Transcriptome analysis of conventional dendritic cells (cDCs) introduced into the thymic microenvironment of re-aggregated thymic organ cultures (RTOCs)

cDCs home from the periphery into the thymus. In order to determine the phenotypic and functional changes introduced by the thymic microenvironment to these thymus-homing cDCs, RNA-based next generation sequencing (RNAseq) was performed with splenic (sp-DC) and thymic conventional DCs (t-DCs) re-isolated from RTOCs after two days of culture. Methods: RTOCs were generated from single-cell suspensions of thymi isolated from E14.5 to E16.5 fetuses of Foxp3hCD2 reporter mice (BALB/c background), and sp‑DCs or t‑DCs sorted from 4-8 weeks old female CD45.1xBALB/c mice were introduced. After two days of culture, 1‑2x103 cDCs were re-isolated from RTOCs as CD45.1+Lin-CD11chi by FACS, and total RNA for transcriptional profiling by low-input RNAseq was isolated. Total RNA from sp-DC and t-DCs isolated ex vivo from 4-8 weeks old female CD45.1xBALB/c mice was used as control. RNAseq was performed on an Illumina HiSeq2500 system. Differential gene expression between the four different conditions was computed from the read counts. Results: The number of differentially expressed genes between ex vivo isolated sp-DC and t-DC was drastically reduced comparing sp-DCs and t-DCs re-isolated from RTOCs. This finding implies that the thymic microenvironment within an RTOC modulates the gene expression in sp‑DCs, conferring a transcriptomic profile that more closely resembled the one from t‑DCs. Conclusion: The thymic microenvironment modulates the transcriptome of thymus-homing peripheral cDCs. RNAseq of sp-DC and t-DCs re-isolated from RTOCs two days after introduction, comparison to ex vivo isolated sp-DC and t-DC

经典树突状细胞（conventional dendritic cells, cDCs）可从外周迁移至胸腺。为探究胸腺微环境对这类胸腺归巢型cDCs的表型与功能产生的影响，研究人员对体外培养2天后从重组胸腺器官培养（RTOCs）中重新分离的脾脏来源经典树突状细胞（sp-DC）与胸腺来源经典树突状细胞（t-DCs）开展了基于RNA的下一代测序（RNAseq）。
方法：本研究以BALB/c背景的Foxp3hCD2报告小鼠的E14.5至E16.5胎鼠胸腺制备单细胞悬液，构建RTOCs，并引入从4~8周龄雌性CD45.1×BALB/c小鼠中分选得到的sp-DC或t-DC。培养2天后，通过荧光激活细胞分选（FACS）以CD45.1+Lin-CD11chi的表型从RTOCs中重新分离得到1×10³~2×10³个cDCs，并提取总RNA用于低起始量RNAseq的转录组分析。以从4~8周龄雌性CD45.1×BALB/c小鼠中离体分离的sp-DC与t-DCs的总RNA作为对照。测序实验在Illumina HiSeq2500平台上完成。基于测序读段计数计算四种不同实验组间的差异基因表达水平。
结果：离体分离的sp-DC与t-DCs之间的差异基因数量，相较于从RTOCs中重新分离的sp-DCs与t-DCs而言显著降低。该结果表明，RTOCs中的胸腺微环境可调控sp-DCs的基因表达，使其转录组特征更接近t-DCs。
结论：胸腺微环境可调控胸腺归巢型外周cDCs的转录组。对引入后2天从RTOCs中重新分离的sp-DC与t-DCs进行RNAseq，并与离体分离的sp-DC及t-DC进行比较。