Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: Excessive intestinal tissue injury and regeneration are linked by IFNγ and T reg cells. a BALB/c mice received 9 Gy TBI followed by allo-BMT (C57BL/6 J donors) of BM ± allogeneic T cells (low dose: 0.1 × 10 6 T cells; medium dose: 0.5 × 10 6 T cells; high dose: 2.5 × 10 6 T cells). Correlation between maximum weight loss (day 7) and peak weight recovery 2 weeks after allo-BMT. The data were pooled from 3 independent experiments. b Ex vivo small intestine (SI) organoid regeneration rate ( = number of established organoids on day 4 after starting ex vivo organoid culture divided by the number of cultured crypts, see Fig. for details) on day 7 after allo-BMT in mice co-transplanted with increasing numbers (low, medium, high) of allogeneic T cells or ( c ) increasing doses of TBI conditioning prior to allo-BMT with a medium dose of T cells. The data were pooled from 3 independent experiments. d Survival and e weight loss of individual mice after they received TBI (9 Gy) followed by allo-BMT (C57BL/6 J donors) with Ifng +/+ BM and Ifng +/+ or Ifng −/− T conv cells (C57BL/6 J donors). Pooled data from 2 independent experiments. f Intestinal FITC-dextran permeability assay two weeks (d16) after allo-BMT. Pooled data from 3 experiments. g Allo-BMT with WT BM and a low or high dose of Ifng +/+ or Ifng −/− T cells. Ex vivo SI organoid regeneration rate. Pooled data from 4 experiments. h SI intraepithelial leukocytes were isolated one week after allo-BMT and analyzed via flow cytometry after 4 h of restimulation in vitro. Frequency of FoxP3 + CD4 + donor (H-2K b+ ) T reg cells among all live CD45 + immune cells. i Allo-BMT with BM and 1 × 10 6 T cells ± ruxolitinib treatment (30 mg/kg body weight, administered orally twice daily from day –1 prior to allo-BMT until the day before analysis). The intraepithelial donor T reg cells are depicted on day 7 after allo-BMT. j Allo-BMT with BM and a low or high dose of T cells ± ruxolitinib treatment from d-1 onward (30 mg/kg body weight, administered orally twice daily). Ex vivo SI organoid regeneration rate on day 7 after allo-BMT. Pooled data from 4 independent experiments. The data of the control group, which included a low dose of T cells and a high dose of T cells, are also shown in Fig. 1g. The data in Fig. 1b, c, f–j are presented as the means ± S.E.M.s and were analyzed via unpaired two-tailed t tests or Kruskal‒Wallis tests (Fig. 1b, c, g, j) with Dunn’s multiple comparisons test for multiple comparisons. The number of biological replicates ( n ), indicating the number of mice analyzed, is shown in the figure for all individual experiments

Article Snippet: Blocking antibodies (InVivoMAb anti-mouse IL-10R (CD210), InVivoMAb anti-mouse IFNγ, InVivoMAb anti-human IFNγ, all Bioxcell) were added at a concentration of 10 μg/mL at the onset of the co-culture.

Techniques: Ex Vivo, Cell Culture, FITC-Dextran Permeability Assay, Isolation, Flow Cytometry, In Vitro, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: T reg cells adapt to intestinal epithelial tissue injury via IFNγ expression. a A published scRNA-seq dataset (GEO accession number: GSE223798 ) was used. Briefly, donor T reg cells were expanded in vitro, analyzed (input T reg cells) or supplied to recipient mice and then extracted from the respective tissues. Plots of single cells in UMAP space for all experimental conditions, colored by their origin or b colored by their IFNγ expression (library size normalized and log-transformed UMI counts). c IFNγ expression (library-size normalized and log-transformed UMI counts) in tissue and input T reg cells (Input n = 3068, Colon n = 14,862, Liver n = 10,153, Spleen n = 8606, 3 mice). Differential expression testing was performed with the Wilcoxon test, and the resulting p -values were corrected for multiple testing with the Bonferroni method. d Exemplary image of chip cytometry of intestinal biopsies of human allo-HSCT recipients. Upper image: overlay of all indicated markers in a larger area (scale bar = 50 µm). Lower images: high-resolution images of single markers and their overlay for three representative single cells (scale bar = 10 µm). e IFNγ expression (library-size normalized UMI counts) of T reg cells was analyzed via scRNA-seq of cells isolated from large intestinal biopsies of allo-HCST recipients ( n = 22 patients, with n = 208 identified T reg cells) or control patients who did not undergo allo-HSCT ( n = 5 patients with n = 34 identified T reg cells). f C57BL/6 J Ifng +/+ or Ifng −/− mice received abdominal irradiation (ABI, 5 × 4.5 Gy/day from day 0 until day 4), and body weight was monitored. Pooled data from 2 experiments. Statistical comparisons are shown for the day of weight nadir (day 6) and for days 9, 11, and 13 of the recovery phase. g Intestinal FITC-dextran permeability assay on day 7 following ABI (5 × 4.5 Gy/day). Pooled data from 2 independent experiments. h Ex vivo SI organoid regeneration rate on day 7 following ABI in Ifng +/+ or Ifng −/− mice. Pooled data from 2 experiments. i SI and large intestine (LI) intraepithelial leukocytes were analyzed by flow cytometry. The cells were isolated at different time points (7, 15, and 30 days) following ABI of the C57BL/6 J WT mice. Control mice without an ABI were pooled and are shown as day 0 after ABI. The graphs show the percentages of T reg cells among all live CD45 + immune cells. Pooled data from 7 experiments. j Graph showing the percentage of IFNγ + T reg cells among all live CD45 + intraepithelial leukocytes in C57BL/6 J WT mice on day 15 after the start of ABI, as analyzed by flow cytometry after in vitro restimulation. The data were pooled from 3 independent experiments. The data are presented as the means ± S.E.M.s and were analyzed via unpaired two-tailed t tests, Mann‒Whitney tests ( e , h ), or ordinary one-way ANOVA (with Dunn’s multiple comparisons test) for multiple comparisons. The data presented in ( g ) were analyzed via an unpaired one-tailed t test on the basis of the hypothesis of increased injury in Ifng −/− mice, as suggested by the results shown in ( f ). The number of biological replicates ( n ), indicating the number of mice analyzed, is shown in the figure for all individual experiments

Article Snippet: Blocking antibodies (InVivoMAb anti-mouse IL-10R (CD210), InVivoMAb anti-mouse IFNγ, InVivoMAb anti-human IFNγ, all Bioxcell) were added at a concentration of 10 μg/mL at the onset of the co-culture.

Techniques: Expressing, In Vitro, Transformation Assay, Quantitative Proteomics, Chip Cytometry, Isolation, Control, Irradiation, FITC-Dextran Permeability Assay, Ex Vivo, Flow Cytometry, Two Tailed Test, One-tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: T reg cell-mediated intestinal organoid growth requires epithelial IFNγ receptor signaling. a Relative organoid growth of murine SI organoids co-cultured with allogeneic and stimulated (IL-2 + beads) CD4 + CD25 - T conv cells or CD4 + CD25 + T reg cells MACS-isolated from splenocytes of WT C57BL/6 J mice (detailed description of “relative organoid growth” in the methods section and Fig. ). The dotted line represents the growth of control organoids cultured without any T cells. b Relative organoid growth of murine SI organoids co-cultured with allogeneic T conv or T reg cells isolated from Ifng +/+ or Ifng −/− mice. Relative organoid growth of murine SI organoids co-cultured with c allogeneic WT T reg cells in the presence of IFNγ blocking antibody (anti-IFNγ = α-IFNγ) (control organoids were cultured without any T cells but were also stimulated with IL-2 and α-IFNγ) d Murine SI organoids isolated from Ifngr1 +/+ or Ifngr1 −/− mice (C57BL/6 J) were co-cultured with allogeneic WT T reg cells (BALB/c) to analyze relative organoid growth and e mean size (area) after co-culture. Area of murine SI organoids on day 6 after co-culture; number of measured organoids: untreated Ifngr1 +/+ ( n = 167) and Ifngr1 −/− ( n = 163) organoids; Ifngr1 +/+ ( n = 116) and Ifngr1 −/− ( n = 113) organoids with T reg cells. f Exemplary images (converted to grayscale) of organoids after co-culture. g Relative murine SI organoid growth after co-culture with flow cytometry-sorted (CD4 + CD25 hi eGFP-Foxp3 + ) T reg cells ± α-IFNγ (left panel), and h IFNγ expression of T reg cells after removal from co-culture on day 4 and 4 h restimulation (eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors) (right panel). Violin plots ( e ) showing the distribution of values with medians and quartiles indicated. All other data are presented as the mean ± S.E.M. and were analyzed via an unpaired two-tailed t test or ordinary one-way ANOVA (with Dunn’s multiple comparisons test) for multiple comparisons. The number of biological replicates ( n ), indicating the number of separate organoid culture experiments, is shown in the figure for all individual experiments

Article Snippet: Blocking antibodies (InVivoMAb anti-mouse IL-10R (CD210), InVivoMAb anti-mouse IFNγ, InVivoMAb anti-human IFNγ, all Bioxcell) were added at a concentration of 10 μg/mL at the onset of the co-culture.

Techniques: Cell Culture, Isolation, Control, Blocking Assay, Co-Culture Assay, Flow Cytometry, Expressing, Cell Stimulation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: T reg cell-mediated IL-10 and IFNγ co-stimulation promotes murine and human intestinal organoid growth and repair from injury. a Relative growth of murine SI WT organoids co-cultured with allogeneic T reg cells isolated from Ifng +/+ or Ifng −/− mice ± stimulation with 0.25 ng/mL rIFNγ. The dotted line represents the growth of control organoids cultured without any T cells. b Allo-BMT with WT BM plus WT T reg cells and Ifng +/+ or Ifng −/− T conv cells. Intestinal FITC-dextran permeability assay 2 weeks (d16) after allo-BMT. Pooled data from 3 experiments . c Relative organoid growth of murine SI WT organoids co-cultured with allogeneic WT T reg cells ± anti-IL-10R antibody (α-IL10R) or d stimulated with ± 10 ng/mL rIL-10 and ± 0.25 ng/mL rIFNγ in the absence of T reg cells. Relative organoid growth of human patient-derived LI organoids co-cultured with e allogeneic T conv or T reg cells, f with allogeneic T reg cells ± α-IFNγ, or g stimulated ± rIL-10 ± rIFNγ. Left panel: rIFNγ 0.25 ng/mL; right panel: rIFNγ 5 ng/mL. h Relative growth of murine SI organoids after in vitro irradiation ± cytokine stimulation or i ) subsequent co-culture with syngeneic T reg cells. j Ifng +/+ or Ifng −/− mice received ABI (5 × 4.5 Gy/day from day 0 until day 4), and body weight was monitored (treatment schedule is depicted in Fig. ; α-IL10-R injections on day-1, day 3, 7, 10, and 14). Pooled data from 2 experiments. k WT mice received ABI (5×4.5 Gy/day from day 0 until day 4) ± α-IFNγ ± α-IL-10R at the indicated time points (the treatment schedule is depicted in Fig. ; group day 1: injections on days 1, 3, 6, 9, 13, and 16; group day 6: injections on days 6, 9, 13, and 16). Pooled data from 2 experiments. Body weight during regeneration (day 11) was analyzed via ordinary one-way ANOVA plus Fisher’s LSD. The data were analyzed via two-tailed or one-tailed methods ( i , on the basis of the hypothesis of improved T reg -mediated regeneration, as suggested by the results shown in Fig. , ( d , h ). Unpaired t-test or ordinary one-way ANOVA for multiple comparisons (with Dunn’s multiple comparisons test) and are presented as the means ± S.E.M. The number of biological replicates ( n ), indicating the number of separate organoid culture experiments or the number of mice analyzed, is shown in the figure for all individual experiments

Article Snippet: Blocking antibodies (InVivoMAb anti-mouse IL-10R (CD210), InVivoMAb anti-mouse IFNγ, InVivoMAb anti-human IFNγ, all Bioxcell) were added at a concentration of 10 μg/mL at the onset of the co-culture.

Techniques: Cell Culture, Isolation, Control, FITC-Dextran Permeability Assay, Derivative Assay, In Vitro, Irradiation, Co-Culture Assay, Two Tailed Test, One-tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: T reg cells promote organoid growth via mTORC1 and Myc activation in ISCs. Murine SI organoids were co-cultured with CD25 high FoxP3-GFP + T reg cells alone or in the presence of IFNγ and IL-10R blocking antibodies, or were stimulated with rIFNγ + rIL-10 for 4 days, and subsequently analyzed via scRNA-Seq. The data were pooled from 3 independent experiments. a Plots of single cells in UMAP space for all experimental conditions, colored by SingleR cell type annotation. b Venn diagrams indicating the overlap of upregulated pathways between experimental conditions and control organoids. Only gene sets/pathways significantly upregulated when controlling for an FDR of 10% were considered. c Heatmap of Pearson correlation coefficients obtained from correlating GSEA-derived NESs (normalized enrichment scores) between conditions. The values indicate correlations of regulated gene sets/pathway activation between different conditions in the indicated cell types. Only gene sets/pathways significantly up- or downregulated when controlling for an FDR of 10% were considered. d Dot plot of the GSEA results of selected pathways/gene sets for different cell types and treatments (vs. control). Dots are colored according to the negative log 10 of the GSEA q value (FDR), and the sign indicates the direction of the regulation (up positive, down negative). The size of the dots corresponds to the GSEA NES. Gene sets/pathways are derived from the Hallmark (H) and Reactome (R) gene set collections of MSigDB. e Relative organoid growth of SI WT organoids ± stimulation with 0.25 ng/mL rIFNγ and rIL-10 and ± the mTOR inhibitor rapamycin (1 µg/mL) or control (DMSO), f ± myc inhibition (compound 10058-F4, 100 µM/mL) or control (DMSO), and g ± co-cultured T reg cells ± myc inhibition ± mTOR inhibitor. The dotted line represents the growth of control organoids without stimulation. The number of biological replicates ( n ), indicating the number of separate organoid culture experiments, is shown in the figure. The data were analyzed via ordinary one-way ANOVA for multiple comparisons and are presented as the means ± S.E.M.s

Article Snippet: Blocking antibodies (InVivoMAb anti-mouse IL-10R (CD210), InVivoMAb anti-mouse IFNγ, InVivoMAb anti-human IFNγ, all Bioxcell) were added at a concentration of 10 μg/mL at the onset of the co-culture.

Techniques: Activation Assay, Cell Culture, Blocking Assay, Control, Derivative Assay, Inhibition

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: IFNγ and IL-10 compensate for the depletion of epithelial growth factors. Murine SI organoids were cultured under normal growth conditions (ENR) or EGF-depleted conditions (NR + anti-EGF antibody) and stimulated with the indicated cytokines. a Representative images, b quantification of organoid size on day 6 of culture, c relative organoid growth (number of organoids after first passage, compared with control conditions) ( n = 4 independent experiments), and d relative organoid growth during long-term culture and several passages ( n = 3 independent experiments). e Relative organoid growth of murine SI organoids as described above ± mTOR or myc inhibitors ( n = 4 independent experiments). f Human LI organoids were cultured under optimal (WENR) or EGF-depleted (WNR) conditions and stimulated with the indicated cytokines ± mTOR or myc inhibitors. The area of viable organoids per image (used as a surrogate marker for organoid size) was determined on day 6, and g ) the relative organoid growth (number of viable organoids) was determined after the first passage ( n = 4 independent experiments). h Murine SI organoids were stimulated for 16 h with the indicated cytokines (representative plots). i The cell cycle phase was analyzed to distinguish proliferating (G1/2/S/M phase) and non-proliferating (G0 phase) cells ( n = 6 independent experiments). The proportion of proliferating cells was statistically analyzed. j Murine SI organoids were stimulated for 5 days with the indicated cytokines, and the abundance of Lrg5+ ISCs among all viable epithelial cells (EpCAM + ) and k the number of proliferating (EdU + ) cells among all viable epithelial cells were determined via flow cytometry ( n = 5 independent experiments). l Human LI organoids were subjected to Wnt-depleted conditions (ENR) and stimulated with the indicated cytokines or Wnt. Organoid size on day 6 of culture ( n = 7 independent experiments). m Human LI organoids were cultured under Wnt-depleted conditions and stimulated with the indicated cytokines or Wnt. The number of viable organoids was determined on day 6 of culture ( n = 4 independent experiments). n Murine SI organoids were cultured and mechanically disrupted. One hundred organoids were seeded into culture and stimulated with the indicated cytokines immediately ( < 5 min) or after 90 min. The number of viable organoids was determined on day 6 of culture ( n = 6–15 engraftment culture wells from 3–6 independent experiments). o Analysis of Ifng +/+ and Ifng −/− mice on day 7 after starting ABI (5 × 4.5 Gy/day from day 0 until day 4). The number of Ki-67 + epithelial cells within SI epithelial crypt cells was quantified, and p representative immunohistochemistry images are shown. Data from 2 independent experiments with n = 9 Ifng +/+ mice and n = 9 Ifng −/− mice were pooled, and a total of 131 crypts were analyzed. q The number of Lgr5 + (Lgr5-GFP + ) ISCs within small intestinal epithelial crypts was quantified, and r representative in situ hybridization images are shown. Data from 2 independent experiments with n = 7 Ifng +/+ mice and n = 9 Ifng −/− mice were pooled, and a total of 993 crypts were analyzed. Violin plots ( o , q ) showing the distribution of values, with medians (solid lines) and quartiles (dotted lines) indicated. All the other data are presented as the means ± S.E.M. p values were calculated via two-tailed t tests or ordinary one-way ANOVA for multiple comparisons

Article Snippet: Blocking antibodies (InVivoMAb anti-mouse IL-10R (CD210), InVivoMAb anti-mouse IFNγ, InVivoMAb anti-human IFNγ, all Bioxcell) were added at a concentration of 10 μg/mL at the onset of the co-culture.

Techniques: Cell Culture, Control, Marker, Flow Cytometry, Immunohistochemistry, In Situ Hybridization, Two Tailed Test