Objective To prepare CD318-specific monoclonal antibodies and evaluate their specificity, affinity, and application in immunological detection, laying the foundation for the development of CD318-targeted antibody drugs. MethodsCD318 protein was expressed and purified, and was used as an antigen to immunize mice, then mice with higher antiserum titers were screened. We prepared CD318-specific monoclonal antibodies through cell fusion and monoclonal screening, and the specificity, affinity, and application of the obtained monoclonal antibodies in immunological assays were evaluated. Then we constructed a CD318/CD3-targeting bispecific antibody and assessed its impact on T-cell cytotoxicity. Results Thirteen monoclonal antibodies were successfully generated, with the hybridoma clone 13-8-G2 exhibiting the highest titer, strongest specificity, and broadest applicability. The antibody was identified as an IgG1 isotype with a kappa light chain. The variable region of the light chain measured 318 bp, while the heavy chain variable region was 357 bp, yielding an affinity constant of approximately 7.68×10⁹. The specificity of CD318 was confirmed using flow cytometry and immunofluorescence assays. Additionally, a CD318/CD3-targeting bispecific antibody was constructed using the variable regions of this CD318 monoclonal antibody, which demonstrated enhanced T-cell cytotoxicity. Conclusion High-affinity and highly specific CD318 monoclonal antibodies were successfully prepared, laying a foundation for the development of therapeutic antibodies targeting CD318.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice ; Antibodies, Bispecific/immunology* ; Humans ; Mice, Inbred BALB C ; Antibody Specificity/immunology* ; CD3 Complex/immunology* ; Antigens, CD/genetics* ; T-Lymphocytes/immunology* ; Hybridomas/immunology* ; Female

OBJECTIVETo construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.

METHODShCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32-CD96. The expression of hCD96om was induced by IPTG in BL21(DE3) cells, and the expression product was identified by Western blotting. The anti-hCD96 polyclonal antibody was prepared by immunization of rabbits with the fusion protein. The specificity of anti-hCD96 antibody was determined by Western blotting.

RESULTShCD96om protein was expressed in E.coli BL21(DE3) cells in the form of inclusion body, with a relative molecular mass around 37 kD. Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 kD hCD96om fusion protein.

CONCLUSIONThe CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.

Animals ; Antibodies ; immunology ; metabolism ; Antigens, CD ; biosynthesis ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; biosynthesis ; Immunization ; Leukemia, Myeloid, Acute ; immunology ; Molecular Sequence Data ; Neoplastic Stem Cells ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.
Animals ; Anticarcinogenic Agents ; administration & dosage ; therapeutic use ; Antigens, CD ; analysis ; Breast Neoplasms ; drug therapy ; immunology ; pathology ; surgery ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chemotherapy, Adjuvant ; Female ; Flavanones ; administration & dosage ; therapeutic use ; Humans ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Interferon-gamma ; biosynthesis ; immunology ; Interleukin-2 ; biosynthesis ; immunology ; Lung Neoplasms ; drug therapy ; immunology ; prevention & control ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism

In addition to CD4+CD25+Foxp3+ regulatory T (T(reg)) cells which protect against autoimmune tissue injury, IL-17-producing CD4+ T (Th17) cells have been recently described and shown to play a crucial role in autoimmune injury. It appears that there is a reciprocal developmental pathway between Th17 and T(reg) cells. Although IL-17 is known to be associated with allograft rejection, the cellular source of IL-17 and the nature of Th17 in the context of allograft rejection remain unknown. In the current study, the dynamics of T(reg) and IL-17-producing cells after syngeneic and allogeneic transplantation were examined using a wild-type murine cardiac transplantation model. Ly6G+ cells were found to produce IL-17 during the early postoperative period and CD8+ as well as CD4+ T cells were also found to produce IL-17 during alloimmune response. Graft-infiltrating Ly6G+, CD4+, and even CD8+ cells were found to express IL-17 highly compared to those in spleen. Although the frequencies of Th17 and T(reg) were found to gradually increase in both syngeneic and allogeneic recipients, Th17/T(reg) ratios were significantly higher in recipients with allograft rejection than in syngeneic recipients. In conclusion, IL-17 is produced by neutrophils during the early postoperative period and subsequently by Th17 and CD8+ T cells during allograft rejection. Th17/T(reg) imbalance is associated with the development of allograft rejection. This study would provide basic information on Th17 biology for future investigation in the field of transplantation.
Animals ; Antigens, CD/biosynthesis ; Autoimmunity ; Forkhead Transcription Factors/biosynthesis ; Graft Rejection/immunology/*metabolism ; Heart Transplantation ; Interleukin-17/immunology/*secretion ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Neutrophils/immunology/*metabolism/pathology ; T-Lymphocyte Subsets/immunology/*metabolism ; T-Lymphocytes, Regulatory/immunology/*metabolism ; Time Factors ; Transplantation Immunology

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.
Animals ; Antigens, CD/immunology/metabolism ; Bacterial Outer Membrane Proteins/*pharmacology ; Cells, Cultured ; Female ; Histocompatibility Antigens Class II/immunology/metabolism ; Host-Pathogen Interactions ; Interleukin-12/biosynthesis ; Interleukin-6/biosynthesis ; Legionella pneumophila/*immunology/metabolism ; Legionnaires' Disease/immunology/metabolism ; Lipoproteins/*pharmacology ; Macrophage Activation/drug effects/immunology ; Macrophages, Peritoneal/drug effects/immunology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Toll-Like Receptor 2/*metabolism ; Tumor Necrosis Factor-alpha/biosynthesis

OBJECTIVETo investigate the expressions of CD1a and CD83 of Langerhans cells (LC) in the lesions of epidermodysplasia verruciformis (EV) patients.

METHODSWe used immunohistochemical method to detect the expressions of CD1a and CD83 in the lesions of 10 patients with EV lesions and in the skins of 10 normal subjects.

RESULTSNo CD83 + LCs was detected in all EV patients and normal controls, but CD1a + LC was found in all cases. The quantity of CD1a + LCs in the lesions of EV patients was significantly lower than that in the normal skin (P < 0.01); furthermore, the distribution of LCs in EV lesions was uneven.

CONCLUSIONThe functions of LCs may be inhibited in EV patients.

Antigens, CD ; biosynthesis ; genetics ; Antigens, CD1 ; biosynthesis ; genetics ; Epidermodysplasia Verruciformis ; immunology ; pathology ; Humans ; Langerhans Cells ; immunology ; Leukocyte Immunoglobulin-like Receptor B1 ; Receptors, Immunologic ; biosynthesis ; genetics ; Skin ; immunology ; pathology

Animals ; Antigens, CD ; immunology ; Cytokines ; metabolism ; Hepacivirus ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Hepatitis C Antibodies ; biosynthesis ; immunology ; Hepatitis C Antigens ; immunology ; Humans ; Killer Cells, Natural ; immunology ; T-Lymphocytes ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; metabolism ; Viral Proteins ; immunology

OBJECTIVETo investigate whether there is an association between the expression of B7-H1 in HBV transgenic mice and the immune tolerance to HBV.

METHODST cells stimulatory capacity of DC was analyzed using mixed lymphocyte reaction. Expression of MHC-II, CD80, CD86, B7-H1 on DC was detected by Flow Cytometry. IL-2, IFNgamma, IL-10 production of T cells were determined by using ELISA. B7-H1 mRNA and protein expression in liver tissue were detected by RT-PCR and Western blotting respectively.

RESULTSThe ability of DC cells from HBV transgenic mice to stimulate T cell proliferation was significantly impaired compared with DC cells from control mice (t = 16.674, 19.674, 21.712, P less than 0.01). Expression of MHC-II, CD80 on DC was markedly decreased in transgenic mice (t = 7.910, 6.413, P less than 0.05). Meanwhile, the expression of CD86 and B7-H1 on DC cells in HBV transgenic mice were not significantly different from that in control mice. The levels of IL-2, IFNgamma, IL-10 in supernatant of T cells was significantly lower compared with controls (t = 18.712, 18.712 and 11.683, P less than 0.05). There was no significant difference in B7-H1 expression at mRNA and protein levels in liver tissue compared with controls.

CONCLUSIONSFunctional defect of DC, partly due to decreased expression of MHC-II, CD80, but not related to B7-H1 expression, is the cause for immune tolerance to HBV in HBV transgenic mice.

Animals ; Antigens, CD ; biosynthesis ; genetics ; Cell Proliferation ; Cytokines ; biosynthesis ; Dendritic Cells ; immunology ; metabolism ; Flow Cytometry ; Hepatitis B virus ; genetics ; immunology ; Histocompatibility Antigens Class II ; metabolism ; Immune Tolerance ; Liver ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA, Messenger ; genetics ; metabolism ; Spleen ; immunology ; metabolism ; T-Lymphocytes ; immunology ; metabolism

Neutrophils are also known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. In this study, neutrophils were cultivated in vitro in the presence or absence of compounds modulating their survival in an attempt to characterize the expression profile of the DC markers. Higher MHC-II, CD80, CD86, CD83, and CD40 expression levels were detected on the surface of the cultured neutrophils for 24 h than on the freshly isolated cells. The annexin V-positive cells showed a higher expression level of the DC markers than the annexin V-negative cells. The population of neutrophils double stained with annexin V and the DC markers increased after being incubated with agonistic anti-Fas Ab. LPS, the anti-apoptotic compound, decreased the CD86 and MHC-II expression levels but 50-60% of the DC marker-positive cells were detected in the annexin V-positive cells. In contrast, CD80, CD86, CD83, and HLA-DR mRNA levels increased in the GM-CSF-treated neutrophils but not in the anti-Fas Ab-treated neutrophils. T cell proliferation was inhibited by co-culturing them with anti-Fas Ab- or LPS-treated neutrophils at a high neutrophil:T cell ratio. However, the superantigen-mediated T cell proliferation was increased by the LPS-treated neutrophils but decreased by the anti-Fas Ab-treated neutrophils. There was a lower level of interferon-gamma production in the T cells co-cultured with anti-Fas Ab-treated neutrophils than with the LPS-treated neutrophils. This suggests that apoptotic neutrophils express DC markers on their surface and the differential expression of DC markers might have a detrimental effect on the immune reaction.
Antigen Presentation ; Antigens, CD/biosynthesis ; Antigens, CD95/pharmacology ; Antigens, Differentiation/*biosynthesis ; *Apoptosis ; Cells, Cultured ; Dendritic Cells/*metabolism ; Humans ; Lipopolysaccharides/pharmacology ; Lymphocyte Activation ; Neutrophils/*metabolism/physiology ; T-Lymphocytes/immunology

OBJECTIVE: To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets (NPIs) transfected with AAV-hCTLA4-Ig. METHODS: Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT-PCR and immunocytochemistry. The levels of IL-2, IFN-gamma, and TNF-alpha in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group. RESULTS: The expressions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group (P<0.01). There was no significant difference in the response of glucose-stimulated insulin release between the transgene group and the control group (P>0.05). CONCLUSION AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.
Animals ; Animals, Newborn ; Antigens, CD ; biosynthesis ; genetics ; Antigens, Differentiation ; biosynthesis ; genetics ; CTLA-4 Antigen ; Cells, Cultured ; Dependovirus ; genetics ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunohistochemistry ; Interferon-gamma ; analysis ; Interleukin-2 ; analysis ; Islets of Langerhans ; cytology ; immunology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Transfection ; Tumor Necrosis Factor-alpha ; analysis