Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Vα14-Jα18 TCRα gene

Abstract

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT- IntroductionNatural killer T (NKT) cells are characterized by their expression of an invariant receptor encoded by V␣14-J␣18 in mice 1 and by V␣24-J␣18 in humans. 2,3 Because the invariant receptor is used only by NKT cells and not by conventional T cells, NKT cells are a distinct lineage from conventional T cells. NKT cells produce both T helper 1 (Th1) and Th2 cytokines, mediating strong adjuvant activity through their Th1 cytokine production essential for protective responses against tumors 4-6 and pathogens 7-9 and also protecting autoimmune disease development through their Th2 cytokine production. 10,11 Despite the importance of this cell type in the immune system, the identity of NKT progenitor cells and their subsequent development remain poorly understood. In previous reports, cells with NKT-cell potential were detected in the CD4CD8 double-positive (DP) thymocyte population, 12,13 indicating that NKT cells are branched off from conventional ␣␤T-cell precursors at the DP stage in the thymus. [12][13][14] It has also been shown that the early immature CD4 NKT cells produce only interleukin-4 (IL-4), but their potential to produce interferon ␥ (IFN-␥) is acquired at a later developmental stage. 15,16 However, it is still possible that NKT cells might be derived from a precursor population distinct from that of conventional ␣␤T cells, because it has previously been shown that NKT precursor cells express on their cell surface granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), 17 which is known to be a unique marker for myeloid cell lineages but not for lymphoid cells. In addition, because the invariant V␣14 receptor is used only by NKT cells and not by conventional T cells, NKT cells may be a distinct lineage from conventional ␣␤T cells. 18 Embryonic stem (ES) cells are a powerful model system in which to study in vitro lymphocyte differentiation, addressing the questions on the cells with NKT precursor potential and the ability of NKT cells to produce both Th1 and Th2 cytokines during their development. For example, embryoid bodies generated from ES cells contain CD34 ϩ cells that develop into lymphocytes when cocultured with OP9 stromal cells plus appropriate cytokines. 19 However, in most cases, ES cells generate B and NK cells, but not T or NKT cells on OP9 coculture. 20,21 To overcome these problems, OP9 stromal cells transduced with Notch ligand delta-like1 (OP9/Dll-1) are used for the directed differentiation of ES cells to T-cell lineages. Induction of Notch signals directs stem cells to differentiate into immature DP T cells and inhibits B-cell development, indicating that Notch signaling is required as a proximal event in T-cell commitment from progenitors. 22,23 Another approach is to use cloned ES (NKT-ES) cells established by nuclear transfer of a cell with a rearranged T-cell receptor (TCR) gene. 24,25 Interestingly...

Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells

“…In the 20-day culture system, iPSCs cultured on OP9/Dll-1 give rise to NKT cells bearing immature phenotypes similar to CD4 + CD8 + DP thymocytes in vitro as shown in Figure 2, while iPSC-derived NKT cells in the switch culture showed mature CD4 + or double-negative (DN) NKT cell phenotypes (Supplemental Figure 3B). These are similar to those obtained by the ESC culture as described (13). Therefore, Notch signaling inhibits phenotypic maturation of NKT cells.…”

“…In fact, the iPSC-derived NKT cells obtained in the 25-day culture showed the ability to produce cytokines at levels similar to those of splenic NKT cells ( Figure 2D and Figure 4G). In particular, IFN-γ production was significantly high in the 25-day culture system using OP9/Dll-1 ( Figure 4G and Figure 2D), although, similar to ESC-derived NKT cells (13), iPSCderived NKT cells mainly produced IL-4 but not IFN-γ in the 20-day culture system using OP9/Dll-1 (Supplemental Figure 3C). Therefore, IFN-γ production by iPSC-derived NKT cells is apparently due to the duration of the culture.…”

“…We first attempted to investigate the potential of NKT cell development from iPSC-7a and iPSC-7g in the 20-day culture system used for analysis of ESCs described previously (Supplemental Figure 3A) (13). Similar to ESC-derived NKT cells, iPSCderived NKT cells mainly produced IL-4 on OP9/Dll-1 culture for 20 days, while mainly producing IFN-γ in the switching culture on OP9/control starting at day 14 (Supplemental Figure 3B), indicating that iPSCs are potentially the same as ESCs.…”

“…Results from our previous study, in which we used embryonic stem cells (ESC) derived from NKT cell-cloned mice (hereafter called the NKT mouse) established by nuclear transfer from C57BL/6 (B6) hepatic NKT cells, had already suggested that these cells could give rise to NKT cells both in vivo and in vitro (13). Since induced pluripotent stem cells (iPSCs) and ESCs are functionally equivalent in many respects, these observations suggested the feasibility of the clinical potential of iPSCs for NKT cell-targeted adjuvant therapy (14)(15)(16).…”

Type I natural killer T cells: naturally born for fighting

“…Complete absence of NKT cells in mice deficient in recombinase subunits RAG-1 and RAG-2 or the TCR Jα18 segment demonstrated that successful rearrangement of TCRα gene segments Vα14 to Jα18 is absolutely necessary to the subsequent selection of DP thymocytes [25,26] . The signaling and transcription factors mentioned below act as master regulators on the NKT cell lineage, but distinct from conventional T cells [19] .…”