Molecular signature of recent thymic selection events on effector and regulatory CD4+ T lymphocytes - PubMed (original) (raw)

Molecular signature of recent thymic selection events on effector and regulatory CD4+ T lymphocytes

Paola Romagnoli et al. J Immunol. 2005.

Abstract

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.

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Figures

Figure 1

Figure 1. Thymocytes acquire MHC class II molecules from the thymic environment

Lethally irradiated C57Bl/6 hosts were reconstituted with a mixture of bone marrow cells from C57Bl/6 (Thy1.1+) and C57BL/6 MHC II° (Thy1.2+) mice at a 1:1 ratio. Four weeks later, thymocytes were analyzed by four-color flow-cytometry using anti-CD4, anti-CD8, anti-CD25 and anti-IAb. To analyze I-A surface levels on Thy1.1+CD4SP cells, FITC-labeled anti-CD11c, anti-B220 mAbs and FITC-labeled anti-Thy1.2 mAbs were used in addition to FITC-labeled anti-CD8 in the exclusion gate. To analyze I-A surface levels on Thy1.2+CD4SP cells, FITC-labeled anti-Thy1.1 mAb was used in the antibody mixture. A reciprocal staining was performed to analyze I-A surface levels on CD8SP thymocytes. Solid lines indicate I-A staining, dotted lines isotype-matched controls. Bar graphs represent relative I-A expression level on different thymocyte subsets. Error bars indicate SD, n=4 from 2 independent experiments.

Figure 2

Figure 2. Displayed MHC class II levels depend on maturation-stage and CD4/CD8 lineage

A: Total thymocytes from C57Bl/6 mice were analyzed by four-color flow cytometry using anti-CD4, anti-CD8, anti-CD25 and anti-I-Ab (bold line) or isotype-matched control (thin line) abs. To analyze I-A surface levels, FITC-labeled anti-CD11c and anti-B220 mAbs were used in the exclusion gate as described in figure 1. Distinct thymocyte subsets were electronically gated based on CD4/CD8 expression and the respective I-A and 6C3/BP-1 levels were plotted as histograms. B: Bar graphs representing % of I-A positive cells in distinct thymocyte subpopulations (upper panel) and relative I-A expression level on different thymocyte subsets from C57Bl/6 mice (lower panel). All graphic representations of I-A levels were compiled from four independent experiments. Error bars indicate SD, n=10 mice.

Figure 3

Figure 3. MHC class II acquisition is an avidity-dependent, TCR-activation mediated process

FACS-histograms and bar graphs represent relative I-A expression level on different thymocyte subsets from the following mice A: C57Bl/6 mice: DP thymocytes were electronically gated according to their CD69 expression level. B: wt and TCRα° C57BL/6 mice: DP thymocytes, in the histogram the two dotted lines depict the isotype matched controls. C: C57Bl/6 and DBA/2 mice: CD4lowCD8low thymocytes were electronically gated. The bar graph (upper panel) represents relative MHC class II display on double dull thymocytes expressing different TCR Vβ, in bold Vβ reactive to Mmtv-encoded superantigens presented in DBA/2 mice, in normal characters the ones that are not. Lower panels: examples of FACS-histograms of I-A display by Vβ 12+ and Vβ8+ thymocytes. D: C57Bl/6 mice: Thymocytes were electronically gated as indicated and I-A display analyzed by FACS. Lower panel: FACS histograms of CD4SP thymocytes gated on HSA expression as indicated. Upper panel: Relative I-A display by the distinct thymocyte subpopulations. E: DO11.10 TCR transgenic mice (analyzed as in D). In panels A, B, and D the anti-MHC class II mAb used was AF6-120.1, M5/114 in panels C and E. In all graphic representations error bars indicate SD, n=4 mice.

Figure 4

Figure 4. CD4+CD25high Regulatory T cell precursors display higher levels of I-A on their surface than CD4+CD25− cells

A: Total thymocytes from C57Bl/6 mice were analyzed by four-color flow cytometry using anti-CD4, anti-CD8, anti-CD25 and anti-I-Ab mAbs, and distinct thymocyte subsets were electronically gated as described in the legend to figure 2. B: Bar graphs representing relative I-A and TCRβ levels on CD4+CD8int and CD4+CD8− thymocytes, electronically gated based on CD25 expression. Error bars indicate SD, n=8. C: CD4+CD8int and CD4+CD8− thymocytes were electronically gated based on CD25 expression and the respective TCR levels were displayed as histograms. Thick lines indicate TCR expression by CD4+CD8int cells, thin lines that of CD4+CD8− cells. D: CD8-depleted thymocytes were cultured in vitro and, at different time points, I-A levels on CD25− and CD25high CD4SP thymocytes were analyzed. Error bars indicate SD, n=3 mice. Similar results were obtained in two independent experiments.

Figure 5

Figure 5. Regulatory T cell precursors are negatively selected by APC but not by thymic epithelial cells

A: Lethally irradiated C57Bl/6 hosts were reconstituted with bone marrow cells from either MHC II° or wt C57Bl/6 mice. I-A levels on distinct thymocyte subsets were analyzed by flow-cytometry as described in the legend to figure 2. B/C/D: I-A levels on distinct thymocyte subsets from MHC II°→wt and wt→wt chimeras are displayed in bar graphs. Error bars indicate SD, n=5. Results from one same experiment are shown; two more independent experiments gave similar results.

Figure 6

Figure 6. Resting and activated peripheral regulatory CD4+CD25high lymphocytes display higher I-A levels than CD4+CD25− cells

A: I-A surface levels were analyzed on CD25− and CD25high CD4+CD8−CD11c CD11c−B220− lymphocytes isolated from lymph nodes of C57Bl/6 mice. The data are displayed as FACS-histograms (Left panel) and data from multiple experiments shown as bar graphs (middle panel). Right panel: MHC class II expression by lymph node B220+ cells. error bars represent SD, n=5). B: I-A levels on resting (CD69low) and recently activated (CD69hi) CD4+CD25high and CD4+CD25− T lymphocyte subsets from lymph nodes of C57Bl/6 mice. Right panel: Relative I-A expression levels are depicted for 5 individual mice, indicated with distinctive symbols.

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