Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 326, Issue 1-2, Pages 93-115Publisher
ELSEVIER
DOI: 10.1016/j.jim.2007.07.012
Keywords
cryopreservation; T cells; IFN-gamma; HIV; intraceflular cytokine staining
Categories
Funding
- NCRR NIH HHS [M01 RR000083, U54RR023566, M01 RR00083, M01 RR000833-30] Funding Source: Medline
- NIAID NIH HHS [R01 AI052745-04, R21 AI055273-02, K08 AI001698, AI49538, R01 AI052745, R21 AI055273, K08 AI001698-06, AI47062, R01 AI049538, P30 AI027763-15, AI41531, 5P30AI027763, R01 AI047062-09, AI055273, P30 AI027763, AI052745, P30 AI27763, R01 AI047062, U01 AI041531-08, U01 AI041531] Funding Source: Medline
- NIH HHS [DP1 OD000329, DP1 OD000329-04, P30 NH62246, P30 NH59037, DPI OD00329] Funding Source: Medline
- NIMH NIH HHS [P30 MH062246, P30 MH062246-01, P30 MH059037-149010] Funding Source: Medline
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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 lFN-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. (c) 2007 Elsevier B.V. All rights reserved.
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