Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.

In contrast to other solid organ transplantations, liver grafts have tolerogenic properties. Animal models indicate that donor leukocytes transferred into the recipient after liver transplantation (LTX) play a relevant role in this tolerogenic phenomenon. However, the specific donor cell types involved in modulation of the recipient alloresponse are not yet defined. We hypothesized that this unique property of liver grafts may be related to their high content of organ-specific natural killer (NK) and CD56 þ T cells. Here, we show that a high proportion of hepatic NK cells that detach from human liver grafts during pretransplant perfusion belong to the CD56bright subset, and are in an activated state (CD69 þ ). Liver NK cells contained perforin and granzymes, exerted stronger cytotoxicity against K562 target cells when compared with blood NK cells, and secreted interferon-c, but no interleukin-10 or T helper 2 cytokines, upon stimulation with monokines. Interestingly, whereas the CD56bright subset is classically considered as noncytolytic, liver CD56bright NK cells showed a high content of cytolytic molecules and degranulated in response to K562 cells. After LTX, but not after renal transplantation, significant numbers of donor CD56dim NK and CD56 þ T cells were detected in the recipient circulation for approximately 2 weeks. In conclusion, during clinical LTX, activated and highly cytotoxic NK cells of donor origin are transferred into the recipient, and a subset of them mixes with the recirculating recipient NK cell pool. The unique properties of the transferred hepatic NK cells may enable them to play a role in regulating the immunological response of the recipient against the graft and therefore contribute to liver tolerogenicity. Liver Transpl 16:895-908, 2010. V C 2010 AASLD.Received December 23, 2009; accepted March 28, 2010. It is generally recognized that after clinical liver transplantation (LTX), the incidence of chronic rejection is lower than after transplantation of other organ grafts. Furthermore, in about 20% of LTX recipients, immunosuppressive therapy can be withdrawn without occurrence of graft rejection.1 Various animal models are spontaneously tolerant to LTX, even though they reject other organs.2,3 Furthermore, cotransplantation of a liver allograft can prevent rejection of other organ grafts from the same donor. 4,5 The mechanisms responsible for this relative tolerogenicity of the liver have only been partially elucidated. A number of observations in animal models indicate that the immune cells present in the liver graft may play a relevant role in the induction of tolerance. With LTX, so-called passenger leukocytes from the donor are transferred into the recipient and can establish a condition of chimerism of variable proportions and duration.6,7 Independent studies from different groups have shown that in rat models,

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.

Cyclosporin A (CsA), rapamycin (Rapa) and mycophenolic acid (MPA) are frequently used for GVHD prophylaxis and treatment after allogeneic stem cell transplantation (SCT). As NK cells have received great interest for immunotherapeutic applications in SCT, we analyzed the effects of these drugs on human cytokinestimulated NK cells in vitro. Growth

Natural killer (NK) cells have an important function in the antitumor response early after stem cell transplantation (SCT). As part of a prospective randomized phase III study, directly comparing the use of CD3 þ /CD19 þ -depleted peripheral blood stem cell (PBSC) harvests with CD34 þ -selected PBSC harvests in allogeneic human leukocyte antigen-matched SCT, we here show that the use of CD3 þ /CD19 þ -depleted PBSC grafts leads to early NK cell repopulation and reconstitution of the CD56 dim and CD56 bright NK cell subsets, with concomitant high cytolytic capacity. In the CD34 group, this process took significantly longer. Moreover, in the CD3/19 group after reconstitution, a higher percentage of killer immunoglobulin-like receptor-positive NK cells was found. Although similar percentages of CD94-positive NK cells were found in both groups, in the CD34 group, almost all expressed the inhibitory CD94:NKG2A complex, whereas in the CD3/19 group, the inhibitory CD94:NKG2A and the activating CD94:NKG2C complex were equally distributed. This preferential development of NKG2C-expressing NK cells in the CD3/19 group was paralleled by a loss of NKG2A-mediated inhibition of NK cell degranulation. These results show that the use of CD3 þ /CD19 þ -depleted grafts facilitates strong NK cell cytolytic responses directly after SCT, and the rapid emergence of an NK cell receptor phenotype that is more prone to activation.