Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire - PubMed (original) (raw)
Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire
Daniel Gray et al. J Exp Med. 2007.
Abstract
Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire(+) and Aire(-) MEC subsets through flow cytometric analysis of 5-bromo-2'deoxyuridine (BrdU) incorporation. The Aire(+) MEC subset was almost entirely postmitotic and derived from cycling Aire(-) precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire(-) MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire(+) population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a "terminal differentiation" model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire(+) MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.
Figures
Figure 1.
Distinguishing features of two models of TEC differentiation. A schematic diagram of two models of TEC differentiation from precursors (P) into mature MECs (M) in terms of the diversity of PTA expression versus time and differentiation. The terminal differentiation model (model 1) proposes that TEC precursors are Aire−, cycling cells expressing few PTAs that give rise to mature, Aire+, CD80hi, MHC IIhi MECs that express the greatest diversity of PTAs. Conversely, the progressive restriction model (model 2) predicts that precursor TECs are Aire+, cycling cells that express the greatest range of PTAs and differentiate down specific lineages into mature TECs expressing PTAs of terminally differentiated cells.
Figure 2.
Phenotypic analysis of Aire+ MECs. (A) Immunofluorescent staining of Aire+/+ or Aire−/− thymus sections with anti-Aire (green), anti–cytokeratin-5 (K5; red), and DAPI (blue). Bars, 50 μm. (B, top) Dot plots show gating, from left to right, used to distinguish three major TEC subsets (CEC, MEClo, and MEChi) present in thymi from 6-wk-old mice. (bottom) Histograms of Aire expression in TEC subsets from aire+/+ (continuous line) and aire−/− (shaded) mice. Numbers denote the proportion of cells in gated regions. (C) Expression of UEA-1, CD86, and PD-L1 in Aire+ (continuous line) and Aire− (dashed line) MEChi are shown, with percentages of CD86+ and PD-L1+ MEChi in Aire+ (top) and Aire− (bottom) subsets. Plots are representative of two to five experiments.
Figure 3.
Aire+ MECs are postmitotic and turn over rapidly. (A) Mice were pulsed with BrdU, and TECs were analyzed 12 h later for BrdU incorporation. Uninjected controls (shaded) and percentages of BrdU+ cells from injected mice (continuous line) are shown. (B) BrdU decay kinetics in TEC populations after a single pulse (values represent the mean ± SD). (C) BrdU incorporation kinetics in TEC populations during a 2-wk exposure (values represent the mean ± SD). Data are representative of three to five experiments, with each using three to four mice per time point. (D) Adult B6 MEClo were reaggregated with E15.5 B6.H-2g7 stroma with SP thymocytes, and Aire expression on H-2b+ MECs was analyzed during culture. Regions and corresponding values show the proportion of Aire− MEClo, Aire− MEChi, and Aire+ MECs. (inset) BrdU staining on Aire+ (continuous line) and Aire− (dotted line) H-2b+ MECs. Data are representative of three experiments.
Figure 4.
Rapid loss of Aire+ MEChi. After a 2-wk BrdU incorporation period, label retention was assayed 1, 2, and 4 wk after withdrawal in whole TECs (dot plots). Gates were set according to uninjected controls for each experiment. Numbers denote the proportion of cells in gated regions. The bar graph plots the mean (±SD) proportion of maximum incorporation found for MEClo, Aire− MEChi, and Aire+ MEChi from three mice per time point.
Figure 5.
The Aire protein does not directly impinge on proliferation. (A) Dot plots gated on TECs from aire+/+ or aire−/− mice with regions distinguishing CECs, MEClo, and MEChi. Bar graph shows mean (±SD) TEC subset proportions for aire+/+ and aire−/− mice (n = 4). *, P < 0.005. (B) BrdU incorporation by MEChi from aire+/+ or aire−/− mice was analyzed 12 h after pulse (continuous line), compared with uninjected controls (shaded). (right) Bar graph of mean (±SD) percentage of BrdU+ MEChi in aire+/+ or aire−/− mice (n = 5). **, P < 0.05. (C) The 1C6 MEC line was transfected with GFP or Aire-GFP expression constructs, and BrdU was added to the cultures 44 h later. The percentage of GFP+ cells incorporating BrdU is shown for plots representative of two experiments at various time points after transfection (time with BrdU is shown in parentheses). (D) Bar graphs of mean (±SD) percent GFP+ of maximum proportion or cell number (at 2 d) in GFP- or Aire-GFP–transfected cultures over time (n = 3). (E) GFP+ cells were analyzed for Annexin V binding and uptake of DAPI at various time points after transfection. Plots are representative of three experiments, with regions distinguishing viable (Annexin−/DAPI−), early apoptotic (Annexin+/DAPI−), and late apoptotic (Annexin+/DAPI+) MECs.
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