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. Overall design: 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