Environmental and genetic factors controlling selection of T cell repertoires

Chapter 2:• M.E.L. prepared and cultured thymic slices (making this project possible), and sorted and cultured TECs.• C.B. did the initial thymic slice qPCR experiments, performed additional qPCR experiments throughout the project, performed pilot coactivation and co-IP experiments, and contributed intellectually throughout the project in terms of planning and data interpretation.• Importantly, B.M. performed all ChIP assays and related optimization experiments.• R.S.T. performed co-IP experiments and designed the plasmids.• S.K. performed the coactivation assay and designed figure 3A.• A.I. performed bioinformatics to identify the presence of a VDRE in the Rnase1 gene in a mouseChIP-seq study performed in gut epithelial cells.• H.M. provided intellectual guidance and contributed materials throughout the project.• P.A. and J.H.W. designed, planned, and interpreted all the results.J.H.W. recognized the significance of the presence of LXXLL motifs in Aire.• P.A. performed most qPCR experiments, all imaging and flow cytometry experiments, and analysis of published scRNAseq data. Chapter 3:• L.N.Y. bred Cyp27b1 knockout mice and did all tissue harvests, including some very early mornings.• C.B. performed some qPCR experiments, assisted greatly with the setup and sample prep for scRNAseq, and contributed intellectually to experiment design and data interpretation.• H.D. provided code and expertise for initial scRNAseq analyses with Seurat.• Y.A.H. helped with imaging experiments and CCL21 image analysis.• A.I. Prepared DEGs from sequencing data.• M.E.L. contributed intellectually to experimental design.• I.R. and D.G. contributed resources for the project.• H.M. provided intellectual guidance and contributed materials throughout the project.• P.A. and J.H.W. designed, planned, and interpreted all the results.• P.A. performed most imaging and qPCR experiments, all flow cytometry experiments, sample preparation for scRNAseq, and all downstream scRNAseq analyses (RNA velocity, heatmaps, UMAPs, cell cycle analysis, etc.). Chapter 4:• C.S. and D.R. helped with Cryptococcus neoformans harvests.• P.A. and J.M. designed, planned, and interpreted all the results.• P.A. performed all the experiments. List of Abbreviations1,25D-1a,25-dihydroxyvitamin D 25D-25-hydroxyvitamin D 4SP-CD4 single positive 8SP-CD8 single positive Abs-Antibodies AIRE-Autoimmune regulator AMP-Antimicrobial peptide APC -Antigen presenting cell APECED-Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy ASL-Airway surface liquid Bcl-6-B cell lymphoma 6 BCR-B cell receptor Brd4-Bromodomain-containing protein C. neoformans-Cryptococcus neoformans CAMP-Cathelicidin antimicrobial peptide CARD-Caspase-activation and recruitment domain CCL21-Chemokine ligand 21 CCR7-Chemokine receptor 7 CD80-Cluster of differentiation 80 CD-Crohn's disease CDR3-Complementarity determining region 3 ChIP-Chromatin immunoprecipitation ChIP-seq-Chromatin immunoprecipitation sequencing CK5-Cytokeratin 5 CK8-Cytokeratin 8 CLP-Common lymphoid precursor CMJ-Cortico-medullary junction CMP-Common myeloid precursor cTEC-Cortical thymic epithelial cell CYP24A1-gene encoding vitamin D 24hydroxylase CYP27B1-gene encoding 25hydroxyvitamin D 1-hydroxylase CYP2R1-gene encoding vitamin D 25hydroxylase DAMP-Damage associated molecular pattern DBD-DNA-binding domain DBP-Vitamin D-binding protein DC-Dendritic cell DN-Double negative DP-Double positive E.coli-Escherichia coli EAE-Experimental autoimmune encephalomyelitis ETP-Early T-lineage precursors FDA-Food and Drug Administration FDC-Follicular dendritic cell Fgf21-Fibroblast growth factor 21 Fgf7-Fibroblast growth factor 7 (KGF) FoxN1-Forkhead box N1 FoxP3-Forkhead box P3 GATA-3-GATA binding protein 3 GC-Germinal center GC-Tfh-Germinal center follicular helper T H. polygyrus-Heligmosomoides polygyrus H3K4-Histone 3 lysine 4 HEK293-Human embryonic kidney 293 cells HEL-Hen egg lysozyme HIV-Human immunodeficiency virus HLA-I/II-Human leukocyte antigen I/II HSC-Hematopoietic stem cell Hsp65-Heat shock protein 65 HSV-Herpes simplex virus IBD-Inflammatory bowel disease IFN-Interferon gamma Igf1-Insulin-like growth factor 1 ILC-Innate lymphoid cell Il-Interleukin IPA-Ingenuity pathway analysis IRF-Interferon regulatory factor iTreg-induced regulatory T cell IU-International units jTEC-Junctional thymic epithelial cell KLH-Keyhole limpet hemocyanin LBD-Ligand binding domain LCMV-Lymphocytic choriomeningitis virus Lifr-Leukemia inhibitory factor receptor LIP-Lymphopenia-induced proliferation LPS-Lipopolysaccharide LT-Lymphotoxin beta LTi-Lymphoid tissue inducer LXXLL-Leucine, any AA, any AA, leucine, leucine M.Tb-Mycobacterium tuberculosis MAPK-Mitogen-activated protein kinase MEP-Erythroid/megakaryocytic precursor MFI-Mean fluorescence intensity MHC-I/II-Major histocompatibility complex I/II MPP-Multipotent progenitor MS-Multiple sclerosis mTEC-Medullary thymic epithelial cell mTOR-Mammalian target of rapamycin NF-B-Nuclear factor kappa B NHEJ-Non-homologous end joining NK-Natural killer NKT-Natural killer T NLS-Nuclear localization signal NOD-Non-obese diabetic nTreg-Natural regulatory T cell OVA-Ovalbumin P.aeruginosa-Pseudomonas aeruginosa PAMP-Pathogen associated molecular pattern PBMC-Peripheral blood mononuclear cell PD-1-Programmed death 1 pDC-Plasmacytoid dendritic cell Pdpn-Podoplanin PHD-Plant-homeodomain pMHC-peptide major histocompatibility complex PMSF-Phenylmethylsufonyl fluoride PRR-Pattern recognition receptor p-TEFb-Positive transcription elongation factor RAG-Recombination-activating gene RANK-Receptor activator of nuclear factor of kappa B RCT-Randomized controlled trial RFI-Relative fluorescence intensity RoRt-Retinoic acid receptor-related orphan receptor gamma t RSS-Recombination signal sequences RT-Room temperature RXR-Retinoid X receptor SAND-Sp100, Aire-1, NucP41/75, Deaf-1 Sca-1-Stem cells antigen-1 scRNAseq-Single cell RNA sequencing SLE-Systemic lupus erythematosus SLO-Secondary lymphoid organ SNP-Single nucleotide polymorphism SWM-Sperm whale myoglobin T1D-Type 1 diabetes TAC-TEC-Transit amplifying cells-thymic epithelial cells T-bet-T-box transcription factor 21 Tb-Tuberculosis TCR-T cell receptor TdT-Terminal deoxynucleotidyl transferase TEC-Thymic epithelial cell Tfh-Follicular helper T TGF-Transforming growth factor Th-T helper TLR-Toll-like receptor TNFα-Tumor necrosis factor alpha TRA-Tissue-restricted antigen Treg-Regulatory T cell UMAP-Uniform manifold approximation and projection UVB-Ultraviolet B VDJ-Variable, diversity, joining VDRE-Vitamin D response element VDR-Vitamin D receptor VSV-Vesicular stomatitis virus Xid-X-linked immunodeficiency Key points• The vitamin D receptor is expressed in Aire + medullary thymic epithelial cells.• Thymic vitamin D signaling stimulates Aire expression and Aire-dependent gene transcription.• Aire interacts with the Vdr and is a coactivator of Vdr-dependent transcription.

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