Tracing development of the dendritic cell lineage Dendritic cells (DCs) are important components of the immune system that form from the bone marrow into two major cell lineages: plasmacytoid DCs and conventional DCs. See et al. applied single-cell RNA sequencing and cytometry by time-of-flight to characterize the developmental pathways of these cells. They identified blood DC precursors that shared surface markers with plasmacytoid DCs but that were functionally distinct. This unsuspected level of complexity in pre-DC populations reveals additional cell types and refines understanding of known cell types. Science, this issue p. eaag3009 In human blood, the immunological dendritic cell lineage contains many predendritic cell populations. INTRODUCTION Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises multiple subsets, including plasmacytoid DC (pDC) and two functionally specialized lineages of conventional DC (cDC1 and cDC2), whose origins and differentiation pathways remain incompletely defined. RATIONALE As DC are essential regulators of the immune response in health and disease, potential intervention strategies aiming at manipulation of these cells will require in-depth insights of their origins, the mechanisms that govern their homeostasis, and their functional properties. Here, we employed two unbiased high-dimensional technologies to characterize the human DC lineage from bone marrow to blood. RESULTS We isolated the DC-containing population (Lineage−HLA−DR+CD135+ cells) from human blood and defined the transcriptomes of 710 individual cells using massively parallel single-cell mRNA sequencing. By combining complementary bioinformatic approaches, we identified a small cluster of cells within this population as putative DC precursors (pre-DC). We then confirmed this finding using cytometry by time-of-flight (CyTOF) to simultaneously measure the expression of a panel of 38 different proteins at the single-cell level on Lineage−HLA−DR+ cells and found that pre-DC possessed a CD123+CD33+CD45RA+ phenotype. We confirmed the precursor potential of pre-DC by establishing their potential to differentiate in vitro into cDC1 and cDC2, but not pDC, in the known proportions found in vivo. Interestingly, pre-DC also express classical pDC markers, including CD123, CD303, and CD304. Thus, any previous studies using these markers to identify or isolate pDC will have inadvertently included CD123+CD33+ pre-DC. We provide here new markers that can be used to identify unambiguously pre-DC from pDC, including CD33, CX3CR1, CD2, CD5, and CD327. When CD123+CD33+ pre-DC and CD123+CD33− pDC were isolated separately, we observed that pre-DC have unique functional properties that were previously attributed to pDC. Although pDC remain bona fide interferon-α–producing cells, their reported interleukin-12 (IL-12) production and CD4 T cell allostimulatory capacity can likely be attributed to “contaminating” pre-DC. We then asked whether the pre-DC population contained both uncommitted and committed pre-cDC1 and pre-cDC2 precursors, as recently shown in mice. Using microfluidic single-cell mRNA sequencing (scmRNAseq), we showed that the human pre-DC population contains cells exhibiting transcriptomic priming toward cDC1 and cDC2 lineages. Flow cytometry and in vitro DC differentiation experiments further identified CD123+CADM1−CD1c− putative uncommitted pre-DC, alongside CADM1+CD1c− pre-cDC1 and CADM1−CD1c+ pre-cDC2. Finally, we found that pre-DC subsets expressed T cell costimulatory molecules and induced comparable proliferation and polarization of naïve CD4 T cells as adult DC. However, exposure to the Toll-like receptor 9 (TLR9) ligand CpG triggered IL-12p40 and tumor necrosis factor–α production by early pre-DC, pre-cDC1, and pre-cDC2, in contrast to differentiated cDC1 and cDC2, which do not express TLR9. CONCLUSION Using unsupervised scmRNAseq and CyTOF analyses, we have unraveled the complexity of the human DC lineage at the single-cell level, revealing a continuous process of differentiation that starts in the bone marrow (BM) with common DC progenitors (CDP), diverges at the point of emergence of pre-DC and pDC potential, and culminates in maturation of both lineages in the blood and spleen. The pre-DC compartment contains functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset and two CD45RA+CD123lo lineage-committed subsets. The discovery of multiple committed pre-DC populations with unique capabilities opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting. Human DC emerge from BM CDP, diverge at the point of emergence of pre-DC and pDC potential, and culminate in maturation of both lineages in the blood. The pre-DC compartment further differentiates into functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset (early pre-DC), which give rise to both cDC1 and cDC2 through corresponding CD45RA+CD123lo pre-cDC1 and pre-cDC2 lineage-committed subsets, respectively. Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies—single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)—to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

中文翻译：

---

通过整合高维技术绘制人类 DC 谱系

追踪树突细胞谱系的发育 树突细胞 (DC) 是免疫系统的重要组成部分，从骨髓形成两个主要细胞谱系：浆细胞样 DC 和常规 DC。见等。通过飞行时间应用单细胞 RNA 测序和细胞计数来表征这些细胞的发育途径。他们鉴定了与浆细胞样 DC 共享表面标记但功能不同的血液 DC 前体。前 DC 群体中这种出人意料的复杂程度揭示了其他细胞类型并加深了对已知细胞类型的理解。科学，这个问题 p。eaag3009 在人类血液中，免疫树突细胞谱系包含许多前树突细胞群。引言 树突状细胞 (DC) 是协调免疫反应的专业抗原呈递细胞。人类 DC 群体包括多个子集，包括浆细胞样 DC (pDC) 和传统 DC 的两个功能特化谱系（cDC1 和 cDC2），其起源和分化途径仍未完全确定。基本原理 由于 DC 是健康和疾病中免疫反应的重要调节器，旨在操纵这些细胞的潜在干预策略将需要深入了解它们的起源、控制其体内平衡的机制以及它们的功能特性。在这里，我们采用了两种无偏见的高维技术来表征从骨髓到血液的人类 DC 谱系。结果我们从人血中分离出含有 DC 的群体（谱系-HLA-DR+CD135+ 细胞），并使用大规模平行单细胞 mRNA 测序确定了 710 个单个细胞的转录组。通过结合互补的生物信息学方法，我们将这一群体中的一小群细胞鉴定为假定的 DC 前体（pre-DC）。然后，我们使用飞行时间 (CyTOF) 流式细胞术证实了这一发现，同时测量了 38 种不同蛋白质在谱系-HLA-DR+ 细胞上的单细胞水平的表达，并发现 pre-DC 具有 CD123 +CD33+CD45RA+ 表型。我们通过建立它们在体外以体内发现的已知比例分化为 cDC1 和 cDC2 而不是 pDC 的潜力，证实了 pre-DC 的前体潜力。有趣的是，pre-DC 也表达经典的 pDC 标记，包括 CD123、CD303 和 CD304。因此，以前使用这些标记来识别或分离 pDC 的任何研究都将无意中包含 CD123+CD33+ pre-DC。我们在这里提供了可用于从 pDC 中明确识别前 DC 的新标记，包括 CD33、CX3CR1、CD2、CD5 和 CD327。当分别分离 CD123+CD33+ pre-DC 和 CD123+CD33− pDC 时，我们观察到 pre-DC 具有以前归因于 pDC 的独特功能特性。尽管 pDC 仍然是真正的产生干扰素-α 的细胞，但它们报道的白细胞介素 12 (IL-12) 产生和 CD4 T 细胞同种异体刺激能力可能归因于“污染”前 DC。然后，我们询问了前 DC 群体是否同时包含未提交和提交的 pre-cDC1 和 pre-cDC2 前体，如最近在小鼠中所示。使用微流体单细胞 mRNA 测序 (scmRNAseq)，我们发现人类 pre-DC 群体包含对 cDC1 和 cDC2 谱系表现出转录组学启动的细胞。流式细胞术和体外 DC 分化实验进一步确定了 CD123+CADM1−CD1c− 推定的未定型 pre-DC，以及 CADM1+CD1c− pre-cDC1 和 CADM1−CD1c+ pre-cDC2。最后，我们发现前 DC 子集表达了 T 细胞共刺激分子，并诱导了与成年 DC 相当的幼稚 CD4 T 细胞的增殖和极化。然而，与分化的 cDC1 和 cDC2 相比，暴露于 Toll 样受体 9 (TLR9​​) 配体 CpG 会触发早期 pre-DC、pre-cDC1 和 pre-cDC2 产生 IL-12p40 和肿瘤坏死因子-α。不表达TLR9。结论使用无监督的 scmRNAseq 和 CyTOF 分析，我们在单细胞水平上揭示了人类 DC 谱系的复杂性，揭示了从骨髓 (BM) 开始的连续分化过程，具有共同的 DC 祖细胞 (CDP)，在 pre-DC 和 pDC 电位出现时发生分歧，并在血液和脾脏中的两个谱系成熟时达到顶峰。前 DC 隔室包含功能和表型不同的谱系提交亚群，包括一个早期未提交的 CD123+ 前 DC 子集和两个 CD45RA+CD123lo 谱系提交子集。具有独特能力的多个承诺的前 DC 群体的发现为 DC 子集特异性靶向的治疗开发开辟了有希望的新途径。人类 DC 从 BM CDP 中出现，在 pre-DC 和 pDC 电位出现时出现分歧，并在血液中的两个谱系成熟时达到顶峰。前 DC 区室进一步分化为功能和表型不同的谱系承诺亚群，包括一个早期未提交的 CD123+ pre-DC 子集（早期 pre-DC），它们分别通过相应的 CD45RA+CD123lo pre-cDC1 和 pre-cDC2 谱系提交子集产生 cDC1 和 cDC2。树突状细胞 (DC) 是协调免疫反应的专业抗原呈递细胞。人类 DC 群体包括两个主要的功能特化谱系，其起源和分化途径仍未完全确定。在这里，我们结合了两种高维技术——单细胞信使 RNA 测序 (scmRNAseq) 和飞行时间流式细胞术 (CyTOF)——来识别人血 CD123+CD33+CD45RA+ DC 前体 (pre-DC)。Pre-DC 与类浆细胞 DC (pDC) 共享表面标记，但具有以前归因于 pDC 的不同功能特性。追踪 DC 从骨髓到外周血的分化表明，前 DC 隔室包含不同的谱系定向亚群，包括一个早期未定向的 CD123high pre-DC 亚群和两个表现出功能差异的 CD45RA+CD123low 谱系定向亚群。多个承诺的前 DC 群体的发现为 DC 子集特异性靶向的治疗开发开辟了有希望的新途径。