BACKGROUND AND AIMS: Innate immune cells, including dendritic cells (DCs), monocytes (Mono), macrophages (Mac), natural killer (NK), and innate lymphoid cells (ILC), contribute to chronic inflammation in lymphoid tissues. Here, we characterized the innate immune cell landscape in inflamed mesenteric lymph nodes (MLNs) of patients with inflammatory bowel diseases (IBD) at the single-cell level. METHODS: Surgically resected colonic MLNs were obtained from patients with Crohn's disease (CD
n = 3), ulcerative colitis (UC
n = 3), non-inflamed UC (n = 1), and non-IBD (n = 2). CD45+CD3-CD19- non-T/non-B cells were FACS-sorted to capture rare innate immune cells. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) was performed on the BD Rhapsody platform alongside multiparameter flow cytometry staining. RESULTS: CITE-seq analysis unveiled the molecular signature of 11 Mono/Mac/DC (MMDC) and 7 NK/ILC enriched clusters in human MLNs. DC clusters included 3 newly characterized DC clusters such as CD1c/CD163/VCAN/CD64-expressing DC3
AXL-expressing DCs
and a CD103+ DC subset, expressing LTB, S100B, and IL22RA2 (encoding IL22BP). Mono/Mac clusters comprised inflammatory monocytes, which accumulated in IBD compared to non-IBD MLNs. Among NK/ILC clusters, we identified a cytotoxic ILC subset (IL7R, KLRD1, GNLY), previously not reported in MLNs, reminiscent of cytotoxic ILC1-like cells found in inflamed gut mucosa. CONCLUSION: CITE-seq and flow-cytometry analyses of colonic MLNs from patients with active IBD reveal the molecular signature and cell distribution of previously uncharacterized DC and ILC subpopulations in human MLNs. These findings expand our understanding of immune responses during chronic inflammation in IBD.