was established. The human CD99 expressing mouse myeloma cells were then used as the immunogen for immunization of BALB/c mice. As endogenous proteins of mouse myeloma cells possess self-non-immunogenicity for BALB/c mice, after immunization, only the expressed human CD99 molecules induce antibody response. After three immunizations, high titers of mouse anti-CD99 pAbs were successfully produced. The produced pAb specifically reacted to both recombinant human CD99 and native CD99 molecules expressed on human blood cells. The established technology is simple and valuable for the production of pAbs specific to human CD99 membrane proteins which can be used for characterization of the CD99 molecule.

ß3 subunit of Na,K ATPase regulates T cell activation with no involvement of Na,K ATPase activity.

Na,K ATPase plays an important role in the regulation of Na(+) and K(+) ions that are required for normal resting membrane potential and various cellular functions. Na,K ATPase is composed of two subunits, α and ß subunits. Engagement of the ß subunit by an agonistic monoclonal antibody (mAb) P-3E10 inhibited T cell activation and induced the G0/G1 cell cycle arrest. In addition, mAb P-3E10 decreased CD25 expression. The mAb P-3E10, however, did not inhibit the proliferation of cell lines and the phagocytosis activity of phagocytes, and did not interfere with the Na,K ATPase activity. These results indicate that mAb P-3E10 reacts to the ß subunit and, as a consequence, brings about the regulation of the T cell activation without disturbing the Na,K pump activity. By sequential immunoprecipitation, we demonstrated the expression of the ß3 subunit free form apart from the α subunit. In this study, we propose that the ß3 subunits of Na,K ATPase are expressed separately from the α subunit, and play a role in regulation of the immune response.

A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.

Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.