Microvesicles CTLA-4 Isoforms in Serum and Investigation of Soluble and Transmembrane

Expression of the CTLA-4 gene is absolutely required for immune homeostasis, but aspects of its molecular nature remain undefined. In particular, the characterization of the soluble CTLA-4 (sCTLA-4) protein isoform generated by an alternatively spliced mRNA of CTLA4 lacking transmembrane-encoding exon 3 has been hindered by the difficulty in distinguishing it from the transmembrane isoform of CTLA-4, Tm-CTLA-4. In the current study, sCTLA-4 has been analyzed using novel mAbs and polyclonal Abs specific for its unique C-terminal amino acid sequence. We demonstrate that the sCTLA-4 protein is secreted at low levels following the activation of primary human CD4 + T cells and is increased only rarely in the serum of autoimmune patients. Unexpectedly, during our studies aimed to define the kinetics of sCTLA-4 produced by activated human CD4 + T cells, we discovered that Tm-CTLA-4 is associated with microvesicles produced by the activated cells. The functional roles of sCTLA-4 and microvesicle-associated Tm-CTLA-4 warrant further investigation, especially as they relate to the multiple mechanisms of action described for the more commonly studied cell-associated Tm-CTLA-4. T he transmembrane isoform of CTLA-4 (Tm-CTLA-4) receptor plays a crucial role in the downregulation of the immune response and the maintenance of immune homeo-stasis, as shown by the lymphoproliferative syndrome and early lethality of CTLA-4–deficient mice (1–3). Tm-CTLA-4 is expressed by activated T cells, whereas it is constitutively expressed and required for regulatory T cell (Treg) suppression (4–6). At the molecular level, previous studies have provided evidence that an alternatively spliced mRNA of the CTLA-4 gene that lacks exon 3 is expressed in human, mouse, and rat immune cells (7, 8). As a result of splicing between exons 2 and 4, the predicted soluble CTLA-4 (sCTLA-4) isoform does not have a transmembrane domain or the membrane-proximal cysteine residue required for co-valent homodimerization of the conventional Tm-CTLA-4 (9), thereby predicting a secreted, or soluble, isoform of monomeric CTLA-4 (sCTLA-4). The skipping of exon 3 predicts a shift in the reading frame, generating a C-terminal amino acid sequence that distinguishes sCTLA-4 from Tm-CTLA-4 (7). In both human and mouse sCTLA-4, mRNA expression is mainly detected in resting T cells, and its level is similar to that of Tm-CTLA-4 mRNA, whereas, following T cell activation, Tm-CTLA-4 is rapidly upreg-ulated and becomes the predominant transcript (7, 8, 10–12). In humans, single nucleotide polymorphism (SNP) CT60 (rs3087243) in the 39 untranslated region of human CTLA4 is associated with multiple autoimmune diseases, …