CTLA-4 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), also known as CD152, is a cell surface glycoprotein belonging to the immunoglobulin family with a role in regulation of T cell activation (1). Human CTLA-4 is a 223 amino acid (aa) protein with a theoretical molecular weight of 24.6 kDa containing a leader peptide, a IgV-like domain, a transmembrane domain, and a cytoplasmic tail (1,2). CTLA-4 is both structurally and functionally related with another member of the immunoglobulin-related receptor family, CD28 (1-3). CTLA-4 and CD28 receptors are both expressed by CD4+ and CD8+ T cells and share two common ligands, CD80 (B7.1) and CD86 (B7.2), expressed on the surface of antigen presenting cells (APCs) (2,3). While CD28 is present on the plasma membrane of T cells, CTLA-4 is predominantly expressed intracellularly on vesicles in FoxP3+ regulatory T (Treg) cells and activated T cells due to endocytosis (3). While they share ligands, the two receptors have opposing functions in T cell activation; CD28 is involved in activation of T cells, while CTLA-4 functions as a negative regulator of T cell response (2,3). One of the primary functions of CTLA-4 is preventing autoimmunity (1-4).

Similar to programmed cell death protein 1 (PD-1), CTLA-4 is an inhibitory immune checkpoint protein (3,5). Checkpoint blockade immunotherapy using drugs or antibodies to target CTLA-4 is one of the main approaches for cancer treatment (5). A number of drugs targeting CTLA-4, or a combination of CTLA-4/PD-1, have been approved for treatment of various cancers like melanoma, renal cell carcinoma, and colorectal cancer (5). While blocking CTLA-4 in the tumor microenvironment is a promising cancer therapeutic, the absence of CTLA-4 under normal conditions can have deleterious effects. Studies have found that patients with CTLA-4 deficiency or mutations have clinical features associated with autoimmunity and immune dysregulation (4). Treatment options for CTLA-4 deficiency includes immunoglobulin-replacement therapy, corticosteroids, CTLA-4-immunoglobulin (Ig) fusion protein, and, in life-threatening cases, hematopoietic stem cell transplantation (4,6). Additionally, engaging CD80/CD86 with CTLA-4-Ig is a common immunosuppressive treatment for rheumatoid arthritis and kidney transplant recipients (6).

References

1. Romo-Tena, J., Gomez-Martin, D., & Alcocer-Varela, J. (2013). CTLA-4 and autoimmunity: new insights into the dual regulator of tolerance. Autoimmunity reviews, 12(12), 1171-1176. https://doi.org/10.1016/j.autrev.2013.07.002

2. Hosseini, A., Gharibi, T., Marofi, F., Babaloo, Z., & Baradaran, B. (2020). CTLA-4: From mechanism to autoimmune therapy. International immunopharmacology, 80, 106221. https://doi.org/10.1016/j.intimp.2020.106221

3. Rowshanravan, B., Halliday, N., & Sansom, D. M. (2018). CTLA-4: a moving target in immunotherapy. Blood, 131(1), 58-67. https://doi.org/10.1182/blood-2017-06-741033

4. Verma, N., Burns, S. O., Walker, L., & Sansom, D. M. (2017). Immune deficiency and autoimmunity in patients with CTLA-4 (CD152) mutations. Clinical and experimental immunology, 190(1), 1-7. https://doi.org/10.1111/cei.12997

5. Rotte A. (2019). Combination of CTLA-4 and PD-1 blockers for treatment of cancer. Journal of experimental & clinical cancer research : CR, 38(1), 255. https://doi.org/10.1186/s13046-019-1259-z

6. Bluestone, J. A., St Clair, E. W., & Turka, L. A. (2006). CTLA4Ig: bridging the basic immunology with clinical application. Immunity, 24(3), 233-238. https://doi.org/10.1016/j.immuni.2006.03.001

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBP2-07774

• CTLA-4 Antibodies
• CTLA-4 Antibody Pair
• CTLA-4 ELISA Kits
• CTLA-4 Lysate
• CTLA-4 Protein
• CTLA-4 ELISAs
• CTLA-4 Proteins and Enzymes