CD8 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.

CD8, also known as Leu-2 or T8 in human and Lyt2 or Lyt3 in mouse, is a cell surface glycoprotein belonging to the immunoglobulin supergene family (1, 2). CD8 is expressed on cytotoxic T-lymphocytes (T-cells), most thymocytes, between 35-45% of peripheral blood lymphocytes, and a population of natural killer (NK) cells (1, 2). The CD8 molecule consists of disulfide-linked alpha (alpha) and beta (beta) chains that present on T-cells as either CD8alphaalpha homodimers or CD8alphabeta heterodimers (1, 3). Both alpha and beta chains consist of a signaling sequence, an extracellular Ig-like domain, a membrane proximal stalk region, a transmembrane domain, and a cytoplasmic tail (3). Human CD8alpha is processed as 235 amino acids (aa) in length with a theoretical molecular weight of ~26 kDa, while mouse CD8alpha is 247 aa and has a theoretical molecular weight of 27.5 kDa (4, 5). Functionally, CD8 acts as an antigen coreceptor on cytotoxic T-cells and interacts with the major histocompatibility complex (MHC) class I molecules on antigen presenting cells (APCs), mediating cell-cell interactions within the immune system. Conversely, CD4 molecules interact with antigens presented on MHC class II molecules and are activated to become helper T-cells (TH) (1,2). Interestingly, thymocytes can transiently express both CD4 and CD8 during the maturation process (2). Furthermore, the cytoplasmic tail of CD8 has a Lck (lymphocyte-specific protein tyrosine kinase) binding domain where Lck interacts with CD8, initiating a phosphorylation cascade that activates transcription factors and promotes T-cell activation (6). More specifically, CD8alphabeta functions as a T-cell co-receptor, while CD8alphaalpha promotes T-cell survival and differentiation (7).

Given its role in the immune system, CD8-deficiency in T-cells is a hallmark of many diseases and pathologies (8-10). Specifically, CD8+ T-cell deficiency is prevalent in chronic autoimmune diseases including multiple sclerosis, rheumatoid arthritis, ulcerative colitis, Crohn's disease, type 1 diabetes mellitus, and Graves' disease (8). Furthermore, cancers or chronic infection can lead to CD8 T-cell exhaustion as the continual antigen presentation and inflammatory signals eventually cause the CD8+ T-cells to lose functionality (9, 10). However, animal models and clinical studies have suggested that T-cells are capable of being reinvigorated using inhibitory receptor blockade resulting in better disease outcomes and these exhausted T-cells may be a potential therapeutic target (9, 10).

Alternative names for CD8 includes CD antigen: CD8a, CD8 antigen, alpha polypeptide (p32), CD8a molecule, CD8A, Leu2 T-lymphocyte antigen, LEU2, MAL, OKT8 T-cell antigen, p32, T cell co-receptor, T8 T-cell antigen, T-cell antigen Leu2, T-cell surface glycoprotein CD8 alpha chain, and T-lymphocyte differentiation antigen T8/Leu-2.

References

1. Littman D. R. (1987). The structure of the CD4 and CD8 genes. Annual review of immunology. https://doi.org/10.1146/annurev.iy.05.040187.003021

2. Naeim F. (2008). Chapter 2- Principles of Immunophenotyping. Hematopathology. https://doi.org/10.1016/B978-0-12-370607-2.00002-8.

3. Gao, G. F., & Jakobsen, B. K. (2000). Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor. Immunology today. https://doi.org/10.1016/s0167-5699(00)01750-3

4. UniProt (P01732)

5. UniProt (P01731)

6. Kappes D. J. (2007). CD4 and CD8: hogging all the Lck. Immunity. https://doi.org/10.1016/j.immuni.2007.11.002

7. Gangadharan, D., & Cheroutre, H. (2004). The CD8 isoform CD8alphaalpha is not a functional homologue of the TCR co-receptor CD8alphabeta. Current opinion in immunology. https://doi.org/10.1016/j.coi.2004.03.015

8. Pender M. P. (2012). CD8+ T-Cell Deficiency, Epstein-Barr Virus Infection, Vitamin D Deficiency, and Steps to Autoimmunity: A Unifying Hypothesis. Autoimmune diseases. https://doi.org/10.1155/2012/189096

9. Kurachi M. (2019). CD8+ T cell exhaustion. Seminars in immunopathology. https://doi.org/10.1007/s00281-019-00744-5

10. Hashimoto, M., Kamphorst, A. O., Im, S. J., Kissick, H. T., Pillai, R. N., Ramalingam, S. S., Araki, K., & Ahmed, R. (2018). CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions. Annual review of medicine. https://doi.org/10.1146/annurev-med-012017-043208

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 NBP2-11336

• CD8 Antibodies
• CD8 Antibody Pairs
• CD8 ELISA Kits
• CD8 Lysates
• CD8 Proteins
• CD8 Proteins and Enzymes
• CD8 ELISA