Fig 2: Akt2 is associated with the pathology of SLE ARepresentative images of lungs and spleens from WT and Akt2 KO mice treated with TMPD (500 µl per mouse) for 2 weeks, and the corresponding H&E staining of the tissues. Bar, 200 µm.B, CqRT–PCR analysis of the relative Ifnb1 and Ifna4 mRNA expression levels in PICs (n = 5) and PBMCs (n = 4) (B), and immunoblot analysis of IRF3, LaminB1, and GAPDH in the PIC nuclear and cytoplasmic fractions (C) from WT and Akt2 KO mice treated with TMPD for 2 weeks. GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control, respectively.D, ERepresentative images of lipogranulomas in peritoneal cavity (D), H&E staining (E left panel) and immunofluorescence of IgG deposits (E right panel) in the kidney sections were collected from WT and Akt2 KO bone marrow-reconstituted CD45.1 mice 12 weeks after TMPD treatment. Bar, 50 µm.F–HQuantitative analysis of serum anti-dsDNA IgG antibody by ELISA (F, n = 4), mRNA expression levels of Ifnb1 and Ifna4 in PIC by qRT–PCR (G, n = 4) and FACS analysis of the percentages of B220+ and CD19+MHCII+ cells in PBMC (H, n = 4) from WT and Akt2 KO bone marrow-reconstituted CD45.1 mice 12 weeks after TMPD treatment.IqRT–PCR analysis of the relative mRNA expression levels of AKT2 in PBMC isolated from SLE patients (n = 9) or healthy donors (n = 9).JqRT–PCR analysis of the relative mRNA expression levels of AKT2 (left panel) and IFNA (middle panel) or IFNß1 (right panel) in CD14-positive monocytes isolated from SLE patients (n = 9) or healthy donors (n = 9).KImmunofluorescence of IRF3 (Red), AKT2 (Green), DAPI (Blue, nucleus) in CD14+ cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with ImageJ software. Cells (n = 39) were collected from 3 patients. Bar, 2 µm. Data information: *P < 0.05, **P < 0.01, and ****P < 0.0001; using unpaired t-test (B, G–I, J left panel), or two-way ANOVA test (F), or correlation analyses (J middle and right panels, K right panel). Data are pooled from different individuals (I, J, K right panel) or representative of three independent experiments (A–H, K left panel). Error bars (B, F–J, mean ± SD). Source data are available online for this figure.

Fig 3: Spatial positioning of MBCs(A–D) Confocal images of (A and D) the lung, (B) mediastinal lymph node, and (C) spleen sections from Aid-EYFP mice treated as in Figure 1A. Scale bars: left 50 µm and right 25 µm in (A), left 300 µm and right 30 µm in (B), left 250 µm and right 55 µm in (C), and 20 µm in (D). The quantification in (A) displays the minimal distance of B cell clusters to the EpCAM+ cells. Bar charts in (B) and (C) show the proportion of MBCs residing in the indicated zones. MZ, marginal zone; PALS, periarteriolar lymphoid sheaths.(E) Dot plot showing CCR6 and CXCR3 fluorescence intensity for MBCs (left). X and Y positions of MBC subsets are laid out in the B220 representation (middle). The quantification displays the distance of each MBC to the cluster’s centroid (right).(F) Lung images of wild-type and CCR6-deficient Aid-EYFP mice treated as in Figure 1A. Scale bars, 30 µm. The quantification displays the density of YFP+ B cells in B cell clusters. Dots represent individual B cell clusters.(G) Strategy to generate mixed bone marrow chimeras.(H and I) Histograms showing (H) CCR6 and (I) CXCR3 expression in the lung MBCs and non-B cells in chimeras.(J and K) Quantification of MBC numbers after 70 days of primary influenza infection in (J) CCR6- or (K) CXCR3-deficient chimeras.(L and M) Enumeration of ASCs after 4 days of re-challenge in (L) CCR6- or (M) CXCR3-deficient chimeras. The quantification of one representative experiment is shown in the bar charts; each dot represents one mouse. In all panels: mean ± SEM, t test: **p < 0.01, ns, not significant.See also Figure S3.

Fig 4: The Mincle-Syk Axis Contributes to Intestinal Barrier Function(A and B) Reg3g transcripts were measured by qPCR and normalized to Gapdh in the jejunum of the indicated genotypes during the steady state (A) or after L. plantarum gavage, as described in the scheme (B).(C) Quantification of total IgA measured by ELISA in the intestinal lumen of the indicated genotypes.(D) Frequency of IgA+ bacteria by flow cytometry in the indicated genotypes.(E and F) Representative plots and summary graphs of CD45+B220-IgA+ (E) or IgG+ (F) plasmatic cells from the small intestine LP of Mincle-deficient (Clec4e-/-) mice and WT littermate controls.(G) Representative plots and summary graph of CD4+PD-1high T cells from PPs of the indicated genotypes.Data were pooled from at least two independent experiments. Individual mice and the arithmetic mean of each group are indicated. *p < 0.05, **p < 0.01, ***p < 0.001 (A and B: one-way ANOVA and Bonferroni post hoc test; C–H: unpaired two-tailed Student’s t test). See also Figure S6.

Fig 5: Reconstitution of ILC2 or EOS reduces AAA growth in ILC2-deficient Rorafl/flIl7rCre/+ mice. A) Aortic diameter and length, and representative images of Rorafl/flIl7rCre/+ mice treated with or without WT EOS or ILC2 from WT and Il5-/- mice. Power: 1.000. B) ILC2 sorting strategy from WT splenocytes. ILC2 were defined as CD45+Lin- ICOS+CD127+KLRG1+ cells and isolated from CD45.1 transgenic mice. Donor CD45.1+ ILC2 cells were used to monitor their accumulation in Rorafl/flIl7rCre/+ mouse AAA lesions by CD45.1 immunofluorescent staining. C) FACS demonstrated the purity of bone-marrow-derived CD11b+Siglec-F+ EOS after 12 d differentiation. Donor CD45.1+ EOS were also detected in Rorafl/flIl7rCre/+ mouse AAA lesions using CD45.1 immunofluorescent staining. D) FACS analysis of blood donor CD45.1+ ILC2 or EOS in Rorafl/flIl7rCre/+ mice at 7 d after reconstitution and peri-aortic CaPO4 injury. E/F) FACS analysis of donor CD45.1+ EOS in blood and CD45.1+ ILC2 in aorta from Rorafl/flIl7rCre/+ recipient mice at different days (1, 3, 5, 7) after donor cell adoptive transfer. FACS analysis of G) CD11b+Siglec-F+ EOS, H) CD11b+Ly6Chi and CD11b+Ly6Clo monocytes, I) CD11c+MHC-II+ DCs, and J) CD11b+Ly6G+ neutrophils in AAA lesions from Rorafl/flIl7rCre/+ mice with or without receiving donor EOS or ILC2 from WT and Il5-/- mice. Data are mean±SEM, n = 10–25 per group, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test for normal distributed samples (after Shapiro–Wilk test) followed by A,F–I) LSD correction or E) nonparametric Mann–Whitney U test.