scRNAseq of human blood, tonsil, lung and small intestine innate lymphoid cells

Group 1 innate lymphoid cells (ILC1s) are cytotoxic and interferon gamma-producing lymphocytes lacking antigen-specific receptors, which include ILC1s and natural killer (NK) cells. In mice, ILC1s differ from NK cells, as they develop independently of the NK-specifying transcription factor EOMES, while requiring the repressor ZFP683 (ZNF683 in humans) for tissue residency. Here we identify highly variable ILC1 subtypes across tissues through investigation of human ILC1 diversity by single-cell RNA sequencing and flow cytometry. The intestinal epithelium contained abundant mature EOMES− ILC1s expressing PRDM1 rather than ZNF683, alongside a few immature TCF7+PRDM1− ILC1s. Other tissues harbored NK cells expressing ZNF683 and EOMES transcripts; however, EOMES protein content was variable. These ZNF683+ NK cells are tissue-imprinted NK cells phenotypically resembling ILC1s. The tissue ILC1-NK spectrum also encompassed conventional NK cells and NK cells distinguished by PTGDS expression. These findings establish a foundation for evaluating phenotypic and functional changes within the NK-ILC1 spectrum in diseases. Blood samples were collected from discarded leukoreduction system (LRS) chambers from apheresis procedures. Blood lymphocytes were purified via a Ficoll density gradient centrifugation. Lineage negative cells were enriched using the EasySep™ Human Pan-ILC Enrichment Kit (StemCell Technologies). Cells were then stained for sorting or flow cytometry analysis. Tonsils were obtained from elective tonsillectomies (Children’s Hospital in Saint Louis) and provided as surgical waste with no identifiers attached. Tonsils were mechanically disrupted, passed through a 0.0059-inch opening sieve, and filtered extensively through 70-μm cell strainers. CD56+ tonsil cells were enriched via positive selection with CD56 microbeads (Miltenyi), according to the manufacturer’s recommendations. Lineage negative cells were enriched using the EasySep™ Human Pan-ILC Enrichment Kit (StemCell Technologies). Cells were then stained for sorting or flow cytometry analysis. Lung tissue was procured from organ donors and explants from transplant recipients suffering from pulmonary fibrosis. Patients provided informed consent. Lungs from healthy donors and fibrotic recipients were mechanically disrupted and digested in complete RPMI medium containing 1 mg ml–1 Collagenase IV (Sigma, C-5138) at 37 °C for 45 min under agitation. Cells were filtered through 70-μm cell strainers and stained for sorting or flow cytometry analysis. All ileum samples were provided as surgical waste with no identifiers attached on written informed consent to the Digestive Disease Research Cores Center (DDRC) at Washington University. Single-cell suspension of the mucosal tissue from the terminal ileum was separated from the muscular layer and serosa and cut into small pieces. Intraepithelial lymphocyte cells were extracted by rotating the tissue at room temperature for 40 min in Hank's balanced salt solution, 10% FCS, and 5 mM ethylenediaminetetraacetic acid (EDTA). Cells were filtered through 100-μm cell strainers, and dithiothreitol (DTT) was added at a final concentration of 5 mM. After intraepithelial lymphocyte removal, LPL cells were extracted by digesting tissue in complete RPMI medium containing 1 mg ml–1 Collagenase IV at 37 °C for 45 min under agitation. Cells were filtered and subjected to density gradient centrifugation using 40% and 70% Percoll solutions. Cells were collected, sorted, and processed for scRNA-seq or collected and stained for flow cytometry analysis. Some samples were processed and frozen for later flow cytometry analysis. Control patients for the present study were undergoing abdominal surgery for colon cancer or polyposis, which had the non-involved terminal ileum removed, as part of the surgical procedure. All human protocols were approved by the Institutional Review Board (IRB) at Washington University School of Medicine.