title: Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors.
abstract: BACKGROUND
  Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors.
  
  
  METHODS
  Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFalpha+IL-1beta+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells.
  
  
  RESULTS
  Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro.
  
  
  CONCLUSIONS
  Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.
authors.affiliation: BD Biosciences Immunocytometry Systems, 2350 Qume Dr, San Jose, CA 95131, USA. smita_ghanekar@bd.com
authors.forename: Smita A
authors.initials: SA
authors.lastname: Ghanekar
authors.forename: Sonny
authors.initials: S
authors.lastname: Bhatia
authors.forename: Joyce J
authors.initials: JJ
authors.lastname: Ruitenberg
authors.forename: Corazon
authors.initials: C
authors.lastname: DeLa Rosa
authors.forename: Mary L
authors.initials: ML
authors.lastname: Disis
authors.forename: Vernon C
authors.initials: VC
authors.lastname: Maino
authors.forename: Holden T
authors.initials: HT
authors.lastname: Maecker
authors.forename: Cory A
authors.initials: CA
authors.lastname: Waters