Identification and Characterization Of Human NK Lineage Restricted Progenitor (NKP)

Natural Killer (NK) cells, the third lymphoid lineage after T and B cells, are large granular lymphocytes belonging to innate immune system, classically defined as CD3^-CD56⁺CD16⁺ in human. NK cells can kill target cells without prior activation, based on the missing self-hypothesis, either due to the lack of MHC-class I molecule or up-regulation of NKG2D ligands, two properties commonly associated with transformed cells (D.H. Raulet, N. Guerra, 2009). Moreover, by secreting cytokines (such as TNF-α and IFN-γ), NK cells can also activate the response of the adaptive immune system. Experiments in mice have shown that NK cells are primarily responsible for the in vivo elimination of transplanted tumor cells (J. Wu, L. L. Lanier, 2003) and, in human, a direct correlation between low NK cytotoxicity and elevated cancer incidence was reported in a large cohort of Japanese aged over 40 (K. Imai et al., 2000). The important anti-tumor role for alloreactive NK cells has been shown in patients with acute myeloid leukemia where transplanted donor NK cells, expressing mismatch inhibitory receptors, provided graft versus leukemia effect in the absence of graft versus host disease (L. Ruggeri et al., 2006). These and many other findings are the basis of a large number of ongoing studies and clinical trials for NK cell immunotherapy against cancer (M. Terme et al., 2008). However, in contrast to B and T cell development where both cellular and regulatory pathways are well studied, the development of NK cells from hematopoietic stem cells (HSCs) is not well understood and the identity of restricted NK cell progenitor (NKP) is unknown (S. Doulatov et al., 2012).

To identify human NKP, we applied the expression of known early lymphoid markers (CD45RA, CD10, CD7) and cytokine receptors (IL-7R: CD127) important for lymphoid development. Using multicolor flow cytometry, Lin^-CD34⁺CD38⁺CD123^-CD45RA⁺CD7⁺CD10^+/-CD127^+/- populations were sorted from human bone marrow (hBM) and umbilical cord blood (hUCB). Purified candidate NKPs were cultured on OP9, OP9DL1 stroma and in Terasaki cultures in the presence of specific cytokines and generation of CD3^-CD56⁺CD16⁺NKp46⁺ NK cells, CD19⁺ B, CD3⁺ CD4⁺ CD8⁺ T and CD33⁺CD14⁺ myeloid cells was investigated. To study the ability of NKP candidate to generate NK cells after transplantation, purified NKPs were injected into new-born NOD/SCID γc^null (NSG) mice and at 11 weeks after transplantation the phenotype of generated progeny was evaluated. In addition, NK cells generated in stroma cultures and after transplantation were tested for their cytotoxic activity against K562 leukemic cell line in degranulation assay, where the surface expression of membrane glycoprotein LAMP-1 (CD107a) is measured after activation of specific killing receptors.

The Lin^-CD123^-CD34⁺CD38⁺CD45RA⁺CD7⁺CD10⁺CD127^- NKP candidate population sorted from hUCB has a robust NK cell potential in vitro at the single cell level and lacked the ability to generate T, B and myeloid cells. Furthermore, NK cells generated from this candidate NKP were functionally mature: show cytotoxic activity against K562 cells and produce cytokines: IFNγ and TNFα after activation in vitro. Further phenotypic characterization showed that the candidate NKP is highly positive for lymphoid markers CD244 and CD62L, lacks the expression of mature NK cell markers NKp46 and NKG2D, and 50% of NKPs express c-kit and Flt3 receptors. At 11 weeks after transplantation, 9 out of 17 NSG mice injected with 600 Lin^-CD123^-CD34⁺CD38⁺CD45RA⁺CD7⁺CD10⁺CD127^- NKPs had a significant human cells engraftment and only CD3^-CD56⁺NKp46⁺CD16^+/- NK cells were found in the peripheral blood, bone marrow and spleen; whereas 15 out 17 NSG mice injected with CD34⁺ cells were positive for the presence of human B, T, NK and myeloid cells in these tissues. Human NK cells generated after transplantation into NSG mice were functionally active and able to kill K562 leukemic cells. The Lin^-CD123^-CD34⁺CD38⁺CD45RA⁺CD7⁺CD10⁺CD127^- NKP candidate was also found in adult hBM and showed NK-lineage restriction.

Our results indicate that the Lin^-CD123^-CD34⁺CD38⁺CD45RA⁺CD7⁺CD10⁺CD127^- cells, found in human BM and UCB, represent NK-lineage restricted progenitors that generate fully mature functional NK cells.