Isolation, Expansion & Function of Cord Blood Natural Killer Cells

The rate of immune reconstitution (IR) is directly correlated with the number of hematopoietic stem cells (HSC) infused and is particularly delayed in patients undergoing cord blood transplantation (CBT) secondary to the limited numbers of HSC. Thus, methods to increase the number of cord blood (CB) progenitors have the potential to accelerate IR after CBT.  Natural killer (NK) cells play a crucial role in early IR after HCT because they are the first lymphocyte subset to recover after transplant.   CB NK cells have been reported to have incomplete maturation and require activation for effective function. Here, we report a clinically relevant method for ex vivo expansion of NK cells isolated from CB without the use of a stromal layer.  Our group has demonstrated that CB NK cells cultured in the presence of IL-2 and IL-15 results in a multi-log increase in the number of precursors that have a significant increase in cytotoxicity against several target cancer cell lines.   After 21 days in culture, there is a 1.848 ± 0.341 log fold increase in the number of CD3-CD56+ NK cells (range, 0.420 to 3.108) (p<0.001, N=9).  After culture, we also found a significant 2.612 ± 0.310 log fold increase (range, 0.979 to 3.622) (p<0.001, N=9) in the number of CD3+CD56+ NKT cells. Evaluation of cytotoxicity against K562 cells, a chronic myelogenous leukemia, showed there was also a significant increase in cytolytic function at days 14 (31.52 ± 8.317% target cell lysis, p<0.01, N=8) and 21 (44.22 ± 9.866% target cell lysis, p<0.01, N=8) in culture when compared to day of isolation; similar results were seen using Jurkat cells, an acute T-cell lymphoblastic leukemia (T-ALL).  Evaluation of NK cell resistant cells lines was also tested.  While we found an increase in cytotoxicity toward the myeloid lymphoid leukemia cell line (MLL), MV411, after culture, the RS411 (MLL/ALL) and HDLM2 (Hodgkin’s lymphoma) cell lines remained resistant to cytolytic killing (N=3).  Currently, we are investigating the NK cell population(s) responsible for the cytotoxic killing and the corresponding killer cell Ig-like receptor (KIR) ligand/adhesion molecule(s) that may be responsible for function. We hypothesize that using methods to increase the number of CB NK cells have the potential to prevent early relapse, infections and graft versus host disease, as well as facilitate engraftment when administered following CBT.