Orderly and Nonstochastic Acquisition of CD94/NKG2 Receptors by Developing NK Cells Derived from Embryonic Stem Cells In Vitro

Abstract

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1b and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1b on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.

Transient Notch signaling induces NK cell potential in Pax5‐deficient pro‐B cells

“…The deficiency in expression of the other Ly49 family members in vivo could be a consequence of the relatively early time point examined in this experiment, as it is known that some family members, including Ly49A and D, are expressed at relatively late time points after BM reconstitution [25]. An inability to express appropriate levels of the Ly49 family members, with the exception of Ly49E, has also been observed in fetal-as well as embryonic stem cell-derived NK cells [25][26][27] and, very recently, in thymus-derived NK cells (Vosshenrich et al, in press), suggesting that the Pax5 -/-pro-B cell-derived NK cells differentiate along a similar pathway in vitro.…”

Molecular Determinants Regulating the Pairing of NKG2 Molecules with CD94 for Cell Surface Heterodimer Expression

“…In a separate study, ϳ50% of NKG2C-expressing NK cell clones also express NKG2A (44). In addition, a high percentage of embyonic stem cell-derived mouse NK cell clones also coexpress NKG2A and NKG2C (45). It is therefore likely that NKG2A and NKG2C are coexpressed in a significant proportion of rhesus monkey NK cells.…”

A Dual Role for Talin in NK Cell Cytotoxicity: Activation of LFA-1-Mediated Cell Adhesion and Polarization of NK Cells

“…Since homozygous Tln1 KO in mice results in embryonic lethality (36), we generated NK cells from Tln1 Ϫ/Ϫ ES cells (clone A28) and the corresponding WT ES cells (clone HM1) by in vitro differentiation cultures as previously described (32,33). As reported previously (28), the two ES cell lines exhibited different morphologies.…”