OBJECTIVE: To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary infection and explore the possible mechanism. METHODS: Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (=5). In the former two groups, was inoculated intranasal administration to establish mouse models of pulmonary infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 μg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. RESULTS: infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation ( < 0.05) and reduced chlamydia load in the lung tissue ( < 0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells ( < 0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages ( < 0.05) and Th1 cells ( < 0.05). CONCLUSIONS Inhibition of CD96 alleviates pneumonia in -infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity.
Animals ; Antigens, CD ; metabolism ; Chlamydia Infections ; complications ; immunology ; physiopathology ; Chlamydia muridarum ; Interferon-gamma ; genetics ; metabolism ; Killer Cells, Natural ; metabolism ; Lung Injury ; etiology ; genetics ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8 T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.
Adult ; Antigens, CD19 ; metabolism ; Bone Marrow ; metabolism ; CD8-Positive T-Lymphocytes ; immunology ; Female ; Gene Expression Regulation, Leukemic ; Gene Regulatory Networks ; Humans ; Immunotherapy, Adoptive ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; therapy ; RNA, Long Noncoding ; genetics ; metabolism ; Receptors, Antigen, T-Cell ; Transcription Factors ; metabolism ; Transcriptome ; genetics

OBJECTIVETo study the relationship between the expression of peripheral blood HLA-DR, CD4CD25 regulatory T cells, IL-17 and IL-27 with liver damage in children with human cytomegalovirus (HCMV) infection.

METHODSTwenty-one HCMV children with liver damage and twenty-one HCMV children without liver damage were enrolled in this study. The expression of peripheral blood HLA-DR and CD4CD25 regulatory T cells was detected by flow cytometry. Plasma levels of IL-17 and IL-27 were measured using ELISA.

RESULTSThe plasma levels of IL-17 and IL-27 in children with liver damage were significantly higher than in those without liver damage, while the expression of peripheral blood CD4CD25 regulatory T cells was lower than in those without liver damage (P<0.05). Plasma IL-17 and IL-27 levels were negatively correlated with the expression of peripheral blood CD4CD25 regulatory T cells (P<0.01).

CONCLUSIONSImmune imbalance mediated by CD4CD25 regulatory T cells and over-expression of IL-17 and IL-27 may be involved in the pathogenesis of liver damage in children with HCMV infection.

CD4 Antigens ; immunology ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; blood ; complications ; genetics ; Female ; Flow Cytometry ; HLA-DR Antigens ; genetics ; immunology ; Humans ; Infant ; Interleukin-17 ; blood ; genetics ; Interleukin-2 Receptor alpha Subunit ; immunology ; Interleukins ; blood ; genetics ; Liver ; injuries ; metabolism ; Liver Diseases ; blood ; etiology ; immunology ; Male ; T-Lymphocytes, Regulatory ; immunology

Tumor-specific neoantigens have attracted much attention since they can be used as biomarkers to predict therapeutic effects of immune checkpoint blockade therapy and as potential targets for cancer immunotherapy. In this study, we developed a comprehensive tumor-specific neoantigen database (TSNAdb v1.0), based on pan-cancer immunogenomic analyses of somatic mutation data and human leukocyte antigen (HLA) allele information for 16 tumor types with 7748 tumor samples from The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA). We predicted binding affinities between mutant/wild-type peptides and HLA class I molecules by NetMHCpan v2.8/v4.0, and presented detailed information of 3,707,562/1,146,961 potential neoantigens generated by somatic mutations of all tumor samples. Moreover, we employed recurrent mutations in combination with highly frequent HLA alleles to predict potential shared neoantigens across tumor patients, which would facilitate the discovery of putative targets for neoantigen-based cancer immunotherapy. TSNAdb is freely available at http://biopharm.zju.edu.cn/tsnadb.
Antigens, Neoplasm ; metabolism ; Data Analysis ; Databases, Genetic ; Humans ; Immunotherapy ; Mutation ; genetics ; Neoplasms ; genetics ; immunology ; Tumor Suppressor Protein p53 ; genetics ; Urinary Bladder Neoplasms ; genetics

T-cell receptor (TCR)-engineered T cells are a novel option for adoptive cell therapy used for the treatment of several advanced forms of cancer. Work using TCR-engineered T cells began more than two decades ago, with numerous preclinical studies showing that such cells could mediate tumor lysis and eradication. The success of these trials provided the foundation for clinical trials, including recent clinical successes using TCR-engineered T cells to target New York esophageal squamous cell carcinoma (NY-ESO-1). These successes demonstrate the potential of this approach to treat cancer. In this review, we provide a perspective on the current and future applications of TCR-engineered T cells for the treatment of cancer. Our summary focuses on TCR activation and both pre-clinical and clinical applications of TCR-engineered T cells. We also discuss how to enhance the function of TCR-engineered T cells and prolong their longevity in the tumor microenvironment.
Animals ; Antigens, Neoplasm ; immunology ; metabolism ; Humans ; Neoplasms ; immunology ; metabolism ; Receptors, Antigen, T-Cell ; genetics ; metabolism ; T-Lymphocytes ; immunology ; metabolism

BACKGROUND: The interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I regulates natural killer (NK) cell cytotoxicity and function. The impact of NK cell alloreactivity through KIR in liver transplantation remains unelucidated. Since the frequency of HLA-C and KIR genotypes show ethnic differences, we assessed the impact of HLA-C, KIR genotype, or KIR-ligand mismatch on the allograft outcome of Korean liver allografts. METHODS: One hundred eighty-two living donor liver transplant patients were studied. Thirty-five patients (19.2%) had biopsy-confirmed acute rejection (AR), and eighteen (9.9%) had graft failure. The HLA-C compatibility, KIR genotypes, ligand-ligand, and KIR-ligand matching was retrospectively investigated for association with allograft outcomes. RESULTS: Homozygous C1 ligands were predominant in both patients and donors, and frequency of the HLA-C2 allele in Koreans was lower than that in other ethnic groups. Despite the significantly lower frequency of the HLA-C2 genotype in Koreans, donors with at least one HLA-C2 allele showed higher rates of AR than donors with no HLA-C2 alleles (29.2% vs 15.7%, P=0.0423). Although KIR genotypes also showed ethnic differences, KIR genotypes and the number of activating KIR/inhibitory KIR were not associated with the allograft outcome. KIR-ligand mismatch was expected in 31.6% of Korean liver transplants and had no impact on AR or graft survival. CONCLUSIONS: This study could not confirm the clinical impact of KIR genotypes and KIR-ligand mismatch. However, we demonstrated that the presence of HLA-C2 allele in the donor influenced AR of Korean liver allografts.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Female ; Genotype ; Graft Rejection ; Graft Survival ; HLA-C Antigens/*genetics ; Homozygote ; Humans ; Killer Cells, Natural/cytology/immunology ; Ligands ; *Liver Transplantation ; Male ; Middle Aged ; Proportional Hazards Models ; Receptors, KIR/chemistry/*genetics/metabolism ; Republic of Korea ; Tissue Donors ; Transplantation, Homologous

OBJECTIVETo generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.

METHODSThe full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.

RESULTSDNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.

CONCLUSIONRecombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.

Animals ; Antigens, Human Platelet ; immunology ; Autoantibodies ; immunology ; Base Sequence ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; Immunoassay ; methods ; Integrin beta3 ; genetics ; immunology ; metabolism ; Microspheres ; Recombinant Proteins ; immunology ; metabolism ; Reproducibility of Results

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; metabolism ; Carbohydrate Sequence ; Carcinoembryonic Antigen ; genetics ; immunology ; Gene Expression Regulation ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Lipopolysaccharides ; chemistry ; immunology ; Luminescent Measurements ; Mutation ; Neisseria gonorrhoeae ; genetics ; metabolism ; pathogenicity ; Neutrophils ; immunology ; microbiology ; Phagocytosis

OBJECTIVEHepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose.

METHODSReconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method.

RESULTSThe expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies.

CONCLUSIONExpression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.

Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Hepatitis D ; diagnosis ; immunology ; virology ; Hepatitis Delta Virus ; immunology ; Hepatitis delta Antigens ; immunology ; Recombinant Proteins ; genetics ; metabolism