Cryopreservation modulates the detection of regulatory T cell markers

Abstract

Background: Regulatory T cells (Tregs) modulate the host response in infectious diseases and are key mediators of peripheral tolerance. Cryopreservation of peripheral blood mononuclear cells (PBMCs) is commonly used in immunological field studies where access to complex laboratory tests is not feasible. Our objective is to assess the effects of cryopreservation on the flow cytometric detection of surface and intracellular markers of Tregs.Methods: Heparinized venous blood was obtained from 36 healthy individuals and 15 HIV-1 infected subjects. PBMCs were isolated and stained for surface and intracellular markers of Tregs. PBMCs from each subject were cryopreserved in liquid nitrogen with DMSO; these cells were thawed and stained at a later date. All samples were analyzed by flow cytometry. The proportion of Tregs was compared using Wilcoxon signed-rank test.Results: Cryopreservation decreased the proportion of Tregs identified by surface and intracellular markers in healthy individuals and in HIV-1 patients. The proportion of CD41CD251FoxP31 was decreased from 3.13 to 2.16% (P < 0.001) for non-HIV subjects and from 2.68 to 0.94% (P < 0.001) for HIV subjects, compared to fresh samples. Significant reduction was also observed for CD41CD251CD127lo-neg. However, the effect varied considerably between samples. The effect was similar among HIV and non-HIV patients (P 5 0.38).Conclusions: Cryopreservation modulates the detection of surface and intracellular markers of Tregs. These results confirm that research on Tregs, including studies of HIV-1 infected patients, should be carried out prospectively on fresh samples in order to obtain unbiased conclusions. Results using cryopreserved cells should be regarded as only preliminary. V C 2011 International Clinical Cytometry Society

Human secondary lymphoid organs typically contain polyclonally-activated proliferating regulatory T cells

“…47 In the current study, both fresh and cryopreserved samples were used for phenotypic characterization of Tregs. As there is conflicting data about whether or not cryopreservation reduces the percentage of Tregs in samples, 48,49 we analyzed SPL samples both freshly after isolation as well as after at least 1 month of cryopreservation in liquid nitrogen. We found no difference in the percentage of Tregs, their activation status, or their chemokine expression pattern (supplemental Figure 1, available on the Blood Web site).…”

Phenotypical characterization of regulatory T cells in humans and rodents

“…Clone of anti-FoxP3 was PCH101. , specifically in cells from HIV-infected individuals [31]. However, some authors have reported comparable values of T regs in frozen samples and fresh samples, suggesting that these variations could be related to differences between cryopreservation and thawing protocols.…”

CD4+CD25+CD127− assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV‐1 infected viremic and aviremic subjects

“…On the one hand, the heterogeneity of patients' population has to be taken into account (level of viremia, type of progressors, treatments duration : : : ). On the other hand, analytical issues may have contributed to discordances: markers used for Tregs phenotyping, staining of whole blood or purified cells, sample conservation (i.e., cryopreserved or not) (9,10).…”