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

Background Aims: 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.