Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.
Animals ; CTLA-4 Antigen ; genetics ; Cell Proliferation ; HEK293 Cells ; Humans ; Lung Neoplasms ; metabolism ; Programmed Cell Death 1 Receptor ; genetics ; Protein Binding ; Protein Domains ; genetics ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism

OBJECTIVETo investigate the association between CTLA-4 gene polymorphism and Henoch-Schönlein purpura (HSP) in children.

METHODSSixty children who were diagnosed with HSP were enrolled as the case group, consisting of 33 males and 27 females. Thirty healthy children were enrolled as the control group. The patients were further divided into HSP nephritis (HSPN) and non-HSPN groups (n=30 each) according to the presence or absence of nephritis. Polymerase chain reaction-restriction fragment length polymorphism was used to analyze the genotype and allele frequencies at +49 and -1722 loci.

RESULTSAA, AG, and GG genotypes were detected at +49; neither genotype nor allele frequencies showed significant differences between the case and control groups, between the HSPN and non-HSPN groups, and between male and female patients (P>0.05). TT, TC, and CC genotypes were detected at -1722; neither genotype nor allele frequencies showed significant differences between the case and control groups and between male and female patients (P>0.05). There were significant differences in CC genotype frequency and T and C allele frequencies between the HSPN and non-HSPN groups (P<0.05). Combinational analysis of +49 A/G and -1722 T/C showed no significant differences in the genotype frequency between the case and control groups and between male and female patients (P>0.05). GG-CC combination showed a significant difference between the HSPN and non-HSPN groups (P<0.05).

CONCLUSIONSCTLA-4 +49 A/G polymorphism is not associated with HSP. CC genotype and C allele of CTLA-4 -1722 and the combination of GG at +49 A/G and CC at -1722 T/C may be risk factors for HSPN.

CTLA-4 Antigen ; genetics ; Child ; Child, Preschool ; Female ; Genotype ; Humans ; Male ; Polymorphism, Genetic ; Purpura, Schoenlein-Henoch ; genetics

BACKGROUND/AIMS: The immunoregulatory molecules programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with the dysfunction of antiviral effector T-cells, which leads to T-cell exhaustion and persistent viral infection in patients with chronic hepatitis C and chronic hepatitis B. Little is known about the role of PD-1 and CTLA-4 in patients with symptomatic acute hepatitis A (AHA). METHODS: Peripheral blood mononuclear cells were isolated from seven patients with AHA and from six patients with nonviral acute toxic hepatitis (ATH) during the symptomatic and convalescent phases of the respective diseases; five healthy subjects acted as controls. The expression of PD-1 and CTLA-4 on T-cells was measured by flow cytometry. RESULTS: PD-1 and CTLA-4 expression during the symptomatic phase was significantly higher in the T-cells of AHA patients than in those of ATH patients or healthy controls (PD-1: 18.3% vs 3.7% vs 1.6%, respectively, p<0.05; CTLA-4: 23.5% vs 6.1% vs 5.9%, respectively, p<0.05). The levels of both molecules decreased dramatically during the convalescent phase of AHA, whereas a similar pattern was not seen in ATH. CONCLUSIONS: Our findings are consistent with a viral-protective effect of PD-1 and CTLA-4 as inhibitory molecules that suppress cytotoxic T-cells and thereby prevent the destruction of virus-infected hepatocytes in AHA.
Acute Disease ; Adult ; CTLA-4 Antigen/*genetics ; Case-Control Studies ; Female ; Flow Cytometry ; Hepatitis/genetics ; Hepatitis A/*genetics/virology ; Hepatitis A Virus, Human ; Humans ; Male ; *Phenotype ; Programmed Cell Death 1 Receptor/*genetics ; T-Lymphocytes/metabolism

OBJECTIVETo evaluate the association of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) + 49A/G polymorphism with type 1 diabetes mellitus (T1DM) in children.

METHODSPapers about the association of CTLA4+49A/G polymorphism with T1DM in children were collected by searching PubMed, EBSCO, CBM, CNKI, and Wanfang Data. A meta-analysis was performed to examine differences in the genotypes (AG, GG, and GG+AG) and G allele at position 49 of the CTLA-4 gene between a childhood T1DM group and a control group.

RESULTSA total of 10 papers involving 1084 T1DM children and 1338 healthy children were included. The Meta-analysis was performed to evaluate the association of the genotypes (AG, GG, and GG+AG) and the G allele at position 49 of the CTLA-4 gene with T1DM using a fixed effect model according to the heterogeneity test results of all studies. The pooled OR values (95% CI) were 1.13 (0.97-1.33), 1.42 (1.16-1.75), 1.20 (1.03-1.40), and 1.21 (1.09-1.33), suggesting a significant difference in genotypes (AG, GG, and GG+AG) and the G allele at position 49 of the CTLA-4 gene between the two groups.

CONCLUSIONSCTLA-4 +49A/G polymorphism is associated with T1DM in children.

CTLA-4 Antigen ; genetics ; Diabetes Mellitus, Type 1 ; genetics ; Genotype ; Humans ; Polymorphism, Genetic

BACKGROUNDThe transcription factor, repressor of GATA-3 (ROG), can simultaneously suppress the expression of T helper cells (Th1 and Th2) cytokines. Since the suppression of Th2 cytokines by GATA-3 is well understood, it is postulated that there are other molecular targets of ROG that can suppress the expression of the Th1 cytokines. We hypothesized that ROG might suppress the stimulators of T lymphocyte cytokines such as CD3, CD28, and inducible costimulator (ICOS), or indirectly enhance the expression of cytokine suppressors such as T lymphocyte-associated antigen-4 (CTLA-4) and CD45. The objective of this study was to clarify the molecular targets of ROG involved in suppressing Th1 or Th2 cytokines.

METHODSReal-time quantitative PCR (RT-PCR) and Western blotting were performed to evaluate the mRNA and protein levels of CD3, CD28, ICOS, CTLA-4, and CD45 in Th1 and Th2 cells during various levels of ROG expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of interferon-γ (IFN-γ) and interleukin (IL)-4 in culture media of Th1 and Th2 cells.

RESULTSThe results showed that the mRNA and protein levels of ROG were relatively low in Th1 and Th2 cells (P < 0.01). After ROG-pcDNA3.1 transfection, the mRNA and protein level of ROG was significantly elevated, while the expression of ICOS, IFN-γ, and IL-4 was markedly down-regulated (P < 0.01). Conversely, transfection of ROG-siRNA led to inhibition of ROG expression and up-regulation of ICOS, IFN-γ and IL-4 (P < 0.01). However, the expression levels of CD3, CD28, CTLA-4 and CD45 did not change in either ROG-pcDNA3.1 or ROG-siRNA-transfected Th1 and Th2 cells (P > 0.05).

CONCLUSIONIt is concluded that ROG can inhibit the expression of Th1 and Th2 cytokines by down-regulating the expression of ICOS, which might be a potential molecular target for asthma treatment.

Animals ; Blotting, Western ; CD28 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; CTLA-4 Antigen ; metabolism ; Cells, Cultured ; Cytokines ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Inducible T-Cell Co-Stimulator Protein ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocyte Common Antigens ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Real-Time Polymerase Chain Reaction ; Repressor Proteins ; genetics ; metabolism ; T-Lymphocytes ; metabolism ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism

OBJECTIVETo evaluate the association between single nucleotide polymorphisms (SNPs) of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene and susceptibility to cervical cancer.

METHODSOne hundred patients and 100 healthy controls from Hubei province were genotyped for 20 polymorphic loci using Sequenom.

RESULTSThe frequency of rs11571316 G allele and rs5742909 T allele, which are localized in the promoter region, and rs11571319 A allele, which is downstream of the gene, were significantly higher in patients than in controls. Luciferase assay showed that, as the previously reported rs5742909 T allele, rs11571316 G allele could significantly increase the expression of the reporter gene.

CONCLUSIONSNPs in the promoter region of (CTLA4) gene might increase the susceptibility to cervical cancer by increasing (CTLA4) gene expression.

Adult ; Aged ; Alleles ; Antigens, CD ; genetics ; CTLA-4 Antigen ; Case-Control Studies ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Middle Aged ; Polymorphism, Single Nucleotide ; Uterine Cervical Neoplasms ; genetics ; Young Adult

OBJECTIVETo investigate the potential association between factor VIII inhibitor development and polymorphisms of tumor necrosis factor-α (TNF-α)-308 and cytotoxic T-lymphocyte associated protein-4 gene in Chinese Han patients with hemophilia A (HA).

METHODSThe single base change polymorphism in TNF-α and CTLA-4 gene was analyzed in 140 Chinese Han patients with hemophilia A who have been treated with plasma-derived FVIII concentrates and 108 normal controls by using PCR-restrictive fragment length polymorphism (RFLP). All of the HA patients' plasma samples were measured by modified-Nijmegen assay simultaneously.

RESULTSIn HA patients, G/G genotype, G/A genotype and A/A genotype were detected in 118 (84.3%), 18 (12.8%) and 4 cases (2.9%) respectively; C/C genotype, C/T genotype and T/T genotype were detected in 108 (77.2%), 30 (21.4%) and 2 cases (1.4%) respectively. The difference in the genotype frequencies between HA patients and controls was nonsignificant (P > 0.05). Patients who were carriers of homozygotes for A allele had a higher risk of inhibitor development compared with those who were not (OR = 7.519, 95% CI = 3.168 - 17.844). Severe HA patients who were carriers of homozygotes for A allele had a higher risk of inhibitor development compared with those who were not (OR = 8.163, 95% CI = 2.521 - 26.434). There was no statistical difference in the risk of inhibitor development between the patients who were carriers or not (OR = 1.586, 95% CI = 0.729 - 3.450).

CONCLUSIONTNF-α-308 gene polymorphism is significantly associated with inhibitor development in Chinese Han patients with severe hemophilia A. TNF-α gene may be a useful marker and potential modulator of the immune response to replacement therapy for hemophilia A patients.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; CTLA-4 Antigen ; genetics ; Child ; Child, Preschool ; Genotype ; Hemophilia A ; genetics ; Humans ; Infant ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Tumor Necrosis Factor-alpha ; genetics ; Young Adult

OBJECTIVETo study mRNA expression of immune-related genes (Foxp3, GATA3, CTLA4 and T-bet) in peripheral blood of the patients with allergic dermatitis induced by trichloroethylene (TCE).

METHODSThe peripheral blood samples were collected from 8 healthy workers (control group) and 8 patients with allergic dermatitis induced by TCE (case group). Real-time quantitative PCR was applied to detect mRNA expression of immune-related genes (Foxp3, GATA3, CTLA4, T-bet).

RESULTSThe mRNA expression levels of Foxp3, GATA3 and CTLA4 genes increased by 115%, 97% and 241% in case group, as compared with control group (P < 0.01). The mRNA expression level of T-bet gene decreased by 47% in case group, as compared with control group (P < 0.01).

CONCLUSIONThe mRNA expression levels of some immune-related genes changed in patients with allergic dermatitis induced by TCE, those genes may play an important role in TCE-induced allergy.

Adult ; CTLA-4 Antigen ; metabolism ; Case-Control Studies ; Dermatitis, Occupational ; genetics ; immunology ; Female ; Forkhead Transcription Factors ; metabolism ; GATA3 Transcription Factor ; metabolism ; Gene Expression ; Humans ; Male ; RNA, Messenger ; genetics ; T-Box Domain Proteins ; metabolism ; Trichloroethylene ; Young Adult

OBJECTIVETo investigate the association between CTLA-4 +49A/G polymorphisms and the risk of susceptibility to cervical cancer.

METHODSA hospital-based case-control study was conducted. 314 cases with primary cervical cancer and 320 healthy controls were collected and genotyped by PCR-based RFLP for +49A/G polymorphisms in the CTLA-4 gene.

RESULTSThe A allele and AA genotype of CTLA-4 gene were 32.5% and 9.6% in the patients, and 25.8% and 5.6% in the controls, respectively. Subjects with CTLA-4 +49AA genotype conferred a higher risk of cervical cancer (OR = 2.06, 95%CI: 1.10 - 3.85; P = 0.024). However, the correlation between AA genotype in CTLA-4 polymorphisms and clinicopathological characteristics was not significant.

CONCLUSIONThe results of this study suggest that CTLA4 gene is associated with cervical cancer risk and may be a susceptible gene of cervical cancer.

Adenocarcinoma ; genetics ; metabolism ; pathology ; surgery ; Adult ; Aged ; Alleles ; CTLA-4 Antigen ; genetics ; metabolism ; Carcinoma in Situ ; genetics ; metabolism ; pathology ; surgery ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; surgery ; Case-Control Studies ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Middle Aged ; Neoplasm Staging ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Risk Factors ; Uterine Cervical Neoplasms ; genetics ; metabolism ; pathology ; surgery

OBJECTIVETo investigate the effect of (AT)n repeat polymorphism of the 3'untranslated region in cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) gene on CTLA-4 mRNA stability and full length (flCTLA-4) and soluble CTLA4 (sCTLA-4) expression in ulcerative colitis (UC).

METHODSflCTLA-4 mRNA in colonic biopsies and sCTLA-4 mRNA stability in peripheral blood mononuclear cells of UC patients were measured by quantitative PCR and half-life, respectively. The protein expression of flCTLA-4 in colonic biopsies and sCTLA-4 in sera of UC patients were determined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The polymorphism of CTLA-4 (AT)n repeats in 300 UC and 700 age and sex matched healthy controls was genotyped by fluorescent PCR.

RESULTSAmong the UC patients, sCTLA-4 mRNA expression levels were decreased in active disease compared to non-active disease (P= 0.004). Carriers of the longer alleles of the (AT)n repeats expressed lower levels of flCTLA-4 and sCTLA-4 mRNA and sCTLA-4 protein than those of the shorter alleles in UC (all P< 0.01), and mRNA with long (AT)n repeat alleles has shorter half-life than mRNA with short alleles and, hence, are unstable. The frequency of long allele carriers of CTLA-4 (AT)n repeats was significantly higher in UC patients than in the healthy controls (22.0% vs. 6.3%, P< 0.01, OR= 4.21, 95% CI: 2.79-6.33), and associated with extensive colitis (P= 0.008).

CONCLUSIONCTLA-4 gene expression levels were associated with (AT)n repeat polymorphisms in UC patients. The expression of CTLA-4 mRNA and protein were decreased in carriers of the longer alleles of the (AT)n repeats of CTLA-4 gene. This study suggests that CTLA-4 plays an important role in genetic risk and pathophysiology for UC in central China.

Adult ; Antigens, CD ; chemistry ; genetics ; metabolism ; CTLA-4 Antigen ; Case-Control Studies ; Colitis, Ulcerative ; genetics ; metabolism ; Female ; Gene Expression Regulation ; Genotype ; Humans ; Male ; Phenotype ; Polymorphism, Genetic ; RNA Stability ; genetics ; RNA, Messenger ; chemistry ; genetics ; metabolism ; Solubility