Loss of T cell responses following long-term cryopreservation.
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.
Owen
Rachel E
RE
Blood Systems Research Institute, 270 Masonic Avenue, San Francisco, CA 94118, USA. rowen@bloodsystems.org
Sinclair
Elizabeth
E
Emu
Brinda
B
Heitman
John W
JW
Hirschkorn
Dale F
DF
Epling
C Lorrie
CL
Tan
Qi Xuan
QX
Custer
Brian
B
Harris
Jeffery M
JM
Jacobson
Mark A
MA
McCune
Joseph M
JM
Martin
Jeffery N
JN
Hecht
Frederick M
FM
Deeks
Steven G
SG
Norris
Philip J
PJ
D000208:Acute Disease; D017209:Apoptosis / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002908:Chronic Disease; D018920:Coculture Techniques; D015925:Cryopreservation; D003587:Cytomegalovirus / Q000276:immunology; D003586:Cytomegalovirus Infections / Q000276:immunology / Q000473:pathology; D006678:HIV / Q000276:immunology; D015658:HIV Infections / Q000276:immunology / Q000473:pathology; D006801:Humans; D008297:Male; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013997:Time Factors
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