Fig 2: Loss of T2Ls further elevates type 3 immunity during hepatic fibrosis.(A) CCl4 schedule in Il5-tdtomato-Cre and IL-5–deleter (Il5Cre-RFP/Cre-RFP;R26RDTA/DTA) mice: 0.5 μl CCl4/g BW ip, three times per week for 2 or 4 weeks. (B and C) Flow quantification of percentages (B) and total numbers (C) of γδ T cells in livers from Il5-tdtomato-Cre and IL-5–deleter mice treated with vehicle or CCl4. Pooled data from three experiments; n ≥ 4 per group. (D to F) Flow quantification of percentages (D) and total numbers (E) of γδ T cells and percent IL-17+ DN T cells (F) in livers from Il5-tdtomato-Cre and IL-5DTR (Il5Cre-RFP/Cre-RFP;R26RDTR/DTR) mice ± CCl4. Mice received 500 ng of DT every other day. Pooled from two experiments; n ≥ 5 per group. (G to L) Representative flow plots and quantitation of IL-17 [(G) and (H)] and RORγt {[(G) and (I)]; gated on CD45+CD19−CD11b−Thy1.2+NK1.1−CD4−CD8−]} in hepatic γδ T cells and IFN-γ [(G) and (J)], IL-13 [(G) and (K)], and IL-17 {[(G) and (L)]; gated on CD45+CD19−CD11b−Thy1.2+NK1.1−CD4+]} in hepatic CD4+ T cells. n ≥ 5 per group. (M to Q) Flow quantification of percent B cells (M), macrophages (N), CD8+ T cells (O), CD4+ T cells (P), and neutrophils (Q) in livers ± CCl4. Pooled from three experiments; n ≥ 4 mice per group. (R) Total numbers of indicated cells at the steady state. n ≥ 5 per group. (S) Representative flow plots of CD9+TREM+ SAMs in livers from IL-5tdtomato-Cre mice ± CCl4 (4 weeks). (T to W) Percentages [(T) and (V)] and total numbers [(U) and (W)] of CD9+TREM+ SAMs and Ly6C+CX3CRI+ monocytes in livers ± CCl4. Pooled from two experiments; n ≥ 2 per group. (X) BDL schedule. (Y and Z) Percentages (Y) and total numbers (Z) of γδ T cells 14 days post-BDL. Pooled from three experiments; n ≥ 3 per group. Bar graphs indicate the means (±SE). Student’s t test [(B) to (F), (H) to (R), (T) to (W), (Y), and (Z)]. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Fig 3: Depletion of IL-17 reduces advanced hepatic fibrosis.(A) Schematic of IL-17; IL-5 double-deleter mice (Il17aCre; Il5Cre-RFP/Cre-RFP;R26RDTA/DTA) and IL-5 deleter controls (Il5Cre-RFP/Cre-RFP;R26RDTA/DTA). (B) CCl4 treatment schedule: 0.5 μl CCl4/g BW ip, three times per week for 4 weeks. (C and D) Sirius red staining of 5-μm liver sections (C) and quantification (D) after 4-week CCl4 treatment. Pooled from two experiments; n ≥ 4 per group. Scale bar, 200µm. (E) Hepatic hydroxyproline quantification; pooled from two experiments; n ≥ 5 per group. (F) Liver IL-6 expression normalized to 36B4 in 4-week CCl4–treated IL-5–deleter mice and double-deleter mice. n ≥ 8 per group, two experiments. (G) Flow plots showing deletion of RORγt+ cells (upper) and γδ T cells (lower) in double-deleter versus IL-5–deleter mice. (H to J) Flow quantification of neutrophils (H), RORγt cells (I), and γδ T cells (J) in livers of Il5-tdtomato-Cre, IL-5–deleter, and double-deleter mice after 4-week CCl4 treatment. Pooled from two experiments; n ≥ 3 per group. (K) GeoMx workflow. Drawn with BioRender. J. Sbierski-Kind (2025); https://biorender.com/f42j576. (L) Representative staining for Col1 (fibrotic tract), α-SMA (vessels), CD45 (leukocytes) in IL-5–deleter mice, with indicated ROIs for GeoMx. (M) Cell abundances for parenchymal, adventitial, and fibrotic tract areas estimated using SpatialDecon. (N to S) AF (525 genes) [(N) to (P)] and MF [521 genes (65)] scores [(Q) to (S)] in vehicle-treated Il5-tdtomato-Cre mice (gray), CCl4-treated Il5-tdtomato-Cre mice (white), IL-5–deleter (red), and double-deleter (purple) mice. (T to V) Volcano plots of top 10 differentially expressed genes for AF and MF scores (70): CCl4-treated Il5-tdtomato-Cre versus vehicle (T), IL-5–deleter versus Il5-tdtomato-Cre mice (U), and double-deleter versus IL-5–deleter mice (V). (W) tSNE of fibrotic tract, adventitial, and parenchymal areas under indicated conditions. (X) Graphic scheme. Created in BioRender [J. Sbierski-Kind (2025); https://biorender.com/mx6h6dl]. Bar graphs indicate the means (±SE). Student’s t test [(D) and (F)] and one-way ANOVA with Tukey post test [(E) and (H) to (J)]. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. See also fig. S7.

Fig 4: ILC2s and γδ T cells expand in different models of liver fibrosis.(A) Schematic showing CCl4 administration in IL-33mcherry/+ reporter mice, 0.5 μl CCl4/g body weight (BW) ip, three times per week for 4 weeks (4w), relevant to (B) to (N). Pooled data from three independent experiments; n ≥ 4 mice per group, unless otherwise noted. (B and C) Flow cytometry showing percentages (B) and total numbers (C) of ILC2s in livers from vehicle (corn oil)– or CCl4-treated mice. (D to F) Flow cytometry plots (D) and quantification [(E) and (F)] of hepatic IL-5+ lymphocytes gated on CD45+CD11b−CD19−NK1.1−Thy1.2+ from Il5-tdtomato-Cre mice, highlighting ILC2s (IL-5+CD3−CD4−) and TH2 cells (IL-5+CD3+CD4+). Total n ≥ 3 mice per group. ns, not significant. (G to N) Percent (G) and total γδ T cells (H), percent IL-17+ γδ T cells (I) and IL-17+ CD4+ T cells (J), percent (K) and total TH2 cells (L), percent IL-13+CD4+ T cells (M), and IFN-γ+CD4+ T cells (N) in livers from vehicle- or CCl4-treated IL-33mcherry/+ mice. (O) Schematic of BDL in IL-5+ lymphocyte lineage tracker mice (IL-5tdtomato-Cre; Rosa26RFP). (P to S) Percent (P) and total liver IL-5+ ILC2s (Q), percent (R) and total liver γδ T cells (S) 14 days post-BDL or post–sham surgery, pooled from two experiments; n ≥ 2 mice per group. (T to W) Percent IL-17+ γδ T cells (T), IL-17+ CD4+ T cells (U), IL-13+CD4+ T cells (V), and IFN-γ+CD4+ T cells (W) in livers from C57BL/6 mice 14 days post-BDL or postsham, pooled from three independent experiments; n ≥ 2 mice per group. Bar graphs indicate the means (±SE). Student’s t test [(B), (C), (G) to (N), and (P) to (W)] or one-way ANOVA with Tukey post test [(E) and (F)]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. See also fig. S2.

Fig 5: Mapping stromal cell 3D topography in liver fibrosis.(A) Confocal liver image from IL-33mcherry/+ mice stained for GS and sodium dodecyl sulfate (SDS). PV (zone 1) and CV (zone 3). n ≥ 3 mice. Scale bars, 1000 μm. (B) Confocal image from Col1a2CreERT; R26-TdT+ livers. n ≥ 3. Scale bar, 200 μm. (C) Confocal images from Gli1 lineage tracker mice. Scale bars, 500 μm. (D) Confocal images from α-SMA-creERT2; PDGFRαGFP mice. n ≥ 2. Scale bar, 300 μm. (E) Confocal images from aSMA-creERT2 mice. n ≥ 2. Scale bar, 400 μm. (F and G) Confocal images from Cthcr1-creER mice (F) and PDGFRαGFP; IL-33mcherry/+ mice (G). n ≥ 3. Scale bar, 200 μm. (H and I) Confocal images from naïve PDGFRαGFP; IL-33mcherry/+ livers (H) and PDGFRαGFP-IL-33mcherry colocalization (I). n ≥ 3. Scale bar, 200 μm. (J) Confocal image from 4-week-old CCl4-treated IL-33mcherry/+ mice. n ≥ 3. Scale bars, 1000 μm. (K, M, and N) Confocal images from 4-week-old CCl4-treated Co1a2CreERT; R26-TdT+, Gli1 lineage tracker, and α-SMA-creERT2 mice. n ≥ 3 per group. Scale bars, 400 μm. (L) Quantification of PDGFRαGFP+ cells per tissue volume in PDGFRαGFP; Il5tdtomato-Cre/+; Rosa26RFP/+ mice ± CCl4. n = 3 to 5 mice per group. (O, P, R, and T) Confocal images and quantification of Cthrc1+ MFs per tissue volume in 4-week-old CCl4-treated Cthrc1CreERT2; R26-TdT lineage tracker mice. n ≥ 3 per group. Scale bars, 50 μm (R) and 20 μm (T). (Q) CD45+ leukocytes <25 μm from Col1a2creERT2; R26-TdT lineage-traced cells. n = 4, two experiments. (S) Confocal images from 4-week-old CCl4-treated Acta2CreERT2; R26-TdT; PDGFRαGFP mice. n ≥ 2. Scale bars, 200 μm (left) and 20 μm (right). (U and V) Confocal images from PDGFRαGFP; IL-33mcherry/+ mice after 4-week CCl4 treatment (U) or 2-week BDL (V), with colocalization highlighted. n ≥ 3 per group. Scale bars, 200 μm. Bar graphs indicate the means (±SE). *P ≤ 0.05 and ****P ≤ 0.0001. Student’s t test (L and P). See also fig. S1.