title: Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing.
abstract: Cryopreservation of peripheral blood mononuclear cells (PBMC) from animal model studies and clinical trials is utilized as a primary method for long-term storage of PBMC for future in vitro and in vivo applications. The objective of this study was to define the mechanistic pathways involved in cryopreservation-induced apoptosis of CD4+ T-cells in PBMC, and to evaluate a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved PBMC. Using cryopreserved PBMC samples isolated from naïve and Simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, frozen PBMC showed significantly increased levels of apoptosis-induced CD4+ T-cell death compared to fresh PBMC over a 5-day culture period as detected by Annexin V/PI and trypan blue staining. Mechanistic studies using a broad-spectrum caspase inhibitor z-VAD demonstrated a crucial involvement of caspases in cryopreservation-induced apoptosis of CD4+ T-cells. Furthermore, the ability of z-VAD to inhibit both mitochondrial membrane perturbation and apoptotic cell death implicated the involvement of caspase-mediated mitochondrial membrane damage in cryopreservation-induced apoptosis of CD4+ T-cells. Due to their known properties to promote T-cell survival and inhibit apoptosis, we evaluated the ability of IL-2, IL-4, and IL-7 combination cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4+ T-cells. The cytokine treatment resulted in a significant inhibition (p<0.01) of apoptosis-induced cell death and rescued CD4+ T-cell survival (p<0.01) in the cryopreserved cells. Efficient rescue of cryopreserved CD4+ T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.
authors.affiliation: Department of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261, USA.
authors.forename: Surojit
authors.initials: S
authors.lastname: Sarkar
authors.forename: Vandana
authors.initials: V
authors.lastname: Kalia
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Montelaro
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000201:enzymology; D020169:Caspases / Q000378:metabolism*; D015925:Cryopreservation / Q000379:methods*; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000201:enzymology; D008253:Macaca mulatta; D008928:Mitochondria / Q000378:metabolism
chemical_list: D007376:Interleukin-2; D015851:Interleukin-7; D015847:Interleukin-4; D020169:Caspases