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Objective Lipopolysaccharide (LPS), a component of gram-negative bacteria, is a potent innate immune stimulus. The interaction of LPS with innate immune cells induces the production of proinflammatory cytokines and chemokines, thereby leading to the control of infection. In the present study, we investigated the effect of a wide range of LPS concentrations on the regulation of various proinflammatory cytokines and chemokines in human primary monocytes and T lymphocytes. Results We demonstrated that a very low concentration of LPS could regulate the production of cytokines and chemokines in monocytes but not T lymphocytes. Unexpectedly, very low concentrations of LPS (0.0025 and 0.005 ng/mL) could induce TNF-α and IL-6 production, respectively, in monocytes. Our findings provide evidence that in the presence of monocytes, even very low endotoxin contamination could induce cytokine production. We suggest that the recombinant proteins used to investigate immune functions must be thoroughly screened for endotoxins using a highly sensitive method.

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.

The immune system comprises a complex network of cells that continuously change during activation, infection, and the maintenance of balance. Immunophenotyping offers valuable insights into the regulation of immune responses. We systematically characterized the expression profile of CD99 ligands across distinct immune cell subsets using both conventional and high-dimensional flow cytometry. CD99 ligands were detected on NK cells and monocytes under both resting and IL-2-activated conditions, with non-classical monocytes and CD56 Dim NK cells exhibiting the highest expression levels. Notably, ligand expression in these subsets was further enhanced following IL-2 activation. In contrast, T lymphocytes (CD3+) displayed low basal levels of CD99 ligand expression, which increased modestly upon activation. Cellular activation was accompanied by an expansion of specific immune phenotypes characterized by elevated CD99 ligand expression alongside the upregulation of activation markers such as CD69 and CD137. Collectively, these findings suggest that the expression of the CD99 ligands may serve as an indicator of immune activation and demonstrate subset-specific regulation, particularly in response to IL-2 stimulation. These findings have revealed the distinct expression patterns of CD99 ligands, emphasizing their crucial role in modulating immune responses.

Cannabinoid receptor 2 (CB2) of the cannabinoid system is predominantly expressed on immune cells and involved in a diverse range of immune functions. However, the role of CB2 in immunoregulation is still controversial. One of the challenges in the detailed characterization and functional study of CB2 is the lack of CB2-specific monoclonal antibodies (mAbs). We aimed to produce mAbs against a native form of human CB2 using human CB2 expressing mouse myeloma cells as immunogens. Non-viral vector expression system was used to generate stable human CB2-expressing mouse myeloma cells and were utilized as an immunogen for mouse immunization. Hybridoma technique was employed in the production of mAbs. The produced mAbs were verified by flow cytometry and western blotting. Using a non-viral vector expression system, myeloma clones, which stable expressed human CB2, were generated and used as immunogen for antibody production. Following mouse immunization process, the anti-CB2 polyclonal antibodies were induced. By hybridoma technique, a mAb against CB2 could be generated. This mAb reacted to CB2-expressing THP-1 cells, but not to non-CB2-expressing SH-SY5Y cells. By western blotting, the generated anti-CB2 mAb reacted with a 42 kDa protein presented in lysates of CB2-expressing THP-1 cells, but not with non-CB2-expressing SH-SY5Y cell lysates. A new approach using human CB2 expressing myeloma cells as immunogen for production of anti-CB2 mAb was developed. The generated anti-human CB2 mAb is regarded as a valuable tool for CB2 characterization. Moreover, the developed technique can be applied to produce other antibodies of interest.

CD99 is a surface molecule expressed on various cell types including cancer cells. Expression of CD99 on multiple myeloma is associated with CCND1-IGH fusion/t(11;14). This translocation has been reported to be a genetic hallmark of mantle cell lymphoma (MCL). MCL is characterized by overexpression of cyclin D1 and high tumor proliferation. In this study, high expression of CD99 on MCL cell lines was confirmed. Our generated anti-CD99 monoclonal antibody (mAb), termed MT99/3, exerted potent antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities against mantle B-cell lymphoma without direct cytotoxic effects. The anti-tumor activities of mAb MT99/3 were more effective in MCL than in other B-cell lymphomas. Moreover, in a mouse xenograft model using Z138 MCL cell line, treatment of mAb MT99/3 reduced tumor development and growth. Our study indicated that mAb MT99/3 is a promising immunotherapeutic candidate for mantle cell lymphoma therapy.

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.

Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided. Immunity to one serotype of dengue virus can worsen disease following exposure to another serotype, a process called ‘antibody-dependent enhancement’. Grimes and colleagues characterize the function and structural basis of an unusual, potent and broadly neutralizing antibody that lacks such activity.

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.

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) are components in the endocannabinoid system that play significant roles in regulating immune responses. There are many agonists for the cannabinoid receptors; however, their effects on T cell regulation have not been elucidated. In the present study, we determined the effects of the CB1 selective agonist ACEA and the CB2 selective agonist GW833972A on T cell responses. It was found that both agonists impaired anti-CD3 monoclonal antibody induced T cell proliferation. However, ACEA and GW833972A agonists down-regulated the expression of activation markers on CD4+ and CD8+ T cells and co-stimulatory molecules on B cells and monocytes in different manners. Moreover, only GW833972A suppressed the cytotoxic activities of CD8+ T cells without interfering in the cytotoxic activities of CD4+ T cells and NK cells. In addition, the CB2 agonist, but not CB1 agonist, caused the reduction of Th1 cytokine production. Our results demonstrated that the CB1 agonist ACEA and CB2 agonist GW833972A attenuated cell-mediated immunity in different mechanisms. These agonists may be able to be used as therapeutic agents for inducing T cell hypofunction in inflammatory and autoimmune diseases.