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Beta-selection: abundance of TCRbeta-/gammadelta- CD44- CD25- (DN4) cells in the foetal thymus.

Hager-Theodorides AL, Rowbotham NJ, Outram SV, Dessens JT, Crompton T - Eur. J. Immunol. (2007)

Bottom Line: We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta.Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis.Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, UK.

ABSTRACT
Expression of TCRbeta and pre-TCR signalling are essential for differentiation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double-positive (DP) stage. Thymocyte development in adult Rag1, Rag2 or TCRbetadelta-deficient mice is arrested at the DN3 stage leading to the assumption that pre-TCR signalling and beta-selection occur at, and are obligatory for, the transition from DN3 to DN4. We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta. These foetal icTCRbeta-/gammadelta- DN4 cells were T lineage as determined by expression of Thy1 and icCD3 and TCRbeta DJ rearrangement. In addition, in the foetal Rag1-/- thymus, a normal percentage of DN4 cells were present. In wild-type mice after hydrocortisone-induced synchronisation of differentiation, the majority of DN4 cells that first emerged did not express icTCRbeta/gammadelta, showing that adult thymocytes can also differentiate to the DN4 stage independently of pre-TCR signalling. Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis. Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.

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Proliferation and apoptosis of wild-type foetal thymocytes. (A) DNA content analysis by PI nuclear staining of sorted CD44–CD3–CD4–CD8–B220–Thy1.2+ TCRβ–/γδ– CD25+ (DN3, top left histogram) and CD25– (DN4, top right histogram) and TCRβ+ DN3 (bottom left histogram) and DN4 (bottom right histogram) cells. Percentages of cells in the S/G2/M phases of the cell cycle are shown. (B) Intracellular expression of the active form of caspase-3 in DP cells where apoptosis was induced by incubation with anti-CD3 antibody for 2 h. (C) Intracellular expression of the active form of caspase-3 in foetal and adult DN4 thymocytes. Histograms on the left show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ (bottom) E16.5 foetal CD44–CD25–CD3–CD4–CD8–B220– Thy1.2+ (DN4) thymocytes and histograms on the right show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ adult DN4 populations. Percentages of active caspase-3 positive cells are shown.
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fig04: Proliferation and apoptosis of wild-type foetal thymocytes. (A) DNA content analysis by PI nuclear staining of sorted CD44–CD3–CD4–CD8–B220–Thy1.2+ TCRβ–/γδ– CD25+ (DN3, top left histogram) and CD25– (DN4, top right histogram) and TCRβ+ DN3 (bottom left histogram) and DN4 (bottom right histogram) cells. Percentages of cells in the S/G2/M phases of the cell cycle are shown. (B) Intracellular expression of the active form of caspase-3 in DP cells where apoptosis was induced by incubation with anti-CD3 antibody for 2 h. (C) Intracellular expression of the active form of caspase-3 in foetal and adult DN4 thymocytes. Histograms on the left show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ (bottom) E16.5 foetal CD44–CD25–CD3–CD4–CD8–B220– Thy1.2+ (DN4) thymocytes and histograms on the right show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ adult DN4 populations. Percentages of active caspase-3 positive cells are shown.

Mentions: A gradual decrease in the proportion of icTCRβ– DN4 cells during embryogenesis suggests that the foetal DN4 TCRβ+ subset expands faster than the DN4 TCRβ– subset. To test whether this is due to increased proliferation, we sorted E16.5 foetal DN3 and DN4 cells based on the intracellular expression of β and γδ TCR and analysed proliferation. In a typical experiment, propidium iodide DNA staining showed that approximately four-times more TCRβ+ DN3 (47.91%) and DN4 (41.06%) cells were in cell cycle than in the TCRβ–/γδ– DN3 (10.67%) or DN4 (11.22%) subset (Fig. 4A), consistent with a pre-TCR induced signal for proliferation [5, 14, 18–21].


Beta-selection: abundance of TCRbeta-/gammadelta- CD44- CD25- (DN4) cells in the foetal thymus.

Hager-Theodorides AL, Rowbotham NJ, Outram SV, Dessens JT, Crompton T - Eur. J. Immunol. (2007)

Proliferation and apoptosis of wild-type foetal thymocytes. (A) DNA content analysis by PI nuclear staining of sorted CD44–CD3–CD4–CD8–B220–Thy1.2+ TCRβ–/γδ– CD25+ (DN3, top left histogram) and CD25– (DN4, top right histogram) and TCRβ+ DN3 (bottom left histogram) and DN4 (bottom right histogram) cells. Percentages of cells in the S/G2/M phases of the cell cycle are shown. (B) Intracellular expression of the active form of caspase-3 in DP cells where apoptosis was induced by incubation with anti-CD3 antibody for 2 h. (C) Intracellular expression of the active form of caspase-3 in foetal and adult DN4 thymocytes. Histograms on the left show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ (bottom) E16.5 foetal CD44–CD25–CD3–CD4–CD8–B220– Thy1.2+ (DN4) thymocytes and histograms on the right show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ adult DN4 populations. Percentages of active caspase-3 positive cells are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651467&req=5

fig04: Proliferation and apoptosis of wild-type foetal thymocytes. (A) DNA content analysis by PI nuclear staining of sorted CD44–CD3–CD4–CD8–B220–Thy1.2+ TCRβ–/γδ– CD25+ (DN3, top left histogram) and CD25– (DN4, top right histogram) and TCRβ+ DN3 (bottom left histogram) and DN4 (bottom right histogram) cells. Percentages of cells in the S/G2/M phases of the cell cycle are shown. (B) Intracellular expression of the active form of caspase-3 in DP cells where apoptosis was induced by incubation with anti-CD3 antibody for 2 h. (C) Intracellular expression of the active form of caspase-3 in foetal and adult DN4 thymocytes. Histograms on the left show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ (bottom) E16.5 foetal CD44–CD25–CD3–CD4–CD8–B220– Thy1.2+ (DN4) thymocytes and histograms on the right show active caspase-3 expression in TCRβ–/γδ– (top) and TCRβ+ adult DN4 populations. Percentages of active caspase-3 positive cells are shown.
Mentions: A gradual decrease in the proportion of icTCRβ– DN4 cells during embryogenesis suggests that the foetal DN4 TCRβ+ subset expands faster than the DN4 TCRβ– subset. To test whether this is due to increased proliferation, we sorted E16.5 foetal DN3 and DN4 cells based on the intracellular expression of β and γδ TCR and analysed proliferation. In a typical experiment, propidium iodide DNA staining showed that approximately four-times more TCRβ+ DN3 (47.91%) and DN4 (41.06%) cells were in cell cycle than in the TCRβ–/γδ– DN3 (10.67%) or DN4 (11.22%) subset (Fig. 4A), consistent with a pre-TCR induced signal for proliferation [5, 14, 18–21].

Bottom Line: We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta.Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis.Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, UK.

ABSTRACT
Expression of TCRbeta and pre-TCR signalling are essential for differentiation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double-positive (DP) stage. Thymocyte development in adult Rag1, Rag2 or TCRbetadelta-deficient mice is arrested at the DN3 stage leading to the assumption that pre-TCR signalling and beta-selection occur at, and are obligatory for, the transition from DN3 to DN4. We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta. These foetal icTCRbeta-/gammadelta- DN4 cells were T lineage as determined by expression of Thy1 and icCD3 and TCRbeta DJ rearrangement. In addition, in the foetal Rag1-/- thymus, a normal percentage of DN4 cells were present. In wild-type mice after hydrocortisone-induced synchronisation of differentiation, the majority of DN4 cells that first emerged did not express icTCRbeta/gammadelta, showing that adult thymocytes can also differentiate to the DN4 stage independently of pre-TCR signalling. Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis. Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.

Show MeSH
Related in: MedlinePlus