Reduced Number of Thymoma CTLA4-Positive Cells Is Associated With a Higher Probability of Developing Myasthenia Gravis

Genetic variation in ICOS regulates mRNA levels of ICOS and splicing isoforms of CTLA4

ABSTRACTObjectives: To investigate the association of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) with immune thrombocytopenia (ITP).Methods: A case–control association analysis of 277 Chinese Han children was performed. The tagging variants rs11571315 and rs3087243 in the CTLA4 gene were detected using polymerase chain reaction-restriction fragment length polymorphism method. The expression quantitative trait loci (eQTL) analysis and quantitative real-time polymerase chain reaction were performed to determine the relationship of CTLA4 with ITP.Results: Neither SNP was significantly different between case and control groups in either the genotypic or allelic distribution. The eQTL analysis results indicated that in the spleen, the rs3087243 was significant with the expression of CTLA4. The rs11571315 has similar results. Interestingly, the transcript level of CTLA4 was found to significantly decrease in patients with ITP.Discussion: The autoimmune and gene etiology is implicated in the pathogen of ITP. The CTLA4 is important for negative regulation of T-cell activation, and CTLA-4 gene has been identified as a risk factor for some autoimmune diseases. However, association studies of ITP and CTLA4 gene have obtained conflicting results. This is the first study to systematically investigate the association of CTLA4 with ITP in Chinese Han children.Conclusions: The CTLA4 gene is suggested to correlate with ITP through its abnormal expression level instead of gene site mutation.

Co-stimulatory molecules of CD28, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and the newly identified inducible co-stimulator (ICOS) are expressed on cell surfaces and provide regulatory signals for T-cell activation. Their genes are candidate susceptibility genes for type 1 diabetes because they co-localize to Chromosome 2q33 with the IDDM12 locus. After determining the genomic structure and screening for polymorphisms of the ICOS gene, we performed association studies between newly identified polymorphisms of the ICOS gene, together with known polymorphisms of CD28 and CTLA-4 genes, and type 1 diabetes. The 49A/G dimorphism in exon 1 and the (AT)n in the 3' untranslated region of the CTLA-4 gene were significantly associated with type 1 diabetes. Evaluation of the CTLA-4 49A-3'(AT)n 86-bp haplotype frequency in patients and controls confirmed the results from the analysis of each polymorphic site. Dimorphism in intron 3 of the CD28 gene was associated with type 1 diabetes only in the early-onset group. In contrast, there was no association with the microsatellite polymorphisms in the ICOS gene or dimorphisms in the promotor region of CTLA-4. Of the three genes encoding co-stimulatory molecules, the CTLA-4 gene appears to confer risks for the development of type 1 diabetes.

Determination of mir-155 and mir-146a expression rates and its association with expression level of TNF-α and CTLA4 genes in patients with Behcet’s disease

Objective: To investigate the survival and death risk factors of mesothelioma cases stratified by the expression levels of CD8 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) , providing new clue to evaluate disease progression and clinical outcome. Methods: This was a retrospective case report, which included 47 clinically and pathologically confirmed mesothelioma cases on November 2016. Their clinical and pathological information, asbestos exposure history and survival data were collected. Infiltrated lymphocyte, 5-methylcytosine (5-mC) , CTLA-4, CD8 and Ki-67 antigen were detected using hematoxylin-eosin staining and immunohistochemistry. Survival time and death risk factors of mesothelioma patients with different CD8 and CTLA-4 protein expression characteristics were analyzed. And analyze the influence of Ki-67 expression on the survival of patients with different CD8 and CTLA-4 protein and gene expression characteristics. Results: Among the 47 cases, 63.8% (30/47) had low/medium level of infiltrated lymphocyte. The immunohistochemistry scores of CTLA-4, CD8, 5-mC and Ki-67 were 92.97 (54.95, 120.65) , 72.41 (36.62, 89.82) , 11.09 (3.40, 52.89) and 5.88 (2.41, 11.48) , respectively. Patients with CD8(high) CTLA-4(high) had higher 5-mC level than those with CD8(high) CTLA-4(low) (P<0.01) . The median survival time of 27 cases was 0.83±0.29 year. The median survival times of those with CD8(high) CTLA-4(high) and CD8(high) CTLA-4(low) were 0.58±0.51 year and 0.83±0.30 year, respectively (P=0.521) . The immunohistochemistry score of Ki-67 ≥5.88 was an independent death risk factor for patients with CD8(high) CTLA-4(low) (HR=8.40, P=0.01) . Under different CD8 and CTLA-4 protein expression characteristics, in the patients with CD8(high) CTLA-4(low), the median survival times of those with high and low Ki-67 expression were 0.57±0.11 years and 2.31±0.46 years, respectively (P<0.01) . Under different CD8 and CTLA-4 mRNA expression characteristics, in the patients with CD8(high) CTLA-4(low), the median survival times of those with high and low Ki-67 mRNA expression were 1.20±0.36 years and 3.38±0.43 years, respectively (P=0.018) . Conclusion: Mesothelioma case with high CD8 but low CTLA-4 content might coexist DNA hypomethylation. In the presence of high Ki-67 expression, their survival time appears to be shortened with increased death risk.

Objective: To investigate the distribution of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (+49 A/G) gene variants and the association of these variants with the clinical and laboratory findings in Egyptian children with Type-1 Diabetes (T1D). Methods: A case control study was done for 104 Egyptian children with T1D and 78 age and sex matched healthy control. CTLA-4 (+49 A/G) gene polymorphism typing was done by PCR amplification followed by restriction fragment length polymorphism (RFLP) method. Results: CTLA-4 G allele and GG homozygous genotype were significantly increased in T1D patients than in control group (P = 0.047, P = 0.048 respectively). There is no statistical difference between patient with optimal diabetic control (HbA1c < 8.5) and poor control (HbA1c ≥ 8.5) as regarding the CTLA-4 gene variant. The CTLA-4 GG genotype was statistically associated with younger age of patients (P = 0.027) and younger age of presentation (P = 0.036). Insignificant association was found between CTLA-4 alleles / genotypes and diabetic complications. Conclusion: The CTLA-4 +49 GG homozygous genotype is associated with T1D in Egyptian children especially with younger age of onset and in younger patients, and not associated with grades of diabetic control or diabetic complication.

CTLA4 gene polymorphism in Italian patients with colorectal adenoma and cancer

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is an important negative regulator of T-cell response and its genetic association with type 1 diabetes (T1D) has recently been demonstrated. The frequent co-association of autoimmune diseases (AID) and the implication from multiple genome scans that the CTLA4 gene region is a general autoimmune region, led us to study the role of CTLA4 in independent cohorts of T1D, coeliac disease (CD) and rheumatoid arthritis (RA) patients. We present independent data that confirm the association of CTLA4 in Dutch patients with juvenile onset T1D and show differential association of CTLA4 with CD and RA. The CTLA4 gene polymorphisms were tested for association in 350 T1D, 310 CD, 520 RA patients and 900 controls. In addition, 218 families were tested by the transmission disequilibrium test (TDT). T1D patients showed the highest association with the MH30*G: -1147*C: +49*G: CT60*G: JO37_3*G (haplotype 2) in both a case/control cohort (P=0.002, OR=1.42) and by TDT (P=0.02, OR=1.43). In contrast, this haplotype showed no association in the RA and CD cohorts. However, we observed an increased frequency of the MH30*G: -1147*T: +49*A: CT60*G: JO37_3*A (haplotype 3) in the CD patients diagnosed at a young age (OR=1.6, P=0.026, P (c)=0.052). Furthermore, when T1D and CD patients were stratified based on the HLA risk, the T1D susceptible CTLA4 haplotype 2 was over-represented in the high HLA-risk T1D and CD groups. In conclusion, we confirmed association between CTLA4 haplotype 2 and T1D in the Dutch population. Association with another CTLA4 haplotype (haplotype 3) was confirmed for CD, but only in those patients who had an early age of expression. No effect was found between RA and CTLA4. The association of the CTLA4 haplotype 2 with the high-risk HLA genotype in T1D and CD, which share DQ2 as the one of high-risk alleles, might provide a clue to understanding the common genetic background of AID.

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Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a potent immunoregulatory molecule that downregulates T-cell activation and thus influences the antitumor immune response. CTLA-4 polymorphisms are associated with various cancers, and CTLA-4 mRNA/protein increased expression is found in several tumor types. However, most of the studies are based on peripheral blood mononuclear cells, and much less is known about the relationship between CTLA-4 expression, especially gene expression, and its polymorphic variants in cancer tissue. In our study we assessed the distribution of CTLA-4 two polymorphisms (+49A/G and −318C/T), using TaqMan probes (rs231775 and rs5742909, resp.), and CTLA-4 gene expression in real-time PCR assay in non-small-cell lung cancer (NSCLC) tissue samples. The increased CTLA-4 expression was observed in the majority of NSCLC patients, and it was significantly correlated with TT genotype (−318C/T) and with tumor size (T2 versus T3 + T4). The presence of G allele and GG genotype in cancer tissue (+49A/G) was significantly associated with the increased NSCLC risk. Additionally, we compared genotype distributions in the corresponding tumor and blood samples and found statistically significant differences. The shift from one genotype in the blood to another in the tumor may confirm the complexity of gene functionality in cancer tissue.

Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is considered as a negative regulator of T cell activation and its role in maintaining immune tolerance is well established. The present case-control study aimed to investigate the CTLA-4 +49 A/G, -1661 A/G, -318 C/T and -1722 T/C single nucleotide polymorphisms (SNPs) and predisposition to recurrent pregnancy loss (RPL) in Gaza Strip - Palestine. The study was performed on 200 women with a history of two or more pregnancy losses (case group) and 200 control women with at least two live births and without any previous history of abortion. PCR-based restriction fragment length polymorphism (RFLP-PCR) method was used for genotyping CTLA-4 polymorphisms. Study results revealed that there is no significant association between the allele/genotype frequencies of the four investigated CTLA-4 SNPs and RPL. This trend remained true under dominant, co-dominant and recessive models. The A\G genotype of -1661 A\G polymorphism was higher in patients (45%) as compared to controls (39.5%) but without statistical significance. The minor allele frequencies (MAFs) of the CTLA-4 gene polymorphisms in the patient/control group were as follows: +49A>G: 0.22/0.22, -318 C>T: 0.15/0.11, -1661 A>G: 0.30/0.26 and -1722 T>C: 0.08/0.08. The four investigated CTLA-4 polymorphisms do not contribute to the risk of RPL in the study population. Testing other CTLA-4 gene polymorphisms and the level of CTLA-4 expression in RPL patients is recommended.

Myasthenia gravis (MG) is an autoimmune disease of striated muscle tissue mediated by autoantibodies. MG is often treated with thymectomy. Protein tyrosine phosphatase non-receptor 22 (PTPN22) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have recently been found to be the genes that predispose to autoimmune diseases. The mechanisms of PTPN22 and CTLA-4 single nucleotide polymorphisms in resected thymomas and thymuses in MG remain unclear. In the present study, 90 patients with thymomas, including 44 patients with MG, 46 patients without MG, and 35 MG patients without thymoma were studied, with 50 healthy people as the controls. The +1858C>T polymorphism of the PTPN22 gene and the 49A>G polymorphism of the CTLA-4 gene were investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The PTPN22 -1123G>C polymorphism at the promoter site was genotyped using single allele-specific primer polymerase chain reaction (SASP-PCR). The PTPN22 +1858C>T polymorphism was not significantly different between the patients and the controls. Statistically significant differences in the allelic and genotypic frequencies of PTPN 22 -1123G>C and CTLA-4 49A>G were observed between the MG(+)-thymoma group and the controls (P = 0.000, 0.003), but not between the MG(-)-thymoma and MG-thymoma(-) groups and the controls (P = 0.192/P = 0.214 and P = 0.067/P = 0.254). Statistically significant differences in allelic and genotypic frequencies of the 49A>G for CTLA-4 were observed between the MG(+)-thymoma group and the controls (P = 0.000, P = 0.003), but not between the MG(-)-thymoma and MG-thymoma(-) groups and the controls (P = 0.077/P = 0.261 and P = 0.058/P = 0.058). Individuals with the PTPN22 CC genotype and the CTLA-4 G alleles had an increased risk of developing paraneoplastic MG (odds ration [OR]= 4.722, 95% confidence interval [CI]: 1.460-15.277) compared with those with the PTPN22 G allele and the CTLA-4 AA genotype. The results show an association between the PTPN22 1123G>C genotype and thymoma-associated MG, with significant synergy with the CTLA-4 G alleles.

Background:Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T lymphocytes and has been shown to be associated with a number of autoimmune diseases. The present study...

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

Recent evidence has pointed out that the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression is a poor prognosis factor. However, the implications of CTLA-4 expression on circulating inflammatory mediators are unclear for breast cancer. Tumor biopsies and blood samples were collected from 117 breast cancer patients. Oxidative stress parameters were evaluated in plasma samples by measuring the lipoperoxidation profile and nitric oxide metabolites (NOx). Interleukins 12 (IL-12) and 4 (IL-4) were assessed by ELISA. CTLA-4 expression was determined by immunofluorescence assessed by its labeling in tumor-infiltrating leukocytes (TILs) or breast tumors. Correlations between CTLA-4 expression in breast tumors with TCD4/TCD8 infiltrating lymphocyte and inflammation-related genes were performed using data from TIMER 2.0/TCGA databases (n = 2160). CTLA-4 expression in TILs significantly correlated to triple-negative breast tumors. Patients carrying CTLA-4-positive tumors exhibited lower plasmatic NOx levels, and those expressing CTLA-4 in TILs had reduced levels of IL-12 in plasma. No changes in either IL-4 or lipid peroxidation profiles were detected concerning any CTLA4 status. Compared to the Luminal A ones, oxidative stress parameters and cytokines were observed in patients bearing triple-negative tumors. CTLA-4 expression in all breast cancer subtypes positively correlated to TCD4/TCD8 lymphocyte infiltrates, as well as to the pro-inflammatory genes IL12A, IL4, NFKB1, NFKB2, NOS1, NOS2, and NOS3. CTLA-4 expression in both tumor and TILs can affect the systemic inflammatory status of breast cancer patients, especially antitumor molecules such as IL-12 and NOx that correlate to more aggressive disease.