Soluble Cytotoxic T Lymphocyte Antigen 4 Research Articles

The role of cytotoxic T-Lymphocyte antigen-4+49A/G gene polymorphism in cutaneous leishmaniasis

BackgroundCutaneous Leishmaniasis (CL) is a parasitic disease caused by intracellular protozoa belonging to the Leishmania genus. In endemic areas, only a proportion of exposed subjects develop the disease under almost similar circumstances, reflecting the role of genetic inheritance in resistance and susceptibility to infection. This study aimed To evaluate the association of cytotoxic T-lymphocyte antigen-4 (CTLA-4)+49G/A single nucleotide polymorphism (SNP) with incidence and severity of CL. MethodsThis cross-sectional study includes 110 patients with confirmed CL (60 newly diagnosed and 50 patients undergoing treatment) and 60 healthy subjects of similar age and sex. The CTLA-4 gene fragment corresponding to CTLA-4+49G/A polymorphism was amplified and genotyped using tetra primer amplification refractory mutation- polymerase chain reaction system (TARMS-PCR) methods. Soluble CTLA-4 (sCTLA-4) was estimated in the serum using an enzyme-linked immunosorbent assay (ELISA). ResultsThe GG genotype of CTLA-4+49G/A polymorphism was significantly more common in controls than in patients (OR = 0.11, 95% CI = 0.02–0.58, p = 0.009). At allelic level, G allele was much more common in controls than in patients (30.83% vs. 17.73%) with a significant difference (OR = 2.07, 95% CI = 1.23–3.48, p = 0.006). However, there was no significant difference in the frequency of genotypes and alleles between newly diagnosed and treated patients. Median serum concentration of sCTLA-4 in newly diagnosed patients was 72.6 pg/ml (range 15.6–127 pg/ml) which was higher than either controls (median = 16.3 pg/ml, range 0.8–48.5 pg/ml) or treated patients (median = 17.9 pg/ml, range 2.9–74.7 pg/ml) with highly significant differences, while there was no significant difference between controls and treated patients. The median sCTLA-4 level was comparable across genotypes of the CTLA-4+49G/A polymorphism, with no significant difference. ConclusionsCollectively, these results show the protective role of allele G of the SNP CTLA-4+49G/A against CL and increased serum sCTLA-4 in newly diagnosed CL patients, which may be used as an additional diagnostic tool. Different genotypes of the CTLA-4+49G/A polymorphism have no effect on sCLTA-4 serum levels.

Targeting co-stimulatory molecules in autoimmune disease.

Therapeutic targeting of immune checkpoints has garnered significant attention in the area of cancer immunotherapy, in which efforts have focused in particular on cytotoxic T lymphocyte antigen 4 (CTLA4) and PD1, both of which are members of the CD28 family. In autoimmunity, these same pathways can be targeted to opposite effect: to curb the over-exuberant immune response. The CTLA4 checkpoint serves as an exemplar, whereby CTLA4 activity is blocked by antibodies in cancer immunotherapy and augmented by the provision of soluble CTLA4 in autoimmunity. Here, we review the targeting of co-stimulatory molecules in autoimmune diseases, focusing in particular on agents directed at members of the CD28 or tumour necrosis factor receptor families. We present the state of the art in co-stimulatory blockade approaches, including rational combinations of immune inhibitory agents, and discuss the future opportunities and challenges in this field.

Association of the soluble CTLA4 with schizophrenia: an observational study

Abstract Objective: The immune system is involved in the pathogenesis of schizophrenia; here, we aimed to explore the relationship of cytotoxic T lymphocyte antigen 4 (CTLA4) with schizophrenia. Methods: CTLA4 gene structure was first analyzed, and then rs231779, rs733618, rs231775 and rs3087243 were selected as tag single nucleotide polymorphisms for the linkage disequilibrium blocks in CTLA4 in the Chinese Han population to study expression quantitative trait loci of CTLA4 gene in normal brain tissue. Additionally, membrane CTLA4 (mCTLA4) and soluble CTLA4 (sCTLA4) mRNA expression levels were evaluated in peripheral blood mononuclear cells from 65 first-episode schizophrenia patients and 61 healthy controls. This study was approved by the Bioethics Committee of corresponding research institutes (approval No. 20150016) on March 6, 2015 and the principles of the Declaration of Helsinki. Results: After applying Bonferroni correction to the P values, only the minor C allele of rs733618 was significantly associated with increased expression of total CTLA4 (P Bonf. = 0.019), but not mCTLA4 (P Bonf. = 0.115), in the hippocampus. The sCTLA4 expression was significantly decreased in the peripheral blood mononuclear cells of schizophrenia patients compared with healthy controls, while mCTLA4 was not. Conclusion: These results suggest that the soluble form of CTLA4 may be associated with schizophrenia and that lower sCTLA4 expression may increase the risk of developing schizophrenia.

Prognostic Value of CD200 Expression and Soluble CTLA-4 Concentrations in Intermediate and High-Risk Myelodysplastic Syndrome Patients.

Objective:This study was designed in order to identify the prognostic relevance of CD200 expression and soluble Cytotoxic T-lymphocyte antigen-4 (CTLA-4) levels in myelodysplastic syndrome (MDS) patients. Methods:The study included 57 MDS (37 intermediate and 20 high risk) patients and 10 controls. For all of included patients; CD200 expression was identified by flowcytometry on CD33 positive cells and soluble CTLA-4 (CD152) concentration was determined by ELISA. Results: CD200 positive expression was detected in 32/57 (56.1%) of MDS cases, the mean serum CTLA-4 concentrations were significantly higher in MDS patients as compared to controls (P<0.01). Significant association between high CD200 positive expression; high CTLA-4 concentration levels and MDS risk stages being higher in high risk MDS group as compared to intermediate risk one (P < 0.01). After 36-month follow-up; the subgroup of MDS patients with high expression of CD200; and high serum CTLA-4 concentrations showed high death rate and high frequency of acute myeloid leukemia transformation. Conclusions: CD200 positive expression could be considered as a new prognostic marker for risk stratification of MDS patients. CD200 expression may exert its effect through upregulation of CTLA-4.

Zoledronate decreases CTLA-4 in vivo and in vitro independently of its action on bone resorption.

Acute phase response (APR) following intravenous zoledronate (ZOL) administration is related to activation and increased proliferation of γδ T cells, attributed to the molecular mechanism of action of nitrogen-containing bisphosphonates (N-BPs). ZOL, however, has also been reported to inhibit the proliferation of regulatory T cells in vitro and to reduce the expression of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), a negative regulator of T cell activation that is increased in patients with autoimmune diseases. There are, however, no data on the relationship between ZOL treatment and soluble(s)CTLA-4 either in vivo in relevant patient populations or in vitro with the use of assays relevant to the mechanism of action of N-BPs. The objectives of the present study were firstly, to characterize the ZOL-induced APR in patients with inflammatory rheumatic diseases (IRDs) and its relationship with changes in circulating sCTLA-4 and secondly, to investigate the effects of ZOL on CTLA-4 production and expression by peripheral blood mononuclear cells (PBMCs). We studied 10 postmenopausal women with IRDs treated with intravenous ZOL 5mg. Five women experienced APR (APR+) associated with significant decreases in blood lymphocytes and increases in granulocytes and serum CRP. Serum sCTLA-4 values were increased in all patients before ZOL administration and decreased significantly 72h after the ZOL infusion (from 30.0±2.9 to 6.3±1.8ng/ml; p<0.001) with no differences between APR+ and APR- patients. Consistent with the results of the in vivo study, ZOL (1μM) decreased the production of sCTLA-4 by 87% and 57% after 3 and 5days in cultures of peripheral blood mononuclear cells (PBMCs) in vitro, respectively, and inhibited the expression of both cytoplasmic and membrane-bound CTLA-4. Our results reveal a novel immunoregulatory action of ZOL that is not related to its action on bone resorption but might be associated with reported clinically significant extraskeletal outcomes of ZOL treatment.

Soluble Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) in Chronic Myeloid Leukemia and its Association with Total and Differential Leukocytes Count

Soluble Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) in Chronic Myeloid Leukemia and its Association with Total and Differential Leukocytes Count

Cytotoxic T- Lymphocyte Antigen-4 (CTLA4) Gene Expression and Urinary CTLA4 Levels in Idiopathic Nephrotic Syndrome.

To detect Cytotoxic T- Lymphocyte Antigen-4 (CTLA4) single nucleotide polymorphisms (SNPs) at +49A/G (rs231775) and -318C/T (rs5742909) positions in children with idiopathic nephrotic syndrome (INS) and also assay urinary soluble CTLA4 (sCTLA4) levels in children with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS) and steroid sensitive nephrotic syndrome (SSNS) in remission. The study included 59 patients of INS (MCD-23, FSGS-15 and SSNS in remission-21) and 35 healthy controls. The CTLA4 SNPs profiling was done in peripheral blood mononuclear cells and urinary sCTLA4 level was assayed by ELISA kit. Although frequency of homozygous +49 GG (rs4553808) genotype (26.3% vs. 11.4%; p = 0.231) and G allele (52.6% vs. 40%; p = 0.216) were found to be higher in INS as compared to controls, the differences were statistically non-significant. Genotypes GG, AG, AA and alleles A and G frequencies were comparable among MCD, FSGS and controls. SNP at -318 C/T (rs5742909) did not show homozygous TT genotype both in INS as well as controls. Median urinary sCTLA4/creatinine level was significantly higher in MCD as compared to FSGS (p = 0.027), SSNS in remission (p = 0.001) and controls (p = 0.003). The positive associations of +49 GG genotype and G allele in patients with nephrotic syndrome were not observed. The frequencies did not differ significantly among MCD, FSGS and controls. Urinary sCTLA4 level was significantly increased in MCD; suggesting its possible role in the pathogenesis of disease.

The expression of mCTLA-4 in skin lesion inversely correlates with the severity of psoriasis

BackgroundPsoriasis is a chronic inflammatory disease characterized by epidermal hyperplasia and increased T cell infiltration. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a key factor that affects T cell function and immune response. However, whether the expression of CTLA-4 affects the severity of psoriasis is still unknown. ObjectiveThe aim of the project was to investigate the correlation between the expression of CTLA-4 and the severity of psoriasis. MethodsThe plasma soluble CTLA-4 levels and membrane CTLA-4 expression were measured by enzyme-linked immunosorbent assay and immunohistochemistry analysis in mild, moderate and severe psoriasis patients, respectively. Imiquimod-induced mouse model of psoriasis was treated with CTLA-4 immunoglobulin fusion protein (CTLA-4 Ig) or anti-CTLA-4 antibody. Epidermal thickness and infiltrating CD3+ T cell counts were evaluated. ResultsThe plasma soluble CTLA-4 levels had no significant difference among mild, moderate, and severe patients (p > 0.05). However, the membrane CTLA-4 expression in skin was significantly higher in mild psoriasis patients compared to moderate and severe psoriasis patients (17652.86 ± 18095.66 vs 6901.36 ± 4400.77 vs 3970.24 ± 5509.15, p < 0.001). Furthermore, in imiquimod-induced mouse model of psoriasis, the results showed that mimicking CTLA-4 function improved the skin phenotype and reduced epidermal thickness (172.87 ± 28.25 vs 245.87 ± 36.61 μm, n = 6, p < 0.01) as well as infiltrating CD3+ T cell counts (5.09 ± 3.45 vs 13.45 ± 4.70, p < 0.01) compared to control group. However, blocking CTLA-4 function aggregated the skin phenotype including enhanced epidermal thickness and infiltrating CD3+ T cell counts compared to control group. ConclusionThese results indicated that the expression of mCTLA-4 in skin lesion inversely correlated with the severity of psoriasis and CTLA-4 might play a critical role in the disease severity of psoriasis.

High levels of soluble CTLA-4 are present in anti-mitochondrial antibody positive, but not in antibody negative patients with primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic autoimmune cholestatic liver disease frequently characterized by anti-mitochondrial autoantibodies (AMA). A minority of patients are AMA-negative. Cytotoxic-T-Lymphocyte-Antigen-4 (CTLA-4) is a surface molecule expressed on activated T-cells delivering a critical negative immunoregulatory signal. A soluble form of CTLA-4 (sCTLA-4) has been detected at high concentrations in several autoimmune diseases, and its possible functional meaning has been suggested. We aimed to evaluate sCTLA-4 concentration in sera of patients with PBC and to correlate it to immunological abnormalities associated with the disease. Blood samples were collected from 82 PBC-patients diagnosed according to international criteria (44 AMA-positive/MIT3-positive and 38 AMA-negative-MIT3-negative), and 65 controls. sCTLA-4 levels were evaluated by ELISA and Western blot. Increased sCTLA-4 concentrations were found in all AMA-positive PBC-patients, but in none of the AMA-negative ones, nor in normal controls or in controls with unrelated liver diseases. sCTLA-4 presence was associated with autoantibodies against MIT3, but not with nuclear autoantibodies (sp100, gp210). This is the first study to demonstrate that levels of sCTLA-4 are elevated in sera of PBC patients. However, they are clearly restricted to patients with AMA positivity, suggesting an immunological difference with respect to AMA-negative ones.

Clinical Benefit from Ipilimumab Therapy in Melanoma Patients may be Associated with Serum CTLA4 Levels.

Stage IV metastatic melanoma patients historically have a poor prognosis with 5–10% 5-year survival. Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte antigen 4 (CTLA4), is one of the first treatments to provide beneficial durable responses in advanced melanoma. However, less than 25% of those treated benefit, treatment is expensive, and side effects can be fatal. Since soluble (s) CTLA4 may mediate inhibitory effects previously ascribed to the membrane-bound isoform (mCTLA4), we hypothesized patients benefiting from ipilimumab have higher serum levels of sCTLA4. We found that higher sCTLA4 levels correlated both with response and improved survival in patients treated with ipilimumab in a small patient cohort [patients with (n = 9) and without (n = 5) clinical benefit]. sCTLA4 levels were statistically higher in ipilimumab-treated patients with response to ipilimumab. In contrast, sCTLA4 levels did not correlate with survival in patients who did not receive ipilimumab (n = 11). These preliminary observations provide a previously unrecognized link between serum sCTLA4 levels and response to ipilimumab as well as to improved survival in ipilimumab-treated melanoma patients and a potential mechanism by which ipilimumab functions.

Development of a Novel Fusion Toxin Reagent for Targeting Antigen Presenting Cells In Vivo: DT390-CTLA-4

Targeted cell therapies are possible through the generation of chimeric recombinant proteins that combine a toxin, such as Diptheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the chimeric protein to the cell of interest, the DT is internalized which results in protein synthesis inhibition and subsequent cell death. The Cytotoxic T lymphocyte antigen-4 (CTLA-4), also known as CD152, is expressed on T helper cells and functions to down-regulate T cells via binding to CD80/CD86 (B7 complex) on antigen presenting cells (APC). Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80/CD86, has potential for induction of peripheral tolerance in autoimmune disease and organ transplantation. Currently, CTLA-4-Ig has proven to be an effective reagent for blocking these interactions in vivo. In our lab, we have a unique large animal model, the MGH MHC-defined miniature swine, for studying immune regulation and transplantation tolerance. We hypothesized that linking DT to CTLA-4 may provide a novel approach for in vivo depletion of APCs. We have expressed and purified recombinant soluble porcine CTLA-4 both with and without N-glycosylation in Yeast Pichia Pastoris for in vivo use in our large animal model. The glycosylated and non-N-glycosylated versions of this recombinant protein competitively inhibit the binding of an anti-CD80 antibody to a CD80 expressing porcine B cell lymphoma cell line (13271) with equal affinity (KD = 13nM). When linked to a truncated diphtheria toxin the non-N-glycosylated version of this recombinant protein proved to efficiently inhibit protein synthesis in target cells after 18 hours and significant cell death was observed after 52 hours in vitro. We are currently analyzing the effects of this DT-linked non-N-glycosylated version of recombinant CTLA-4 in vivo.

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on P. pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ∼2 to ∼8mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD=13nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model.

CTLA4CT60 Single-Nucleotide Polymorphism Is Associated with Slovenian Inflammatory Bowel Disease Patients and Regulates Expression ofCTLA4Isoforms

We have evaluated functional polymorphism (rs3087243; in literature known also as CTLA4 CT60) in the cytotoxic T lymphocyte antigen 4 (CTLA4) gene, previously associated with several autoimmune diseases, for potential association with inflammatory bowel diseases (IBD). In addition, we investigated correlations between CTLA4 CT60 polymorphism and CTLA4 gene expression in peripheral blood lymphocytes and colon biopsies from IBD patients. We genotyped CTLA4 CT60 polymorphism in 266 healthy control subjects and 481 IBD patients and found statistically lower frequency of CTLA4 CT60 AA genotype in IBD patients (13.72%) compared to control subjects (23.31%; p&#x2009;=&#x2009;0.001, odds ratio [OR]&#x2009;=&#x2009;0.504) as well as lower allele frequency of minor A allele in IBD patients (0.346) compared to control subjects (0.461, p&#x2009;&lt;&#x2009;0.001, OR&#x2009;=&#x2009;0.623). The association was confirmed with both major forms of IBD, Crohn's disease, and ulcerative colitis (UC), but was slightly stronger in UC patients, particularly when we compared allele frequency of A allele in UC patients (0.299) and control subjects (0.461, p&#x2009;&lt;&#x2009;0.001, OR&#x2009;=&#x2009;0.500). We found lower expression of the CTLA4 gene in blood lymphocytes from IBD patients compared to control subjects (p&#x2009;&lt;&#x2009;0.001) and higher CTLA4 expression in biopsies taken from inflamed part of the colon compared to noninflamed part of the colon (p&#x2009;=&#x2009;0.021). We found lower expression of soluble CTLA4 isoform than membrane-bound full-length isoform in peripheral blood lymphocytes from IBD patients compared to control subjects (p&#x2009;=&#x2009;0.010) and in lymphocytes from IBD patients with CTLA4 CT60 GG genotype compared to IBD patients with AA genotype (p&#x2009;=&#x2009;0.034). Our genotype and gene expression data suggest that CTLA4 plays a role in IBD pathogenesis. Polymorphism CTLA4 CT60 contributes to genetic susceptibility to IBD in Slovenian population and regulates expression of CTLA4 isoforms.

CTLA4 −1661A/G and 3′UTR long repeat polymorphisms are associated with ulcerative colitis and influence CTLA4 mRNA and protein expression

Reduced cytotoxic T-lymphocyte antigen 4 (CTLA4) expression has been proposed as a risk for autoimmunity. CTLA4 polymorphisms have been associated with several autoimmune diseases, including ulcerative colitis (UC). In this study, we performed genotyping for CTLA4 -1661A/G, -1722T/C and 3' untranslated region (AT)n repeat polymorphisms in 300 Chinese UC patients and in 700 healthy controls, and evaluated the effects of polymorphisms on full-length (flCTLA4) and soluble CTLA4 (sCTLA4) expression in UC patients. The frequency of the -1661G allele was higher in UC patients than in healthy controls (16.5 vs 11.4%, P=0.003, odds ratio (OR)=1.53, 95% confidence interval (95% CI): 1.17-2.01). The prevalence of (AT)n repeats of the CTLA4 gene carrying long alleles (≥116 bp) was more common in UC patients than in healthy controls (22.0 vs 6.3%, P<0.001, OR=4.21, 95% CI: 2.79-6.33), and was associated with extensive colitis (P=0.008). Among UC patients, long-allele carriers expressed lower levels of flCTLA4 and sCTLA4 mRNA and sCTLA4 protein than did short-allele carriers (P<0.001, P<0.001, P<0.001, respectively). CTLA4 gene -1661A/G and long 3' untranslated region (AT)n repeat polymorphisms are associated with UC in Central China. This is likely from decreased expressions of sCTLA4 mRNA and sCTLA4 protein. Our study suggests that CTLA4 has an important role in susceptibility for UC in Central China.

Association of Plasma Soluble CTLA-4 with Lung Function and GenePolymorphism in Chinese Asthmatic Children

Background: Cytotoxic T lymphocyte antigen 4 (CTLA-4) is known to downregulate the T_H2 immune response. Recent studies have suggested an association of CTLA-4 polymorphisms with allergic diseases. We investigated the effects of single nucleotide polymorphisms (SNPs) of CTLA-4 on asthma traits and plasma sCTLA-4 in 298 Chinese asthmatic children and 175 controls. Methods: Plasma sCTLA-4, total and allergen-specific IgE concentrations were measured by enzyme immunoassay. Six SNPs, namely –1147CT, +49AG, CT60, JO31, JO30 and JO27_1, in CTLA-4 were genotyped by restriction fragment length polymorphism. Results: Plasma sCTLA-4 was negatively associated with FEV₁/FVC (r = –0.146, p = 0.036) among our asthmatic patients. Analysis of locus-locus interaction by generalized multifactor dimensionality reduction showed that –1147CT was the best model for plasma sCTLA-4 with a cross-validation consistency of 10 out of 10 and a prediction error of 40.9% (p < 0.001). Multivariate regression analysis confirmed the association between plasma sCTLA-4 concentration with –1147CT among the 6 SNPs tested (p = 0.002) after adjustment for gender and age. The plasma sCTLA-4 concentration was significantly lower in patients homozygous for the C allele than in T allele carriers (p = 0.001). There was also a significant association between the most common haplotypes with low sCTLA-4 in asthmatics. We could not find any significant association between plasma total IgE, atopy and lung function with the 6 SNPs after Bonferroni correction. Conclusions: Plasma sCTLA-4 is associated with lung function and –1147CT polymorphism in Chinese asthmatic children. This may help to identify CTLA-4 signaling as a potential therapeutic target in asthma.

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.

CTLA-4 polymorphisms and clinical outcome after allogeneic stem cell transplantation from HLA-identical sibling donors.

CTLA-4 is an inhibitory molecule that down-regulates T-cell activation. Although polymorphisms at CTLA-4 have been correlated with autoimmune diseases their association with clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has yet to be explored. A total of 5 CTLA-4 single-nucleotide polymorphisms were genotyped on 536 HLA-identical sibling donors of allo-HSC transplants. Genotypes were tested for an association with patients' posttransplantation outcomes. The effect of the polymorphisms on cytotoxic T-lymphocyte antigen 4 (CTLA-4) mRNA and protein production were determined in 60 healthy control participants. We observed a reduction in the mRNA expression of the soluble CTLA-4 isoform in the presence of a G allele at CT60 and +49. Patients receiving stem cells from a donor with at least 1 G allele in position CT60 had worse overall survival (56.2% vs 69.8% at 5 years; P = .001; hazard ratio [HR], 3.80; 95% confidence interval [CI], 1.75-8.22), due to a higher risk of relapse (P = .049; HR, 1.71; 95% CI, 1.00-2.93). Acute graft-versus-host disease (aGVHD) was more frequent in patients receiving CT60 AA stem cells (P = .033; HR, 1.54; 95% CI, 1.03-2.29). This is the first study to report an association between polymorphisms at CTLA-4 and clinical outcome after allo-HSCT. The CT60 genotype influences relapse and aGVHD, probably due to its action on CTLA-4 alternative splicing.

Allelic effects on gene regulation at the autoimmunity-predisposing CTLA4 locus: a re-evaluation of the 3′ +6230G&gt;A polymorphism

Genetic variation at a linkage disequilibrium block encompassing the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene influences susceptibility to autoimmunity, but identifying the polymorphism(s) responsible for this effect has been challenging. Recently, a single-nucleotide polymorphism (SNP) located 3' to the known polyadenylation site of CTLA4 (+6230G>A) and strongly associated with autoimmune disease was reported to regulate levels of soluble CTLA4 isoform (sCTLA4) but not the full-length isoform. The purpose of the present study is to define the mechanistic effect of the 3'SNP on the isoforms of CTLA4 (alternative splicing vs polyadenylation vs effects on RNA stability). However, using allele-specific single-nucleotide primer extension, we found no difference between mRNA transcripts derived from either +6230G>A allele in 11 heterozygous individuals, in either of the two known CTLA4 isoforms. We also found no effect of this polymorphism on ICOS (inducible costimulator), a putative downstream target. In addition, repeated attempts at 3' RACE (3'rapid amplification of cDNA ends) were unsuccessful in amplifying any contiguous sequence past the known CTLA4 polyadenylation site and no such sequence was found in the EST databases. We conclude that the mechanism of the observed association of the +6230 SNP with autoimmune disease remains to be determined, but does not involve modulation of steady-state mRNA of any known CTLA4 isoform.

CTLA4 polymorphisms are associated with vitiligo, in patients with concomitant autoimmune diseases

The cytotoxic T lymphocyte antigen4 (CTLA4) gene plays a critical role in the control of T cell activation. The gene encodes a surface molecule with inhibitory effects on activated T cells. Several studies have disclosed an association between the previously known variants of the CTLA4 gene and autoimmune disorders, but no study has as yet found any definite association between vitiligo and the CTLA4 polymorphisms. A recent study identified new candidate susceptibility polymorphisms in this region, associated with differential gene splicing and thereby the relative abundance of soluble CTLA4. To assess these new polymorphisms in patients with vitiligo, we genotyped 100 vitiligo patients and 140 healthy controls from the UK, for these novel polymorphisms. No association was found in patients with isolated vitiligo, but a significant association was seen in patients with vitiligo and other autoimmune diseases. The results indicate that the polymorphisms in the CTLA4 gene region confer susceptibility to vitiligo when occurring together with other autoimmune diseases, but not in patients with isolated vitiligo. This raises the possibility that there are two distinct forms of vitiligo where only a subgroup of patients may have a disease caused by the autoimmune destruction of melanocytes.

CTLA-4–Ig Activates Forkhead Transcription Factors and Protects Dendritic Cells from Oxidative Stress in Nonobese Diabetic Mice

Prediabetes and diabetes in nonobese diabetic (NOD) mice have been targeted by a variety of immunotherapies, including the use of a soluble form of cytotoxic T lymphocyte antigen 4 (CTLA-4) and interferon (IFN)-γ. The cytokine, however, fails to activate tolerogenic properties in dendritic cells (DCs) from highly susceptible female mice early in prediabetes. The defect is characterized by impaired induction of immunosuppressive tryptophan catabolism, is related to transient blockade of the signal transducer and activator of transcription (STAT)1 pathway of intracellular signaling by IFN-γ, and is caused by peroxynitrite production. Here, we show that soluble CTLA-4 imparts suppressive properties to DCs from early prediabetic NOD female mice through mechanisms that rely on autocrine signaling by IFN-γ. Although phosphorylation of STAT1 in response to IFN-γ is compromised in those mice, CTLA-4 obviates the defect. IFN-γ–driven expression of tryptophan catabolism by CTLA-4–immunoglobulin is made possible through the concomitant activation of the Forkhead Box class O (FOXO) transcription factor FOXO3a, induction of the superoxide dismutase gene, and prevention of peroxynitrite formation.