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Delta-like 4 is indispensable in thymic environment specific for T cell development.

Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S - J. Exp. Med. (2008)

Bottom Line: Further analysis showed that the double-negative cell fraction was lacking T cell progenitors.The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4.These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Isehara 259-1193, Japan. hozumi@is.icc.u-tokai.ac.jp

ABSTRACT
The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

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No T cells, but substantial B cells, appear in the thymus with Dll4- epithelium. (A) Thymic cellularity (mean ± SD) of fetus (E15.5; left, n = 3; right, n = 5), neonate (N.B.; left, n = 5; right, n = 3), 4-wk-old (4W, n = 4), or 8-wk-old (8W, n = 3) Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed, shaded columns) mice compared with Dll4lox/lox (Dll4-floxed, open columns) mice. Asterisks indicate unpaired Student's t test; P < 0.001. (B) Flow cytometry of thymocytes from young adult (4W) Dll4lox/lox mice with (bottom, FoxN1Cre:Dll4-floxed) or without (top, Dll4-floxed) FoxN1-Cre transgene. Thymocytes were stained with monoclonal antibodies to surface molecules as shown. Numbers in the profiles indicate the relative percentages, in CD4−CD8− cells (right, CD4+CD8 vs. Thy1.2), for each corresponding quadrant or fraction. (C) The ETP population was not observed in Dll4lox/loxFoxN1-Cre (4W) mice. Thymocytes from Dll4lox/lox (top) or Dll4lox/loxFoxN1-Cre mice (bottom) were stained for lineage markers (Lin.: CD4, CD8, CD3, B220, CD19, Gr1, CD11b, TER119), CD44, CD25, CD24, and c-kit. Profiles are shown with the gate (Lin.−, middle; Lin.−CD44+CD25− as DN1, right). Numbers on the plots represent the frequency of cells lying in the indicated regions within the gate. The ETP population is identified as Lin.−CD44+CD25−CD24− or lowc-kit+ cells (DN1a+b). DN1c and DN1–3 populations are also shown in the plots. Data are representative of three experiments.
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fig4: No T cells, but substantial B cells, appear in the thymus with Dll4- epithelium. (A) Thymic cellularity (mean ± SD) of fetus (E15.5; left, n = 3; right, n = 5), neonate (N.B.; left, n = 5; right, n = 3), 4-wk-old (4W, n = 4), or 8-wk-old (8W, n = 3) Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed, shaded columns) mice compared with Dll4lox/lox (Dll4-floxed, open columns) mice. Asterisks indicate unpaired Student's t test; P < 0.001. (B) Flow cytometry of thymocytes from young adult (4W) Dll4lox/lox mice with (bottom, FoxN1Cre:Dll4-floxed) or without (top, Dll4-floxed) FoxN1-Cre transgene. Thymocytes were stained with monoclonal antibodies to surface molecules as shown. Numbers in the profiles indicate the relative percentages, in CD4−CD8− cells (right, CD4+CD8 vs. Thy1.2), for each corresponding quadrant or fraction. (C) The ETP population was not observed in Dll4lox/loxFoxN1-Cre (4W) mice. Thymocytes from Dll4lox/lox (top) or Dll4lox/loxFoxN1-Cre mice (bottom) were stained for lineage markers (Lin.: CD4, CD8, CD3, B220, CD19, Gr1, CD11b, TER119), CD44, CD25, CD24, and c-kit. Profiles are shown with the gate (Lin.−, middle; Lin.−CD44+CD25− as DN1, right). Numbers on the plots represent the frequency of cells lying in the indicated regions within the gate. The ETP population is identified as Lin.−CD44+CD25−CD24− or lowc-kit+ cells (DN1a+b). DN1c and DN1–3 populations are also shown in the plots. Data are representative of three experiments.

Mentions: Without the Dll4 expression in the TECs (FoxN1Cre:Dll4-floxed), the number of cells obtained from the thymus was significantly decreased (1/3–1/25) compared with that from the thymus in littermates (Dll4-floxed) during the developmental process (Fig. 4 A). Flow-cytometric analysis of 4-wk-old mouse thymus revealed that almost all cells were composed of CD4 and CD8 DN cells expressing neither Thy1, TCR αβ, nor γδ with CD3 in FoxN1Cre:Dll4-floxed mice, in comparison to the substantial presence of DP or CD4 and CD8 single-positive T cells expressing Thy1 and TCR–CD3 complex in Dll4-floxed mice (Fig. 4 B). These DN cells were mainly immature B cells expressing CD19 and, partially, surface IgM, and showed identical phenotype to that seen in BM, but not in the periphery (Fig. 4 B). If DN cells are subdivided into four fractions based on CD44 and CD25 expression, the DN2 and DN3 stages are defined as being CD25 highly positive with TCR gene recombinations. Although more than half of the DN cells belonged to DN2/3 in the control thymus, no CD25-positive cells were detected in FoxN1Cre:Dll4-floxed mice (Fig. 4 C, middle). It was recently reported that the DN1 stage was further distinguished into DN1a, b, and c by the expression of CD24 and c-kit (Fig. 4 C, right), showing that the DN1a+b fraction contained the earliest T cell progenitors (ETPs) and that DN1c possessed differentiation potential for B cell lineage (17). In the thymus of FoxN1Cre:Dll4-floxed mice, few DN1a+b cells, but a substantial amount of DN1c cells, were detected (Fig. 4 C). Similar phenotype was observed in the fetal thymus, where NK cells normally developed, and in the newborn thymus (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20080134/DC1). In peripheral lymphoid tissues such as spleen and lymph node of the mutant mice, only a few mature T cells were detected (Fig. S4). These findings first indicated that the Notch signaling induced by Dll4 on the thymic epithelium is essential for the intrathymic immigrant cells to differentiate into T lineage cells, and that the immigrants tend to differentiate into B lineage cells without the Dll4-mediated Notch signaling. They also indicated that the maintenance of DN1a+b immediately after HPCs have migrated, and further differentiation in the thymus, require the Notch signaling provided by Dll4.


Delta-like 4 is indispensable in thymic environment specific for T cell development.

Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S - J. Exp. Med. (2008)

No T cells, but substantial B cells, appear in the thymus with Dll4- epithelium. (A) Thymic cellularity (mean ± SD) of fetus (E15.5; left, n = 3; right, n = 5), neonate (N.B.; left, n = 5; right, n = 3), 4-wk-old (4W, n = 4), or 8-wk-old (8W, n = 3) Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed, shaded columns) mice compared with Dll4lox/lox (Dll4-floxed, open columns) mice. Asterisks indicate unpaired Student's t test; P < 0.001. (B) Flow cytometry of thymocytes from young adult (4W) Dll4lox/lox mice with (bottom, FoxN1Cre:Dll4-floxed) or without (top, Dll4-floxed) FoxN1-Cre transgene. Thymocytes were stained with monoclonal antibodies to surface molecules as shown. Numbers in the profiles indicate the relative percentages, in CD4−CD8− cells (right, CD4+CD8 vs. Thy1.2), for each corresponding quadrant or fraction. (C) The ETP population was not observed in Dll4lox/loxFoxN1-Cre (4W) mice. Thymocytes from Dll4lox/lox (top) or Dll4lox/loxFoxN1-Cre mice (bottom) were stained for lineage markers (Lin.: CD4, CD8, CD3, B220, CD19, Gr1, CD11b, TER119), CD44, CD25, CD24, and c-kit. Profiles are shown with the gate (Lin.−, middle; Lin.−CD44+CD25− as DN1, right). Numbers on the plots represent the frequency of cells lying in the indicated regions within the gate. The ETP population is identified as Lin.−CD44+CD25−CD24− or lowc-kit+ cells (DN1a+b). DN1c and DN1–3 populations are also shown in the plots. Data are representative of three experiments.
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Related In: Results  -  Collection

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Show All Figures
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fig4: No T cells, but substantial B cells, appear in the thymus with Dll4- epithelium. (A) Thymic cellularity (mean ± SD) of fetus (E15.5; left, n = 3; right, n = 5), neonate (N.B.; left, n = 5; right, n = 3), 4-wk-old (4W, n = 4), or 8-wk-old (8W, n = 3) Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed, shaded columns) mice compared with Dll4lox/lox (Dll4-floxed, open columns) mice. Asterisks indicate unpaired Student's t test; P < 0.001. (B) Flow cytometry of thymocytes from young adult (4W) Dll4lox/lox mice with (bottom, FoxN1Cre:Dll4-floxed) or without (top, Dll4-floxed) FoxN1-Cre transgene. Thymocytes were stained with monoclonal antibodies to surface molecules as shown. Numbers in the profiles indicate the relative percentages, in CD4−CD8− cells (right, CD4+CD8 vs. Thy1.2), for each corresponding quadrant or fraction. (C) The ETP population was not observed in Dll4lox/loxFoxN1-Cre (4W) mice. Thymocytes from Dll4lox/lox (top) or Dll4lox/loxFoxN1-Cre mice (bottom) were stained for lineage markers (Lin.: CD4, CD8, CD3, B220, CD19, Gr1, CD11b, TER119), CD44, CD25, CD24, and c-kit. Profiles are shown with the gate (Lin.−, middle; Lin.−CD44+CD25− as DN1, right). Numbers on the plots represent the frequency of cells lying in the indicated regions within the gate. The ETP population is identified as Lin.−CD44+CD25−CD24− or lowc-kit+ cells (DN1a+b). DN1c and DN1–3 populations are also shown in the plots. Data are representative of three experiments.
Mentions: Without the Dll4 expression in the TECs (FoxN1Cre:Dll4-floxed), the number of cells obtained from the thymus was significantly decreased (1/3–1/25) compared with that from the thymus in littermates (Dll4-floxed) during the developmental process (Fig. 4 A). Flow-cytometric analysis of 4-wk-old mouse thymus revealed that almost all cells were composed of CD4 and CD8 DN cells expressing neither Thy1, TCR αβ, nor γδ with CD3 in FoxN1Cre:Dll4-floxed mice, in comparison to the substantial presence of DP or CD4 and CD8 single-positive T cells expressing Thy1 and TCR–CD3 complex in Dll4-floxed mice (Fig. 4 B). These DN cells were mainly immature B cells expressing CD19 and, partially, surface IgM, and showed identical phenotype to that seen in BM, but not in the periphery (Fig. 4 B). If DN cells are subdivided into four fractions based on CD44 and CD25 expression, the DN2 and DN3 stages are defined as being CD25 highly positive with TCR gene recombinations. Although more than half of the DN cells belonged to DN2/3 in the control thymus, no CD25-positive cells were detected in FoxN1Cre:Dll4-floxed mice (Fig. 4 C, middle). It was recently reported that the DN1 stage was further distinguished into DN1a, b, and c by the expression of CD24 and c-kit (Fig. 4 C, right), showing that the DN1a+b fraction contained the earliest T cell progenitors (ETPs) and that DN1c possessed differentiation potential for B cell lineage (17). In the thymus of FoxN1Cre:Dll4-floxed mice, few DN1a+b cells, but a substantial amount of DN1c cells, were detected (Fig. 4 C). Similar phenotype was observed in the fetal thymus, where NK cells normally developed, and in the newborn thymus (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20080134/DC1). In peripheral lymphoid tissues such as spleen and lymph node of the mutant mice, only a few mature T cells were detected (Fig. S4). These findings first indicated that the Notch signaling induced by Dll4 on the thymic epithelium is essential for the intrathymic immigrant cells to differentiate into T lineage cells, and that the immigrants tend to differentiate into B lineage cells without the Dll4-mediated Notch signaling. They also indicated that the maintenance of DN1a+b immediately after HPCs have migrated, and further differentiation in the thymus, require the Notch signaling provided by Dll4.

Bottom Line: Further analysis showed that the double-negative cell fraction was lacking T cell progenitors.The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4.These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Isehara 259-1193, Japan. hozumi@is.icc.u-tokai.ac.jp

ABSTRACT
The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

Show MeSH
Related in: MedlinePlus