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

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... tides, 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.