Figure 2. The Rroid locus is necessary for NK cell function.

A. IFN-γ production in splenic NK cells. Splenocytes were stimulated for 4h ex vivo with 20ng/mL IL-12, a combination of IL-12 and 20ng/mL IL-18, or with media alone (U/S). Data are representative of three independent experiments.

B. NK cell degranulation as measured by CD107a staining in splenocytes activated by anti-NK1.1 for 4h ex vivo. Data are representative of two independent experiments.

C. IFN-γ production in liver NK cells (left) or ILC1s (right). Liver lymphocytes were stimulated for 4h ex vivo with 20ng/mL IL-12, a combination of IL-12 and 20ng/mL IL-15/IL-15Rα complex, or media alone (U/S, n=3–4 mice per group). Data are representative of two independent experiments.

D. Expression of Il12rb2 by qPCR in liver NK cells and ILC1s. 2500 NK cells and ILC1s were sorted from the liver and subjected to 1-step qRT-PCR. Data are normalized to Hprt1 expression. (n=3 mice per group).

E. Experimental outline of the RMA-S transfer system to probe NK cell function in vivo. 5×10⁶ CFSE-labeled RMA cells (MHC-I⁺) and 15×10⁶ CTV-labeled RMA-S (MHC-I⁻) cells were combined were injected intravenously into recipients, and splenocytes were analyzed 16h after injection.

F. Gating strategy for identifying fluorescently-labeled RMA and RMA-S cells in the spleen (left). Ratio of RMA-S:RMA cell numbers (right, n=6 mice from two pooled independent experiments).

G. Listeria monocytogenes (Lm) burden in the liver (left) and spleen (right). Mice were infected intravenously with 3×10⁴ CFU of Listeria and analyzed 3d post infection (n=8–9 mice per group). Data are representative of two independent experiments.

H. Absolute numbers of ILC1 (left) and NK cells (right) in the livers of Rroid^−/− mice and controls 3d post infection with Listeria (n=4–5 mice per group).

I. Production of IFN-γ from splenic NK cells in mice 3d post infection with Listeria. Cells were stimulated 4h with 20ng/mL IL-12, a combination of IL-12 and 20ng/mL IL-18, or with media alone. (n=4–5 mice per group).

J. Absolute numbers of eosinophils (CD45⁺ CD11c⁻ Siglec-F⁺) in the bronchoalveolar lavage (BAL) and lung parenchyma of mice challenged intranasally with 30μg papain every 24h for 5 days (n=4–5 mice per group).

K. ILC2 cytokine production from mice challenged with papain. Lung lymphocytes were stimulated 4h with PMA/Ionomycin and gated on CD45⁺ CD3, CD5, CD19⁻ CD90.2⁺ T1/ST2⁺ cells (n=5 mice per group).

L. Enumeration of Peyer’s patches in the small intestine of Rroid^+/+ and Rroid^−/− mice (n=10 mice per group pooled from three independent experiments).

M. IL-22 production in small intestine ILC3s. Lymphocytes from small intestine were stimulated 4h with media alone (U/S), IL-23 (20ng/mL), or IL-23 and IL-1β (10ng/mL) (n=4 mice per group). Representative of two independent experiments.

*p≤0.05, **p≤0.01, ***p≤0.001. Two-tailed t-test. All error bars represent SEM. See also Figure S2.