An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury

Abstract

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

Graphical abstract

Figure 1.

ATG16L1 in the intestinal epithelium protects against lethal GVHD mediated by RIPK1 and RIPK3. (A) Survival of Atg16L1^f/f and Atg16L1^ΔIEC mice receiving a chemotherapy conditioning regimen and transplanted with 5 × 10⁶ T-cell–depleted BM cells, with or without 4 × 10⁶ splenic T cells from donor LP/J mice. (B) Disease scores (see “Methods”) evaluated every 7 days after allo-HCT in (A). (C) Survival of chemotherapy-pretreated f/f Ripk3^−/− and Atg16L1^ΔIEC × Ripk3^−/− (ΔIEC Ripk3^−/−) mice transplanted with 5 × 10⁶ T-cell–depleted BM cells, with or without 4 × 10⁶ splenic T cells from donor LP/J mice. (D) Disease scores evaluated every 7 days after allo-HCT in (C). (E) Survival of chemotherapy-pretreated Atg16L1^f/f (f/f) and Atg16L1^ΔIEC (ΔIEC) mice that received GSK547 or control chow and were transplanted with 5 × 10⁶ T-cell–depleted BM cells and 4 × 10⁶ splenic T cells from donor LP/J mice. GSK547 was started 10 days before allo-HCT and continued until the end of the study. (F) Disease scores evaluated every 7 days after allo-HCT in (E). Data points in A, C, and E represent individual mice and are the combined results of 2 experiments performed independently. Data points in B, D, and F are mean disease scores of viable mice. Bars represent means ± standard error of the mean. For disease score, the area under the curve was determined for each mouse. *P < .05, ****P < .0001 analysis of variance with Tukey’s multiple-comparison test. HCT, hematopoietic cell transplantation; ns, not significant.

Figure 2.

ATG16L1 prevents intestinal GVHD by inhibiting epithelial necroptosis. (A-D) Mice receiving BM and T cells from donor LP/J mice as in Figure 1 were euthanized on day 28 after allo-HCT and analyzed for signs of intestinal GVHD (n = 11 [Atg16L1^f/f; f/f], n = 12 [Atg16L1^ΔIEC; ΔIEC], n = 8 [f/f Ripk3^−/−], and n = 8 [ΔIEC Ripk3^−/−]). (A) Colon length. (B) Pathology score of small intestine, colon, liver, and skin. Representative images (C) and quantification (D) of hematoxylin and eosin (H&E), TUNEL, and cleaved caspase 3 staining. Arrowheads indicate Paneth cells or IECs positive for the indicated markers. Scale bars, 10 µm. At least 50 crypts were quantified per mouse. Data points in A, B, and D represent individual mice. Bars represent mean ± standard error of the mean, and ≥2 independent experiments were performed. *P < .05, **P < .01, ***P < .001, ****P < .0001. SI, small intestine.

Figure 3.

Allogeneic T cells induce cell death in intestinal organoids with autophagy gene mutations. Representative images (A), viability (B), and size (C) of small intestinal organoids from B6-background Atg16L1^f/f (f/f) and Atg16L1^ΔIEC (ΔIEC) mice cocultured for 48 hours with 1 × 10⁵ splenic T cells separately harvested from B6, B10.BR, and LP/J mice. n = 3 mice each. Arrowheads indicate dead organoids. Scale bars, 400 µm. (D) Viability of organoids from B6-background Atg4B^−/− and Atg16L1^T316A mice cocultured for 48 hours with 1 × 10⁵ splenic T cells separately harvested from B10.BR mice; n = 3 mice each. (E) Viability of small intestinal organoids from f/f and ΔIEC mice cocultured for 48 hours with FACS-sorted 1 × 10⁵ CD4⁺ or 7 × 10⁴ CD8⁺ T cells from B10.BR and LP/J mice; n = 3 mice each. Representative images (F) and number of T cells associated with organoid (G). At least 50 organoids were analyzed per group. T cells were stained with CellBrite Green (green) before coculture, and propidium iodide (PI; red) was added to the culture medium at the beginning to stain dead organoids/T cells. Scale bars, 25 µm; n = 3 mice each. Data points in B, D, and E are mean of technical replicates, and data points in C and F represent individual organoids. Bars represent mean ± standard error of the mean, and ≥2 independent experiments were performed. *P < .05, **P < .01, ***P < .001, ****P < .0001. ns, not significant; WT, wild-type.

Figure 4.

Allogeneic T cells induce TNF-α–mediated necroptosis in intestinal organoids. (A) Fold change in indicated cytokines in culture supernatants from Figure 3B. Each value is normalized to nonstimulated samples; n = 3 mice each. (B) Viability of small intestinal organoids treated or not with anti–TNF-α and/or anti–IFN-γ antibody and cocultured with B10.BR T cells for 48 hours; n = 3 mice each. Representative images of cocultured small intestinal (C) and colonic (E) organoids. Arrowheads denote dead organoids. Scale bars, 100 µm. Viability of small intestinal (D) and colonic (F) organoids from B6-background Atg16L1^f/f (f/f), Atg16L1^ΔIEC (ΔIEC), f/f Ripk3^−/−, and ΔIEC Ripk3^−/− mice cocultured for 48 hours with B10.BR T cells; n = 3 mice each. Data points in A, B, D, and F are mean of technical replicates. Bars represent mean ± standard error of the mean, and ≥2 independent experiments were performed. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 5.

Loss of viability in ATG16L1-deficient intestinal organoids is associated with an IFN signature. (A) Unsupervised clustering based on expression of most variable genes by genotype and treatment with 20 ng/mL TNF-α for 2 hours. n = 4 replicates per group, each replicate was derived from separate mice. (B) Heat map of genes with a twofold change in Atg16L1^ΔIEC (ΔIEC) over Atg16L1^f/f (f/f) organoids. ISGs are highlighted with red and bold. (C) Pathway analysis of genes differentially expressed between f/f and ΔIEC naive organoids. (D) Quantitative reverse-transcription polymerase chain reaction (RT-PCR) measurement of indicated ISG expression normalized to actb in small intestinal organoids from B6 mice that were treated or not with 100 nM ruxolitinib at day 3. n = 3 mice each. (E) Viability of small intestinal organoids stimulated with 20 ng/mL TNF-α and/or 100 nM ruxolitinib for 48 hours. n = 3 mice each. (F) Western blot analysis of cell death–related proteins at day 3. f/f and ΔIEC organoids cultured with or without 100 nM ruxolitinib were treated with 20 ng/mL TNF-α for 2 hours. Blots are representative of ≥2 independent repeats. (G) Viability of small intestinal organoids stimulated with 20 ng/mL TNF-α and/or 500 μM 2-aminopurine (2-AP) for 48 hours. n = 3 mice each. Representative images (H) and viability (I) of small intestinal organoids from Atg16L1^ΔIEC mice transduced with lentiviruses encoding shRNAs targeting Eif2ak2 or a nonspecific control and treated or not with 20 ng/mL TNF-α for 48 hours; n = 3 mice each. Scale bars, 1 mm. Data points in D, E, G, and I are mean of technical replicates. Bars represent mean ± standard error of the mean, and ≥2 independent experiments were performed. **P < .01, ***P < .001, ****P < .0001.

Figure 6.

Development of an ex vivo intestinal GVHD model using human intestinal organoids and peripheral T cells. (A) Representative images of human small intestinal organoids cocultured for 8 hours with syngeneic (syn) or allogeneic (allo) human T cells. Sorted T cells were stained with CellBrite Green (green) before coculture, and PI (red) was added into the culture medium at the beginning to stain dead organoids. Scale bars, 25 µm. Viability of human small intestinal organoids from 20 patients (supplemental Table 3) at 48 hours after stimulation with 50 ng/mL TNF-α or post coculture with allogeneic and syngeneic T cells (B) or with only allogeneic T cells (C). (D) Viability of human colonic organoids from 4 patients (supplemental Table 2) at 48 hours after stimulation with 50 ng/mL TNF-α or post coculture with allogeneic and/or syngeneic T cells. At least 2 independent experiments were performed. *P < .05, **P < .01, ***P < .001, ****P < .0001. Pt., patient.

Figure 7.

Intestinal organoids derived from ATG16L1^T300Ahomozygous individuals exhibit heightened susceptibility to TNF-α and allogeneic T cells. (A) Proportion of human small intestinal organoids from Figure 6B and 6C that were susceptible (displayed >50% lethality) to recombinant TNF-α (left panel) or allogeneic T cells (right panel). n = 14 (nonrisk) and n = 6 (T300A/T300A). Statistical significance was validated with Fisher’s exact test. (B) Combined organoid viability in A; n = 14 (nonrisk) and n = 6 (T300A/T300A). Data points represent an average viability of individual organoids in Figure 6. Representative images (C) and viability (D) of human small intestinal organoids stimulated or not with 50 ng/mL TNF-α, 100 nM ruxolitinib, 1 μM GSK547, 20 μM necrostatin-1s (Nec-1s), or 2 μM necrosulfonamide (NSA) for 48 hours. Scale bars, 400 µm. Data points are mean of technical replicates. At least 2 independent experiments were performed. ***P < .001, ****P < .0001.