title: The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells.
abstract: BACKGROUND
  The role of CD4(+) regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs.
  
  
  METHODS
  HIV-uninfected (HIV-) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b(+) and CD39(+) Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared.
  
  
  RESULTS
  Percentages of CD120b(+) Tregs 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 4h of in vitro culture. In contrast, expression of CD39 on Tregs 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.
  
  
  CONCLUSIONS
  The data suggest that percentages of CD120b(+) Tregs and CD39(+) Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV- and HIV+ men. However, for measurement of CD120b(+) Tregs 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.
authors: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA. Electronic address: wzhang32@jhu.edu.
authors: Weiying
authors: W
authors: Zhang
authors: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA. Electronic address: tnilles1@jhu.edu.
authors: Tricia L
authors: TL
authors: Nilles
authors: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA. Electronic address: jjohns20@jhu.edu.
authors: Jacquett R
authors: JR
authors: Johnson
authors: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA. Electronic address: jmargol1@jhu.edu.
authors: Joseph B
authors: JB
authors: Margolick
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation; D015331:Cohort Studies; D015925:Cryopreservation; D015658:HIV Infections / Q000276:immunology / Q000473:pathology; D006801:Humans; D016131:Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011446:Prospective Studies; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism*
chemical_list: D015415:Biomarkers
keywords: CD120b; CD39; CD4(+) regulatory T cells; HIV; PBMC cryopreservation