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An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis.

Hettmann T, DiDonato J, Karin M, Leiden JM - J. Exp. Med. (1999)

Bottom Line: However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals.The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor.Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Pathology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
To examine the role of nuclear factor (NF)-kappaB in T cell development and activation in vivo, we produced transgenic mice that express a superinhibitory mutant form of inhibitor kappaB-alpha (IkappaB-alphaA32/36) under the control of the T cell-specific CD2 promoter and enhancer (mutant [m]IkappaB-alpha mice). Thymocyte development proceeded normally in the mIkappaB-alpha mice. However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals. The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4(+)CD8(+); DP) thymocytes from the mIkappaB-alpha mice were resistant to alpha-CD3-mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by gamma-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mIkappaB-alpha thymocytes maintained high level expression of bcl-xL after alpha-CD3 treatment. Taken together, these results demonstrated important roles for NF-kappaB in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-kappaB is required for the alpha-CD3-mediated apoptosis of DP thymocytes through a pathway that involves the regulation of the antiapoptotic gene, bcl-xL.

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T cell development in mIκB-α transgenic mice. Single cell suspensions of thymocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice  were stained with PE–α-CD4 and FITC–α-CD8 (A), or PE–α-TCR-α/β and FITC–α-CD3 (B) antibodies and analyzed by flow cytometry. Percentages of  cells in the individual subpopulations are shown in the lower right quadrant. (C) Splenocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice were  stained with 7-amino actinomycin D, PE–α-CD4, and FITC–α-CD8, and were analyzed by flow cytometry. Total numbers (× 106 cells) of live CD4+ and  CD8+ splenic T cells were determined from at least three mice in each group. The data is shown as mean ± SD.
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Figure 3: T cell development in mIκB-α transgenic mice. Single cell suspensions of thymocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice were stained with PE–α-CD4 and FITC–α-CD8 (A), or PE–α-TCR-α/β and FITC–α-CD3 (B) antibodies and analyzed by flow cytometry. Percentages of cells in the individual subpopulations are shown in the lower right quadrant. (C) Splenocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice were stained with 7-amino actinomycin D, PE–α-CD4, and FITC–α-CD8, and were analyzed by flow cytometry. Total numbers (× 106 cells) of live CD4+ and CD8+ splenic T cells were determined from at least three mice in each group. The data is shown as mean ± SD.

Mentions: To investigate T cell development in the mIκB-α mice, thymocytes and peripheral T cells from transgenic and wild-type animals were analyzed by flow cytometry using antibodies specific for a variety of developmentally regulated T cell surface antigens (Fig. 3). Thymi from the mIκB-α animals contained normal populations of DN, DP, and SP cells. Levels of CD3 and TCR-α/β expression were also indistinguishable on wild-type and mIκB-α thymocytes (Fig. 3 B), as were expression of TCR-γ/δ and heat-shock antigen (data not shown). Thus, expression of IκB-αA32/36 did not appear to perturb thymocyte ontogeny. Total numbers of SP peripheral T cells in both the spleen and the lymph nodes were normal in mIκB-α mice as were the numbers of splenic CD4+ T cells. However, as described previously by Boothby at al. and Esslinger et al. (33, 46), the numbers of peripheral CD8+ T cells were reduced by ∼50% in mIκB-α mice (Fig. 3 C).


An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis.

Hettmann T, DiDonato J, Karin M, Leiden JM - J. Exp. Med. (1999)

T cell development in mIκB-α transgenic mice. Single cell suspensions of thymocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice  were stained with PE–α-CD4 and FITC–α-CD8 (A), or PE–α-TCR-α/β and FITC–α-CD3 (B) antibodies and analyzed by flow cytometry. Percentages of  cells in the individual subpopulations are shown in the lower right quadrant. (C) Splenocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice were  stained with 7-amino actinomycin D, PE–α-CD4, and FITC–α-CD8, and were analyzed by flow cytometry. Total numbers (× 106 cells) of live CD4+ and  CD8+ splenic T cells were determined from at least three mice in each group. The data is shown as mean ± SD.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887697&req=5

Figure 3: T cell development in mIκB-α transgenic mice. Single cell suspensions of thymocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice were stained with PE–α-CD4 and FITC–α-CD8 (A), or PE–α-TCR-α/β and FITC–α-CD3 (B) antibodies and analyzed by flow cytometry. Percentages of cells in the individual subpopulations are shown in the lower right quadrant. (C) Splenocytes from mIκB-α transgenic (Tg) and wild-type (WT) mice were stained with 7-amino actinomycin D, PE–α-CD4, and FITC–α-CD8, and were analyzed by flow cytometry. Total numbers (× 106 cells) of live CD4+ and CD8+ splenic T cells were determined from at least three mice in each group. The data is shown as mean ± SD.
Mentions: To investigate T cell development in the mIκB-α mice, thymocytes and peripheral T cells from transgenic and wild-type animals were analyzed by flow cytometry using antibodies specific for a variety of developmentally regulated T cell surface antigens (Fig. 3). Thymi from the mIκB-α animals contained normal populations of DN, DP, and SP cells. Levels of CD3 and TCR-α/β expression were also indistinguishable on wild-type and mIκB-α thymocytes (Fig. 3 B), as were expression of TCR-γ/δ and heat-shock antigen (data not shown). Thus, expression of IκB-αA32/36 did not appear to perturb thymocyte ontogeny. Total numbers of SP peripheral T cells in both the spleen and the lymph nodes were normal in mIκB-α mice as were the numbers of splenic CD4+ T cells. However, as described previously by Boothby at al. and Esslinger et al. (33, 46), the numbers of peripheral CD8+ T cells were reduced by ∼50% in mIκB-α mice (Fig. 3 C).

Bottom Line: However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals.The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor.Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Pathology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
To examine the role of nuclear factor (NF)-kappaB in T cell development and activation in vivo, we produced transgenic mice that express a superinhibitory mutant form of inhibitor kappaB-alpha (IkappaB-alphaA32/36) under the control of the T cell-specific CD2 promoter and enhancer (mutant [m]IkappaB-alpha mice). Thymocyte development proceeded normally in the mIkappaB-alpha mice. However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals. The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4(+)CD8(+); DP) thymocytes from the mIkappaB-alpha mice were resistant to alpha-CD3-mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by gamma-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mIkappaB-alpha thymocytes maintained high level expression of bcl-xL after alpha-CD3 treatment. Taken together, these results demonstrated important roles for NF-kappaB in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-kappaB is required for the alpha-CD3-mediated apoptosis of DP thymocytes through a pathway that involves the regulation of the antiapoptotic gene, bcl-xL.

Show MeSH
Related in: MedlinePlus