Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells. Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells

Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-Seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-Seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ propDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs, and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s Overall design: For transposon integration site cloning, bone marrow cells and splenocytes were isolated from the Pdzk1ip1CreERR26FlipJump mice at weeks 20 post tamoxifen induction. HSC, DC subsets (pDC, cDC1 and cDC2) and Immature B were sorted from isolated bone marrow cells and splenocytes. Splenocytes from Cx3cr1CreERYFP R26FlipJump mice were obtained on days 8 post tamoxifen induction. GFP+ YFPlo/- DC subsets (pDC, cDC1 and cDC2) were sorted. GFP+ DC subsets (pDC, cDC1 and cDC2) from Flt3L cultured Cx3cr1CreERYFPR26FlipJump mouse BM cells were sorted. For CITE-seq experiments, HSC-derived Flt3+ progenitors were isolated from the Pdzk1ip1CreERR26FlipJump mice at weeks 3, 4 or 5 following tamoxifen induction. Cx3cr1+ progenitors were isolated from Cx3cr1CreERYFP R26FlipJump at days 2 and 5 post tamoxifen induction. For bulk RNA-seq experiments, the pDC with GFP or without GFP was sorted from Cx3cr1CreERYFPR26FlipJump mouse BM cells at 8 days post tamoxifen induction.

树突状细胞（dendritic cells, DCs）包括常规树突状细胞（conventional DCs, cDCs，分为cDC1与cDC2两个亚群）以及浆细胞样树突状细胞（plasmacytoid DCs, pDCs）的发育起源迄今仍未阐明。本研究通过诱导型谱系示踪联合克隆DNA条形码技术、单细胞转录组与表型分析（CITE-Seq），探究了未经实验干预的成年小鼠体内的DC发育过程。对骨髓中Cx3cr1阳性造血祖细胞的诱导型谱系示踪结果显示，该类祖细胞可同时分化为包括pDCs、cDC1s与cDC2s在内的所有DC亚群。对造血干细胞（hematopoietic stem cells, HSCs）及Cx3cr1阳性造血祖细胞的克隆谱系示踪揭示，cDC1s与pDCs之间存在克隆共享关系，而两种cDC亚群之间、pDCs与B细胞之间则无此类克隆共享现象。据此，对分化中的HSCs与Cx3cr1阳性造血祖细胞的CITE-Seq分析鉴定出了pDC发育的渐进阶段，其中包括与淋巴样祖细胞截然不同的Cx3cr1阳性、Ly-6D阳性前体DC（propDCs）。上述结果揭示了pDCs与cDCs的共同起源，并提出了修订后的DC发育模型：pDCs与cDC1s共享克隆亲缘关系。实验设计：针对转座子整合位点克隆实验，于他莫昔芬诱导20周后从Pdzk1ip1CreERR26FlipJump小鼠中分离骨髓细胞与脾脏细胞，并从上述分离样本中分选造血干细胞（HSC）、DC亚群（pDC、cDC1与cDC2）及未成熟B细胞；于他莫昔芬诱导8天后获取Cx3cr1CreERYFP R26FlipJump小鼠的脾脏细胞，分选出GFP+ YFPlo/- DC亚群（pDC、cDC1与cDC2）；同时从经Flt3L培养的该小鼠骨髓细胞中分选GFP+ DC亚群（pDC、cDC1与cDC2）。CITE-seq实验中，于他莫昔芬诱导第3、4或5周后从Pdzk1ip1CreERR26FlipJump小鼠中分离HSC来源的Flt3+祖细胞，并于诱导第2、5天后从Cx3cr1CreERYFP R26FlipJump小鼠中分离Cx3cr1阳性造血祖细胞。批量RNA-seq实验则于他莫昔芬诱导8天后，从Cx3cr1CreERYFPR26FlipJump小鼠骨髓细胞中分选GFP阳性与阴性的pDC。