Splenocytes of untreated or poly(I:C) treated RAG1^−/− and RAG1^−/−β2i/MECL-1^−/−β5i/LMP7^−/− (RAG1^−/−IS^−/−) mice were analyzed for the expression of H2-K^b on DCs (CD11c^hiCD11b^int). (A) Representative histogram of untreated mice showing H2-K^b expression on DCs. (B) H2-K^b mean fluorescent intensity (MFI) on DCs of individual mice. Data are representative of three independent experiments, n = 4–6 mice per group. Statistical analysis was performed using a Mann-Whitney U test. *, P<0.05.

Splenocytes of congenic CD45.1 (wt) and CD45.2 β2i/MECL-1^−/−β5i/LMP7^−/− (IS^−/−) mice were 1∶1 mixed based on total cell numbers, and transferred i.v. into untreated or anti-asialo GM1 treated CD45.1.2 recipients, that were subsequently infected i.n. with influenza virus or left uninfected. Recipient mice were sacrificed 8 days later and spleens were analyzed. (A) Representative FACS plots showing gating strategies and percentages of recovered CD45.1⁺ (wt) and CD45.2⁺ (IS^−/−) cells of CD19⁺ cells in the different groups: uninf – NK (uninfected, anti-asialo GM1 treated), uninf (uninfected, untreated), inf – NK (influenza virus infected, anti-asialo GM1 treated), inf (influenza virus infected, untreated). (B) Representative FACS plots gated on TCRβ⁻ cells showing staining for DX5 and NKp46 on splenocytes from untreated and anti-asialo GM1 treated mice. Cells in the gate are DX5⁺NKp46⁺ NK cells. (C) Ratio of CD45.2 (IS^−/−)/CD45.1 (wt) calculated by dividing absolute numbers of CD19⁺ CD45.2⁺ (IS^−/−) cells by absolute numbers of CD19⁺ CD45.1⁺ (wt) cells. Results are representative for 2 independent experiments with 5–6 mice per group. Statistical analysis was performed using a Mann-Whitney U test. **, P<0.01.

(A–C) Splenocytes of RAG1^−/− and RAG1^−/−β2i/MECL-1^−/−β5i/LMP7^−/− (RAG1^−/−IS^−/−) mice were analyzed by flow cytometry (A) Frequencies of NK cells (DX5⁺NKp46⁺) as percentage of total lymphocytes. (B,C) Expression of CD11b and CD27 on DX5⁺NKp46⁺ NK cells. (B) Representative FACS plots and (C) graph showing percentages of subsets for individual mice for the regions indicated in (B). (D–H) Splenocytes of RAG1^−/− and RAG1^−/−IS^−/− mice were incubated on plates coated with anti-NKG2D and anti-NKp46, in the presence or absence of IL-2, or left unstimulated. Frequencies of IFNγ⁺, LAMP-1⁺ NK cells and CD69 mean fluorescent intensity (MFI) on NK cells were determined by flow cytometry. (D) Representative FACS plots showing IFNγ⁺ and LAMP-1⁺ NK cells. (E–H) Bars showing the percentages of IFNγ⁺LAMP-1⁺ NK cells (E), IFNγ⁺ NK cells (F), LAMP-1⁺ NK cells (G), and CD69 MFIs on NK cells (H). Results are shown as mean ± S.E.M. Data are representative for 2 independent experiments with 6 mice per group. Statistical analysis was performed using Mann-Whitney U test. *, P<0.05.

CFSE-labeled splenocytes of untreated β2i/MECL-1−/−β5i/LMP7−/− (IS−/−; CFSElow) and of poly(I:C) treated IS−/− (CFSEhigh) mice were mixed 1∶1 and transferred i.v. into untreated or anti-asialo GM1 treated wt recipients. Recipient mice were sacrificed 17 h later and spleens were analyzed. (A) Representative FACS plots showing gating strategies. (B) Ratios of poly(I:C) treated (CFSEhigh) and untreated (CFSElow) IS−/− cells calculated by dividing absolute numbers of B220+CFSEhigh cells by absolute numbers of B220+CFSElow cells. Results represent one experiment with 6 mice per group.