Alternative Splice Forms of CTLA-4 Induced by Antisense Mediated Splice-Switching Influences Autoimmune Diabetes Susceptibility in NOD Mice

Abstract

Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.

MECHANISMS IN ENDOCRINOLOGY: Alternative splicing: the new frontier in diabetes research

“…Studies in NOD mice and other models have shown that differential expression of CTLA-4 splice variants impacts on T cell function and the overall immune response. Thus, while splice variants lacking the transmembrane domain exacerbate the autoimmune pathology, variants lacking the ligand-binding domain are protective (83, 84, 85). Importantly, AS is more frequent in the critical MHC region (a region that accumulates more than 40% of the genetic risk associated with T1D (86)) than it is genome wide (87).…”

“…The identification of splice variants acting as β cell autoantigens, modulators of immune response or β cell survival, may lead to the development of novel therapeutic strategies for T1D based on splicing modulation. In line with this possibility, the use of an AON-targeted splice-switching approach against the CTLA-4 gene, which modulates T cell activation and proliferation, reduced the incidence of insulitis and diabetes in diabetes-prone NOD mice (84). Another potential therapeutic target to enhance β cell survival is the short isoform of the pro-apoptotic BH3-protein Bim (Bim S).…”

Investigation of CTLA-4-318C/T gene polymorphism in cases with type 1 diabetes of Azerbaijan, Northwest Iran

“…Indeed, high serum levels of soluble CTLA-4 have been reported in type-1 diabetes patients [47]. Over expression of soluble CTLA-4 in mice can lead to the induction and exacerbation diabetes [48,49].…”

Abnormalities in alternative splicing in diabetes: therapeutic targets

“…Isoforms of this protein are generated by AS due to the presence of a single nucleotide polymorphism. This gives rise to the normal full length protein and a shorter soluble sCTLA4 that lacks the transmembrane domain encoded by exon 4 (Gerold et al 2011, Mourich et al 2014. Another isoform is the result of the absence of the ligand binding domain encoded by exon 2 and is called liCTLA-4.…”

“…NOD mice have been used to study how these different isoforms can affect T cell response. NOD mice expressing the sCTL4 isoform are more prone to getting type 1 diabetes, while mice expressing liCTLA4 are more resistant to the getting the disease (Mourich et al 2014). This is due to the activation of the T cells as those lacking receptors with a ligand binding site showed reduced activity while the soluble CTLA4 has an increased ability to activate T cells.…”