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. 2009 Sep 28;206(10):2121-30.
doi: 10.1084/jem.20091033. Epub 2009 Sep 8.

TCR-dependent differentiation of thymic Foxp3+ cells is limited to small clonal sizes

Monica W L Leung  1 , Shiqian Shen, Juan J Lafaille
Affiliations

"VSports最新版本" TCR-dependent differentiation of thymic Foxp3+ cells is limited to small clonal sizes

Monica W L Leung et al. J Exp Med. .

Abstract

Numerous studies have highlighted the importance of high-affinity interactions between T cell receptors (TCRs) and their ligands in the selection of Foxp3(+) regulatory T cells (T reg cells). To determine the role of the TCR in directing T cells into the Foxp3(+) lineage, we generated transgenic (Tg) mice expressing TCRs from Foxp3(+) cells. Initial analyses of the TCR Tg mice crossed with RAG-deficient mice showed that the percentage of Foxp3(+) cells was very low. However, intrathymic injection and bone marrow chimera experiments showed a saturable increase of the Foxp3(+) population when T reg TCR Tg cells were present in low numbers VSports手机版. Furthermore, when analyzing whole thymi of T reg TCR Tg RAG-deficient mice, we found significantly more Foxp3(+) cells than in conventional T cell TCR Tg mice. Our results indicate that although the TCR has an instructive role in determining Foxp3 expression, selection of Foxp3(+) individual clones in the thymus is limited by a very small niche. .

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Figures

Figure 1.
Figure 1.
T reg TCR Tg mice have a low percentage of Foxp3+ cells in the thymus. (A) Schematic of two approaches used to choose T reg TCR for generating Tg mice (see Material and methods for more details). (B) Foxp3 flow cytometry analysis of A12-end and A9-end RAG+ splenocytes expressing different levels of Vα2. (C) Analysis of thymic Foxp3+ cells in T reg TCR RAG-deficient Tg lines. Foxp3 analysis was gated on CD4+ CD8− Vβ6+ Vα2+ cells. Numbers in the quadrant represent the percentage of cells of the indicated quadrant. Results are representative of at least three independent experiments with three or more mice per group.
Figure 2.
Figure 2.
T reg monoclonal cells are present in high percentages but low absolute numbers upon intrathymic injection or generation of BM chimeras. (A) ∼5 × 106 Thy1.2 A12-end BM cells were injected intrathymically into 4–6-wk-old Thy1.1 WT hosts. 14 d after transfer, the mice were sacrificed and 10% of the thymus was analyzed. Representative Foxp3 stainings of gated on Thy1.2+ cells are shown on the left, and the quantification of recovered Foxp3+ cell numbers is shown on the right. (B) ∼50 × 106 cells total thymocytes were injected intrathymically into 4–6-wk-old Thy1.1 WT hosts. 14–20 d after transfer, individual thymus single cell suspensions were depleted with anti-CD8 beads before analyzed with flow cytometry. Representative Foxp3 staining of Thy1.2+ gated cells are shown on the left, and the quantification of Thy1.2+ CD4+ Foxp3+ cells from respective donor Tg lines is shown on the right. MBP and TB are T conv TCR Tg, whereas 2P-CD4 and A12-end are T reg TCR Tg. Asterisk indicates P < 0.05 compared with MBP. Each symbol represents an individual mouse from two independent experiments. The bar indicates the mean percentage of CD4+ Foxp3+ cells. (C) Mixed BM chimeras at a 3:1 ratio of Thy1.1 WT/Thy1.2 A12-CD4 RAG-deficient BM cells into RAG-deficient hosts were analyzed 10 wk after injection; 10% of the thymus was analyzed in the FACS. Representative Foxp3 staining gated on Thy1.2+ cells from different levels of reconstitution hosts are shown on the left, and the quantification of CD4+ CD25+ Foxp3+ cells relative to total Thy1.2+ cells recovered is shown on the right. Results are representatives of two independent experiments.
Figure 3.
Figure 3.
Foxp3+ cells are present in the thymus of T reg TCR Tg mice at higher numbers than in T conv TCR Tg mice. 95% of whole thymi were depleted with CD8 beads before being analyzed using flow cytometry, whereas the remaining 5% was stained and analyzed undepleted. (A) Representative Foxp3 staining of WT and T reg TCR Tg RAG-deficient (2P-CD4) mice gated on CD4+ Vβ6+ Vα2+ thymocytes after CD8 depletion. (B) CD24 and CD69 staining of CD4+ Foxp3+ (line) and CD4+ Foxp3− (shaded) populations of WT and T reg TCR Tg RAG-deficient (2P-CD4) mice. (C) Quantification of absolute numbers of thymic TCR+ CD4+ CD25+ Foxp3+ cells in respective T reg TCR Tg RAG-deficient. A two-tailed Student's t test was used to calculate the indicated p-values. Each symbol represents an individual mouse from four independent experiments (A12-end, n = 6; A12-CD4, n = 7; 2P-CD4, n = 2; T-Bmc, n = 4; T-Bsf, n = 3; WT, n = 4). Bars indicate the mean number of CD4+ TCR+ CD25+ Foxp3+ cells in each group.
Figure 4.
Figure 4.
T reg TCR Tg mice generated using the CD4 constructs have increased numbers of Foxp3+ T cells in the peripheral lymphoid organs. (A) Analysis of Foxp3+ cells in T reg TCR Tg lines in pooled inguinal/auxillary/brachial peripheral lymph nodes. Foxp3 analysis was gated on Vβ6+ Vα2+ CD4+ B220− cells. (B) Percentage of Vβ6+ Vα2+ CD4+ CD25+ Foxp3+ cells in T reg TCR Tg lymph nodes. At least three independent experiments of n = 3 per group are represented. (C) CD62L and CD44 staining of Vβ6+ Vα2+ CD4+ Foxp3− and Foxp3+ cells in peripheral blood. Results are representative of three independent experiments. (D) CD25− T cells from WT and T reg TCR Tg mice were labeled with CFSE and transferred into RAG1-deficient recipients. After 2 wk, pooled lymph nodes were collected and Foxp3 expression was analyzed by flow cytometry. Data is representative of three independent experiments with n = 3 per group.
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References

    1. Apostolou I., Sarukhan A., Klein L., von Boehmer H. 2002. Origin of regulatory T cells with known specificity for antigen.Nat. Immunol. 3:756–763 - PubMed
    1. Baldwin T.A., Sandau M.M., Jameson S.C., Hogquist K.A. 2005. The timing of TCR- α expression critically influences T cell development and selection.J. Exp. Med. 202:111–121 doi:10.1084/jem.20050359 - DOI - PMC - PubMed
    1. Bennett C.L., Brunkow M.E., Ramsdell F., O'Briant K.C., Zhu Q., Fuleihan R.L., Shigeoka A.O., Ochs H.D., Chance P.F. 2001a. A rare polyadenylation signal mutation of the FOXP3 gene (AAUAAA—>AAUGAA) leads to the IPEX syndrome.Immunogenetics. 53:435–439 doi:"V体育2025版" 10.1007/s002510100358 - DOI - PubMed
    1. Bennett C.L., Christie J., Ramsdell F., Brunkow M.E., Ferguson P.J., Whitesell L., Kelly T.E., Saulsbury F.T., Chance P.F., Ochs H.D. 2001b. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.Nat. Genet. 27:20–21 doi:10.1038/83713 - DOI - PubMed
    1. Bensinger S.J., Bandeira A., Jordan M.S., Caton A.J., Laufer T.M. 2001. Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4+25+ immunoregulatory T cells.J. Exp. Med. 194:427–438 doi:10.1084/jem.194.4.427 - DOI - PMC - PubMed

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