Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2

Abstract

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2-/- mice that exhibit defective NK cell development. In the Id2-/- FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2-/- FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2-/- fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2-/- and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Figure 1

Selective developmental defect in NK cells of the Id2^−/− fetal thymus. (A) Cells of 17-dpc FT were stained with CD3-, NK1.1-, CD4-, and CD8-specific mAbs. Total thymocyte numbers were 1.5 ± 0.9 × 10⁶, 1.8 ± 0.6 × 10⁶ and 0.7 ± 0.6 × 10⁶ for Id2^+/+, Id2^+/−, and Id2^−/− FT, respectively. (B) Cells of 14-dpc FT were stained with FITC-conjugated mAbs specific for TER119, Mac-1, Gr-1, B220, NK1.1, CD3, CD4, and CD8. Only FL-1-negative cells were analyzed for expression of CD44 vs. CD122 and CD44 vs. CD25. Total thymocyte numbers were 5.6 ± 1.4 × 10⁴, 3.2 ± 1.4 × 10⁴, and 1.5 ± 1.3 × 10⁴ for Id2^+/+, Id2^+/−, and Id2^−/− FT, respectively. Numbers represent the percentage of cells in the gated population and in each quadrant in A and B.

Figure 2

Id2^−/− fetal thymocytes give rise to T cells but not NK cells in IL-2-supplemented fetal thymus organ culture. Lin⁻CD44⁺CD25⁻ FT cells at 14 dpc were sorted, and 100 cells were cultured with a dGuo-treated FT lobe (B6Ly5.1) in the presence of IL-2 (25 units/ml), IL-7 (50 units/ml), and stem cell factor (10 ng/ml). After 10 days of culture, cells in each well were analyzed by flow cytometry. Representative flow cytometric profiles for expression of indicated markers on Ly5.1⁻ cells were shown. Cell recovery was 2.4 × 10⁴, 3.0 × 10⁴, and 2.0 × 10⁴ cells for Id2^+/+, Id2^+/−, and Id2^−/−, respectively.

Figure 3

Abrogated NK cell development of Id2^−/− fetal thymocytes. Single CD44⁺CD25⁻CD122⁻ FT cells at 14 dpc were picked up under microscopical visualization and were seeded into wells containing a dGuo-treated FT lobe. After 10 days of culture under HOS conditions, the numbers of the different types of progenitors detected among 50 CD44⁺CD25⁻CD122⁻ cells were scored. Error bars indicate SEM (n = 4 in both genotypes). The flow cytometric profiles of cells generated from p-NK, p-T, and p-T/NK were essentially same as those presented in our previous report (10).

Figure 4

Reverse transcription–PCR analysis for the gene expression of Id and E proteins in subpopulations of fetal thymus. Wild-type thymocytes at 15 dpc were fractionated, based on cell surface phenotype, into developmental subsets. Lin⁻ TN CD122⁺ cells in FT are exclusively NK lineage committed, as previously reported (10). mRNA was prepared from sorted cells (as indicated at the Top of the figures) and reverse-transcribed. The cDNA was amplified with specific primers for each gene indicated. Reverse transcription–PCR and PCR products of 14-dpc embryonic head total RNA served as positive and negative controls, respectively, in each reaction.

Figure 5

Schematic representation of Id2 function in the lineage restriction of NK cells. Bipotent T/NK precursors first colonize the fetal thymus. At the stage of CD44⁺CD25⁻, some FT cells start to express Id2, which inactivates bHLH factors, probably E proteins such as HEB and E2A gene products. These cells are committed to the NK cell lineage and express CD122/IL-2Rβ. At present, the order of CD122 expression and NK cell commitment is uncertain, although our data suggest that commitment precedes CD122 expression.