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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.


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.