Use of stem cell radiation chimeras to analyze how domains of specific proteins impact on murine NK cell development in vivo.

Although the use of mutant mice has been extremely useful in identifying those proteins and molecules specifically required for the development of NK cells, the establishment of a well-defined protocol to replicate in vitro the major steps corresponding to the process of NK cell differentiation and maturation has enabled us to dissect the molecular events governing certain aspects of NK cell development. This chapter describes a protocol that combines both the use of mutant mice and the in vitro bone marrow (BM) culture system for examining the role of proteins and their putative signaling domains in NK cell development. BM-derived Lin-c-kit(+) stem cells expressing the protein of interest are first cultured for 6 days in a cocktail of cytokines that promote lymphoid development. The semi-differentiated cells are then transplanted into mice to complete their development in vivo. While all hematopoietic lineages can develop from these transplanted cells, we focus primarily on assessing the effect of the protein on the production of NK cells, as well as the acquisition of Ly49 receptors. The most prevalent advantage of this method is the ability to potentially link signaling regulators to known aspects of NK cell development.

A role for lymphotoxin in the acquisition of Ly49 receptors during NK cell development.

NK cells lyse tumor, virus-infected and allogeneic cells through a recognition system involving inhibitory and activating receptors, among which are the Ly49 molecules that recognize MHC class I proteins. To date, little is known about the regulation of Ly49 expression during NK cell development. In this study we report that the acquisition of Ly49 receptors by NK cells is significantly reduced in lymphotoxin (LT) alpha-deficient mice, whereas it is increased in LTalpha transgenic mice. Treating normal mice with LTbetaR-Ig fusion protein reduced Ly49 expression, indicating that regulation of Ly49 receptor expression occurs through the engagement of membrane LT to LTbetaR, and not soluble LT to TNFR. In addition, when LTalpha(-/-) mice were treated exogenously with recombinant IL-15, NK cell numbers as well as Ly49 acquisition were restored to wild-type levels. Finally, using real-time PCR analyses of bone marrow cells obtained from LT-deficient or transgenic mice, we show a direct correlation between LTbetaR activation and increased IL-15 transcription. These data suggest that LTbetaR-mediated signals regulate Ly49 expression at least in part through the activation of IL-15.

Murine natural killer cell progenitors and their requirements for development.

Molecules that are essential to natural killer (NK) cell development have been identified mostly through characterizing knock-out mice that exhibit NK deficiencies. Such studies have shown that the interaction of membrane lymphotoxin (LT) on NK cells with its receptor on stromal elements is necessary for inducing a permissive microenvironment for NK development. Also, transcription factors such as Id2, interferon regulatory factors-1 (IRF-1), IRF-2, and Ets-1 are indispensable while PU.1 has a somewhat selective role. In addition, recent studies have identified T/NK progenitors (T/NKPs) in the fetal liver that precede migration to the fetal thymus as well as the earliest committed NK precursors in the bone marrow.

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro.

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-1(b) 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-1(b) 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.