Loss of T cell responses following long-term cryopreservation

J Immunol Methods. 2007 Sep 30;326(1-2):93-115. doi: 10.1016/j.jim.2007.07.012. Epub 2007 Aug 8.


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-gamma 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-gamma responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-gamma 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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Disease
  • Apoptosis / immunology
  • B-Lymphocytes / immunology
  • Cell Line
  • Cell Line, Transformed
  • Cells, Cultured
  • Chronic Disease
  • Coculture Techniques
  • Cryopreservation*
  • Cytomegalovirus / immunology
  • Cytomegalovirus Infections / immunology
  • Cytomegalovirus Infections / pathology
  • HIV / immunology
  • HIV Infections / immunology
  • HIV Infections / pathology
  • Humans
  • Male
  • T-Lymphocytes* / cytology
  • T-Lymphocytes* / immunology
  • T-Lymphocytes* / metabolism
  • Time Factors