A distinct human cell type expressing MHCII and RORγt 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 RORγt, 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. RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota specific Tregs. While RORγt+ 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 RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ 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 RORγt, 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. 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.

过往研究已对多种表达淋巴系转录因子RORγt的小鼠抗原呈递细胞（antigen-presenting cells, APCs）进行了特征描述，这类细胞具有独特的表型特征，参与外周调节性T细胞（regulatory T cells, Tregs）的诱导以及针对菌群与自身抗原的免疫耐受调控。此类抗原呈递细胞包含Janus细胞与Thetis细胞亚群，其中部分亚群表达自身免疫调节因子（autoimmune regulator, AIRE）。RORγt阳性MHCII阳性3型先天淋巴细胞（type 3 innate lymphoid cells, ILC3）也被证实参与菌群特异性Treg的分化调控。尽管学界已对小鼠体内的RORγt阳性APCs开展了大量研究，但人类体内此类细胞亚群的身份与功能仍不明确。本研究通过整合单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与单细胞ATAC测序（single-cell ATAC sequencing, scATAC-seq），鉴定出一类罕见的、具有树突状细胞（dendritic cell, DC）特征的RORγt阳性细胞亚群。我们将这类细胞命名为RORγt阳性DC样细胞（RORγt+ dendritic cell-like cells, R-DC-like），它们具有DC形态，表达MHC II类分子复合物，与所有已报道的DC及ILC3亚群均存在显著差异，但与DC2及ILC3在转录与表观遗传层面具有相似性。我们已建立体外分离与扩增此类细胞的实验方案，可实现其功能特征的解析。R-DC-like细胞可在体外增殖，持续表达RORγt，并可分化为CD1c阳性DC2样细胞（CD1c+ DC2-like cells），同时可刺激同种异体T细胞的增殖。人类R-DC-like细胞的发现——其具有增殖潜能且可向CD1c阳性DC2样细胞发生可塑性分化——将推动学界进一步探究其对免疫稳态、炎症与自身免疫的调控作用。本研究的样本取自择期扁桃体切除术患者的扁桃体组织，经机械解离后用于后续分析；回肠样本则取自手术废弃组织。研究采用多种富集方法从样本中分离特定细胞群。从骨髓或外周血中通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）纯化的CD34阳性细胞（CD34+ cells），在表达绿色荧光蛋白（green fluorescent protein, GFP）的OP9-Delta-like1细胞共培养体系中，分别采用两种培养条件进行15天体外培养：其一为2% FLT3L上清液、20ng/ml干细胞因子（stem cell factor, SCF）与20ng/ml粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）；其二为0.5% FLT3L上清液、20ng/ml SCF与20ng/ml白细胞介素7（interleukin-7, IL-7）。培养第15天时，依据细胞活性与CD45表达对细胞进行分选，随后进行scRNA-seq。本研究严格遵循华盛顿大学医学院机构审查委员会（Institutional Review Board, IRB）批准的人类研究伦理方案。分选后的细胞采用10X Genomics平台3'端测序试剂盒进行测序，其中肺组织样本仅使用试剂盒版本2与版本3进行测序。