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

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.

Figure 1.

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 CDR2^hi, EpCAM^int, and mTECs as CDR2⁻, EpCAM^hi. The corresponding histological staining patterns of both Abs are shown. Thymic DCs are defined as CD11c^int/hi, HLA-DR^hi, EpCAM^neg 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).

Figure 2.

Thymic expression of tumor-associated antigens. Expression of selected tumor-associated antigens in thymic stromal cells of five thymi was assessed by RT-PCR. Expression in each case was confined to mTECs. Note interindividual variations and the difference in signal strength compared with control tissues. The amount of input cDNA was normalized according to signals obtained for GAPDH in a titration experiment (not depicted).

Figure 3.

Comparative global gene expression in thymic stromal cells. The relative expression levels as detected by gene arrays are depicted as scatter plots of two experiments (blue and red). (a) Signal strength of genes present or marginally present in mTECs in both comparisons and present, marginally present, or absent in cTECs. (b) Signal strength of genes present or marginally present in cTECs in both comparisons and present, marginally present, or absent in mTECs. (c) Signal strength of genes present or marginally present in mTECs in both comparisons and present, marginally present, or absent in DCs. Note the higher proportion of genes specific for mTECs (absent in cTECs) and their low expression levels. (d) Quantitative analysis of relative gene expression in mutual comparisons between cTECs, mTECs, DCs, and mature thymocytes. Numbers above bars denote the frequency of genes overexpressed in each case. The gene pool overexpressed in mTECs versus cTECs (red bar) has been analyzed in more detail. For details of calculations, see Materials and Methods. TC, thymocytes.

Figure 4.

Genomic distribution of mTECs overexpressed genes. (a) Chromosomal assignment of genes overexpressed in mTECs versus cTECs. Pooled data from a female and a male donor have been analyzed. The relative distribution of all transcripts present on the chip with known chromosomal assignment was normalized to 1 for each chromosome. The absolute number of genes overexpressed in mTECs per chromosome is indicated above the bars. The relative distribution of mTECs genes shows no particular chromosomal preferences. n.d., not determined. (b) Distribution of the number of neighbors in 10,000 random gene lists that are located on the same chromosome within a distance of 200 kb (calculated from transcription start). The median and the 95th percentile of the distribution are marked by blue and green lines, respectively. The actually observed number in the list of 415 genes overexpressed in mTECs is indicated by a red line. (c) Number of clusters of size 2, 3, 4, and 5 within a window of 10 consecutive genes. The gray bars refer to the number in the list of genes overexpressed in mTECs and the white bars to the number in 1,000 random gene lists. The SD among these 1,000 lists is indicated by the error bar. (d) Clustered expression of 10 promiscuously expressed genes in mTECs within 5 Mbp on chromosome 1. SELENBP1, selenium binding protein 1; S100 A, calcium binding protein A; FLG, filaggrin; IVL, involucrin; SPRR1B, small proline rich protein 1B; NPR1, atrial natriuretic peptide receptor A; MUC1, mucin 1.