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4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor that is primarily expressed on activated T cells and professional antigen-presenting cells. In this study, the expression pattern of 4-1BB on immunology cells and tumor cells was explored by flow cytometry using newly generated three anti-4-1BB monoclonal antibodies (mAbs; 6F9, 7D6, and 1G11), which bind to distinct 4-1BB epitopes. Compared with the available 4-1BB mAb 4B4-1 that recognized 4-1BB on activated T cells and monocytes, the novel mAbs also could recognize 4-1BB on some cancer cell lines, particularly on lung cancer cell lines such as SPC-A-1, H446, H460, and H1299 by flow cytometry analysis, western blot, and RT-PCR. Immunohistochemistry staining showed the 4-1BB was expressed on lung tumor tissue (33/35) but not on normal lung tissue (3/3). It was determined that 4-1BB was strictly expressed on lung cancer cells, which may provide information on the 4-1BB signal in tumor immunology mechanism.

CD40, a member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule during the immune response. Here, we report a blocking mouse antihuman CD40 monoclonal antibody, mAb 3G3, of which the specificity was verified by flow cytometry and Western blot. It was shown by competition test that 3G3 bound to a different site (epitope) of CD40 from the reported CD40 mAbs, including clone mAb89, 3B2, and 5C11. It was also found that mAb 3G3 could inhibit homotypic aggregation of Daudi cells induced by the agonistic anti-CD40 mAb 5C11. Furthermore, mAb 3G3 effectively inhibited the proliferation of peripheral blood mononuclear cells in mixed lymphocyte reaction assay. Finally, a sensitive and specific soluble CD40 (sCD40) ELISA kit was established by matching mAb 3G3 with 5C11, and it was found that the levels of sCD40 in sera from patients with immune disorders such as hyperthyroidism, chronic nephritis, and rheumatoid arthritis were obviously higher than those from normal individuals. Thus, this blocking anti-CD40 mAb provides a novel tool for the study of CD40.

Human B7-H3, a novel member of B7 family, has two isoforms (2IgB7-H3 and 4IgB7-H3). As costimulatory functions of both isoforms are not clarified, there has been much discussion on their expression patterns, T-cell responses, etc. This study generated two specific mouse anti-human 2IgB7-H3 monoclonal antibodies (mAbs) (7D7 and 10F1), whose specificities are quite different from those of the available B7-H3 mAb (21D4) by competition assay. The use of antibodies indicated that B7-H3 was found to have different expression patterns on monocyte-derived dendritic cells, that is the isoform 2IgB7-H3 is tended to express on immature dendritic cells (iDCs), whereas 4IgB7-H3 could be detected in the whole process of maturation of dendritic cells. The isoform 4IgB7-H3 was shown in the immunohistochemistry assay to be more widely expressed than 2IgB7-H3 in human benign and malignant hepatic tissues. Furthermore, flow cytometry and reverse transcription-polymerase chain reaction indicated that 4IgB7-H3 rather than 2IgB7-H3 was the major isoform on many human tumor cell lines. In addition, both 2IgB7-H3- and 4IgB7-H3-transfected cells could promote T-cell proliferation, which could be blocked by 7D7 mAb. These two novel antibodies may shed light on the function of the two B7-H3 isoforms.

B and T lymphocyte attenuator (BTLA) has been recently identified as a new inhibitory receptor of the CD28 superfamily, with similarities to cytotoxic T lymphocyte activation antigen (CTLA)-4 and programmed death (PD)-1. Engagement of BTLA on T lymphocytes can profoundly reduce the T cell receptor (TCR)-mediated activation. In this study, we generated four monoclonal antibodies (mAbs) against human BTLA. Using the produced mAb 8H9, the BTLA molecule was found to distinctly express on many subgroups of immunocytes and show a regulatory expression, which was in accordance with its unique ligand herpes virus entry mediator (HVEM) in the process of T cell activation. In addition, the expression of BTLA was increased in the CD4(+) and CD8(+) T cells of pleural fluid in lung cancer patients. Furthermore, we showed that the BTLA-induced negative signals could be triggered by mAb 7D7. Cross-linking of BTLA with mAb 7D7 suppressed T lymphocyte proliferation, downregulated the expression of T cell activation marker CD25, and inhibited the production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10.

Programmed death-1 ligand-1 (PD-L1), a member of the B7 family of costimulatory molecules, plays an important role in the regulations of the cellular and humoral immune responses. In this study, two mouse anti-human PD-L1 monoclonal antibodies named 10E10 and 2H11 were successfully generated and further characterized. Monoclonal antibody 10E10 bound to distinct PD-L1 epitope comparing an available anti-PD-L1 monoclonal antibody on a series of malignant cell lines, activated T lymphocytes, B lymphocytes and dendritic cells. Then, by using immunohistochemistry staining with monoclonal antibody 2H11, the expression of PD-L1 was found in human gastric carcinoma specimens but not in normal or gastric adenoma tissues. Additional data show that PD-L1 can be regarded as a decisive factor in evaluating gastric carcinoma prognosis and anti-human PD-L1 monoclonal antibody 10E10 could inhibit T-cell apoptosis induced by tumor-associated PD-L1. Taken together, these results showed that the two functional mouse anti-human PD-L1 monoclonal antibodies we generated might be of great value for further exploration of the costimulatory molecule regulating network and immunointervention for tumor immunotherapy.

2IgB7-H3 has recently been identified as a new member of the B7 family. Its expression at the protein level remains largely unknown due to the lack of the specific monoclonal antibody (mAb). To characterize the expression of 2IgB7-H3, we newly generated two mouse antihuman 2IgB7-H3 mAbs (4H7 and 21D4). We found the constitutive expression of 2IgB7-H3 on a series of tumor cell lines. Furthermore, the expression was examined on monocyte-derived dendritic cells (Mo-DCs) and DCs from CD34(+) hematopoietic progenitor cells (HPC) by means of mAb staining. The results showed that 2IgB7-H3 was expressed on Mo-DCs at a high and stable level during differentiation in vitro. With the maturation of DCs from CD34(+) HPCs, the expression of the molecule was upregulated. However, the 2IgB7-H3 was not expressed on fresh isolated T and B lymphocytes, monocytes, or CD34(+) HPCs. These results suggested that 2IgB7-H3 may be a valuable surface antigen for the detection of DCs.

OX40 ligand (OX40L), a molecule originally identified as human gp34, is an important co-stimulatory molecule during immune response. In this study, we report on five functional mouse anti-human OX40L monoclonal antibodies named as 9H10, 4C12, 8D10, 4H4 and 1G1, characterized by means of flow cytometry, Western blot and competition assay. These monoclonal antibodies bound to distinct OX40L epitopes on activated B cells and dendritic cells (DCs) and two of them could suppress the proliferation of T lymphocytes co-stimulated by mature DCs. Furthermore, we demonstrated that our monoclonal antibodies, such as 9H10 and 4C12, could trigger OX40L reverse signal that enhanced IgG production of B cells and promoted maturation of DCs as evidenced by the upexpression of CD80, CD86, CD83 and CXCR4 and monoclonal antibody 9H10 could also promote anti-CD40 monoclonal-antibody-stimulated DCs in order to induce T cells to secrete more interleukin-2 and interferon-gamma, which suggested that OX40L signals could strengthen the effect of CD40 signals on promoting Th1 differentiation.

CD40 ligand, a 33-kDa cell membrane molecule, a member of the tumor necrosis factor superfamily, is an important costimulatory molecule during immune response. Here, we report on two functional mouse anti-human CD40L monoclonal antibodies 1B1 and 4F1 characterized by flow cytometry, Western blotting, and competition assay. The antibodies bound to distinct CD40L epitopes and therefore resulted in different bioactivity. Both antibodies could induce CD4+ T-cell alloantigenic hyporesponsiveness ex vivo. The antibodies were matched to develop a two-site enzyme-linked immunosorbent assay (ELISA) for soluble CD40L (sCD40L). Using this ELISA assay, we found major differences between plasma and serum sCD40L levels. Because the count of platelet sharply decreased in aplastic anemia (AA) and idiopathic thrombocytopenic purpura (ITP), we further analyzed the sCD40L concentration in the plasma of AA and ITP patients. The results showed that the sCD40L in serum was much lower than that of healthy subjects. These data demonstrate that platelets seem to be a major contributor to sCD40L, though not the only source of sCD40L in serum.