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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-naive pediatric patients with Crohn\u2019s disease (pediCD;\n                    <jats:italic>n<\/jats:italic>\n                    = 14), matched repeat biopsies (pediCD-treated;\n                    <jats:italic>n<\/jats:italic>\n                    = 8) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGIDs;\n                    <jats:italic>n<\/jats:italic>\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-naive 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-naive 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-naive pediCD and on-treatment adult CD. We identify that anti-TNF treatment pushes the pediatric cellular ecosystem toward an adult, more treatment-refractory state. Our study jointly leverages a treatment-naive cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict Crohn\u2019s disease trajectory.\n                  <\/jats:p>","DOI":"10.7554\/elife.91792","type":"journal-article","created":{"date-parts":[[2023,11,23]],"date-time":"2023-11-23T10:31:20Z","timestamp":1700735480000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.91792","source":"Crossref","is-referenced-by-count":3,"title":["Concerted changes in the pediatric single-cell intestinal ecosystem before and after anti-TNF blockade"],"prefix":"10.7554","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0575-6475","authenticated-orcid":true,"given":"Hengqi Betty","family":"Zheng","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00cvxb145","id-type":"ROR","asserted-by":"publisher"}],"name":"Division of Gastroenterology and Hepatology, Seattle Children's Hospital and University 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