A distinct human cell type expressing MHCII and ROR?t with dual characteristics of dendritic cells and type 3 innate lymphoid cells

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor ROR?t, which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the autoimmune regulator AIRE. ROR?t+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota specific Tregs. While ROR?t+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of ROR?t+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term ROR?t+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express ROR?t, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity towards CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity. Overall design: Tonsils were obtained from elective tonsillectomies, mechanically disrupted, and processed for analysis. Ileum samples were obtained from surgical waste. Different enrichment methods were used for isolating specific cell populations from the samples. FACS-sorted purified CD34+ cells from bone marrow or peripheral blood were cultured for 15 days in the presence of GFP-expressing OP9-Delta-like1 cells and either 2% of FLT3L supernatant, 20ng/ml SCF and 20ng/ml GM-CSF or 0.5% of FLT3L supernatant, 20ng/ml SCF and 20ng/ml IL-7. Cells were sorted at day 15 for viability and CD45 expression and subjected to scRNAseq. The study followed approved human protocols by the Institutional Review Board (IRB) at Washington University School of Medicine.The sorted cells were then subjected to sequencing using the 10X Genomics Platform 3' with chemistry version 2 and version 3 for lung samples only.