The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

Background: The role of CD4⁺ regulatory T cells (T_regs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of T_regs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of T_regs. Methods: HIV-uninfected (HIV-) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b⁺ and CD39⁺ T_regs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared. Results: Percentages of CD120b⁺ T_regs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4 h of in vitro culture. In contrast, expression of CD39 on T_regs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor. Conclusion: The data suggest that percentages of CD120b⁺ T_regs and CD39⁺ T_regs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV- and HIV+ men. However, for measurement of CD120b⁺ T_regs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.