Abstract Background Crohn’s disease (CD) is a chronic inflammatory bowel disorder driven in part by dysregulated immune responses to intestinal microbes. Although impaired barrier function and antimicrobial antibodies appear years before diagnosis, the immune changes preceding CD onset remain unclear. This study comprehensively profiles the peripheral immune landscape in the preclinical phase to identify immune signatures associated with CD risk. Methods In a nested case-control cohort of the GEM Project, which prospectively follows healthy first-degree relatives (FDRs) of CD patients, 80 FDRs who developed CD (pre-CD) were matched by age, sex, follow-up duration, and geographic location with FDRs who remained healthy (HMC, n = 310). PBMCs collected at recruitment were analyzed by mass cytometry (CyTOF) using 40 phenotypic and functional markers. Immune subsets were identified by automated unbiased clustering, and their associations with future CD, faecal calprotectin (FCP), serum proteomics (Olink®), and antimicrobial antibodies (Prometheus®) were assessed using conditional logistic regression, generalized estimating equations, or partial Spearman correlation as appropriate. ROC analyses evaluated performance of immune subsets to stratify future CD cases from controls. Results Among 64 unbiased immune cell clusters, 20 showed associations with future CD onset (p = 0.00003-0.05), revealing a distinct preclinical immune landscape marked by expansion of myeloid and contraction of lymphoid populations. Subgroup analysis based on median follow-up time to diagnosis revealed an early expansion of RORγt+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and CCR9+ dendritic cells more than 2.5 years prior to CD onset, preceding a marked peripheral loss of α4β7+ activated naïve-like T and B cells observed within 2.5 years before diagnosis. The early increase in these myeloid subsets correlated positively with the antimicrobial antibodies ASCA- and OmpC-IgA, suggesting a microbial trigger. Although RORγt+ PMN-MDSCs remained elevated throughout the preclinical phase, proteomic analysis revealed a shift from negative (e.g., CLEC7A, CLEC4G, CXCL12) to positive (e.g., CLEC4D, CXCL9, OSM) associations with inflammatory proteins, indicating a temporal transition from regulatory to proinflammatory phenotype approaching diagnosis. Finally, RORγt+ PMN-MDSCs showed the highest performance for predicting CD risk (AUC=0.821). Conclusion This study delineates a potential preclinical immune cascade where microbial activation drives expansion of innate myeloid cells followed by peripheral loss of adaptive lymphocytes. This highlights RORγt+ PMN-MDSCs as potential early target for CD risk stratification and possible immune intervention. Conflict of interest: Thakur, Bhupesh Kumar: No conflict of interest Bharali, Biju: No conflict of interest Turner, Dan: Consultation fee: Janssen, Pfizer, Ferring, Abbvie, Takeda, Prometheus Biosciences, Lilly, SorrisoPharma, Boehringer Ingelheim, Galapagos, BMS, AlfaSigma, Merck, Gentech Research support: Janssen, Abbvie, Takeda, Pfizer Royalties: Shaare Zedek Medical Center, Hospital for Sick Children Griffiths, Anne: No conflict of interest Panaccione, Remo: Grant: Abbvie, Janssen, Pfizer, Takeda Other: Consultant for: Abbott, AbbVie, Abbivax, Alimentiv (formerly Robarts), Amgen, AnaptysBio, Arena Pharmaceuticals, AstraZeneca, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Celltrion, Cosmos Pharmaceuticals, Eisai, Elan, Eli Lilly, Ferring, Galapagos, Fresenius Kabi, Genentech, Gilead Sciences, Glaxo-Smith Kline, JAMP Bio, Janssen, Merck, Mylan, Novartis, Oppilan Pharma, Organon, Pandion Pharma, Pendopharm, Pfizer, Progenity, Prometheus Biosciences, Protagonist Therapeutics, Roche, Sandoz, Satisfai Health, Shire, Sublimity Therapeutics, Spyre Therapeutics, Takeda Pharmaceuticals, Theravance Biopharma, Trellus, Union Biopharma, Viatris, Ventyx, UCB Speaker’s Fees for: AbbVie, Amgen, Arena Pharmaceuticals, Bristol-Myers Squibb, Celgene, Eli Lilly, Ferring, Fresenius Kabi, Gilead Sciences, Janssen, Merck, Organon, Pfizer, Roche, Sandoz, Shire, Takeda Pharmaceuticals Advisory Boards for: AbbVie, Alimentiv (formerly Robarts), Amgen, Arena Pharmaceuticals, AstraZeneca, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Eli Lilly, Ferring, Fresenius Kabi, Genentech, Gilead Sciences, Glaxo-Smith Kline, JAMP Bio, Janssen, Merck, Mylan, Novartis, Oppilan Pharma, Organon, Pandion Pharma, Pfizer, Progenity, Protagonist Therapeutics, Roche, SandozShire, Sublimity Therapeutics, Takeda Pharmaceuticals, Ventyx. Murthy, Sanjay: No conflict of interest Steinhart, Hillary: No conflict of interest Jacobson, Kevan: No conflict of interest Martin, Alberto: No conflict of interest Lee, Sun-Ho: Grant: Sun-Ho Lee is a recipient of the Imagine/ Canadian Institute of Health Research/ Canadian Association of Gastroenterology Fellowship Award. He is also a recipient of the Mount Sinai Hospital Department of Medicine Fellowship Award. Turpin, Williams: No conflict of interest Croitoru, Kenneth: No conflict of interest
Thakur et al. (Thu,) studied this question.