Toll-like receptor signaling in thymic epithelium controls monocyte-derived dendritic cell recruitment and Treg generation

The development of thymic regulatory T cells (Treg) is mediated by Aire-regulated self-antigen presentation on medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), but the cooperation between these cells is still poorly understood. Here we show that signaling through Toll-like receptors (TLR) expressed on mTECs regulates the production of specific chemokines and other genes associated with post-Aire mTEC development. Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirpα+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines. Consistently, the cellularity of CD14+moDC is diminished in mice with MyD88-deficient TECs, in which the frequency and functionality of thymic CD25+Foxp3+ Tregs are decreased, leading to aggravated mouse experimental colitis. Thus, our findings describe a TLR-dependent function of mTECs for the recruitment of CD14+moDC, the generation of Tregs, and thereby the establishment of central tolerance.


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Sample size
No statistical methods were used to predetermine sample size. Sample size were not calculated prior to performing experiment. To minimized the number of mice models used, sample size were determined to be sufficient to detect differences between groups. All comparisons had minimum of 3 data points. Non of the data points used was generated as technical replicate.
Data exclusions The only data excluded from the manuscript is from the RNAseq and single-cell RNAseq experiments. In the case of RNAseq, genes that were not expressed in at least two samples were discarded. Cells in single-cell RNAseq data sets were excluded according to the quality control used in Seurat R Package. The exclusion criteria were pre-established by Seurat R Package.

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All data used in this study were replicated (minum 2 independent experiments were used). All attempts for replication were successful.
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Investigators were not blinded as non-subjective measures were used (i.e. frequencies, total numbers of cell populations, weights,..). Bioinformatics were blinded for RNAseq and single-cell RNAseq library preparation and analysis. Blinded investigators were used for counting the dendritic cells in medullary and cortical regions of the thymus in microscopic slices.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Most of the antibodies used are commercially available, and were validated by the manufacturer. Antibodies were tested in the laboratory using on known positive and negative controls and titrated. The Anti-mouse CD45RB-Biotin was generated in house. The function and specificity of this antibody was tested using positive and negative controls. FMO or a particular Isotype control staining was used to determine the staining positivity. Hrádek, Czech Republic. Both GF and SPF mice were subject to the SSNIFF V1124-300 diet. Thymic cell populations were isolated from 3-6 week old mice with the exception of newborn mice (4 days old) used in Supplementary Fig. 7b. For the purpose of BM chimera experiments, 5-6 week old mice were irradiated and analysed between 11-13 weeks of age. Comparative analysis used age-matched cohorts regardless of sex and caging. Where possible, littermates were used as the controls. For the purpose of tissue isolation, mice were euthanized by cervical dislocation.

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Methodology Sample preparation
Thymic antigen presenting cells, TECs and DCs, were isolated as follows. Thymus was minced into small pieces and treated with Dispase II (Gibco), dissolved in RPMI at concentration 0.1 mg.ml-1. Tissue was homogenized by pipetting and after 10 minutes of incubation (37°C), the supernatant was collected and the reaction was stopped by adding 3% FSC and 2nM EDTA. The process was repeated until all thymic fragments were digested. For thymic epithelial cells isolation, the whole thymic cell suspension was depleted of CD45+ cells by CD45 microbeads staining (Miltenyi biotec). Thymic dendritic cells were isolated using MACS enrichment for CD11c+ cells through staining with biotinylated CD11c antibody, followed by Ultrapure Anti-Biotin microbeads staining (Miltenyi biotec). For isolation of T-cell, thymus, peripheral lymph nodes (pLN), mesenteric lymph nodes (mLN) or spleen were mechanically mashed through 40μm Cell strainer (Biologix) and cell suspensions were passed through 50μm filters (Sysmex). The resulting cell suspension was spun down (4°C, 400g, 10 minutes) and erythrocytes were removed using ACK lysis buffer.