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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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Analysis of Rag1–/– thymocyte subsets. (A) CD44 and CD25 expression in E15.5 and adult Rag1–/– thymocytes. Top panel are FSC/SSC dot plots profiles of E15.5 and adult thymocytes; live gates are shown. Profiles in the lower panel show cells that fell in the live gate and were negative for anti-CD3/CD4/CD8 antibody staining. CD44 against CD25 dot plots and percentages of cells in each quadrant are given for E15.5 (left) and adult (right). (B) Graph representing the percentages of thymocytes in the DN3 and DN4 subsets in E15.5, E16.5, E17.5, juvenile (3-week-old) and adult Rag1–/– mice. (C) Absolute number of DN3 and DN4 cells in the E15.5, E16.5 and adult Rag1–/– thymi. Bars represent the mean of at least three mice. Cells were gated positive for Thy1.2 staining, negative for CD4, CD8 and CD44 expression and positive or negative for CD25 surface expression, respectively. (D) Expression of lymphocyte markers in E15.5 DN4 Rag1–/– thymocytes. Histograms show the expression of HSA, icCD3, NK1.1, B220 in CD44–CD25– cells and the percentage of CD44–CD25– cells positive for each marker are shown. Cells were gated negative for CD25, CD44, CD3, CD4 and CD8.
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fig02: Analysis of Rag1–/– thymocyte subsets. (A) CD44 and CD25 expression in E15.5 and adult Rag1–/– thymocytes. Top panel are FSC/SSC dot plots profiles of E15.5 and adult thymocytes; live gates are shown. Profiles in the lower panel show cells that fell in the live gate and were negative for anti-CD3/CD4/CD8 antibody staining. CD44 against CD25 dot plots and percentages of cells in each quadrant are given for E15.5 (left) and adult (right). (B) Graph representing the percentages of thymocytes in the DN3 and DN4 subsets in E15.5, E16.5, E17.5, juvenile (3-week-old) and adult Rag1–/– mice. (C) Absolute number of DN3 and DN4 cells in the E15.5, E16.5 and adult Rag1–/– thymi. Bars represent the mean of at least three mice. Cells were gated positive for Thy1.2 staining, negative for CD4, CD8 and CD44 expression and positive or negative for CD25 surface expression, respectively. (D) Expression of lymphocyte markers in E15.5 DN4 Rag1–/– thymocytes. Histograms show the expression of HSA, icCD3, NK1.1, B220 in CD44–CD25– cells and the percentage of CD44–CD25– cells positive for each marker are shown. Cells were gated negative for CD25, CD44, CD3, CD4 and CD8.

Mentions: The presence of icTCRβ– DN4 cells in the first wave of differentiation of wild-type foetal thymocytes led us to ask whether DN4 cells are present in Rag1–/– foetal thymi. We analysed Rag1–/– foetal thymi at E15.5, E16.5, E17.5, thymi at 3 weeks postnatally (juvenile) and adult thymi and observed a substantial DN4 population on all embryonic days tested (Fig. 2A, B), whereas the percentage of DN4 thymocytes was extremely small in the adult thymus of Rag1–/– mice as previously reported [10]. In a typical experiment, 31.12% of the thymocytes were DN4 on E15.5 compared to only 0.76% of the adult thymocytes. The percentage of DN4 thymocytes gradually decreased over time until they were virtually undetectable in the adult thymus (Fig. 2B). Correspondingly, the percentage of DN3 cells increased substantially from E15.5 to E16.5 and thereafter the thymus consisted mainly of DN3 thymocytes (Fig. 2B). As expected, no TCRβ expression or DP thymocytes were observed at any stage of embryonic or postnatal development in Rag1–/– mice (data not shown). Although the percentage of Rag1–/– DN4 thymocytes gradually decreased over time after E15.5, the absolute number of DN4 cells did not change appreciably, whereas the number of DN3 cells increased (Fig. 2C).


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)

Analysis of Rag1–/– thymocyte subsets. (A) CD44 and CD25 expression in E15.5 and adult Rag1–/– thymocytes. Top panel are FSC/SSC dot plots profiles of E15.5 and adult thymocytes; live gates are shown. Profiles in the lower panel show cells that fell in the live gate and were negative for anti-CD3/CD4/CD8 antibody staining. CD44 against CD25 dot plots and percentages of cells in each quadrant are given for E15.5 (left) and adult (right). (B) Graph representing the percentages of thymocytes in the DN3 and DN4 subsets in E15.5, E16.5, E17.5, juvenile (3-week-old) and adult Rag1–/– mice. (C) Absolute number of DN3 and DN4 cells in the E15.5, E16.5 and adult Rag1–/– thymi. Bars represent the mean of at least three mice. Cells were gated positive for Thy1.2 staining, negative for CD4, CD8 and CD44 expression and positive or negative for CD25 surface expression, respectively. (D) Expression of lymphocyte markers in E15.5 DN4 Rag1–/– thymocytes. Histograms show the expression of HSA, icCD3, NK1.1, B220 in CD44–CD25– cells and the percentage of CD44–CD25– cells positive for each marker are shown. Cells were gated negative for CD25, CD44, CD3, CD4 and CD8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Analysis of Rag1–/– thymocyte subsets. (A) CD44 and CD25 expression in E15.5 and adult Rag1–/– thymocytes. Top panel are FSC/SSC dot plots profiles of E15.5 and adult thymocytes; live gates are shown. Profiles in the lower panel show cells that fell in the live gate and were negative for anti-CD3/CD4/CD8 antibody staining. CD44 against CD25 dot plots and percentages of cells in each quadrant are given for E15.5 (left) and adult (right). (B) Graph representing the percentages of thymocytes in the DN3 and DN4 subsets in E15.5, E16.5, E17.5, juvenile (3-week-old) and adult Rag1–/– mice. (C) Absolute number of DN3 and DN4 cells in the E15.5, E16.5 and adult Rag1–/– thymi. Bars represent the mean of at least three mice. Cells were gated positive for Thy1.2 staining, negative for CD4, CD8 and CD44 expression and positive or negative for CD25 surface expression, respectively. (D) Expression of lymphocyte markers in E15.5 DN4 Rag1–/– thymocytes. Histograms show the expression of HSA, icCD3, NK1.1, B220 in CD44–CD25– cells and the percentage of CD44–CD25– cells positive for each marker are shown. Cells were gated negative for CD25, CD44, CD3, CD4 and CD8.
Mentions: The presence of icTCRβ– DN4 cells in the first wave of differentiation of wild-type foetal thymocytes led us to ask whether DN4 cells are present in Rag1–/– foetal thymi. We analysed Rag1–/– foetal thymi at E15.5, E16.5, E17.5, thymi at 3 weeks postnatally (juvenile) and adult thymi and observed a substantial DN4 population on all embryonic days tested (Fig. 2A, B), whereas the percentage of DN4 thymocytes was extremely small in the adult thymus of Rag1–/– mice as previously reported [10]. In a typical experiment, 31.12% of the thymocytes were DN4 on E15.5 compared to only 0.76% of the adult thymocytes. The percentage of DN4 thymocytes gradually decreased over time until they were virtually undetectable in the adult thymus (Fig. 2B). Correspondingly, the percentage of DN3 cells increased substantially from E15.5 to E16.5 and thereafter the thymus consisted mainly of DN3 thymocytes (Fig. 2B). As expected, no TCRβ expression or DP thymocytes were observed at any stage of embryonic or postnatal development in Rag1–/– mice (data not shown). Although the percentage of Rag1–/– DN4 thymocytes gradually decreased over time after E15.5, the absolute number of DN4 cells did not change appreciably, whereas the number of DN3 cells increased (Fig. 2C).

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