Agonist ligands expressed by thymic epithelium enhance positive selection of regulatory T lymphocytes from precursors with a normally diverse TCR repertoire

Abstract

CD4+CD25+ regulatory T lymphocytes play a crucial role in inhibition of autoimmune pathology. In accordance with this physiological role, it is now well established that the repertoire of these lymphocytes is strongly enriched in autospecific cells. However, despite extensive investigation, the thymic mechanisms involved in development of regulatory T cells remain incompletely defined. To address the issue of selection of regulatory T cell precursors in mice with a naturally diverse TCR repertoire, we have analyzed development of superantigen-specific regulatory T cells in hemopoietic chimeras in which endogenous super-antigens are exclusively presented by thymic epithelial cells. Our results demonstrate that recognition of agonist ligands expressed by thymic epithelium does not lead to deletion but substantially enhances development of mature regulatory T cells. Interestingly, also development of a small subpopulation of CD25-expressing T cells lacking expression of the transcription factor Foxp3, thought to be autospecific, is enhanced by expression of the agonist ligand on thymic epithelium. Based on quantitative arguments, we propose that commitment to the regulatory T cell lineage is not dictated by the specificity of precursors, but that recognition of the agonist ligand expressed by thymic epithelium substantially enhances their positive selection.

Figure 1. SAg presented by TE enhance generation of CD4SP CD25^high regulatory T cells

(A) Analysis of CD25-expression by electronically gated CD4SP thymocytes. (B) Total numbers of thymocytes (left panel), percentage of CD4SP cells among total thymocytes (middle panel), and percentage of CD25^high cells among CD4SP thymocytes (right panel), in indicated chimeras. (C) Flow-cytometry analysis of TCR Vβ expression by electronically gated CD4SP CD25⁻ and CD25^high thymocytes in indicated chimeras. (D) Percentages of CD4SP CD25⁻ and CD25^hi thymocytes expressing indicated Vβ in the distinct chimeras. ***, p < 0.001; **, p < 0.01; ns, not significant; Student’s t test. Error bars indicate SD (B6→B6 and DBA/2→DBA/2: n = 3, MHC°→B6 and MHC°→DBA/2: n = 6).

Figure 2. SAg-specific CD4SP Foxp3⁺ T cell-differentiation is enhanced by endogenous sAg presented by TE

(A) Analysis of Foxp3-expression by electronically gated CD4SP thymocytes. (B) Percentage of Foxp3-expressing cells among CD4SP thymocytes in indicated chimeras. (C) Percentages of thymocytes expressing indicated Vβ among CD4SP Foxp3⁻ and Foxp3⁺ cells in indicated chimeras. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant; Student’s t test. Error bars indicate SD (n≥4).

Figure 3. TE-expressed sAg differentially affect CD4⁺ T cell development

(A) Electronically gated CD4SP thymocytes were subdivided into five different populations according to their CD25 and Foxp3 expression levels. (B). Percentages of thymocytes expressing indicated Vβ among the CD4SP Foxp3⁺ thymocyte subpopulations indicated in the distinct chimeras. (C) Percentages of thymocytes expressing indicated Vβ among CD25⁻ and CD25⁺ CD4SP Foxp3⁻ thymocyte subpopulations in the distinct chimeras. ***, p<0.001; **, p<0.01; *, p < 0.05; ns, not significant; Student’s t test. Error bars indicate SD (n≥4).

Figure 4. Positive selection of conventional T cells is not modulated by TE-expressed sAg

(A) Thymocytes were analyzed by flow-cytometry using antibodies specific for CD4, CDS, CD69, and indicated Vβ. FACS plots are electronically gated as indicated. (B) Percentages of CD69⁺ DP thymocytes expressing intermediate levels of indicated Vβ in the distinct chimeras.