CTLA-4 expression, regulation and associations in autoimmune myasthenia gravis

Abstract

Myasthenia gravis (MG) is a neuromuscular disease with muscle weakness due to an autoimmune attack against the nicotinic acetylcholine receptor (nAChR) on the skeletal muscle endplate. Cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) plays a global inhibitory role in the immune system and has a crucial role in autoimmunity.

We focused our research on the CTLA-4 molecule, exploring its expression, regulation, and genetic variations, especially in association with MG. Single nucleotide polymorphisms (SNPs) contribute most significantly to the variation in a population.

To determine an association between the genetic polymorphisms of the Ctla-4 and MG, the A/G SNP in CDS 1 at position +49 (A/G+49) and a C/T SNP in promoter at position 318 (C/T-318) were analyzed. The allele G+49 and genotype G/G+49 were more frequently present in MG patients with thymoma. Patients with this genotype also had signs of immune activation manifested as elevated levels of serum IL-Beta and a higher percentage of CD28 4, T lymphocytes. Ctla-4 variations may thus contribute to the pathophysiology of MG. The expression of CTLA-4 was investigated in patients with MG. We found that the patients had low expression of CTLA-4 by peripheral T cells and a decreased response to ConA. Moreover, the patients had higher serum levels of soluble CTLA-4 (sCTLA-4) which were positively correlated with the concentration of anti-nAChR antibodies.

The functional correlations to genetic polymorphisms are most interesting. By constructing Ctla-4 promoter reporters, we demonstrated that the T-318 allele was associated with a higher promoter activity than the C-318 allele. To define the function of the microsatellite polymorphism in the 3'-UTR, we analyzed allele-specific expression in individuals heterozygous for (AT)n of different lengths, and found that mRNA expression of CTLA-4 from alleles with longer AT repeats was less than mRNA from shorter alleles. The difference in allele expression was due to alteration in mRNA stability. The degradation of mRNA was faster in individuals homozygous for the longer AT repeats than in those homozygous for the shorter alleles.

The expression profile of CTLA-4 in humans has not been completely elucidated. We found a constitutive expression of CTLA-4 by peripheral T cells, mainly in the cytoplasm. IL-2 upregulated both intracellular and surface expression of CTLA-4 in a dose-dependent manner. More than half of the CTLA-4+ T cells expressed CD25. Also human monocytes expressed intracellular CTLA-4 and surface expression of CTLA-4 was achieved by IFN-gamma treatment. CTLA-4 expression was induced by PMA and IFN-gamma in the monocytic cell lines U937 and THP-1. CTLA-4 engagement by antibodies inhibited cell proliferation and down-regulated expression of the activation markers CD86, CD54, HLA-DR and HLA-DQ. CTLA-4 engagement blocked NF-kappaB and AP-I transcription factor activities. Thus, CTLA-4 might act early to reduce the activity of several key nuclear transcription factors important for monocyte activation. CTLA-4 might thus contribute to the manifestation of MG due to abnormal expression caused by genetic variations. The properties of CTLA-4 should be further investigated with regard to its therapeutic potentials.