Concerted changes in the pediatric single-cell intestinal ecosystem before and after anti-TNF blockade

Crohn’s disease is an inflammatory bowel disease (IBD) commonly treated through anti-TNF blockade. However, most patients still relapse and inevitably progress. Comprehensive single-cell RNA-sequencing (scRNA-seq) atlases have largely sampled patients with established treatment-refractory IBD, limiting our understanding of which cell types, subsets, and states at diagnosis anticipate disease severity and response to treatment. Here, through combining clinical, flow cytometry, histology, and scRNA-seq methods, we profile diagnostic human biopsies from the terminal ileum of treatment-naïve pediatric patients with Crohn’s disease (pediCD; n=14), matched repeat biopsies (pediCD-treated; n=8) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGID; n=13). To resolve and annotate epithelial, stromal, and immune cell states among the 201,883 baseline single-cell transcriptomes, we develop a principled and unbiased tiered clustering approach, ARBOL. Through flow cytometry and scRNA-seq, we observe that treatment-naïve pediCD and FGID have similar broad cell type composition. However, through high-resolution scRNA-seq analysis and microscopy, we identify significant differences in cell subsets and states that arise during pediCD relative to FGID. By closely linking our scRNA-seq analysis with clinical meta-data, we resolve a vector of T cell, innate lymphocyte, myeloid, and epithelial cell states in treatment-naïve pediCD (pediCD-TIME) samples which can distinguish patients along the trajectory of disease severity and anti-TNF response. By using ARBOL with integration, we position repeat on-treatment biopsies from our patients between treatment-naïve pediCD and on-treatment adult CD. We identify that anti-TNF treatment pushes the pediatric cellular ecosystem towards an adult, more treatment-refractory state. Our study jointly leverages a treatment-naïve cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict Crohn’s disease trajectory. ### Competing Interest Statement J.O.-M. reports compensation for consulting services with Cellarity and Tessel Biosciences. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Third Rock Ventures, Ochre Bio, FL82, Senda Biosciences, Relation Therapeutics, Empress Therapeutics, IntrECate Biotherapeutics, and Dahlia Biosciences unrelated to this work. A.K.S. has received research support from Merck, Novartis, Leo Pharma, Janssen, the Bill and Melinda Gates Foundation, the Moore Foundation, the Pew-Stewart Trust, Foundation MIT, the Chan Zuckerberg Initiative, Novo Nordisk and the FDA unrelated to this work. Dr. Kean is on the scientific advisory board for HiFiBio and Mammoth Biosciences. She reports research funding from Kymab Limited, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals. She reports consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gilead Sciences, Vertex Pharmaceuticals, and Takeda Pharmaceuticals. Dr. Kean reports grants and personal fees from Bristol Myers Squibb that are managed under an agreement with Harvard Medical School. N.F., B.K., S.C., S.J., Y.Y., M.D., M.F.W., S.H., G.D.K., A.H., W.K.L., S.H., Y.W. are employees and shareholders of Regeneron Pharmaceuticals, Inc. L.A. is a consultant for Takeda Pharmaceuticals. G.W. reports Research funding from Abbvie, Jansen, Takeda, Allakos; SAB for Abbvie, Bristol Myers Squibb; DSMB for Abbvie. D.L.S., is the co-founder, CM, president of NiMBAL Health. S.B.S. declares the following interests: Scientific advisory board participation for Pfizer, Lilly, IFM therapeutics, Merck, Pandion, and Takeda Inc., and grant support from Pfizer, Novartis, Amgen, Takeda Consulting for Hoffman La Roche and Amgen. A.K.S., L.S.K., J.O.-M., H.B.Z., K.K. and B.A.D., are co-inventors on a provisional patent application relating to methods of stratifying and treating IBD. ### Clinical Trial NCT03369353 ### Funding Statement J.O.-M was supported by the Richard and Susan Smith Family Foundation, the AGA Research Foundation AGA-Takeda Pharmaceuticals Research Scholar Award in IBD AGA2020-13-01, the HDDC Pilot and Feasibility P30 DK034854, the Food Allergy Science Initiative, the Leona M. and Harry B. Helmsley Charitable Trust, The Pew Charitable Trusts Biomedical Scholars, The Broad NextGen Award, The Mathers Foundation, The Manton Foundation, and The New York Stem Cell Foundation. L.S.K is supported by NIH P01 1P01HL158504, R01 5R01HL095791, U19 U19AI051731, and by the Helmsley Charitable Trust. V.N. was supported by the International mobility of research, technical and administrative staff of research organizations (CZ.02.2.69/0.0/0.0/18_053/0016981). A.K.S. was supported, in part, by the Searle Scholars Program, the Beckman Young Investigator Program, a Sloan Fellowship in Chemistry, and the NIH (5U24AI118672, 2R01HL095791). V.M. reports research support from Novartis. V.T. Is supported by ASTCT New Investigator Award and CIBMTR/Be the Match Foundation Amy Strelzer Manasevit Research Program Award. S.B.S. is supported by NIH grants P30 DK034854 and RC2 DK122532, and the Helmsley Charitable Trust. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Pediatric patients less than 20 years of age with suspected inflammatory bowel disease were enrolled on the PREDICT Study (ClinicalTrials.gov# [NCT03369353][1]). Enrollment took place between November 9, 2017 to December 21, 2018 in accordance with the Fred Hutch Institutional Review Board (Protocol #9730, ethical approval given) with written informed consent and assent when applicable. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data and Code Availability Single-cell RNA-seq data can be visualized via the Single Cell Portal links for each atlas of FGID https://singlecell.broadinstitute.org/single\_cell/study/SCP1422/predict-2021-paper-fgid and pediCD https://singlecell.broadinstitute.org/single\_cell/study/SCP1423/predict-2021-paper-cd. The cell-by-gene matrices will be available with the peer-reviewed version of this manuscript. The raw human FASTQ files will be available from the Broad controlled access repository DUOS with the peer-reviewed version of this manuscript. Please contact the authors for further information. All original code has been assembled as the ARBOL package and deposited at GitHub and is publicly available together with this manuscript for both R and Python versions at: https://github.com/jo-m-lab/ARBOL and https://github.com/jo-m-lab/ARBOLpy and https://jo-m-lab.github.io/ARBOL/ARBOLtutorial.html. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03369353&atom=%2Fmedrxiv%2Fearly%2F2023%2F05%2F11%2F2021.09.17.21263540.atom