Fig 2: Induced ILC2 depletion exacerbates AAA growth in ICOS-T mice. A) Scheme of DTx injection and surgical procedure. B) Aortic diameter and length, and representative images from ICOS-T mice treated with PBS or DTx to induce ILC2 depletion. Powers: 0.560 and 0.539. C) Representative FACS analysis of bone-marrow and splenic Lin-ICOS+ cells in ICOS-T mice treated with or without IL33 for 3 d to stimulate ILC2 production, and then with or without DTx injection for 5 d to verify depletion efficiency. D) Lesion Myh11-positive SMC areas. Scale: 25 µm. E) Immunofluorescent double staining of a-actin and Ki67 to detect lesion SMC proliferation. Scale: 25 µm. FACS detected F) CD11b+Siglec-F+ EOS, G) Siglec-F immunostaining detected EOS (scale: 50 µm), and H) FACS detected CD11b+Ly6Chi and CD11b+Ly6Clo monocytes in AAA lesions from ICOS-T mice with or without ILC2 depletion. Representative images for panels (B) and (D)–(H) are shown to the left. Data are mean±SEM, n = 12 and 15, *p < 0.05, **p < 0.01, nonparametric Mann–Whitney U test.

Fig 3: ILC2 accumulation in mouse CaPO4 injury-induced AAA lesions and PaLN. A) Establishment of ILC2 gating strategy with splenocytes. Splenocytes were enriched using a lineage cell depletion kit. B) Representative FACS images of ILC2 from sham and CaPO4 injury-induced AAA lesion from Il7rCre mice. C) FACS determined lesion CD45+Lin-KLRG1+CD127+ICOS+ ILC2 percentage in live CD45+ cells and ILC2 absolute number per AAA lesion. D) Representative FACS images of PaLN ILC2 from Il7rCre sham mice and Il7rCre AAA mice. E) FACS determined PaLN CD45+Lin-KLRG1+CD127+ICOS+ ILC2 percentage in live CD45+ cells and ILC2 absolute number per AAA lesion. Data are mean±SEM, n = 3–4 per group, *p < 0.05, **p < 0.01, Mann-Whitney U test.

Fig 4: The Deficiency of TCF1 and LEF1 Leads to a Severe Reduction of aTregs(A) Expression-expression plot of transcriptomes of WT Tregs isolated from Foxp3YFP-Cre/wtTcf7wt/wtLef1wt/wt mice and dKO Tregs from Foxp3YFP-Cre/wtTcf7fl/flLef1fl/fl mice. Superimposed is the bulk Treg gene signature. Numbers depict the Treg signature genes over- or under-represented in dKO versus WT samples.(B) GSEA analysis of the “hallmark” gene sets deposited in MSigDB. Both p values and FDR q values were shown. NES, normalized enrichment score.(C) Volcano plots showing the comparisons between WT and dKO Tregs. The T cell activation gene signature (red) was superimposed. The numbers of T cell activation signature genes that were over- or under-represented in each comparison and the p value (by ?2 test) were shown.(D) The proliferation of WT and dKO Tregs in Spleen, pLNs, and mLNs measured by Ki67 staining.(E) Row-normalized heatmap of the expression of bulk Treg signature genes across r-, a-, and e-Treg subtypes (left). Superimposing each sub-cluster defined on the left to the FC plot comparing dKO versus WT Tregs (right).(F) The percentages of Treg subsets in dKO and WT littermate controls using the gating strategy defined in Figure S5E.(G) Representative fluorescence-activated cell sorting (FACS) plots showing the gating strategy and the compositions of Treg pool based on the expression of Helios, ICOS, and CD62L in WT and dKO mice (top). The percentages and numbers of each Treg subsets in Helios+ Treg compartment (bottom).(H) The effect of retroviral transduction of Tcf7 on Treg differentiation in vitro. Experimental settings (top) and statistical data of Treg subsets in control (pMIG) and Tcf7-transduction groups (bottom).The data are representative of one experiment with n = 2 replicates for each Treg subsets (A–C and E) or n = 2 (F–H), n = 3 (D) independent experiments. n = 3 mice in each group. Shown are mean ± SEM. p values in (C) were calculated using the chi-square (?2) test. p values in (D) and (F)–(H) were calculated using the two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., non-significant.See also Figure S5.

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.