Fig 2: NK cell specification, maturation, and effector functions require mTORC1 activity. a Cumulative ratio and enumeration of NK cells (CD3-CD19-NK1.1+) present in BM, spleen, and peripheral lymph nodes (pLNs) from control (Rptorfl/+/Ncr1-Cre+) versus Raptor?NK (Rptorfl/fl/Ncr1-Cre+) mice. b Flow cytometric analysis and cumulative frequencies of NK cell (CD3-CD19-NK1.1+NKp46+) subsets found in the BM, spleen, and pLNs from control versus Raptor?NK mice. c Statistical quantification of IFN-?+ splenic NK cells (CD3-CD19-NK1.1+NKp46+) and the MFI of IFN-? among IFN-?+ NK cells following stimulation with PMA and ionomycin in the presence of GolgiPlug for 6 h. d Flow cytometric analysis and cumulative frequencies of CD107a+ cells among total splenic NK cells (CD3-CD19-NK1.1+NKp46+) and the indicated subpopulations thereof from control versus Raptor?NK mice after coculture with Yac-1 targets in the presence of GolgiStop for 6 h. e The cumulative ratio of RMA-S cell rejection 18 h after intraperitoneal injection of a 1:1 mixture of RMA and RMA-S cells into each mouse. f, g Flow cytometric analysis and cumulative data depicting the expression of perforin and granzyme B (f), CD98 and CD71, and uptake of 2-NBDG (g) within total splenic NK cells (CD3-CD19-NK1.1+NKp46+) and the indicated subpopulations thereof. The MFI was calculated relative to total NK cells or to the CD27SP subset of NK cells from control littermates. CD27SP, DP, and CD11bSP represent CD27+CD11b-, CD27+CD11b+, and CD27?CD11b+ NK cell subsets, respectively. Each dot represents one mouse, and the experiments were replicated 3 (c), 4 (g), 5 (a, b), 7 (d), or 9 (f) times; error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; unpaired two-tailed Student’s t test with Welsh’s correction (a–d, f, g); generalized linear models (e). Each of the shapes represents a matched pair of littermates in an individual experiment, and the horizontal red line represents the median (e)

Fig 3: ILC2s promote skeletal muscle eosinophilia(A) Immunofluorescence staining of mdx quadriceps showing IL-5 (Red5), Siglec-F, DAPI, and IL-33 expression. Nuclei were stained with DAPI.(B) Concatenated TSNE flow plots of live CD45+Thy1+ muscle cells from DTA- and DTA+ mice. Grey indicates CD45+Thy1+ cells; red indicates CD45+Thy1+Lin-KLRG1+ ILC2s.(C) Representative contour plots showing mdx muscle Eos (F4/80+CD11b+Siglec-F+). Same samples used in (B).(D and E) Quantification of flow cytometry data in (B) and (C), showing the number of ILC2s (D) and Eos (E) in vehicle (Veh)- and IL-2c/IL-33-treated (Treated) DTA- (-) and DTA+ (+) mice. n = 3–10.(F) Expression of eosinophilia-promoting factors in muscle of DTA- and DTA+ mice treated with vehicle or IL-2c/IL-33. n = 4–8.(G) Number of Eos detected by flow cytometry in the quadriceps of DTA+ mice following adoptive transfer of IL-5-/- and IL-5+/+ ILC2s. No ILC2 indicates non-injected muscle.(H–L) Expression of fibrotic factors in hamstring muscle of DTA- and DTA+ mice treated vehicle or IL-2c/IL-33. n = 4.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using an unpaired Welch’s t test (D and E) or one-way ANOVA with Bonferroni correction (F–L). All mice were analyzed at 4 weeks of age. Error bars indicate SEM.

Fig 4: Rab27-dependent transfer of miR-146a to myeloid and T cells in vivo.(A) Schematic of BM chimera and representative flow plots of CD45.2+CD3+ sorted cells from the mLN. (B) miR-146a expression within sorted mLN and cLP CD11b+ cells, as well as mLN and cLP CD4+ T cells (n = 6). One-way ANOVA with Tukey’s multiple-comparison test comparing the column means. (C) Schematic of mTmG gut explant experiment with ImageStream technology. (D) Representative images of green puncta on mTomato+CD3+CD4+ cells from CD11c-Cre mTmG mLNs. Representative images of green puncta on mTomato+ CD11b+ cells from CD11c-Cre mTmG mLNs. (E) Representative flow cytometry plots of P3/4 monocytes from F. (F) Percentage of mGFP+mTomato+ myeloid populations within the mLNs and cLP. (G) Representative flow cytometry plots of CD4+ T cells from H. (H) Percentage of mGFP+mTomato+ lymphocyte populations within the mLNs and cLP. Unpaired 2-tailed t test for each cell type in F and H. *P < 0.05, **P < 0.005, ***P < 0.0005. See also Supplemental Figure 4.

Fig 5: More durable expression of pDNA LNPs and extended transgene expression duration by co-delivery of anti-inflammatory siRNA.a, b Whole-body bioluminescence imaging of female BALB/c mice (6–8 weeks) at different time points after i.v. administration of a single dosage of LNPs containing 25 µg of Luc pDNA per mouse or 5 µg of Luc mRNA per mouse for mRNA LNPs (n = 3, 21 mice in total). A mRNA LNP with the same composition as the ONPATTRO® was used as a control. c–e Whole-body bioluminescence imaging of female BALB/c mice (6–8 weeks) at different time points post-administration (25 µg Luc pDNA per mouse, 2.5 µg siRNA for each transcription factor per mouse (n = 3, 12 mice in total). f The levels of transcription factors of treated mice were determined by ELISA at 7 days post-administration with single dosage. (female BALB/c mice (6–8 weeks), n = 3, 15 mice in total) g FACScan was used to determine the infiltrating inflammatory monocytes (CD45+CD11b+ cells) in the liver after treatments. (n = 3) Data above are presented as mean±S.E.M. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test (two-sided) for Fig. 7c, e, f and g. Statistical P-values: No significance: NS; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Without specific indications, the label above each group indicates the statistical comparison with the PBS control group. Source data are provided as a Source Data file.