Levels Of sCTLA-4 Research Articles

Serum Immune Activation Markers Are Persistently Increased in Patients with HIV Infection after 6 Years of Antiretroviral Therapy despite Suppression of Viral Replication and Reconstitution of CD4+T Cells

The effect of long-term antiretroviral therapy on serum immune activation markers was assessed in a cohort of 63 patients before and after 6 years of boosted lopinavir-based antiretroviral therapy. High levels of most markers were associated with lower CD4(+) T cell counts at baseline and at year 6, with the exception of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4); high levels of sCTLA-4 were associated with higher CD4(+) T cell counts at year 6. Abnormalities of serum immune activation markers persisted after 6 years of ART but probably had different causes. Further investigation of the clinical usefulness of assaying immunoglobulin A, neopterin, and sCTLA-4 levels to assess the effectiveness of treatments for human immunodeficiency virus (HIV) disease are warranted.

Soluble CTLA-4 receptor an immunological marker of Graves' disease and severity of ophthalmopathy is associated with CTLA-4 Jo31 and CT60 gene polymorphisms

Graves' disease (GD) is an autoimmune disorder with genetic and environmental background. CTLA-4 is a candidate gene for thyroid autoimmunity. Increased serum levels of soluble CTLA-4 (sCTLA-4) were found in some autoimmune diseases. The aim of the study was to evaluate the relation between sCTLA-4 level and clinical manifestation of Graves' ophthalmopathy (GO), thyroid status, and CTLA-4 gene polymorphisms. Serum sCTLA-4 concentrations were determined in 93 GO patients and 93 healthy controls. In the GO group, CTLA-4 gene was genotyped in five polymorphic sites: g.319C>T, c.49A>G, CT60 by means of PRC-RFLP, Jo31, and g.*642AT(8_33) by means of minisequencing assay. Serum sCTLA-4 level was significantly higher in the GO group than in controls (median: 7.94 vs 0.00 ng/ml, P=0.000001). This level was higher in severe than in nonsevere GO (median: 10.3 vs 5.6 ng/ml, P=0.01). sCTLA-4 concentration was related neither to the activity of GO nor to thyroid function. Elevated sCTLA-4 levels were observed in carriers Jo31[G] allele (genotype GG+GT) as compared with subjects with an absence of the [G] allele (TT genotype; median: 9.18 vs 4.0 ng/ml, P=0.02). Also patients possessing CT60[G] allele (genotype GG+GA) had higher serum sCTLA-4 levels than subjects who lack the [G] allele (AA genotype; median: 8.73 vs 2.28 ng/ml, P=0.03). It was shown for the first time that increased serum concentration of sCTLA-4 correlate with the severity of GO. Genetic variation in the CTLA-4 gene region in GD patients at least partially determines the level of sCTLA-4.

CTLA4 gene polymorphisms and soluble CTLA4 protein in Behcet’s disease

Cytotoxic T lymphocyte antigen 4 (CTLA4; CD152) is a costimulatory molecule expressed on activated T cells that plays a key inhibitory role during T lymphocyte activation. The gene encoding for CTLA4 has been suggested as a candidate for conferring susceptibility to autoinflammatory diseases. We investigated the polymorphisms of the CTLA4 gene [promoter region (-1722 T/C, -1661 A/G and -318 C/T) and exon 1 (+49 G/A)] and the differences of serum soluble sCTLA4 levels in 285 patients with Behcet's disease (BD) and 287 controls. The frequency of the CTLA4 -1661 GG genotype was significantly higher in BD patients than in controls [P = 0.019, odds ratio (OR) = 5.2, 95% confidence interval (CI) = 1.13-23.86]. Also, the genotype frequency for CTLA4 -1722 TC was significantly higher (P = 0.014, OR = 1.8, 95% CI = 1.13-2.99), while CTLA4 -1722 CC was significantly lower (P = 0.018, OR = 0.4, 95% CI = 0.20-0.87) in BD patients with ocular lesions compared with patients without this symptom. Serum sCTLA4 levels in BD patients were significantly lower, especially in BD patients with the CTLA4 +49 G allele, than those in healthy controls (P < 0.05). Although our understanding of the role of the CTLA4 gene and its protein product in BD is incomplete, these results suggest that single nucleotide polymorphisms of the promoter and exon regions in the CTLA4 gene are candidates that predispose to BD and that sCTLA4 may be related to the immunological abnormalities and disease expressions associated with BD.

A functional soluble form of CTLA-4 is present in the serum of celiac patients and correlates with mucosal injury

Celiac disease (CD) is a multifactorial disorder influenced by environmental, genetic and immunological factors. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune disorders. A native soluble cytotoxic T-lymphocyte-associated protein-4 (sCTLA-4), lacking of transmembrane sequence, has been described in several autoimmune diseases. We aimed to evaluate the presence of increased sCTLA-4 concentration in the serum of patients with CD and the possible immunoregulatory function. Blood samples were collected from 160 CD patients; sCTLA-4 levels were evaluated by ELISA, western blot and reverse transcription-PCR. The capability of serum sCTLA-4 to modulate T-lymphocyte proliferation in vitro was evaluated by two-way mixed leukocyte reaction assay. We demonstrated high levels of sCTLA-4 in serum of untreated celiac patients. Additionally, we observed that sCTLA-4 concentrations are related to gluten intake and that a correlation between autoantibodies to tissue transglutaminase and sCTLA-4 concentration exists. Moreover, sCTLA-4 levels correlate with the degree of mucosal damage. Conversely, no correlation between sCTLA4 levels and the HLA-related risk was observed. Finally, we show that sCTLA-4 from sera of CD patients displays functional activities. These results strongly suggest a regulation of sCTLA-4 synthesis depending on the presence or absence of dietary gluten and imply a possible immunomodulatory effect on cytotoxic T lymphocyte functions. In gluten-exposed patients, serum sCTLA-4 levels might provide insight about mucosal injury.

The Soluble CTLA-4 Receptor and its Emerging Role in Autoimmune Diseases

CTLA-4, expressed by activated T and B lymphocytes, is a co-stimulatory receptor transducing a potent inhibitory signal. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. A soluble form of cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4) was recently found and shown to possess CD80/CD86 binding activity and in vitro immuno-regulatory functions. sCTLA4 is generated by alternatively spliced mRNA. Low levels of sCTLA-4 have been detected in normal human serum, but increased serum levels have been observed in several autoimmune diseases (e.g. Graves’ disease, myasthenia gravis, systemic lupus erythematosus, type 1 diabetes, systemic sclerosis, celiac disease and autoimmune pancreatitis disease). The biological significance of increased sCTLA-4 serum level has not been fully clarified. On one hand, we can hypothesize that sCTLA-4 can specifically inhibit the early T-cell activation by blocking the interaction of CD80/CD86 with the costimulatory receptor CD28. On the other hand, higher levels of sCTLA-4 could compete with membrane-bound CTLA-4 for CD80/CD86, in later T lymphocytes activation phase, causing a reduction of inhibitory signaling. Thus, this doubleedged nature of CD80/CD86 blocking by sCTLA-4 may result in different outcomes during the clinical course of the dis-

Interactions between programmed death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) gene polymorphisms in type 1 diabetes

Aim To explore the contribution of the PD-1 gene polymorphisms involved in T1D as well as the relationship between the PD-1/ CTLA-4 genes and soluble CTLA-4 concentrations. Patients and methods 261 incident cases of T1D and 280 healthy children less 15 years old were included in this study. Haplotypes for polymorphisms of the PD-1 and CTLA-4 genes were determined by PCR and RFLP methods. Screening for soluble CTLA-4 was done using an ELISA assay. Statistical analysis was performed using the online SHESIS package. Results Our results show that sCTLA-4 levels were higher in T1D than in controls (2.99 ± 1.7 ng/ml versus 1.43 ± 0.31 ng/ml, p < 0.001). The allele dosage of CTLA-4 on PD-1 haplotypes, showing a significant modified effect of G carriers over AA genotype on the sCTLA-4 concentrations (5.48 ± 2.09 ng/ml versus 3.27 ± 1.30 ng/ml, p < 0.03 in T–C haplotype) and (1.92 ± 0.79 ng/ml versus 3.41 ± 1.10 ng/ml, p < 0.02 in C–T haplotype). Conclusion Consistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 is elevated in T1D. Our data suggest a possible gene dosage effect of “G” CTLA-4 carriers on sCTLA-4 over the possible protective or susceptible effect conferred by PD-1 haplotypes.

Increased production of soluble CTLA-4 in patients with spondylarthropathies correlates with disease activity

IntroductionSpondylarthropathies (SpA) are characterized by abnormal immune responses including T cell activation. Cytotoxic T lymphocyte associated molecule-4 (CTLA-4) is involved in down-regulating immune responses. A soluble form of CTLA-4 (sCTLA-4), resulting from an alternative splicing, has been identified and was found increased in several autoimmune diseases. Here, we evaluated circulating levels of sCTLA-4 as a marker of immune dysregulation in SpA. Intracellular CTLA-4 and levels of CTLA-4 transcript expression in peripheral blood lymphocytes (PBL) were also studied.MethodsSera from 165 patients with SpA were evaluated for sCTLA-4 measurements. Results were compared with those from 71 patients with rheumatoid arthritis (RA) and 88 healthy subjects. In 32 patients with SpA, 22 patients with RA and 15 healthy controls, we analyzed the intracellular CTLA-4 expression in CD4+ T cells, CD8+ T cells, activated (HLA-DR+Foxp3-) CD4+ T cells, CD4+ regulatory (CD25+Foxp3+) T cells and in CD3 negative cells by flow cytometry. Expression of the full length (coding for membrane CTLA-4) and spliced form (coding for sCTLA-4) of CTLA-4 transcripts in PBL were analyzed by quantitative real-time polymerase chain reaction (QRT-PCR).ResultsHigh levels of sCTLA-4 were found in the SpA group compared to the RA group and healthy controls (P < 0.0001). Soluble CTLA-4 serum levels strongly correlated with clinical index of disease activity BASDAI (r = 0.42, P < 0.0001) and C-reactive protein (CRP) levels (r = 0.17, P = 0.037). In contrast to RA patients, SpA patients did not exhibit changes in intracellular CTLA-4 expression in the different PBL subsets tested. Finally, the SpA group showed a preferential expression of the spliced CTLA-4 mRNA (P = 0.0014) in PBL.ConclusionsSpA patients exhibit high levels of circulating sCTLA-4 that may result from an alternative splicing of CTLA-4 transcripts. This may influence immune activation and regulation in SpA.

Biochemical analysis of CTLA-4 immunoreactive material from human blood

BackgroundCTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear.MethodsMolecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material.ResultsMass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins.ConclusionWe have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.

Lack of association between sCTLA-4 levels in human plasma and common CTLA-4 polymorphisms

BackgroundCytotoxic T lymphocyte antigen-4 (CTLA-4) is an important downregulatory molecule expressed on both T and B lymphocytes. Numerous population genetics studies have documented significant associations between autoimmune diseases and single nucleotide polymorphisms (SNPs) within and around the CTLA-4 region of chromosome 2 in man. Furthermore, circulating levels of a soluble form of CTLA-4 (sCTLA-4) have been reported in a variety of autoimmune mediated diseases. Despite these findings, the relationship between levels of sCTLA-4 protein, mRNA transcript levels, and SNPs within the CTLA-4 region have not been clearly defined. In order to further clarify this relationship, we have tested four different SNPs within the CTLA-4 region among subjects whom are negative (n = 53) versus positive (n = 28) for sCTLA-4.ResultsOur data do not support a clear association between sCTLA-4 levels and any of the four SNPs tested.ConclusionThe variation in the SNPs tested does not appear to effect sCTLA-4 protein levels, despite reports that they affect sCTLA-4 mRNA.

Genomic regulation of CTLA4 and Multiple Sclerosis

The cytotoxic T lymphocyte antigen 4 gene (CTLA4) is a critical regulator of T-cell activation and it is an important therapeutic target for cancer and autoimmune diseases. Here, we analyzed the genomic regulation of CTLA4 gene expression in order to identify single nucleotide polymorphisms (SNPs) that affect its expression and splicing, and to assess their association with Multiple Sclerosis (MS). We analyzed 152 healthy subjects and 146 patients with MS, of which 52 controls and 51 patients were used for gene expression analysis. We genotyped 17 SNPs in the CTLA4 gene using the SNaPshotTM Multiplex Kit, and in addition gene expression of the soluble (sCTLA4) and full length (flCTLA4) isoforms was quantified by real-time PCR, while protein levels of sCTLA4 were measured by ELISA. We found that the SNPs at − 1577, + 6230, + 10242, + 10717 and + 12310 influence CTLA4 expression and the combination of the − 1577 GG and + 6230 GG genotypes provokes the strongest decrease in CTLA4 gene expression. We found that the SNP at − 658 only acted as a regulatory SNP in patients with MS, suggesting the existence of epigenetic changes due to this disease. We also identified a decrease in CTLA4 gene expression levels in patients receiving chemotherapy, although no association was observed between MS and any of the polymorphisms studied. In conclusion, we have identified several SNPs in the CTLA4 gene and studied their influence on its genetic regulation. The involvement of CTLA4 in the pathogenesis of MS may be subtle and related to the functional changes in its pathway rather than predisposing genetic polymorphisms.

Differences in the Contribution of the CTLA4 Gene to Susceptibility to Fulminant and Type 1A Diabetes in Japanese Patients

OBJECTIVE—To examine the contribution of the CTLA4 gene in the susceptibility to fulminant type 1 diabetes and compare it with classic type 1A diabetes.RESEARCH DESIGN AND METHODS—We genotyped the +49G>A and CT60G>A variants of the CTLA4 gene in fulminant type 1 diabetic patients (n = 55), classic type 1A diabetic patients (n = 91), and healthy control subjects (n = 369). We also assessed serum levels of the soluble form of CTLA4 (sCTLA4).RESULTS—The +49GG and CT60GG genotypes were associated with type 1A diabetes (P < 0.001). In contrast, the CT60AA genotype, but not the +49G>A variation, was associated with fulminant type 1 diabetes (P < 0.05), especially in patients carrying HLA-DR4 (P < 0.01). Serum levels of sCTLA4 were significantly decreased in patients with fulminant type 1 diabetes (P < 0.05).CONCLUSIONS—These results suggest that CTLA4 CT60 affects the genetic susceptibility to fulminant type 1 diabetes. Furthermore, the contribution of CTLA4 to disease susceptibility is distinct between fulminant type 1 diabetes and classic type 1A diabetes.

Association of Autoimmune Pancreatitis With Cytotoxic T-lymphocyte Antigen 4 Gene Polymorphisms in Japanese Patients

Autoimmune pancreatitis (AIP) is characterized by high serum IgG4 concentrations, lymphoplasmacytic inflammation, and a favorable response to corticosteroid treatment. Although the HLA DRB1*0405-DQB1*0401 haplotype and Fc receptor-like 3 polymorphisms have been associated with AIP, the role of other genetic factors is largely unknown. As cytotoxic T-lymphocyte antigen 4 (CTLA4) polymorphisms have recently been linked with several autoimmune diseases, we sought to determine if CTLA4 polymorphisms and serum sCTLA4 levels were associated with AIP as well. Five CTLA4 polymorphisms, located at -1722, -658, and -318 in the promoter, +49 in exon 1, and +6230 in the 3' untranslated region, were genotyped in 59 patients with AIP and 102 healthy subjects. Serum sCTLA4 levels were also determined in cohorts of 52 patients and 32 controls. Compared with healthy subjects, we found a significant increase in the +6230 G/G genotype (64%vs 42%, odds ratio [OR] 2.48, P= 0.011) in AIP patients. Haplotype 2, which had the +6230A, was associated with AIP resistance (OR 0.49, P= 0.011). The +49A/A and +6230A/A genotypes were associated with an enhanced risk of relapse (OR 5.45, P= 0.038 and OR 12.66, P= 0.022). Additionally, median serum sCTLA4 levels were significantly higher in patients with AIP (8.9 ng/mL) compared with healthy subjects (2.9 ng/mL, P < 0.001). The +6230 G/A polymorphism did not influence sCTLA4 levels in AIP patients. Our findings suggest that AIP is associated with a genetic polymorphism in CTLA4 and is positively correlated with serum sCTLA4 levels.

Decrease of CD4 +CD25 highFoxp3 + regulatory T cells and elevation of CD19 +BAFF-R + B cells and soluble ICAM-1 in myasthenia gravis

Myasthenia gravis (MG) is caused by T-cell-dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokine and chemokine sICAM-1 and to analyze the phenotypes of CD4 + and CD8 + regulatory cells as well as the expression of BAFF-R on CD19 + B cells in peripheral blood from 75 MG patients and 50 healthy controls. There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-β and sCTLA-4 in both sera and culture supernatants between MG patients and healthy controls. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Although the populations of CD8 +CD28 − and CD8 +CD122 + regulatory T cells were not different between MG patients and healthy controls, MG patients exhibited the decrease of CD4 +CD25 highFoxp3 + regulatory T cells and the increase of CD19 +BAFF-R + B cells, revealing that MG patients should display the dysfunction of T cell balance and the activation of B cell maturation.

Soluble CTLA-4 in autoimmune thyroid diseases: Relationship with clinical status and possible role in the immune response dysregulation

CTLA-4 molecule, expressed by activated T and B lymphocytes, transduces an inhibitory signal. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. The aim is to evaluate the augmented sCTLA-4 serum levels in different autoimmune thyroid diseases when compared with normal donors or with non-autoimmune hyperthyroidism and to investigate the functional activities and suggest the possible pathogenetic role of sCTLA-4. We demonstrate the presence of a soluble form of CTLA-4 in 59/90 sera from patients with autoimmune thyroid diseases (both Graves’ disease and autoimmune thyroiditis). sCTLA-4 levels were not related to specific clinical manifestations, such as clinical thyroid status (hypo- or hyperthyroidism), circulating thyroid hormones, or other clinical features (ophthalmopathy). sCTLA-4 production does not seem to be affected by disease evolution during time. We showed that sCTLA-4 from sera of patients with thyroid autoimmunity is able to bind its physiological ligands CD80/CD86 and displays functional activities on different in vitro systems (T-cell proliferation induced by specific soluble antigens, bi-directional mixed lymphocyte reaction). In conclusion, we demonstrate an increment of sCTLA-4 in serum of patients with autoimmune thyroid diseases. Its possible pathogenetic role during autoimmune processes can be speculated: sCTLA-4 can specifically inhibit the early T-cell activation by blocking the interaction of CD80/CD86 with the co-stimulatory receptor CD28. Conversely, higher levels of sCTLA-4 could compete with membrane-bound CTLA-4 for CD80/CD86, in later T lymphocytes activation phase, causing a reduction of inhibitory signaling.

CT60 genotype does not affect CTLA-4 isoform expression despite association to T1D and AITD in northern Sweden.

BackgroundPolymorphisms in and around the CTLA-4 gene have previously been associated to T1D and AITD in several populations. One such single nucleotide polymorphism (SNP), CT60, has been reported to affect the expression level ratio of the soluble (sCTLA-4) to full length CTLA-4 (flCTLA-4) isoforms. The aims of our study were to replicate the association previously published by Ueda et al. of polymorphisms in the CTLA-4 region to T1D and AITD and to determine whether the CT60 polymorphism affects the expression level ratio of sCTLA-4/flCTLA-4 in our population.MethodsThree SNPs were genotyped in 253 cases (104 AITD cases and 149 T1D cases) and 865 ethnically matched controls. Blood from 23 healthy individuals was used to quantify mRNA expression of CTLA-4 isoforms in CD4+ cells using real-time PCR. Serum from 102 cases and 59 healthy individuals was used to determine the level of sCTLA-4 protein.ResultsHere we show association of the MH30, CT60 and JO31 polymorphisms to T1D and AITD in northern Sweden. We also observed a higher frequency of the CT60 disease susceptible allele in our controls compared to the British, Italian and Dutch populations, which might contribute to the high frequency of T1D in Sweden. In contrast to previously published findings, however, we were unable to find differences in the sCTLA-4/flCTLA-4 expression ratio based on the CT60 genotype in 23 healthy volunteers, also from northern Sweden. Analysis of sCTLA-4 protein levels in serum showed no correlation between sCTLA-4 protein levels and disease status or CT60 genotype.ConclusionAssociation was found between T1D/AITD and all three polymorphisms investigated. However, in contrast to previous investigations, sCTLA-4 RNA and protein expression levels did not differ based on CT60 genotype. Our results do not rule out the CT60 SNP as an important polymorphism in the development of T1D or AITD, but suggest that further investigations are necessary to elucidate the effect of the CTLA-4 region on the development of T1D and AITD.

The soluble forms of CD28, CD86 and CTLA‐4 constitute possible immunological markers in patients with abdominal aortic aneurysm

The T cell co-stimulatory factors CD28 and CTLA-4 and their ligands CD80 and CD86 occur as receptors on T cells and antigen-presenting cells and also in soluble forms in the circulation. We determined the levels of soluble co-stimulatory molecules in patients with abdominal aortic aneurysm (AAA) and normal individuals. We further correlated these soluble co-stimulatory molecules to other clinical parameters of importance such as age of the patient, presence of hypertension, size of the aneurysm and levels of matrix metalloproteinases-9 and C-reactive protein. This case-control study was designed to quantify the circulating levels of soluble co-stimulatory molecules by an in-house enzyme linked immunosorbent assay. A total of 314 subjects participated in the study including 100 patients and 214 normal controls. The statistical analysis was performed by Mann-Whitney test and Spearman's correlation rank test. Our results show increased plasma levels of sCD28, sCD86 (P = 0.0001) and decreased plasma levels of sCTLA-4 (P = 0.0018) in the patients compared with normal individuals. The levels of these factors were not related to the age of the patient, size of aneurysm or levels of C-reactive protein in plasma. There was, however, a significant inverse relationship between the concentrations of sCTLA-4 and sCD80 with matrix metalloproteinase-9. We suggest that soluble co-stimulatory molecules serve as biomarkers for the estimation of immune activation in AAA patients.

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

Genetic and functional studies suggest that polymorphism in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) and inducible costimulator (ICOS) genes, both reported to harbour autoimmune susceptibility loci, could regulate the immune activation through affecting their expression and splicing of CTLA4. To address this, we studied expression of CTLA4 and ICOS and the role of polymorphisms in the gene region by measuring the relative amounts of transcripts, including the soluble CTLA4 (sCTLA4) splicing isoform in healthy volunteers. We combined a physiologically relevant in vitro activation for human CD4 + T lymphocytes and a quantitative RT-PCR. The susceptibility allele CT60G in CTLA4 gene was confirmed to be associated with a decreased amount of sCTLA4, but only in resting cells. During the T cell activation two genetic variants in ICOS gene, IVS1+173T/C and c.1624C/T, affected expression of CTLA4 isoforms and ICOS, respectively. We could not confirm that the level of sCTLA4 is down-regulated following T lymphocyte activation, instead the levels of CTLA4 splicing isoforms correlated to each others. Our results indicate that genetic variation in this gene region regulates the expression of both CTLA4 and ICOS and not only the splicing of sCTLA4 as suggested earlier.

Relationship of CTLA-4 exon 1 A49-->G polymorphism with sCTLA-4 and Th1/Th2 bias in idiopathic dilated cardiomyopathy

To investigate the association of cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene exon 1 A49-->G polymorphism with the genetic susceptibility to idiopathic dilated cardiomyopathy (IDC) in Chinese Han nationality. Peripheral blood samples were collected from 48 patients with IDC, 31 males and 17 females, and 50 sex- and age-matched normal controls. ELISA was used to examine the cytokines: sCTLA-4, gamma-interferon (IFN-gamma), and interleukin-4 (IL-4)with the ratio of IFN-gamma/IL-4 as an indicator for Th1/Th2 bias. PCR-RFLP was used to analyze the A/G polymorphism of CTLA-4 exon 1 A49-->G. The relationship of CTLA-4 genotype and alleles frequencies with sCTLA-4, IFN-gamma and IFN-gamma/IL-4 was evaluated by linear regression analysis. Compared with the normal controls, the frequencies of GG genotype (0.6042 and 0.7396, P = 0.012) and the G allele (0.36 and 0.56, P = 0.008) were significantly increased in the patients with IDC. Increased serum sCTLA-4 was found in the IDC group compared with the controls (1.87 microg/L +/- 1.06 microg/L vs. 0.54 microg/L +/- 0.19 microg/L, P < 0.05). IFN-gamma was significantly lower in the IDC group than in the control group (16 ng/L +/- 6 ng/L vs. 30 ng/L +/- 10 ng/L, P < 0.05). The ratio of IFN-gamma/IL-4 was significantly in the IDC group than in the control group (1.63 +/- 0.50 vs. 3.01 +/- 0.89, P < 0.05). No statistically difference was found in the IL-4 level between the two groups. Linear regression analysis manifested significant interrelationship between the GG genotype, G allele frequencies and serum sCTLA-4 (r = 0.57, P = 0.021), IFN-gamma/IL-4 ratio (r = 0.42, P = 0.028) in the IDC group. While no correlation was found for AA, AG genotype and the A allele frequency. CTLA-4 gene exon 1 A49-->G substitution is associated with an increased IDC genetic susceptibility, which implicates that the CTLA-4 gene may have a significant role in IDC, possibly via a Thr-->Ala change in CTLA-4 signal peptide, with a result of functional change of sCTLA-4. The bias of Th1/Th2 paradigm is associated with the increased sCTLA-4 level under certain background of immunogenicity.

Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes

BackgroundCytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have been shown to be associated with several autoimmune diseases including type-1 diabetes (T1D). The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that is believed to control the processing and production of soluble CTLA-4 (sCTLA-4).MethodsWe therefore determined sCTLA-4 protein levels in the sera from 82 T1D patients and 19 autoantibody positive (AbP) subjects and 117 autoantibody negative (AbN) controls using ELISA. The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye terminating sequencing reaction.ResultsHigher levels of sCTLA-4 were observed in T1D (2.24 ng/ml) and AbP (mean = 2.17 ng/ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these subjects becoming significant with age (p = 0.02). However, we found no correlation between sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes.ConclusionConsistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 may be a risk factor for T1D. However, our results do not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.

Serum soluble CTLA-4 levels are increased in diffuse cutaneous systemic sclerosis.

To determine serum levels of soluble cytotoxic T-lymphocyte associated molecule-4 (sCTLA-4) and clinical association in patients with systemic sclerosis (SSc). Serum sCTLA-4 levels from 32 patients with diffuse cutaneous SSc (dSSc) and 27 patients with limited cutaneous SSc (lSSc) were examined by enzyme-linked immunosorbent assay (ELISA). For a longitudinal study, 211 sera from 30 SSc patients were analysed (follow-up 2.1-7.0 yr). Serum sCTLA-4 levels were elevated in dSSc patients compared with normal controls (n = 41), lSSc patients and patients with active systemic lupus erythematosus (SLE) (n = 23). By contrast, sCTLA-4 levels in patients with lSSc or SLE were normal. SSc patients with elevated sCTLA-4 levels had a shorter disease duration and more frequent presence of digital pitting scars, contracture of phalanges, diffuse pigmentation, pulmonary fibrosis and decreased percentage vital capacity (%VC) than those with normal sCTLA-4 levels. sCTLA-4 levels correlated positively with the extent of skin fibrosis, serum IgG levels and anti-topoisomerase I antibody levels. In a longitudinal study, sCTLA-4 levels decreased on a parallel with improvement of skin sclerosis in five dSSc patients. Skin sclerosis did not improve in two of six dSSc patients with high sCTLA-4 levels throughout the follow-up, while the remaining four patients showed improvement of skin sclerosis. These results suggest that sCTLA-4 correlates with disease severity and activity of SSc and that sCTLA-4 plays a role in immunological abnormalities of SSc, since sCTLA-4 may augment humoral immune responses as well as T-cell responses by interfering with B7-CTLA-4 interactions that induce negative signals in T and B cells.