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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 in DN4 population after HC treatment. (A) The left panel is the percentages of icTCRβ–/γδ– and icTCRβ+ DN4 cells positive for high level of intracellular expression of cyclin B1, indicative of cells at the S/G2/M cell cycle stages. Bars represent the mean of five mice analysed 3 days (open bars) and 4 days (black bars) after HC treatment. The right panel is representative histogram overlay of intracellular cyclin B1 staining of icTCRβ–/γδ– (full histogram) and icTCRβ+ (empty histogram) DN4 cells 4 days after HC treatment. (B) Intracellular expression of the active form of caspase-3 in DN4 thymocytes after HC treatment. Histograms show caspase-3 expression in TCRβ–/γδ– (shaded histograms) and TCRβ+ (open histograms) DN4 thymocytes after 4 days (left), 6 days (middle) and 7 days (right). Percentages of active caspase-3-positive cells are shown.
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fig06: Proliferation and apoptosis in DN4 population after HC treatment. (A) The left panel is the percentages of icTCRβ–/γδ– and icTCRβ+ DN4 cells positive for high level of intracellular expression of cyclin B1, indicative of cells at the S/G2/M cell cycle stages. Bars represent the mean of five mice analysed 3 days (open bars) and 4 days (black bars) after HC treatment. The right panel is representative histogram overlay of intracellular cyclin B1 staining of icTCRβ–/γδ– (full histogram) and icTCRβ+ (empty histogram) DN4 cells 4 days after HC treatment. (B) Intracellular expression of the active form of caspase-3 in DN4 thymocytes after HC treatment. Histograms show caspase-3 expression in TCRβ–/γδ– (shaded histograms) and TCRβ+ (open histograms) DN4 thymocytes after 4 days (left), 6 days (middle) and 7 days (right). Percentages of active caspase-3-positive cells are shown.

Mentions: To assess cell cycle status in the DN4 population, we compared cyclin B1 expression in the TCRβ–/γδ– and TCRβ+ DN4 thymocytes at 3 and 4 days after HC treatment (Fig. 6A). Approximately four times more cells in the TCRβ+ DN4 compartment showed high intensity cyclin B1 staining (Fig. 6A), indicative of cells in the S/G2/M stages of the cell cycle, than in the TCRβ–/γδ– DN4 population, indicating that TCRβ expression (and hence pre-TCR signalling) induces expansion.


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 in DN4 population after HC treatment. (A) The left panel is the percentages of icTCRβ–/γδ– and icTCRβ+ DN4 cells positive for high level of intracellular expression of cyclin B1, indicative of cells at the S/G2/M cell cycle stages. Bars represent the mean of five mice analysed 3 days (open bars) and 4 days (black bars) after HC treatment. The right panel is representative histogram overlay of intracellular cyclin B1 staining of icTCRβ–/γδ– (full histogram) and icTCRβ+ (empty histogram) DN4 cells 4 days after HC treatment. (B) Intracellular expression of the active form of caspase-3 in DN4 thymocytes after HC treatment. Histograms show caspase-3 expression in TCRβ–/γδ– (shaded histograms) and TCRβ+ (open histograms) DN4 thymocytes after 4 days (left), 6 days (middle) and 7 days (right). 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

fig06: Proliferation and apoptosis in DN4 population after HC treatment. (A) The left panel is the percentages of icTCRβ–/γδ– and icTCRβ+ DN4 cells positive for high level of intracellular expression of cyclin B1, indicative of cells at the S/G2/M cell cycle stages. Bars represent the mean of five mice analysed 3 days (open bars) and 4 days (black bars) after HC treatment. The right panel is representative histogram overlay of intracellular cyclin B1 staining of icTCRβ–/γδ– (full histogram) and icTCRβ+ (empty histogram) DN4 cells 4 days after HC treatment. (B) Intracellular expression of the active form of caspase-3 in DN4 thymocytes after HC treatment. Histograms show caspase-3 expression in TCRβ–/γδ– (shaded histograms) and TCRβ+ (open histograms) DN4 thymocytes after 4 days (left), 6 days (middle) and 7 days (right). Percentages of active caspase-3-positive cells are shown.
Mentions: To assess cell cycle status in the DN4 population, we compared cyclin B1 expression in the TCRβ–/γδ– and TCRβ+ DN4 thymocytes at 3 and 4 days after HC treatment (Fig. 6A). Approximately four times more cells in the TCRβ+ DN4 compartment showed high intensity cyclin B1 staining (Fig. 6A), indicative of cells in the S/G2/M stages of the cell cycle, than in the TCRβ–/γδ– DN4 population, indicating that TCRβ expression (and hence pre-TCR signalling) induces expansion.

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