Differentiation of regulatory Foxp3+ T cells in the thymic cortex - PubMed (original) (raw)

Differentiation of regulatory Foxp3+ T cells in the thymic cortex

Adrian Liston et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Regulatory Foxp3(+) T cells (T(R)) are indispensable for preventing autoimmune pathology in multiple organs and tissues. During thymic differentiation T cell receptor (TCR)-ligand interactions within a certain increased affinity range, in conjunction with gammac-containing cytokine receptor signals, induce Foxp3 expression and thereby commit developing thymocytes to the T(R) lineage. The contribution of distinct MHC class II-expressing accessory cell types to the differentiation process of Foxp3(+) thymocytes remains controversial, because a unique role in this process has been ascribed to either thymic dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC). Furthermore, it was suggested that the thymic medulla, where the bulk of the negative selection of self-reactive thymocytes takes place, provides a specialized microenvironment supporting T(R) differentiation. Here, we report that the cortex, as defined by cortical thymic epithelial cells (cTEC), is sufficient for supporting T(R) differentiation. MHC class II expression restricted to both cTEC and mTEC or to cTEC alone did not significantly affect the numbers of Foxp3(+) thymocytes. Furthermore, genetic or pharmacologic blockade of thymocyte migration resulted in a prominent accumulation of Foxp3(+) thymocytes in the cortex, demonstrating that secondary signals required for Foxp3 up-regulation exist in the cortex. Our results suggest that mTEC or tDC do not serve as a cell type singularly responsible for T(R) differentiation and that neither the cortex nor the medulla exclusively provides an environment suitable for Foxp3 induction. Instead, multiple accessory cell types probably contribute to the thymic generation of regulatory Foxp3(+) T cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Hemopoietic MHC class II expression is dispensable for commitment to the Foxp3+ lineage or gain of suppressor function. (a) B6 mice were irradiated and reconstituted with either Foxp3GFP or MHC class II–deficient Foxp3GFP BM. Eight weeks after reconstitution, commitment to the Foxp3+ lineage was analyzed by flow cytometry; representative flow profiles are shown (n = 10,6). (b) Percentages of SP thymocytes that express Foxp3 and (c) absolute number of Foxp3+ SP thymocytes from Foxp3GFP → B6 and Ab1null Foxp3GFP → B6 chimeras (mean ± standard deviation). (d) CD11c-Cre transgenic mice crossed with IAb flx/flx mice showed highly efficient ablation of MHC class II expression on thymic CD11c+ cells (black line = Cre−; red line = Cre+). Representative profiles (n = 3): (e) flow cytometric analysis of the absolute number of CD4 SP thymocytes, (f) the percentage of CD4 SP thymocytes that express Foxp3, and (g) the absolute number of Foxp3+ SP cells (mean ± standard deviation).

Fig. 2.

Fig. 2.

TCR–MHC interactions in the medulla are not necessary for Foxp3+ TR cell commitment. Foxp3+ TR cell differentiation was compared in wild type, Ab1null, and Ab1null K14-Aβb transgenic mice. (a) Representative flow cytometric profiles showing wild type, Ab1null, and Ab1null K14-Aβb transgenic CD4, CD8, and Foxp3 expression in the DP, early SP, SP, and CD4+ splenocyte populations. (b) Percentage of thymocytes expressing Foxp3 at the DP, early SP, and SP stages, for wild type (n = 19, black bar), Ab1null (n = 10, white bar), and Ab1null K14-Aβb transgenic (n = 12, gray bar) mice. (c) Localization of Foxp3+ cells (Foxp3, green) in the thymic cortex (CDR1/6C3, blue) and medulla (unlabeled) of wild type (top), Ab1null (middle), and Ab1null (bottom) K14-Aβb transgenic mice. Results are representative of four experiments. (d) Average absolute number of thymocytes expressing Foxp3 at the DP, early SP, and SP stages.

Fig. 3.

Fig. 3.

DP Foxp3+ cells are postselection. (a) Foxp3+ DP cells (line) compared with Foxp3− DP cells (shaded) for CD69 expression. (b) Proportion of Foxp3+ cells among DP CD69− cells (Left) and DP CD69+ cells (Right). Results are representative of three experiments. (c) Proportion of Foxp3+ cells among CCR7− DP cells (Left) and CCR7+ DP cells (Right). (d) Percentage of CCR7− DP and CCR7+ DP cells that are Foxp3+ (mean ± standard deviation, n = 5). (e) Representative histograms and (f) average (mean ± standard deviation, n = 5) of CCR7 expression on Foxp3− DP cells (solid gray area), Foxp3+ DP cells (black line), Foxp3− SP cells (blue line), and Foxp3+ SP cells (red line). (g) CD25 expression on Foxp3− DP (red line), Foxp3− SP (blue line), Foxp3+ SP (black line), and Foxp3+ DP (solid gray area) cells (representative of n = 10).

Fig. 4.

Fig. 4.

Retention of SP cells in the thymic cortex does not impede Foxp3 commitment. The thymuses of PT-treated mice and CCR7-deficient mice were analyzed for Foxp3+ T cell location. (a) CD4-CD8 profiles and Foxp3 expression within DP and SP populations for untreated and PT-treated mice (representative sections, n = 6). (b) Thymic sections from untreated and PT-treated mice stained for Foxp3 (green) and CDR1/6C3 (cortex, blue) (representative sections, n = 6). (c) Thymic sections from wild type and _Ccr_−/− mice stained for Foxp3 (green) and CDR1/6C3 (cortex, blue). (d) Number of Foxp3+ thymocytes in Ly5.1 Foxp3GFP BM, Ly5.1 Foxp3GFP + Ly5.2 _Ccr7_−/− mixed BM, and Ly5.2 _Ccr7_−/− BM chimeras, for Ly5.1 (wild type, black bar) and Ly5.2 (_Ccr7_−/−, white bar) cells, corrected for degree of BM chimerism. (e) Number of Foxp3+ cells in the cortex of Ly5.1 Foxp3GFP BM, Ly5.1 Foxp3GFP + Ly5.2 _Ccr7_−/− mixed BM, and Ly5.2 _Ccr7_−/− BM chimeras, using Foxp3 (Left) or GFP (Right). n = 5. (f) Thymic sections from Ly5.1 Foxp3GFP + Ly5.2 _Ccr7_−/− mixed BM chimeras, stained for CDR1/6C3 (cortex, blue) and either GFP (green; left) or Foxp3 (green; right) (representative sections, n = 5).

Fig. 5.

Fig. 5.

Product–precursor relationship between DP and SP Foxp3+ thymocytes. (a) Representative flow profiles of regenerated GFP+ cells in Foxp3wt/DTR-GFP mice treated with two doses of DT at day −1 and day 0 and traced for reconstitution of GFP+ cells. (b) Mean ± standard deviation (n = 3, 4, 4, 4, 3, 2) for the absolute number of Foxp3+ DP and Foxp3+ SP cells. Day 0.1 represents mice injected with DT at day −1 and at −2 h. Short and long dashes indicate numbers of SP and DP GFP+ cells, respectively, in uninjected mice. (c) Expression of CD8 (Left) and Foxp3Thy1.1 (Right) on Ly5.1+Ly5.2- thymocytes (shaded) and purified Ly5.2 DP Foxp3Thy1.1+ thymocytes 18 h after intrathymic injection into Lys5.1 mice.

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