title: A novel method to expand large numbers of CD56(+) natural killer cells from a minute fraction of selectively accessed cryopreserved cord blood for immunotherapy after transplantation.
abstract: OBJECTIVE
  Umbilical cord blood transplantation (UCBT) is increasingly used to treat acute leukemias. UCB units are thawed and infused in their entirety at transplant, precluding later use as immunotherapy to prevent or treat leukemia relapse.
  
  
  METHODS
  We developed a device that selectively thaws only 1 mL of the UCB unit, leaving the remaining UCB unit cryopreserved for subsequent transplantation. We also show that large numbers of CD56(+) natural killer (NK) cells can be expanded from these 1-mL fractions of selectively accessed UCB. Immunomagnetic depletion of CD3(+) cells of the 1-mL fraction was performed, and the cells were subsequently stimulated with irradiated Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) and set to culture in media containing interleukin (IL)-2.
  
  
  RESULTS
  When a 1:20 ratio of total nucleated cells to EBV-LCL feeder cells was used, day-21 and day-35 NK cell cultures initiated from 1 mL of UCB contained a median of 430 × 10(6) (range: 44-4321 × 10(6)) and 6092 × 10(6) (range: 165-20947 × 10(6)) CD3(-)CD56(+) NK cells. These cells expressed high levels of CD161, LFA-1, CD69, NKG2D, NKp30, NKp44, NKp80 and NKp46. UCB-derived NK cells were highly cytotoxic against K562 leukemia cells, although cytotoxicity was slightly lower than in expanded PBMC-derived NK cells.
  
  
  CONCLUSIONS
  We have developed and optimized a strategy to selectively access a small fraction from cryopreserved UCB and show that large numbers of CD56(+) cells can be expanded from this selectively accessed fraction. This strategy presents a method to explore whether early adoptive transfer of NK cells expanded from the same UCB unit used for transplantation can prevent leukemic relapse and decrease graft-versus-host disease after UCBT.
authors.affiliation: Division of Hematology, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Sumithira
authors.initials: S
authors.lastname: Vasu
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Berg
authors.affiliation: Medical Oncology Branch, National Cancer Institute, Bethesda, Maryland, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Davidson-Moncada
authors.affiliation: Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Tian
authors.affiliation: American Flouroseal Corporation, Gaithersburg, Maryland, USA.
authors.forename: Herb
authors.initials: H
authors.lastname: Cullis
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: childsr@nhlbi.nih.gov.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Childs
mesh_terms: D019002:CD56 Antigen / Q000378:metabolism; D049109:Cell Proliferation; D036101:Cord Blood Stem Cell Transplantation; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D004867:Equipment Design; D061252:Feeder Cells; D005312:Fetal Blood / Q000166:cytology*; D006086:Graft vs Host Disease / Q000517:prevention & control; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology
chemical_list: D019002:CD56 Antigen; C522918:NCAM1 protein, human
keywords: cryopreserved cord blood; expanded NK cells; ex vivo expansion; natural killer cells; umbilical cord blood