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. Author manuscript; available in PMC: 2018 Sep 19.
Published in final edited form as: Immunity. 2017 Sep 19;47(3):435–449.e8. doi: 10.1016/j.immuni.2017.08.012

Figure 3. Rroid promotes the maturation of group 1 ILCs.

Figure 3

A. Rroid expression in NK cells and progenitors. Lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), and NK cell progenitors (NKP) sorted from bone marrow; CD27+, double positive (DP, CD27+, CD11b+), and CD11b+ NK cells sorted from spleen and Rroid expression was quantified by qPCR (LMPP, CLP, and NKP populations are n=2 of 5 pooled mice each, and splenic NK cells are n=3). Normalized to Hprt expression.

B. Absolute numbers of LMPP, CLP, pre-NK progenitor (pNKP), and refined NK progenitor (rNKP) populations in bone marrow (n=3 mice per group). Data are representative of two independent experiments.

C. Gating strategy for identifying common helper ILC progenitors (CHILP) in the bone marrow (left). Absolute numbers of CHILP in Rroid+/+ and Rroid−/− mice (right; n=4 mice per group). Representative of two independent experiments.

D. Histogram depicting the frequency±SEM of ILC progenitors marked by PLZF expression in CLP, pNKP, and rNKP populations (left), and absolute numbers of PLZF+ cells within pNKP and rNKP subsets (right). Shaded histograms are GFP-negative controls (n=3–4 mice per group from two pooled experiments).

E. Absolute numbers of bone marrow NK cells and ILC1s (n=3 mice per group). Data are representative of three independent experiments.

F. Schematic (left) and flow plots (right) depicting maturation pathway in splenic NK cells in Rroid+/+ and Rroid−/− mice. Gated on CD45.2+ CD3, CD5− NK1.1+ NKp46+ cells.

G. Frequency (left) and absolute numbers (right) of NK cell maturation in the spleen of Rroid+/+ and Rroid−/− mice (n=4 mice per group). Data are representative of four independent experiments.

H. BrdU incorporation (left) and Annexin V staining (right) of maturing splenic NK cells. Mice were injected i.p. every 12h with 1mg BrdU for 3d. Splenocytes were either fixed and probed for BrdU incorporation or stained with Annexin V to determine cell viability (n=3–4 mice per group). Data are representative of two independent experiments.

I. Flow cytometry plot (left) and frequency (right) of maturing splenic NK cells in competitive bone marrow chimeras analyzed 8 weeks post-transfer (n=8 mice per group). Data are pooled from two independent experiments.

*p≤0.05, **p≤0.01, ***p≤0.001. Two-tailed t-test. All error bars represent SEM. See also Figure S3.