Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance

Abstract

The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire(+) (i.e., GFP(+)) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire(+) and Aire(-) mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.

Figure 1.

Establishment of Aire/GFP knock-in mice. (A) Targeted insertion of the GFP gene into the Aire gene locus by homologous recombination. SspI, SspI restriction site. (B) Southern blot analysis of genomic DNA from offspring of Aire/GFP knock-in mice. Tail DNA was digested with SspI and hybridized with the 3′ probe shown in A. (C) Concomitant expression of GFP (green) and endogenous mouse Aire (red) assessed by immunohistochemistry of a thymus section from an Aire^+/gfp mouse. Cells positive for Aire staining but negative for GFP expression are marked with arrows. Bar, 20 μm. One representative experiment from a total of four repeats is shown.

Figure 2.

Altered morphology and distribution of mTECs committed to express Aire in the absence of functional Aire protein. (A and B) mTECs active in Aire gene transcription were visualized by immunohistochemistry with anti-GFP Ab (green). The medullary region was identified by staining with UEA-1 (A) or anti-EpCAM mAb (B; red). Bars, 100 μm. One representative experiment from a total of five repeats is shown. (C) Enlargement of the staining with anti-GFP Ab from A for demonstration of altered morphology and distribution of mTECs committed to express Aire in Aire^gfp/gfp mouse thymus. There were more GFP⁺ cells with globular shapes (bottom, arrows) in Aire^gfp/gfp thymus than in Aire^+/gfp thymus. GFP⁺ cells from Aire^+/gfp thymus were enriched at the cortico-medullary junction (top), whereas GFP⁺ cells from Aire^gfp/gfp thymus tended to be localized more evenly within each medulla or even enriched at the center of the medulla (bottom). Bars, 100 μm. One representative experiment from a total of five repeats is shown. (D) Morphological changes in the shape of GFP⁺ cells from Aire^gfp/gfp mouse thymus demonstrated in C were analyzed statistically. Each circle corresponds to the relative cell shape complexity of a single GFP⁺ cell calculated with a computer program (see Materials and methods). A total of 80 and 88 GFP⁺ cells from Aire^+/gfp and Aire^gfp/gfp thymi, respectively, were evaluated. Red lines represent mean values. Two mice for each group were analyzed, and similar results were obtained from a total of three repeats.

Figure 3.

Reduced numbers of terminally differentiated mTECs in the absence of Aire. (A) Involucrin-expressing mTECs (green) were scattered within the thymic medulla (red; stained with anti-EpCAM Ab) of Aire-sufficient mice. Bar, 50 μm. (B) Numbers of involucrin-expressing mTECs were reduced in Aire-deficient mice at 4 (left) and 8 (middle) wk of age. Numbers of involucrin-expressing mTECs in Aire-sufficient mice declined at 11 wk of age (right). Each circle corresponds to the mean number of involucrin-expressing mTECs per section examined in individual mice. Detailed information for the mice examined from a total of five experiments is presented in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20080046/DC1). (C) Hassall's corpuscle-like structures seen in Aire-sufficient mouse thymus stained with anti-involucrin Ab (green) together with anti-EpCAM Ab (red). These discrete and larger involucrin-expressing structures were scarcely detectable in Aire-deficient mouse thymus. Bar, 20 μm. One representative experiment from a total of five repeats is shown.

Figure 4.

Global alteration of mTEC phenotypes in the absence of Aire. (A) Detection of GFP-expressing cells from thymic stroma by flow cytometric analysis. CD45⁻ thymic stromal cells were analyzed for the expression of GFP together with binding of UEA-1. Percentages of cells from each fraction are indicated below. (B) mTECs committed to express Aire were larger than mTECs noncommitted to express Aire, irrespective of the presence of Aire protein. FSC/SSC profiles of mTECs committed to express Aire were altered in the absence of functional Aire protein (top). Each FSC/SSC profile was obtained by back gating the corresponding fractions from A based on the expression of GFP and UEA-1. (C) CD80 and MHC class II expression levels were higher in mTECs committed to express Aire than in mTECs noncommitted to express Aire, irrespective of the presence of functional Aire protein. Filled profiles in green and gray are from GFP⁺ and GFP⁻ mTECs, respectively. (D) CD80 and MHC class II expression from mTECs committed to express Aire were indistinguishable between Aire^+/gfp and Aire^gfp/gfp mice (left) but were reduced in mTECs noncommitted to express Aire in the absence of functional Aire protein (right). Filled profiles in gray and green lines are from Aire^+/gfp and Aire^gfp/gfp mice, respectively. Flow cytometric profiles from C were merged for comparison. One representative result from a total of more than five repeats is shown.

Figure 5.

TRA gene expression from mTECs assessed by real-time PCR. Expression of insulin 2, SAP1, CRP, and Aire was examined from each fraction of mTECs sorted on the basis of the flow cytometric profile demonstrated in Fig. 4 A. Color bars corresponding to each fraction are indicated on the right. Aire⁺ mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes (insulin 2 and SAP1), whereas an Aire-independent TRA gene (CRP) was expressed from both Aire⁺ and Aire⁻ mTECs. Aire expression was assessed to verify the proper sorting of each mTEC fraction. Numbers are relative gene expression level compared with that of the Hprt gene. Results are expressed as the mean ± SEM for triplicate wells of one representative experiment from a total of three repeat experiments.

Figure 6.

Expression of the Aire-independent TRA gene GAD67 and of Aire from mTECs in situ. (A) Expression of the GAD67 gene and Aire was detected by immunohistochemistry with anti-GFP Ab (green) and anti-Aire Ab (red), respectively, in thymus sections from GAD67/GFP knock-in mice. Bar, 20 μm. (B) Results obtained as described for A were calculated for a total of 152 mTECs expressing the GAD67 gene and/or Aire. One representative experiment from a total of three repeats is shown.

Figure 7.

Expression of the Aire and GAD67 genes by nonproliferating mTECs. (A) BrdU incorporation by Aire- and GAD67-expressing mTECs was evaluated 4 h after i.p. injection of BrdU into Aire^+/gfp and GAD67^+/gfp mice, respectively. The thymus sections were stained with anti-GFP (green) and anti-BrdU (red) Abs. Bars, 20 μm. (B) p63 (red) was not detected in mTECs expressing the Aire and GAD67 genes (green). Bars, 40 μm. One representative experiment from a total of four repeats is shown.

Figure 8.

Schematic representation of the roles of Aire in mTEC differentiation and TRA gene expression. Aire-expressing cell lineages develop from mTEC progenitor cells through concomitant expression of claudin (26). Expression of Aire-dependent TRA genes, such as insulin 2 and SAP1, can be accomplished in terminally differentiated mTECs showing a dendritic to fibroblastic morphology that have fully matured with the help of Aire protein (marked as Aire-sufficient). Lack of Aire in mTECs results in premature termination of differentiation, although claudin⁺ Aire-expressing cell lineages can still develop and pass the CD80-expressing maturation stage (marked as Aire-deficient). These CD80^hi Aire-less mTECs have a more globular cell shape and lack transcriptional machinery for Aire-dependent TRA genes. Because Aire-independent TRA genes, such as CRP and GAD67, can be expressed before the terminal differentiation stages, lack of Aire has little impact on their expression. The possibility also remains that Aire is necessary for the maintenance of a terminally differentiated state, in which mTECs manifest a dendritic shape with fully competent promiscuous gene expression.