Loss of T cell responses following long-term cryopreservation

• Published in: Journal of immunological methods 2007-09, Vol.326 (1), p.93-115
• Main Authors: Owen, Rachel E., Sinclair, Elizabeth, Emu, Brinda, Heitman, John W., Hirschkorn, Dale F., Epling, C. Lorrie, Tan, Qi Xuan, Custer, Brian, Harris, Jeffery M., Jacobson, Mark A., McCune, Joseph M., Martin, Jeffery N., Hecht, Frederick M., Deeks, Steven G., Norris, Philip J.
• Format: Article
• Language: English
• Subjects: Acute Disease; Apoptosis - immunology; B-Lymphocytes - immunology; Biological and medical sciences; Cell Line; Cell Line, Transformed; Cells, Cultured; Chronic Disease; Coculture Techniques; Cryopreservation; Cytomegalovirus; Cytomegalovirus - immunology; Cytomegalovirus Infections - immunology; Cytomegalovirus Infections - pathology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; HIV; HIV - immunology; HIV Infections - immunology; HIV Infections - pathology; Human immunodeficiency virus; Humans; IFN-γ; Intracellular cytokine staining; Male; Microbiology; Molecular immunology; T cells; T-Lymphocytes - cytology; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; Techniques; Techniques used in virology; Time Factors; Virology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2007.07.012

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4 + T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8 + T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8 + T cell responses to peptides and peptide pools were well preserved. Loss of both CD4 + and CD8 + T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4 + T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4 + T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4 + and CD8 + T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4 + T cell responses and mild skewing of T cell phenotypic marker expression.