Bad can act as a key regulator of T cell apoptosis and T cell development - PubMed (original) (raw)

Bad can act as a key regulator of T cell apoptosis and T cell development

C L Mok et al. J Exp Med. 1999.

Abstract

Bad is a distant relative of Bcl-2 and acts to promote cell death. Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis. We generated bad transgenic mice to study the action of upregulated Bad expression on T cell apoptosis. The T cells from these mice are highly sensitive to apoptotic stimuli, including anti-CD95. The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed. We show that the proapoptotic function of Bad in primary T cells is regulated by Akt kinase and that Bad overexpression enhances both cell cycle progression and interleukin 2 production after T cell activation. These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.

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Figures

Figure 1

Upregulation of Bad expression during thymocyte apoptosis. Total cell lysate from 107 thymocytes per lane was resolved by SDS PAGE, Western blotted, and Bad expression detected by probing with anti-Bad antibody. Cell lysate was prepared from thymocytes immediately after removal of the thymus from C57 Bl/10 mice (Control, 0 h). Lysates were also prepared from thymocytes 5 h after γ-radiation, 5 h with 5 μM dexamethasone, or 5 h in culture alone. The percent apoptosis in the samples before cell lysate preparation is shown. The positive control for Bad expression is a thymocyte lysate from a bad transgenic mouse; equal numbers of cells were not added in this lane. Tubulin expression is a loading control to show that each track contains approximately equivalent amounts of protein.

Figure 2

bad transgene. (A) The mouse bad cDNA including a 5′ HA epitope was cloned into the EcoRI site of the human CD2 VA expression vector. The SalI-XbaI fragment was then isolated for microinjection. Western blot analysis of transgene expression in the two transgenic lines studied was carried out using total cell extract of thymocytes. Equal amounts of protein were loaded in each lane. (B) The blot was probed with the 12CA5 mAb against HA, to detect the presence of the transgene and (C) with a polyclonal anti-Bad antibody to compare the levels of endogenous and transgenic Bad expression.

Figure 2

Figure 3

bad transgenic mice have decreased numbers of mature α/β T cells. (A) FACS® analysis of CD4 vs. CD8 expression of thymocytes and (B) splenocytes from bad transgenic and nontransgenic littermates. Cells were stained with specific anti-CD4 and anti-CD8 antibodies. The subpopulations of CD8+CD4+, CD4+CD8−, and CD4−CD8+ T cells are gated and their percentages given. (C) Histogram indicating the percentage of TCR-γ/δ expressing cells within the CD4−CD8− thymocytes of bad transgenic and nontransgenic littermates. The numbers in parentheses indicate the total number of TCR-γ/δ expressing cells in each thymus.

Figure 3

Figure 4

bad transgenic mice exhibit increased levels of apoptosis. (A) Total thymocytes from bad and nontransgenic (WT) littermates were cultured in vitro for 6 h. Duplicate samples were analyzed from each mouse at each time point indicated. Each value represents the mean ± range of the duplicate determinations. Two individual mice per phenotype are represented. The (+) indicates the presence of the apoptotic stimulus and the (−) indicates its absence. The numbers 1 and 3 refer to mice from the independent bad transgenic lines 1 and 3, respectively. Similar studies were carried out after treatment with either (B) 5 Gy γ-irradiation, (C) 5 μM dexamethasone, or (D) anti-CD95 antibody, together with untreated controls. Similar results were obtained in three independent experiments.

Figure 4

Figure 5

bcl-2 expression partially rescues thymocytes from the proapoptotic effects of bad. Total thymocytes from bad, bcl-2, and bad/bcl-2 heterozygous double transgenic mice were cultured in vitro over 8 h and samples processed at 4-h intervals to determine the percentage of apoptotic cells.

Figure 6

bad perturbs T cell selection. Thymocytes and splenocytes were isolated from F5/bad/RAG-1−/− and F5/RAG-1−/− mice, and subsequently stained with antibodies specific for CD8, CD4, and Vβ11. (A) Dot plots showing CD8 vs. CD4 expression on thymocytes with the percentages of each subpopulation indicated. Vβ11 expression in the gated regions, CD8+CD4+, CD8hiCD4lo, and CD8+CD4−, is shown in the histograms. The shaded region represents F5/RAG-1−/− mice and the solid line represents F5/bad/RAG-1−/− mice. (B) Similarly for splenocytes from mice of the same genotypes, CD4 vs. CD8 expression is shown in the dot plots with a gate on the CD8+CD4− population. Vβ11 expression in the CD8+CD4− cells is shown in the histogram below.

Figure 6

Figure 7

Inhibition of Akt accelerates _bad_-induced apoptosis. (A) Total thymocytes from bad transgenic mice and control littermates of both transgenic lines were cultured in vitro in the presence of 2 μM wortmannin (irreversible inhibitor of PI-3-K) and the percentage of apoptotic cells at each time point was determined as before. (B) Similar studies were carried out with 20 μM LY294002 (competitive inhibitor of PI-3-K).

Figure 7

Figure 8

Akt kinase activity in bad thymocytes is constitutively higher and is inhibited by wortmannin. Total cell lysates were prepared from thymocytes of bad transgenic mice and control littermates which had been cultured for up to 4 h. Akt was immunoprecipitated and an in vitro kinase assay was carried out using histone H2B as substrate. (A) The constitutive (0 h) level of kinase activity and the activity versus time of the nontransgenic control is shown in the left panel, and that of the bad transgenic mice in the right panel. (B) Akt kinase activity of bad transgenic thymocytes is shown in the absence (left) or presence (right) of 2 μM wortmannin over 4 h.

Figure 8

Figure 9

bad transgenic T cells produce higher levels of IL-2 on stimulation with anti-CD3 antibody. T cells purified from the lymph nodes of bad, bcl-2 transgenic mice, and nontransgenic mice were activated with serial dilution of cross-linked anti-CD3 antibody. IL-2 production was determined by CTLL assay and [3H]thymidine incorporation.

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Bad can act as a key regulator of T cell apoptosis and T cell development - PubMed (original) (raw)