Functional Consequences of Human Lymphocyte Cryopreservation

Abstract

In order to understand human inflammatory diseases and to develop and assess new therapeutic strategies targeting leukocyte recruitment to tissue, it is necessary to study human lymphocyte interactions with endothelium. It is often not practical to carry out assays on fresh human samples and therefore cells may be cryopreserved and batched for later study. Furthermore, many forms of adoptive cell therapy use cryopreserved cells that are required to migrate to tissue after infusion in vivo. The consequences of cryopreservation on the adhesion and migration of leukocytes is not known leading us to study the effects of cryopreservation on lymphocyte phenotype, migration, and adhesion. Cryopreservation and subsequent thawing did not alter the proportion of retrieved T cell subsets. Overall levels of expression of β1 or β2 integrins were unaffected but marked changes were observed in other relevant receptors. Expression of CD69, a transmembrane protein that plays a critical role in lymphocyte egress from tissues and the chemokine receptor CXCR4, increased on thawed populations and levels of CD62L and CXCR3 were reduced on thawed cells but restored if cells were allowed to recover after thawing. These changes were associated with modulation of the ability of lymphocytes to migrate across cytokine-stimulated monolayers of endothelium toward recombinant CXCL11 and CXCL12. Thus cryopreservation and thawing of lymphocytes induces changes in their adhesive phenotype and modulates their ability to migrate across endothelial monolayers. These findings have implications for in vitro experimentation and for cell therapy in which cryopreserved cells are expected to migrate when reinfused into patients.

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

“…2, P > 0.5, ANOVA) indicating functional integrity of PBMC after thawing. These data are consistent with previous findings that cryopreservation did not affect integrins on mononuclear cells (8,11).…”

“…2, P > 0.5, ANOVA) indicating functional integrity of PBMC after thawing. These data are consistent with previous findings that cryopreservation did not affect integrins on mononuclear cells (8,11).Overall, based on this limited set of markers, there was no indication for diminution or selection by cryopreservation of PBMC subpopulations, thus confirming and extending previous reports (2,4,12). In addition, our finding that cryopreservation of PBMC from several volunteers apparently did not affect their capacity to interact with activated endothelial cells now lends experimental evidence to the notion that cryopreservation is a useful tool to preserve native human immune cells for long-term or repeated functional analyses, at least regarding dynamic interactions with endothelial cells.…”

Expression of CXCL12 receptors in B cells from Mexican Mestizos patients with systemic lupus erythematosus

“…Cryopreservation of PBMC might also constitute a source of changes in labile phenotypic markers and B-cell subset distribution. Recently, Faint and coworkers showed that cryopreserved sample batching, and subsequent thawing for deferred analysis, did not alter the proportion of lymphocyte subsets but led to an increased CXCR4 expression on lymphocytes [52]. In our hands, the cryopreservation step indeed influenced positively CXCR4 expression on control B cells (Additional file 2: Figure S2).…”

Cryopreservation of human mesenchymal stromal cells expressing TRAIL for human anti-cancer therapy

“…Faint et al. investigated the effect of cryopreservation on lymphocyte migration and adhesion and demonstrated no functional effect but again noted an increased expression of CXCR4 [41] .…”