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Effects of cryopreservation on lymphocyte immunophenotype and function
A Costantini1, S Mancini, S Giuliodoro
1Service of Clinical Immunology, Department of Internal Medicine, Ancona University Medical School, Ancona, Italy.
Insights
Cryopreservation significantly alters peripheral blood mononuclear cells (PBMCs), impacting T cell subsets and chemokine receptor expression. For HIV-infected patients, studies on immune reconstitution or infectivity require fresh samples to avoid altered results.
Area of Science:
- Immunology
- Cell Biology
- Virology
Background:
- Cryopreservation of peripheral blood mononuclear cells (PBMCs) is standard for minimizing variability in phenotypic and functional analyses.
- Limited data exist on cryopreservation's effects on PBMCs, particularly in HIV-infected individuals, where lymphocyte assessment is critical.
Purpose of the Study:
- To compare the effects of cryopreservation on PBMCs from healthy individuals and HIV-infected patients.
- To evaluate cryopreservation-induced changes in T cell subsets, chemokine receptor expression, and antigen-specific responses.
Main Methods:
- Comparison of fresh versus frozen/thawed (F/T) PBMCs from 19 healthy and 21 HIV-infected individuals.
- Flow cytometry analysis of cell surface markers (CD62L, CD45RA, CD45RO, CCR5).
- Assessment of T cell proliferation in response to HIV and recall antigens.
Main Results:
- Cryopreservation decreased CD62L expression, reducing naive and central memory T cells.
- Effector CD8+ T cells increased in healthy subjects but not in HIV-infected patients.
- Significant decline in CC chemokine receptor 5 (CCR5) expression observed.
- HIV-infected patients showed loss of proliferative responses to p24, CMV, and Influenza antigens post-cryopreservation.
Conclusions:
- Cryopreservation induces consistent changes in PBMCs from both healthy and HIV-infected individuals.
- Immunological studies in HIV-infected patients, such as immune reconstitution or in vitro infectivity assays, should utilize fresh samples.
Abstract:
Cryopreservation of isolated peripheral blood mononuclear cells (PBMCs) for phenotypic and functional analyses is considered a standard procedure in order to minimize operator-dependent inter-assay variability and to optimize the use of available resources. However, only few and somewhat conflicting data are presently available on the effects of cryopreservation on PBMCs, especially in samples from HIV-infected patients in which assessment of lymphocyte phenotype and function is of the outmost importance. In this study, we compared fresh versus frozen/thawed (F/T) samples isolated from 19 healthy individuals and 21 HIV-infected patients, showing that cryopreservation induces: (i) a profound decrease of CD62L expression, with a consequent significant decline of the calculated proportions of "naïve" (CD45RA+CD62L+) and "central memory" (CD45RO+CD62L+) T cells; (ii) an increase of the calculated proportions of "effector" CD8+ T cells (CD45RA+CD62L- and CD45RO+CD62L-) in the healthy subjects, while no changes were observed in the HIV-infected group; (iii) a significant decline of CC chemokine receptor 5 (CCR5) expression; (iv) a loss of proliferative responses to some HIV antigens (i.e. p24) and recall antigens [cytomegalovirus (CMV) and Influenza] in HIV-infected patients. We thus conclude that cryopreservation induces a consistent set of changes in PBMCs from both healthy and HIV-infected individuals, and that certain immunological studies of HIV-infected patients (i.e. studies of immune reconstitution following antiretroviral therapy in HIV-infected patients or studies of HIV-infectivity in vitro using CCR5-tropic strains) should be performed on fresh samples.
