Transcriptional comparison of in vitro and in vivo generated human dendritic cells

Dendritic cells (DC) are antigen presenting cells controlling T cell activation. In human, the diversity, ontogeny and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DC (termed as DC3) as an immediate precursor of inflammatory CD88-CD14+CD1c+CD163+FceRI+ DC. DC3 develop via a specific pathway, independent from the cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors, and are activated by GM-CSF. As classical DC, but unlike monocytes, DC3 drove the activation of naïve T cells. In vitro, DC3 displayed a distinctive ability to prime CD8+ T cells expressing a tissue-homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor-b (TGF-ß) signaling. In vivo, DC3 infiltrated luminal breast cancer primary tumors and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Altogether, these findings define DC3 as a lineage of inflammatory DC endowed with a strong potential to regulate tumor immunity. Overall design: Blood monocytes, pDC, AS-DC, CD5+ DC2, CD5- DC2 and DC3 were isolated from healthy donor. Monocytes, DC2 and DC3 were stimulated by a TLR agonist coktail. Naive CD8+ T cells and CXCR3+CD103- and CXCR3+CD103- CD8+ T cells were sorted by flow cytometry at day 5 of in vitro coculture with blood DC3. All cell types were analyzed in triplicates with 200 to 1000 cells per subset.

树突状细胞（Dendritic cells, DC）是一类调控T细胞活化的抗原呈递细胞。在人类中，DC亚群的多样性、个体发育及功能特性尚未完全阐明。本研究鉴定出循环型CD88-CD1c+CD163+ DC（命名为DC3），其为炎性CD88-CD14+CD1c+CD163+FceRI+ DC的直接前体。DC3通过一条特异性通路发育成熟，该通路不依赖于经典DC限制性祖细胞（cDC-restricted progenitor, CDP）与单核细胞限制性祖细胞（monocyte-restricted progenitor, cMoP），且可被粒细胞-巨噬细胞集落刺激因子（Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF）激活。作为经典DC且不同于单核细胞，DC3可驱动幼稚T细胞的活化。在体外实验中，DC3展现出独特的功能能力：可通过转化生长因子-β（TGF-β）信号通路，激活表达组织归巢特征及上皮归巢α-E整合素（CD103）的CD8+ T细胞。在体内环境中，DC3可浸润腔面型乳腺癌原发肿瘤，且DC3的浸润程度与CD8+CD103+CD69+组织驻留记忆T细胞的丰度呈正相关。综上，本研究将DC3定义为一类炎性DC亚群，具备调控肿瘤免疫的强大潜力。

实验设计：从健康供者体内分离血液单核细胞、浆细胞样DC（plasmacytoid DC, pDC）、AS-DC、CD5+ DC2、CD5- DC2及DC3。对单核细胞、DC2与DC3使用Toll样受体激动剂鸡尾酒（TLR agonist cocktail）进行刺激。将幼稚CD8+ T细胞、CXCR3+CD103-及CXCR3+CD103- CD8+ T细胞在与血液DC3体外共培养第5天时，通过流式细胞术分选获得。所有细胞类型均设置三次生物学重复，每个亚群的细胞数为200至1000个。