. 2021 Nov 4;28(11):1922-1935.e5.

doi: 10.1016/j.stem.2021.08.007. Epub 2021 Sep 15.

Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis

Semir Beyaz¹, Charlie Chung², Haiwei Mou², Khristian E Bauer-Rowe³, Michael E Xifaras⁴, Ilgin Ergin², Lenka Dohnalova⁵, Moshe Biton⁶, Karthik Shekhar⁷, Onur Eskiocak², Katherine Papciak², Kadir Ozler², Mohammad Almeqdadi³, Brian Yueh², Miriam Fein², Damodaran Annamalai⁸, Eider Valle-Encinas³, Aysegul Erdemir³, Karoline Dogum³, Vyom Shah², Aybuke Alici-Garipcan², Hannah V Meyer², Deniz M Özata⁹, Eran Elinav¹⁰, Alper Kucukural¹¹, Pawan Kumar¹², Jeremy P McAleer¹³, James G Fox⁸, Christoph A Thaiss⁵, Aviv Regev¹⁴, Jatin Roper¹⁵, Stuart H Orkin¹⁶, Ömer H Yilmaz¹⁷

Affiliations

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA; The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA. Electronic address: beyaz@cshl.edu.
² Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
³ The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA.
⁴ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA.
⁵ Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁶ Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; The Department of Biological Regulation, Weizmann Institute of Science, Rehovot 7610001, Israel.
⁷ Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemical and Biomolecular Engineering, Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
⁸ Division of Comparative Medicine, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
¹⁰ Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel.
¹¹ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
¹² Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
¹³ Department of Pharmaceutical Science and Research, Marshall University School of Pharmacy, Huntington, WV 25701, USA.
¹⁴ The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.
¹⁵ Department of Medicine, Division of Gastroenterology, Duke University, Durham, NC 27710, USA.
¹⁶ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA. Electronic address: stuart_orkin@dfci.harvard.edu.
¹⁷ The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Electronic address: ohyilmaz@mit.edu.

PMID: 34529935
PMCID: PMC8650761
DOI: 10.1016/j.stem.2021.08.007

Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis

Semir Beyaz et al. Cell Stem Cell. 2021.

. 2021 Nov 4;28(11):1922-1935.e5.

doi: 10.1016/j.stem.2021.08.007. Epub 2021 Sep 15.

Authors

Affiliations

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA; The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA. Electronic address: beyaz@cshl.edu.
² Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
³ The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA.
⁴ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA.
⁵ Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁶ Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; The Department of Biological Regulation, Weizmann Institute of Science, Rehovot 7610001, Israel.
⁷ Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemical and Biomolecular Engineering, Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
⁸ Division of Comparative Medicine, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
¹⁰ Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel.
¹¹ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
¹² Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
¹³ Department of Pharmaceutical Science and Research, Marshall University School of Pharmacy, Huntington, WV 25701, USA.
¹⁴ The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.
¹⁵ Department of Medicine, Division of Gastroenterology, Duke University, Durham, NC 27710, USA.
¹⁶ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA. Electronic address: stuart_orkin@dfci.harvard.edu.
¹⁷ The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Electronic address: ohyilmaz@mit.edu.

PMID: 34529935
PMCID: PMC8650761
DOI: 10.1016/j.stem.2021.08.007

Abstract

Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II-) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.

Keywords: MHC-II; antigen presentation; cancer; diet; high-fat diet; intestinal stem cells; microbiome; obesity.

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Conflict of interest statement

Declaration of interests The authors declare no competing financial interests. S.H.O. serves as an advisory board member for Cell Stem Cell. S.B. received research funding from Elstar Therapeutics and Revitope Oncology for research that is not related to this study.

Figures

**Figure 1 |. High Fat Diet reduces MHC-II expression on Lgr5+ intestinal stem cells (ISCs).**
A. A heat map of downregulated genes assessed by bulk RNA-seq in Lgr5-GFP^hi intestinal stem cells (Lgr5+ ISCs) isolated from long-term high fat diet (HFD)-fed mice compared to control mice (n=2). Scale represents computed z-scores of log10 expression values. B. Violin plots demonstrating MHC-II expression in single Lgr5+ ISCs isolated from control (n=171 cells, 2 independent experiments) or HFD mice by single cell RNA-seq (scRNA-seq) (n=144 cells, 2 independent experiments). C. Control (blue) and HFD (red) Lgr5+ ISCs ranked according to their expression of MHC II pathway genes (y-axis). Dashed lines correspond to y-intercepets of −0.2 and 0.75, which are the 25^th and 75^th percentile of scores in HFD cells, used to define MHC-II low (score < −0.2) and MHC-II high (score > 0.75) HFD cells. In contrast, these values correspond to 1^st and 35^th percentile of scores in control cells. D. Heatmap showing differentially expressed (DE) genes (rows) between MHC-II low and MHC-II high HFD ISCs as defined in panel C. Scale represents computed z-scores of log10 expression values. E. Single-molecule *in situ* hybridization of MHC-II (*H2-Ab1*) in control and HFD mice (n=5). F, G. Mean fluorescence intensity (MFI) of MHC-II in Epcam+ intestinal epithelial cells (IECs) from crypts of control and HFD mice (F, n=10 mice, mean ± s.e.m.). Representative flow cytometry histogram plots of MHC-II expression in Epcam+ IECs (G). H. Relative expression of MHC-II (*H2-Ab1*) in Epcam+ IECs isolated from crypts of control and HFD mice (n=10 mice, mean ± s.e.m.). I, J. Mean fluorescence intensity (MFI) of MHC-II in Lgr5+ ISCs from crypts of control and HFD mice (I, n=10 mice, mean ± s.e.m.). Representative flow cytometry histogram plots of MHC-II expression in Epcam+ cells (J). K. Relative expression of MHC-II (*H2-Ab1*) in Lgr5+ ISCs isolated from crypts of control and HFD mice (n=10 mice, mean ± s.e.m.). **L, M.** Frequency of MHC-II+ and MHC-II− Lgr5+ ISCs in control and HFD mice by flow cytometry (L, n=4, mean ± s.d). Representative flow cytometry plots of MHC-II in control and HFD ISCs (M). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 50 μm (E) and 20 μm (E, inset). See also Figure S1 and Table S1.

**Figure 2 |. Intestine-specific deletion of MHC-II does not alter the organoid forming capacity of ISCs.**
A, B. Number of bromodeoxyuridine (BrdU)+ crypt base columnar cells after a 4-hour pulse in MHC-II^L/L; Villin-CreERT2 (vil-iKO) mice after tamoxifen administration (WT: n=3, vil-iKO: n=4, mean ± s.d). Representative images of BrdU immunostain in proximal small intestinal crypts (B). C-E Organoid-initiating capacity of WT and vil-iKO crypts (C, n=5, mean ± s.d). Number of secondary organoids per dissociated crypt-derived primary organoid (D, n=5, mean ± s.d). Representative images of day-5 WT and vil-iKO primary organoids (E). F. Relative expression of MHC-II in dissociated WT and vil-iKO primary organoids at day 5 (n=5, mean ± s.d). G, H. Organoid-initiating capacity of ISCs from WT and MHC-II^L/L; Lgr5-EGFP-IRES-CreERT2 (Lgr5-iKO) mice with and without Paneth cells (P) from WT mice (n=5, mean ± s.d). Representative images of organoids derived from WT and Lgr5-iKO ISCs co-cultured with WT Paneth cells five days after seeding (H). n.s.: not significant, ***P < 0.001 (Student’s t-tests). Scale bars, 100 μm (E, H) and 20 μm (B). See also Figure S2

**Figure 3 |. Intestinal microbiome regulates epithelial MHC-II expression**
A. *In situ* hybridization for *H2-Ab1* in control, HFD, and antibiotic-treated mice in proximal small intestine (n=5 mice). B. Relative expression of MHC-II in Lgr5+ ISCs from control, HFD, and antibiotic-treated mice (n=5, mean ± s.d). C. Single-molecule *in situ* hybridization of MHC-II (*H2-Ab1*) in specific pathogen-free (SPF) and germ-free mice (n=5 mice). D. Relative expression of MHC-II (*H2-Ab1*) in Lgr5+ ISCs from SPF and germ-free mice (n=5, mean ± s.e.m.). E, F. Mean fluorescence intensity (MFI) of MHC-II in Lgr5+ ISCs from SPF and germ-free mice (E, n=5, mean ± s.e.m.). Representative flow cytometry histogram plots of MHC-II expression in Lgr5+ ISCs (F). G. Chao1 index of microbial diversity in Control, HFD, and mice treated with antibiotics for 3 months (n=5, mean ± s.d). H. Volcano plot demonstrating significantly enriched and depleted microbial species in HFD versus control mice (n=5). **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 100 μm (A, C) and 20 μm (A, C, insets). See also Figure S2

**Figure 4 |. *Helicobacter* colonization correlates with epithelial MHC-II expression**
A. Relative abundance of *Helicobacter spp.* in mice housed in clean (H−) room and dirty (H+) room (n=5 mice, mean ± s.e.m.). B. Relative expression of MHC-II (*H2-Ab1*) in Epcam+ cells isolated from crypts of mice housed in H− room or H+ room (n=7, mean ± s.e.m.). C. Principal coordinate analysis (PCoA) of microbial composition in feces of mice housed in H− room or H+ room (n=5). D. Volcano plot demonstrating significantly enriched and depleted microbial species in mice housed in H+ room versus H− room (n=5). E. Relative abundance of *Helicobacter sp.* in mice housed either in H− room (n=10), H+ room (n=3), or after co-housing H− mice (n=10) with H+ mice (n=4) in H+ room (mean ± s.e.m.). F. Relative expression of MHC-II (*H2-Ab1*) in Epcam+ cells isolated from crypts of mice housed either in H− room, H+ room, or after co-housing H− mice with H+ mice in H+ room (n=5, ANOVA). G, H. Mean fluorescence intensity (MFI) of MHC-II in Epcam+ cells isolated from crypts of mice housed either in H− room, H+ room, or after co-housing H− mice with H+ mice in H+ room (G, n=5, mean ± s.e.m., ANOVA). Representative flow cytometry histogram plots of MHC-II expression in Lgr5+ ISCs (H). I. Correlation of MHC-II expression with *Helicobacter* abundance in germ-free mice that were transplanted with fecal content from H− and H+ mice. Unless otherwise indicated *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). See also Figure S2 and Table S2.

**Figure 5 |. PRR and IFN γ signaling regulate epithelial MHC-II expression**
A. Relative expression of pattern recognition receptors (PRR) in control and HFD ISCs (n=3). B. Mean fluorescence intensity (MFI) of MHC-II in Lgr5+ ISCs from vehicle- and TLR2/NOD2 agonist CL429− treated control and HFD mice (n=6 mice, mean ± s.e.m.). C. A heat map of expression levels of IFN γ-induced genes between HFD and control Lgr5+ ISCs by bulk RNA-seq (n=2). Scale represents computed z-scores of log10 expression values. D. Violin plots demonstrating the expression levels of IFN γ-induced genes in control and HFD Lgr5+ ISCs by scRNA-seq. E. IFN γ levels in the intestines of control and HFD mice as measured by ELISA (n=10, mean ± s.e.m.). F. *In situ* hybridization for *H2-Ab1* in vehicle- and JAK1/2 & TBK1/IKKε inhibitor (CYT387)-treated mice in small intestine (n=3). G. Relative expression of MHC-II (*H2-Ab1*) in Lgr5+ ISCs from vehicle- and CYT387-treated mice (n=7, mean ± s.e.m.). H, I. Mean fluorescence intensity (MFI) of MHC-II in Lgr5+ ISCs from vehicle- and CYT387-treated mice (H, n=5, mean ± s.e.m.). Representative flow cytometry histogram plots of MHC-II expression in Lgr5+ ISCs (I). J. Relative expression of MHC-II (*H2-Ab1*) in intestinal organoids-treated with or without CYT387 and/or IFN γ (n=5, mean ± s.e.m.). K. Relative expression of MHC-II (*H2-Ab1*) in Epcam+ cells isolated from crypts of control or IFNGR1 KO (n=5, mean ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). See also Figure S3 and Figure S4

**Figure 6 |. Loss of MHC-II in premalignant ISCs increases tumor initiation.**
**A, B.** Organoid-initiating capacity of control and HFD MHC-II+ and MHC-II− *Apc*^null Lgr5+ ISCs at day 5 (control MHC-II+, n=3, control MHC-II-, n=3, HFD MHC-II+, n=4, HFD MHC-II-, n=4, mean ± s.d.). Representative images of HFD MHC-II+ and MHC-II− *Apc*^null organoids at day 5 (B). C. Tumor initiation rate of orthotopically transplanted MHC-II+ and MHC-II− *Apc*^null Lgr5+ ISCs from HFD mice into immunocompetent syngeneic hosts (n=8). D. Tumor initiation rate of orthotopically transplanted MHC-II+ and MHC-II− *Apc*^null Lgr5+ ISCs from HFD mice into immunodeficient (Rag2 KO) hosts (n=8). **E, F.** Tumor size index in distal colon of mice that received tamoxifen through endoscopy guided tamoxifen injection to induce tumor formation upon loss of APC (E, n=6, MHC-II WT APC KO: villin-Cre-ERT2 APC^L/L, MHC-II^L/+, MHC-II KO APC KO: villin-Cre-ERT2 APC^L/L, MHC-II^L/L). Representative optical colonoscopy images of tumors (F). G. Number of tumors per small intestine in Lgr5-CreERT2 MHC-II^L/+, APC^L/+ (n=9, MHC-II WT APC-het) and Lgr5-CreERT2 MHC-II^L/L, APC^L/+ (n=9, MHC-II KO APC KO) mice 5 months post tamoxifen injection. Unless otherwise indicated, data are mean ± s.e.m from n independent experiments; n.s.: not significant, *P < 0.05, **P < 0.01 (Student’s t-tests). See also Figure S5 and Figure S6

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Comment in

Microbiome effects on immune surveillance of intestinal tumours.
Minton K. Minton K. Nat Rev Immunol. 2021 Nov;21(11):692-693. doi: 10.1038/s41577-021-00629-5. Nat Rev Immunol. 2021. PMID: 34535786 No abstract available.
High fat stems tumor immune surveillance.
Caruso R, Chen GY. Caruso R, et al. Cell Rep Med. 2021 Dec 21;2(12):100483. doi: 10.1016/j.xcrm.2021.100483. eCollection 2021 Dec 21. Cell Rep Med. 2021. PMID: 35028622 Free PMC article.

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Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis

Affiliations

Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials