Fig 2: Rora-Teal Expression Distinguishes ILCs from NK Cells(A) Flow-cytometry gating strategy for ILC1 or ex-ILC3 in the siLP of 5x polychromILC mice.(B) Flow-cytometry analysis of Rora-Teal expression in splenic NK cells (CD3-CD19-NKp46+NK1.1+) and siLP ILC1or exILC3 (gated as shown in A) from 5x polychromILC mice.(C) Flow-cytometry analysis of the indicated cell surface markers in Lin-NKp46+NK1.1+ cells from the spleen, liver, and siLP of Rora+/Teal mice.(D) Flow-cytometry analysis of CD11c, Eomes, and T-bet expression in NK or ILC1 cells from the spleen of Rora+/Teal mice.(E) Flow-cytometry analysis of CD11c, Eomes, and T-bet expression in NK or ILC1 cells from the liver of Rora+/Teal mice.(F) Flow-cytometry analysis of CD11c, Eomes, and T-bet expression in NK or ILC1 cells from the siLP of Rora+/Teal mice.Data are representative of 2 independent experiments.

Fig 3: Generation of Compound 5x polychromILC TF Reporter Mice to Define ILC Lineage Development(A) Flow-cytometry gating strategy for ILC subsets in siLP from TF reporter mice (ILC1 or ex-ILC3: CD45+Lin-IL-7Ra+CD4-KLRG1-NKp46+NK1.1+; ILC2: CD45+Lin-IL-7Ra+CD4-KLRG1+; ILC3: CD45+Lin-IL-7Ra+CD4-KLRG1-NKp46+NK1.1-; CD4-LTi: CD45loLin-IL-7Ra+CD4-KLRG1-NKp46-NK1.1-CCR6+; CD4+LTi: CD45loLin-IL-7Ra+KLRG1-CD4+CCR6+).(B) Flow-cytometry analysis of Rorc-Kat expression in the ILC subsets of the siLP of Rorc+/Kat mice.(C) Flow-cytometry analysis of Id2-BFP expression in the ILC subsets of the siLP of Id2+/BFP mice.(D) Flow-cytometry analysis of Gata3-hCD2 expression in the ILC subsets of the siLP of Gata3+/hCD2 mice.(E) Flow-cytometry analysis of Rora-Teal expression in the ILC subsets of the siLP of Rora+/Teal mice.(F) Schematic for generation of Id2+/BFPGata3+/hCD2Rora+/TealBcl11b+/tdTomRorc+/Kat 5x polychromILC mice.(G) Flow-cytometric comparison of the ILC subsets in siLP of Id2+/BFPGata3+/hCD2Rora+/TealBcl11b+/tdTom (4x polychromILC) mice and Id2+/BFPGata3+/hCD2Rora+/TealBcl11b+/tdTomRorc+/Kat (5x polychromILC) mice.Data are representative of 2 independent experiments; mean ± SEM; not significant (ns), *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s post-hoc test. Please also see Figures S1–S4.

Fig 4: The Qdm-ß2m-pQa-1 single chain trimer (SCT) specifically recognizes the CD94/NKG2A receptor.(A) Spleen lymphocytes isolated from wildtype C57BL/6 or NKG2A-/- knockout mice were stained with PE-labeled pQa-1-dtSCT tetramer (PE-pQa-1 Tet), or PE-labeled streptavidin (PE-SA) as negative control, at room temperature for 1 hr followed by staining with a mixture of fluorochrome labeled antibodies containing either anti-NKG2A/C/E (20D5) or isotype control for 30 min. The stained cells were acquired by BD Canto II and data were analyzed with FlowJo software. Representative data of four (wildtype) and two (NKG2A-/-) independent experiments are shown. NK cells were defined as the live NK1.1+CD3-CD19- population, while CD8 T cells were gated on the live CD19- CD3+CD8+ population.

Fig 5: BTB domain And CNC Homolog 2 (Bach2) expression at different natural killer (NK) cell developmental stages by analysis of Bach2Flag knock-in mouse.(A) Histogram plots of Bach2Flag expression (red) in common lymphoid progenitor (CLP), pre-NK progenitor (pre-NK), and refined NK progenitor (rNKp) as compared to cells from wild-type (WT) mice (gray fill). The geometric MFI (gMFI) number was indicated in the plot. Summary of gMFI was shown (n=5 for each group in three independent experiments). (B) Histogram plots of Bach2Flag expression (red) in NK cell (CD3-CD19-NK1.1+) from bone marrow (BM) and spleen as compared to cells from WT mice (gray fill). Numbers indicate gMFI and summary of gMFI is shown (n=7 or 9 for each group in three to four independent experiments). (C) CD3-CD122+ NK cells from BM and spleen were subdivided into iNK (DX5-NK1.1+) and mNK (DX5+NK1.1+) and analyzed for Bach2Flag expression (red for iNK, blue for mNK) as compared to cells from WT mice (gray fill). Numbers indicate gMFI and summary of gMFI is shown (n=7 for each group in three to four independent experiments). (D) Histogram plot of Bach2Flag expression in CD27+CD11b- (red), CD27+CD11b+ (blue), and CD27+CD11b+ (yellow) NK subsets as compared to cells from WT mice (gray fill). Note that WT plot represents expression of Bach2Flag from CD27-CD11b+ subsets. Numbers indicate gMFI and summary of gMFI is shown (n=6 for each group in three independent experiments). (E) Splenic NK cells were sorted into CD27+CD11b-, CD27+CD11b+ and CD27-CD11b+ subsets. Bach2Flag expression in the subsets was detected using Anti-FLAG M2-Peroxidase (HRP) antibody by western blot. Expression of Actin was used as an internal control. Two individual experiments have been done with one mouse each time. (F) Histogram plot of Bach2Flag expression in splenic NK cell (CD3-CD19-NK1.1+) from mice at 2 weeks’ (red), 5 weeks’ (blue), and 12 weeks’ (orange) age as compared to cells from WT mice (gray fill). Note that WT plot represents expression of Bach2Flag from 12-week age mice. Numbers indicate gMFI and summary of gMFI fold change is shown (n=6 or 11 for each group in three to four independent experiments). (G) Summary of gMFI data for Bach2Flag expression in NK cells from lymph node, spleen, lung, and liver (n=4 or 6 for each group in three independent experiments). NK cells from lymph node, spleen, and lung were gated as CD3-CD19-NK1.1+. NK cells from liver were gated as CD3-CD19-NK1.1+DX5+. Statistical significance was determined by two-way ANOVA (A, C, D, and G), one-way ANOVA, (F) or by Student’s t test (B). Error bars indicate SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant. See Figure 1—figure supplement 1 for gating strategies. Figure 1—source data 1.Bach2 expression in different subsets by western blot.Splenic NK cells were enriched from splenocytes of Bach2Flag mice. Enriched NK cells (CD3-NK1.1+) from Bach2Flag mice were further sorted into CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ subsets. Bach2 expression in the subsets was detected using Anti-FLAG M2-Peroxidase (HRP) antibody by western blot. Expression of Actin was used as an internal control. Splenocytes from wild-type (WT) mice were used as negative control. Splenocytes from Bach2Flag mice were used as positive control. Two individual experiments have been done with one mouse each time.