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9 Jun 2014

Investigation of soluble and transmembrane CTLA-4 isoforms in serum and microvesicles

The role of transmembrane CTLA-4/ CD152 Receptor

Transmembrane Cytotoxic T Lymphocyte Antigen 4 (Tm-CTLA-4) also known as CD152 plays a key role in downregulating the immune response and helping regulate immune homeostasis. Tm-CTLA-4 receptor is expressed on the surface of T cells, which lead the cellular immune attack on foreign as well as self antigens. T cell mediated attack against antigens is regulated by two receptors, CD28 and Tm-CTLA-4, both of which bind CD80/ B7-1 and CD86/ B7-2 on antigen presenting cells (APCs). Stimulation of the CD28 receptor results in the activation of T cells and hence T cell mediated attack against the antigen, whilst stimulation of the Tm-CTLA-4 receptor results in inhibition of the CD28 induced signal resulting in inhibition of the T cell attack. This highly regulated system has become an attractive target for therapeutic intervention with many pre-clinical studies focusing on the effect of altering Tm-CTLA-4 activity to determine the effects on autoimmune diseases, organ transplant rejection and tumor growth.

The role of soluble CTLA-4

An alternative splice variant of the gene encoding CTLA-4 that lacks exon 3 has been described and is predicted to yield a soluble, monomeric isoform termed sCTLA-4, which differs from Tm-CTLA-4 by the lack of a transmembrane domain and the absence of the cysteine residue necessary for homodimerization. Alterative splicing that produces sCTLA-4 results in a reading frame shift that generates a unique C terminal amino acid sequence that distinguishes sCTLA-4 from Tm-CTLA-4.
Although previous studies have suggested the presence of sCTLA-4 in patient sera in the context of a number of autoimmune diseases, the lack of specific high affinity sCTLA-4 antibodies had put into question the validity of these results, an issue addressed in Esposito, L. et al. 2014. Investigation of soluble and Tm-CTLA-4 isoforms in serum and microvesicles. Journal of Immunology, Jul 15;193:889-900.

Custom Polyclonal Antibody Generation

To ascertain the presence of sCTLA-4 in activated T cells and quantify patient serum levels during autoimmune disease, Professor Linda Wicker from the Medical Genetic Department at the University of Cambridge commissioned Cambridge Research Biochemicals (CRB) to raise a polyclonal antibody and two monoclonal antibodies to the unique C terminal amino acid sequence of the sCTLA-4 protein.

Cambridge Research Biochemicals raised a custom rabbit polyclonal antibody corresponding to the C-terminal of human sCTLA-4: ENAPNRARM (9mer). Once the peptide was constructed it was chemically conjugated to a carrier protein and used for immunisation. Once a suitable antibody titre was achieved, the crude anti-serum underwent affinity purification. The purification was a two-step process which involved using a thiopropyl Sepharose affinity column derivatised with the peptide antigen (see image below). The antibody was then passed over a KLH column, depleting the sera from antibodies generated to the carrier protein.

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Custom Monoclonal Antibody Generation

Cambridge Research Biochemicals raised two custom mouse monoclonal cell lines (4B8 and 10D1) that produced antibody specific to the modified C terminal of the sCTLA-4 protein: IAKEKKPSYNRGLSENAPNRARM (23mer). The peptide was conjugated to a carrier protein and used for immunisation. The immunisation protocol CRB implement for monoclonal antibody production is designed to induce a hyperimmune response against the antigen which is measured by ELISA analysis against the free target peptide and/ or the recombinant protein. Once a suitable serum antibody titre was achieved target specific hybridomas were produced by fusing splenocytes from the immunised mice with a myeloma cell line. Once hybridomas were established positive cultures were identified and isolated before undergoing sub-cloning by limiting dilution to isolate cell lines derived that had derived from a single cell. The final cell lines displayed good growth characteristics with a high capacity for antibody production capable of detecting both the free peptide and soluble protein.

The custom polyclonal and monoclonal anti-sCTLA-4 antibodies generated were validated by Western blot and flow cytometry using HeLa cells expressing either recombinant sCTLA-4 or Tm-CTLA-4. All three antibodies displayed specific high affinity binding to human sCTLA-4 and no binding to Tm-CTLA-4. The polyclonal antibody and monoclonal cell line 10D1 also displayed cross reactivity with mouse sCTLA-4 unlike 48B, indicating that the two cell lines produce antibodies to different epitopes within the 23mer peptide target. Esposito, L. et al. 2014. Investigation of soluble and Tm-CTLA-4 isoforms in serum and microvesicles. Journal of Immunology, Jul 15;193:889-900.

The anti-sCTLA-4 antibodies generated will prove to be a useful tool for understanding the functional and clinical importance of CTLA-4 in immune regulation. A thorough understanding of this pathway combined with refined therapeutic approaches will have powerful clinical applications.