Dinucleotide repeat expansion in the CTLA-4 gene leads to T cell hyper-reactivity via the CD28 pathway in myasthenia gravis

Abstract

CD28 is required to promote T cell proliferation and cytokine production, while the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) functions as a negative modulator for T cell activation. We previously reported that alleles with longer PCR products (designated as allele xx) in an (AT)n polymorphism in Ctla-4 are associated with myasthenia gravis with thymoma, while the shortest allele, 86, is negatively associated with the disease. Here, we demonstrate that serum IL-2 sRα increases parallel to the length of (AT)n in Ctla-4. Periphereal blood mononuclear cells (PBMC) from patients with Ctla-4 xx/xx contained higher activity of telomerase than patients bearing Ctla-4 86/86. Blockade of CTLA-4 increased the telomerase activity in PBMC stimulated by acetylcholine receptor in vitro. There was a positive correlation between the expression of CD28 and CTLA-4 on anti-CD3 activated PBMC, suggesting a balance between CD28 and CTLA-4. Cells from patients with Ctla-4 xx/xx had the highest level of T cell proliferative responses upon the addition of anti-CD28 antibodies to the anti-CD3 containing culture system while cells from patients with Ctla-4 86/xx had an intermediate and cells from patients with Ctla-4 86/86 the lowest increase. The current results point to the (AT)n in Ctla-4 as a myasthenia gravis facilitating mutation under certain permissive environments by influencing the T cell reactivity via the CD28 pathway.

Introduction

Activation of naive T cells requires two signals. The first is the antigen-specific activation that involves engagement of the TCR by peptides bound to MHC, and the second from the costimulatory molecules such as CD28 and CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4, CD152). CD28 is required to promote adequate T cell proliferation and cytokine production while CTLA-4 functions as a negative modulator for T cell activation, thus comprising a Yin Yang balance in vivo (Allison and Krummel, 1995). Regulation of expression and function of CTLA-4 and CD28 is critical for the normal function of the immune system. CD28-deficient mice show T cell unresponsiveness (Shahinian et al., 1993, Green et al., 1994), while CTLA-4 mutant mice develop early lethal autoimmunity (Tivol et al., 1995, Waterhouse et al., 1995). Blockade of CTLA-4 using anti-CTLA-4 antibodies enhances the anti-tumour effect of T lymphocytes (Leach et al., 1996). In contrast, CD28 blocking by CTLA-4Ig fusion protein results in a long-lasting survival of organ transplantation in many models (Turka et al., 1992, Levisette et al., 1997), prevents the development of experimental autoimmune encephalomyelitis (Cross et al., 1995, Miller et al., 1995) and experimental autoimmune myasthenia gravis (McIntosh et al., 1998).

The proposed mechanisms by which CTLA-4 suppresses an ongoing immunoresponse in mice are prevention of CD28 binding to its ligand (CD80 and CD86) by competition for ligand access to CD28, antagonizing or aborting the CD28 signal by delivering a inhibitory signal or by ‘stealing’ signal molecules away from CD28 by means of the rapid endocytosis of CTLA-4 (Krummel and Allison, 1995, Linsley et al., 1996), and delivering a signal that antagonizes or aborts the TCR-delivered signal (Fallarino et al., 1998, Lin et al., 1998). However, little is known about these mechanisms in humans.

CD28 is expressed constitutively on mature T cells and upregulated upon activation. CTLA-4 is expressed at very low levels on resting T cells and induced after activation (Linsley et al., 1992, Finn et al., 1997). It is speculated that decreased expression of CTLA-4 may promote autoimmunity, especially in the diseases that have been shown to be associated with a dinucleotide repeat microsatellite in the CTLA-4 gene (Ctla-4) such as Graves’ disease (Yanagawa et al., 1995), insulin-dependent (type I) diabetes mellitus (Nistico et al., 1996, Marron et al., 1997), myasthenia gravis (MG) with thymoma (Huang et al., 1998), and autoimmune hypothyroidism (Kotsa et al., 1997). Alleles with longer PCR products (Fig. 1), up to 128 bp (designated by allele xx), of this polymorphism are positively associated with MG patients having thymoma, a subgroup of patients with relatively severe clinical manifestation (Huang et al., 1998). Recently, Wegener’s granulomatosis, a systemic vasculitis with multiple immune abnormalities, is found to be strongly associated with the (AT)n in Ctla-4 (Huang et al., 1999). Thus, it is of interest to explore the mechanisms behind the associations of these autoimmune disorders to longer alleles of the (AT)n in Ctla-4, a T-cell-related gene.

Levels of circulating IL-2 soluble receptor α chain (IL-2 sRα) are elevated in sera while T cells are activated (Waldmann, 1993). Telomerase, an enzyme responsible in most eukaryotes for replication of the ends of chromosomes, is selectively expressed in human germline and malignant cells (Kim et al., 1994) and normal T lymphocytes during development and after in vitro or in vivo activation (Bodnar et al., 1996; Weng et al., 1996, 1997). It has been shown that signal transduction events in T cells correlate with augmented expression of telomerase activity (Bodnar et al., 1996). Therefore, levels of IL-2 sRα and telomerase activity can be serological and cellular parameters reflecting in vivo T cell activation status. In the present study, both IL-2 sRα in serum and telomerase activity in periphereal blood mononuclear cells (PBMC) from MG patients were examined. Furthermore, the cellular surface expression of CTLA-4 and CD28 detected by flow cytometry, and CD28 pathway determined by T cell proliferation upon anti-CD3 and anti-CD28 stimulation in vitro were investigated to explore the mechanism(s) by which this (AT)n microsatellite influences the T cell activation.