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.
Figures
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. 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. 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. 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. 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. 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.
Similar articles
- Thymic commitment of regulatory T cells is a pathway of TCR-dependent selection that isolates repertoires undergoing positive or negative selection.
Coutinho A, Caramalho I, Seixas E, Demengeot J. Coutinho A, et al. Curr Top Microbiol Immunol. 2005;293:43-71. doi: 10.1007/3-540-27702-1_3. Curr Top Microbiol Immunol. 2005. PMID: 15981475 Review. - Agonist ligands expressed by thymic epithelium enhance positive selection of regulatory T lymphocytes from precursors with a normally diverse TCR repertoire.
Ribot J, Romagnoli P, van Meerwijk JP. Ribot J, et al. J Immunol. 2006 Jul 15;177(2):1101-7. doi: 10.4049/jimmunol.177.2.1101. J Immunol. 2006. PMID: 16818767 Free PMC article. - Peptide specificity of thymic selection of CD4+CD25+ T cells.
Pacholczyk R, Kraj P, Ignatowicz L. Pacholczyk R, et al. J Immunol. 2002 Jan 15;168(2):613-20. doi: 10.4049/jimmunol.168.2.613. J Immunol. 2002. PMID: 11777953 - In vivo maintenance of T-lymphocyte unresponsiveness induced by thymic medullary epithelium requires antigen presentation by radioresistant cells.
Hudrisier D, Feau S, Bonnet V, Romagnoli P, Van Meerwijk JP. Hudrisier D, et al. Immunology. 2003 Jan;108(1):24-31. doi: 10.1046/j.1365-2567.2003.01546.x. Immunology. 2003. PMID: 12519299 Free PMC article. - Thymic involution: implications for self-tolerance.
Hakim FT, Gress RE. Hakim FT, et al. Methods Mol Biol. 2007;380:377-90. doi: 10.1007/978-1-59745-395-0_24. Methods Mol Biol. 2007. PMID: 17876107 Review.
Cited by
- Lymphocytes and Trogocytosis-Mediated Signaling.
Reed J, Reichelt M, Wetzel SA. Reed J, et al. Cells. 2021 Jun 12;10(6):1478. doi: 10.3390/cells10061478. Cells. 2021. PMID: 34204661 Free PMC article. Review. - Trogocytosis-Mediated Intracellular Signaling in CD4+ T Cells Drives TH2-Associated Effector Cytokine Production and Differentiation.
Reed J, Wetzel SA. Reed J, et al. J Immunol. 2019 May 15;202(10):2873-2887. doi: 10.4049/jimmunol.1801577. Epub 2019 Apr 8. J Immunol. 2019. PMID: 30962293 Free PMC article. - CD4+CD25+Foxp3 regulatory T cells and vascular dysfunction in hypertension.
Kassan M, Wecker A, Kadowitz P, Trebak M, Matrougui K. Kassan M, et al. J Hypertens. 2013 Oct;31(10):1939-43. doi: 10.1097/HJH.0b013e328362feb7. J Hypertens. 2013. PMID: 23881298 Free PMC article. Review. - Hematopoietic chimerism and transplantation tolerance: a role for regulatory T cells.
Pasquet L, Joffre O, Santolaria T, van Meerwijk JP. Pasquet L, et al. Front Immunol. 2011 Dec 28;2:80. doi: 10.3389/fimmu.2011.00080. eCollection 2011. Front Immunol. 2011. PMID: 22566869 Free PMC article. - Cellular immunotherapy for ovarian cancer.
Cannon MJ, O'Brien TJ. Cannon MJ, et al. Expert Opin Biol Ther. 2009 Jun;9(6):677-88. doi: 10.1517/14712590902932897. Expert Opin Biol Ther. 2009. PMID: 19456205 Free PMC article. Review.
References
- Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–1164. - PubMed
- Shevach EM. Regulatory T cells in autoimmmunity. Annu Rev Immunol. 2000;18:423–449. - PubMed
- Sakaguchi S. Regulatory T cells: key controllers of immunologic self-tolerance. Cell. 2000;101:455–458. - PubMed
- Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat Immunol. 2001;2:816–822. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Research Materials
Miscellaneous