Different TEC subsets are temporally regulated during postnatal thymus development in wild type mice.

<p>(<b>A</b>) UEA/MHCII→K8/K5 (blue dots) and K8/K5→UEA/MHCII (red dots) overlays were generated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086129#pone-0086129-g002" target="_blank">Figure 2A and B</a> comparing patterns of UEA-1, MHCII, K8 and K5 expression in 1-week-old vs. 6-week-old wild type mice as shown. (<b>B</b>) Quantification of the total numbers of TEC subpopulations in 1-week old and 6-week-old wild type mice within the total gated populations was performed by flow cytometry. Bar graphs represent mean+SEM with n = 9 from three independent experiments. (<b>C</b>) Frozen thymic sections from 1- and 6-week-old wild type were stained with anti-Aire FITC and rhodamine-conjugated UEA-1 and analyzed by fluorescence microscopy. Scale bar = 100 µm.</p

Identification of novel TEC populations by simultaneous expression of cortical and medullary TEC markers.

<p>(<b>A</b> and <b>B</b>) The different cell populations subgated on UEA/MHCII and K8/K5 dot plots from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086129#pone-0086129-g001" target="_blank">Figure 1C</a> were overlaid onto K5/K8 (A) and UEA/MHCII (B) dot plots respectively (panels <b>a–g</b>) to identify cells coexpressing these markers. Panel <b>f</b> in B is a replica of <b>c</b> to show the overlapping populations within gate 3 of the K8/K5 dot plots. (<b>C</b>) Color-coded cell subsets defined in the UEA/MHCII and K8/K5 overlays in A and B were renamed and designated as described in the text and as shown (table). The dot plots show a schematic representation of the overlapping populations defined in A and B. (<b>D</b>) Percentages and total numbers of each cell subset were quantified by flow cytometry. (<b>E</b>) Bar graphs show the proportion of each cell subset characterized in C (color bars) within the total population of cells gated (gray bars). Bar graphs represent mean+SEM. n = 12 data were pooled from at least three independent experiments.</p

RANKL differentially regulates TEC expansion in the post-natal vs. mature thymus.

<p>(<b>A</b>) UEA/MHCII→K8/K5 (blue dots) and K8/K5→UEA/MHCII (red dots) overlays generated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086129#pone-0086129-g002" target="_blank">Figure 2A and B</a> comparing patterns of UEA-1, MHCII, K8 and K5 expression in 1- vs. 6-week-old RANKL-Tg mice. (<b>B</b>) Quantification of the total numbers of TEC subpopulations in 1- and 6-week-old RANKL-Tg type mice was performed by flow cytometry. Bar graphs represent mean+SEM with n = 9 from three independent experiments. (<b>C</b>) Frozen thymic sections from 1- and 6-week-old RANKL-Tg mice were stained with anti-Aire FITC and rhodamine-conjugated UEA-1 and analyzed by fluorescence microscopy. Scale bar = 100 µm.</p

Rare EpCAM1⁺ cells are present in the different TEC cell subsets.

<p>(<b>A</b> and <b>B</b>) EpCAM1⁺ cells gated on CD45⁻ epithelial cells were overlaid on UEA/MHCII and K8/K5 dot plots (blue dots). (<b>C</b>) EpCAM1 and other marker expression levels for each cell subset were analyzed by flow cytometry on histograms. An isotype control was used to differentiate EpCAM1⁺ from EpCAM1⁻ cells. (<b>D</b>) Total numbers of all CD45⁻ cells gated as well as EpCAM1⁺ cells within the different gates were determined by flow cytometry. (E) Nomenclature assignments of the TEC subsets identified. Bar graphs represent the mean+SEM. n = 16, results were pooled from at least three independent experiments.</p

The thymic medulla contains several distinct TEC populations.

<p>(<b>A</b>) Frozen thymic sections from ∼8-week-old mice were stained with K5, K8, MHCII and UEA-1. Arrows point to different TEC subsets as described in the text. Scale bar = 50 µm. (<b>B</b>) Schematic representation of known subsets of mTECs defined by expression/binding levels of MHCII, Aire and UEA-1. (<b>C</b>) Purified thymic epithelial cells from ∼8-week-old mice were stained with anti-CD45, -MHCII, -K5 and -K8 antibodies and biotinylated UEA-1, and analyzed by flow cytometry. Seven populations based on UEA-1 binding and MHCII expression (left dot plot) and six populations based on K5 and K8 expression levels were identified (right dot plot). (<b>D</b>) Isotype controls for UEA, MHCII, K8 and K5 were included in the experiment as shown. (<b>E</b>) The total numbers of the populations within the different gates of UEA/MHCII (1–7) and K8/K5 (1–6) dot plots were quantified by flow cytometry. Bar graphs represent the mean+Standard Error of the Mean (SEM). n = 12, results in C–E were pooled from at least three independent experiments.</p

Aire expression is upregulated in the mature TEC^hi cell subset.

<p>(<b>A</b>) Aire-expressing cells within the CD45⁻ gate were overlaid onto UEA-1/MHCII dot plots of TEC suspensions (right panel, red dots). (<b>B</b>) Aire expression levels within each cell population gated on the UEA/MHCII dot plot in 4A were analyzed by flow cytometry on histograms. (<b>C</b>) The percentages and total numbers of Aire⁺ cells within the different UEA-1/MHCII-gated populations (1–7) were determined by flow cytometry. Bar graphs represent mean+SEM with n = 10 from 3 different experiments. (<b>D</b>) Table shows the name designations of the different subsets based on expression levels of all markers tested.</p

RANKL-mediated medullary expansion inhibits T cell development.

<p>(<b>A and B</b>) Mosaic images of H&E stained thymic sections of 6-week and 3-month old wild type and RANKL-Tg mice showing progressive expansion of the medulla in response to RANKL expression. Scale bar = 500 µm. Micrographs are representative of at least three independent experiments. (<b>C–F</b>). Thymocyte suspensions from 6-week and 3-month old wild type and RANKL transgenic mice were stained with anti-CD4, -CD8, -CD44 and-CD25 antibodies and the total numbers of the different thymocyte subpopulations were analyzed by flow cytometry. Bar graphs represent the mean+SEM<b>.</b> n = 9 for each genotype, results were pooled from at least three independent experiments. DP = double positive; DN1-4 = double negative stages 1–4.</p

RANKL and Traf6 regulate the expansion of TEC subsets.

<p>(<b>A</b>) Mosaic images of H&E sections of thymic lobes from ∼3-month-old RANKL-Tg and Traf6ΔTEC mice show the effect of RANKL expression and Traf6 deletion on medullary architecture. Scale bars = 500 µm<b>.</b> (<b>B</b> and <b>C</b>) UEA/MHCII→K8/K5 (blue dots) and K8/K5→UEA/MHCII (red dots) overlays generated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086129#pone-0086129-g002" target="_blank">Figure 2A and B</a> comparing patterns of UEA-1, MHCII, K8 and K5 expression in wild type, RANKL-Tg and Traf6ΔTEC mice. (<b>D</b> and <b>E</b>) Quantification of the total numbers of TEC subpopulations in wild type and RANKL-Tg (D) or Traf6ΔTEC (cKO) mice (E) within the total gated populations as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086129#pone-0086129-g001" target="_blank">Figure 1C</a>. Bar graphs represent mean+SEM. At least 9 mice were used per genotype from four independent experiments.</p

Model of thymic epithelial cell development.

<p>Production of TEC subsets is temporally regulated in the adult vs. the neonatal thymus. In the adult thymus the P2 cell pool contains cells expressing low levels of K5 which may serve as precursors to both the P3 and P8 populations. Expression of the UEA-1 receptor in the P3 population commits the cells into the mTEC lineage. The P8 population could serve as a bipotent precursor by upregulating the UEA-1 receptor and contributing to the formation of the P5 subset or by driving the cells into the cTEC lineage through an intermediate P9 population that involves K5 downregulation (as it occurs in the most mature mTECs) and upregulation of MHCII and EpCAM1. The large expansion of the P5 population compared to the P4 subset may represent a checkpoint during TEC differentiation after which P5 cells give rise to mature P7 mTECs either directly or indirectly through the P6 subset. Alternatively the P6 subset may represent terminally differentiated post Aire-stage mTEC expressing low levels of MHCII. In the neonatal thymus, the expanded P2 and P3 cell subsets could give rise to the mature P7 population through the same pathway as the adult thymus or directly to promote rapid production of Aire⁺ mTECs (P7) to tolerize the emerging T cell repertoire. Whereas RANKL does not directly regulate the production of CMJ cTECs, Traf6 regulates generation of mTECs either directly or indirectly through CMJ cTECs. Because Traf6 deletion does not interfere with cortex development, other mechanisms contribute to mature cTEC production.</p

Effects of ACC2 deletion on cellular FAO in T cells.

<p>(A) T cell-specific deletion of the <i>ACC2</i> gene; PCR analysis confirms ACC2 deletion in genomic DNA in FACS-purified CD4⁺, CD8⁺, B220⁺, CD11b⁺ cells from <i>ACC2</i>^<i>f/f</i> (WT) and <i>ACC2</i>^<i>f/f</i><i>Cd4-Cre</i> (ACC2ΔT) mice. IL-2 was an internal control. (B) Real-time quantitative PCR analysis of <i>ACC1</i> or <i>ACC2</i> (exon 12) expression from WT or ACC2ΔT CD8⁺ T cells. Results are presented relative to 18S. (C) FACS purified naïve CD8⁺ T cells from WT and ACC2ΔT mice were incubated with ³H-palmitate for 11 hours, and supernatants assayed for production of ³H₂O as detailed in Methods. Analysis was performed in triplicate or quadruplicate. Each dot represents independent experiments, and the graph depicts paired analyses of WT and ACC2ΔT cells performed four times independently (Mixed-effect model, p = 0.0032).</p