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Medullary epithelial cells of the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters.

Gotter J, Brors B, Hergenhahn M, Kyewski B - J. Exp. Med. (2004)

Bottom Line: Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon.Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes.These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany.

ABSTRACT
Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, and cancer-germline genes. Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes. These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression.

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Isolation of distinct stromal cells of the human thymus. (a) Sort regions selected for the isolation of thymic stromal cell subsets; the coexpression profile for CDR2 and EpCAM of low density, CD45 negative trypsin-digested cells is shown. Cortical TECs are defined as CDR2hi, EpCAMint, and mTECs as CDR2−, EpCAMhi. The corresponding histological staining patterns of both Abs are shown. Thymic DCs are defined as CD11cint/hi, HLA-DRhi, EpCAMneg cells of low density. (b) Expression of a selected panel of marker genes was assessed in purified cTECs, mTECs, and DCs of five thymi by RT-PCR. The cell type–specific expression patterns document the purity of the respective subsets. The expression of DC-LAMP in mTECs probably reflects its promiscuous expression rather than contamination by DCs. The amount of input cDNA was normalized according to signals obtained for GAPDH in a titration experiment (not depicted).
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fig1: Isolation of distinct stromal cells of the human thymus. (a) Sort regions selected for the isolation of thymic stromal cell subsets; the coexpression profile for CDR2 and EpCAM of low density, CD45 negative trypsin-digested cells is shown. Cortical TECs are defined as CDR2hi, EpCAMint, and mTECs as CDR2−, EpCAMhi. The corresponding histological staining patterns of both Abs are shown. Thymic DCs are defined as CD11cint/hi, HLA-DRhi, EpCAMneg cells of low density. (b) Expression of a selected panel of marker genes was assessed in purified cTECs, mTECs, and DCs of five thymi by RT-PCR. The cell type–specific expression patterns document the purity of the respective subsets. The expression of DC-LAMP in mTECs probably reflects its promiscuous expression rather than contamination by DCs. The amount of input cDNA was normalized according to signals obtained for GAPDH in a titration experiment (not depicted).

Mentions: A protocol previously established for the isolation of thymic stromal cells in mice has been adapted to purify the corresponding cell types from the human thymus (3). A combination of stepwise enzymatic digestion, density centrifugation, magnetic cell depletion, and multicolor sorting yielded pure populations of mature thymic DCs, cTECs, and mTECs. The differential coexpression of EpCAM and CDR2, a cTEC-specific antigen (19), allowed separation of the latter two cell subsets (Fig. 1 a). Although CDR2 is specifically expressed on cTECs and rare nonepithelial cells of the medulla, EpCAM is expressed both on mTECs and at 10-fold lower levels on cTECs. This expression pattern thus yields cTECs, i.e., CD45−, CDR2hi, EpCAMint, and mTECs, i.e., CD45−, CDR2−, EpCAMhi. Thymic DCs were isolated as HLA-DRhi, CD11c+, EpCAM− cells according to a previously published protocol (18). The cell yield per tissue volume was comparable between mouse and human. The purity of these populations was verified by PCR expression analysis of indicator genes, i.e., FOXN1 was expressed in mTECs and cTECs but not in DCs, AIRE in mTECs, and weakly in DCs and DC-LAMP in DCs and surprisingly also in mTECs (Fig. 1 b). We presume that expression of DC-LAMP in mTECs either reflects promiscuous expression or a physiological feature of mTECs rather than DC contamination. Based on these expression patterns, we regarded these populations as sufficiently pure to pursue gene expression analysis.


Medullary epithelial cells of the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters.

Gotter J, Brors B, Hergenhahn M, Kyewski B - J. Exp. Med. (2004)

Isolation of distinct stromal cells of the human thymus. (a) Sort regions selected for the isolation of thymic stromal cell subsets; the coexpression profile for CDR2 and EpCAM of low density, CD45 negative trypsin-digested cells is shown. Cortical TECs are defined as CDR2hi, EpCAMint, and mTECs as CDR2−, EpCAMhi. The corresponding histological staining patterns of both Abs are shown. Thymic DCs are defined as CD11cint/hi, HLA-DRhi, EpCAMneg cells of low density. (b) Expression of a selected panel of marker genes was assessed in purified cTECs, mTECs, and DCs of five thymi by RT-PCR. The cell type–specific expression patterns document the purity of the respective subsets. The expression of DC-LAMP in mTECs probably reflects its promiscuous expression rather than contamination by DCs. The amount of input cDNA was normalized according to signals obtained for GAPDH in a titration experiment (not depicted).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211762&req=5

fig1: Isolation of distinct stromal cells of the human thymus. (a) Sort regions selected for the isolation of thymic stromal cell subsets; the coexpression profile for CDR2 and EpCAM of low density, CD45 negative trypsin-digested cells is shown. Cortical TECs are defined as CDR2hi, EpCAMint, and mTECs as CDR2−, EpCAMhi. The corresponding histological staining patterns of both Abs are shown. Thymic DCs are defined as CD11cint/hi, HLA-DRhi, EpCAMneg cells of low density. (b) Expression of a selected panel of marker genes was assessed in purified cTECs, mTECs, and DCs of five thymi by RT-PCR. The cell type–specific expression patterns document the purity of the respective subsets. The expression of DC-LAMP in mTECs probably reflects its promiscuous expression rather than contamination by DCs. The amount of input cDNA was normalized according to signals obtained for GAPDH in a titration experiment (not depicted).
Mentions: A protocol previously established for the isolation of thymic stromal cells in mice has been adapted to purify the corresponding cell types from the human thymus (3). A combination of stepwise enzymatic digestion, density centrifugation, magnetic cell depletion, and multicolor sorting yielded pure populations of mature thymic DCs, cTECs, and mTECs. The differential coexpression of EpCAM and CDR2, a cTEC-specific antigen (19), allowed separation of the latter two cell subsets (Fig. 1 a). Although CDR2 is specifically expressed on cTECs and rare nonepithelial cells of the medulla, EpCAM is expressed both on mTECs and at 10-fold lower levels on cTECs. This expression pattern thus yields cTECs, i.e., CD45−, CDR2hi, EpCAMint, and mTECs, i.e., CD45−, CDR2−, EpCAMhi. Thymic DCs were isolated as HLA-DRhi, CD11c+, EpCAM− cells according to a previously published protocol (18). The cell yield per tissue volume was comparable between mouse and human. The purity of these populations was verified by PCR expression analysis of indicator genes, i.e., FOXN1 was expressed in mTECs and cTECs but not in DCs, AIRE in mTECs, and weakly in DCs and DC-LAMP in DCs and surprisingly also in mTECs (Fig. 1 b). We presume that expression of DC-LAMP in mTECs either reflects promiscuous expression or a physiological feature of mTECs rather than DC contamination. Based on these expression patterns, we regarded these populations as sufficiently pure to pursue gene expression analysis.

Bottom Line: Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon.Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes.These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany.

ABSTRACT
Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, and cancer-germline genes. Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes. These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression.

Show MeSH
Related in: MedlinePlus