Immunological Research Unit and Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden

Correspondence to: Prof. A K Lefvert, Immunological Research Unit, CMM L8: 03, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: Ann.Kari.Lefvert@cmm.ki.se

This study was supported by grants the Swedish Research Council, the Gustaf V: s 80-years Foundation, the Swedish Rheumatism Association and the Börje Dahlin Foundation

CTLA-4 is an important negative regulator of the immune system. The regulation of the CTLA-4 gene (Ctla-4) transcription is poorly understood. A single nucleotide polymorphism (SNP) at -318 in the Ctla-4 promoter region is associated with certain autoimmune diseases. Since the -318 SNP occurs in a potential regulatory region, it is conceivable that the C' T transition may affect the expression of Ctla-4. In the present study, we show that the -318T allele is associated with a higher promoter activity than the -318C allele (8.13 ± 0.46 vs 6.87 ± 0.49). The presence of the -318T allele may thus contribute to up regulation of the expression of CTLA-4, and consequently represent one mechanism to inhibit exaggerated immune activity.

Genes and Immunity (2002) 3, 233-234. doi:10.1038/sj.gene.6363869

CTLA-4; costimulator; polymorphism; promoter

CTLA-4 plays an important role in the immune system as a T cell inhibitory factor. This inhibitory signal not only determines whether T cells become activated, but also affects the clonal representation in an immune response. CTLA-4 regulates cell cycle progression rather than directly the induction of apoptosis.¹ CTLA-4 Ig and anti-CTLA-4 have been applied for the treatment of both autoimmune diseases and cancers.

Little is known about the regulatory mechanism for CTLA-4 expression. Polymorphisms in the Ctla-4 are associated with several autoimmune diseases, and polymorphisms at certain locations of the Ctla-4 might conceivably affect its expression. We have described a higher prevalence of thymine at position -318 of the Ctla-4 promoter (-318T) in patients with Wegener's granulomatosis.² Recently, Ligers and colleagues reported that individuals carrying -318T exhibited significantly increased expression both of cell-surface CTLA-4 in activated cells and of Ctla-4 mRNA in non-stimulated cells,³ suggesting a potential role for the C' T transition in the regulation of expression. In the present study, we examined the effect of C' T switch at -318 on the Ctla-4 promoter activity. Our results demonstrated that the -318T allele was associated with a significantly higher promoter activity than the -318C allele.

Results and discussion

To determine whether the C' T transition at position -318 transcriptionally regulates the Ctla-4 gene expression, we made two Ctla-4 promoter reporter constructs which contained C and T alleles, respectively. As shown in Figure 1, the -318T allele was associated with a higher promoter activity than the -318C allele (8.13 ± 0.46 vs 6.87 ± 0.49). This result was also confirmed in the THP-1 cell line transfected with these two reporter constructs, and stimulated with IFN- (5 mg/ml) and PMA (10 ng/ml).

Our allele-specific transcription data indicate that the -318T allele is associated with higher transcriptional activity than the -318C allele. The -318T allele could be considered as protective against autoimmune reactions. Our results could explain the increased expression of CTLA-4³ and the observed associations of the -318T allele to some diseases.⁴

Homozygosity for -318/T in the promoter of Ctla-4 is rarely present in a Caucasian population.^4,5,6,7 In our previous study, the frequencies of individuals with genotypes -318C/C, C/T, and T/T were 86%, 17% and 0%, respectively, in healthy individuals (n = 122) and 69%, 31%, 0%, respectively, in patients with Wegener's granulomatosis (n = 32). When we considered the prevalence of longer (AT)n in the 3'-untranslated region together with the promoter single nucleotide polymorphism (SNP), we found that all patients carrying the -318T allele were homozygous for (AT)n >86 bp alleles.² As the longer (AT)n alleles are associated with decreased mRNA stability and thus decreased protein expression, the -318T mutation should counteract the effects of long (AT)n alleles. The -318T mutation could thus be regarded as protective against increased T cell stimulation.

In conclusion, the C' T transition at position -318 transcriptionally regulates the Ctla-4 gene expression and the T mutation could be considered as protective for autoimmune disease.

1 Chambers CA, Kuhns MS, Egen JG, Allison JP. CTLA-4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Ann Rev Immunol 2001; 19: 565-594.

2 Giscombe R, Wang X, Huang D, Lefvert A. CDS1 and promoter single nucleotide polymorphisms of te CTLA-4 gene in Wegener's Granulomatosis. J Rheumatol 2001; in press.

3 Ligers A, Teleshova N, Masterman T, Huang WX, Hillert J. CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms. Genes Immun 2001; 2: 145-152. MEDLINE

4 Braun J, Donner H, Siegmund T, Walfish PG, Usadel KH, Badenhoop K. CTLA-4 promoter variants in patients with Graves' disease and Hashimoto's thyroiditis. Tissue Antigens 1998; 51: 563-566. MEDLINE

5 Gonzalez-Escribano MF, Rodriguez R, Valenzuela A, Garcia A, Garcia-Lozano JR, Nunez-Roldan A. CTLA4 polymorphisms in Spanish patients with rheumatoid arthritis. Tissue Antigens 1999; 53: 296-300. Article MEDLINE

6 Harbo HF, Celius EG, Vartdal F, Spurkland A. CTLA4 promoter and exon 1 dimorphisms in multiple sclerosis. Tissue Antigens 1999; 53: 106-110. MEDLINE

7 Masterma T, Ligers A, Olerup O, Vrethem M, Hillert J. CTLA-4 dimorphisms in gammopathy-associated peripheral neuropathy. Eur J Neurol 1999; 6: 491-493. MEDLINE

Figure 1 Relative promoter activity of the human Ctla-4 gene in Jurkat T cells. Genomic DNA was used to amplify a 329-bp fragment covering the -318 polymorphic site. The following primers were used: -318C forward: AGG GGT ACC ACT TAG TTA TCC AGA TCC TCA AAG, -318T forward: AGG GGT ACC ACT TAG TTA TCC AGA TCC TTA AAG, and reverse: AG CGC TCG AGC CAG GTT CAG. The amplieid fragments (318C and -318T alleles) were subcloned into a TA cloning vector (PCRII; In Vitrogen, Leek, The Netherlands). The sequence of inserted fragment was confirmed by sequence analysis using the ABI-PRISM Terminator Cycle Ready Reaction Kit (Perkin Elmer, Foster City, CA, USA). Each fragment was then inserted upstream of the firely luciferase reporter gene at the KpnI/XhoI sites of the PGL3-basic vector (Promega, Madison, WI, USA). Jurkat T cells were maintained in complete medium (CM) (RMPI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37°C under 5% CO₂. Transfection experiments were conducted using the FuGENE 6 Transfection Reagent from Roche Molecular Biochemical (Stockholm, Sweden). Twenty-five thousand cells were seeded with 1 mg of constructs and 100 ng of pRL.CMV (a Renilla-based luciferase construct, Promega) per-well. After 24 h, the cells were stimulated with ConA (5 mg/ml), PMA (10 ng/ml) and harvested after 48 h. Dual-Luciferase Report assay system (Promega) was applied according to the manufacturer's instructions. The luciferase activity was measured using a Turner luminometer (TD-20/20). Firefly luciferase activity was normalized to Renilla luciferase activity to correct for variations in transfection efficiency. The relative luciferase levels were obtained from three assays performed in duplicate.