Interferon gamma is required for activation-induced death of T lymphocytes - PubMed (original) (raw)

Interferon gamma is required for activation-induced death of T lymphocytes

Yosef Refaeli et al. J Exp Med. 2002.

Erratum in

Abstract

The effector cytokine interferon gamma (IFN-gamma) may play a role in T cell homeostasis. We have examined the requirement for IFN-gamma in one mechanism that regulates T cell expansion and survival, activation-induced cell death (AICD). CD4(+) T cells lacking IFN-gamma or the Stat1 transcription factor are resistant to AICD. IFN-gamma is required for the production of caspases, and retrovirus-mediated expression of caspase-8 restores the sensitivity of Stat1-deficient T cells to AICD. In vitro, IFN-gamma limits the expansion of T cells that are stimulated through their antigen receptors. Thus, IFN-gamma may function to control the expansion and persistence of T cells by promoting caspase-8-dependent apoptosis.

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Figures

Figure 4.

Figure 4.

IFN-γ regulates the number of cells following antigenic stimulation by affecting apoptosis but not cell division and proliferation. CD4+ T cells were purified from pooled spleen and lymph node suspensions obtained from either C57BL/6 (wild type), IFN-γ−/− mice, or Stat-1−/− mice. (A) The cells were cultured with anti-CD3 and anti-CD28 antibodies for 4 d, rested in medium plus cytokines, as indicated, restimulated with soluble anti-CD3 antibodies at the indicated concentrations. These cultures were pulsed with [H3]thymidine for 12 h, 2 d after initial activation. (B) Freshly isolated cells were labeled with CFSE, activated with antibodies to CD3 and CD28, in the presence of exogenously added cytokines, as indicated. The activated T cells were analyzed by flow cytometry four days after activation.

Figure 1.

Figure 1.

IL-2 and IFN-γ independently regulate AICD. CD4+ T cells were purified from pooled spleen and lymph node suspensions obtained from C57BL/6 (WT), IFN-γ−/−, or Stat1−/− mice (A–C), or from 3A9/+ (wild-type) and 3A9/IL-2−/− mice (D–F). These cells were activated in vitro with anti-CD3 and anti-CD28, with the following cytokines added to the cultures: none (A and D), IL-2 (50 U/ml; B and E), or IFN-γ (10 U/ml; C and F). Activated cells were collected 3 d later, and incubated on anti-CD3 coated plates in the presence of IL-2. Apoptosis was determined by Propidium Iodide staining. The same cells were analyzed for the expression of FasL (G) and Fas (H) by staining and flow cytometry. The filled histograms in G represent resting cells and the empty histograms represent activated T cells. The filled histograms in H represent cells that were stained with an isotype-matched control antibody and the empty histograms represent cells that were stained with anti-Fas.

Figure 2.

Figure 2.

IFN-γ and Stat1 increase the levels of caspase transcripts. CD4+ T cells were purified from pooled lymph node and spleen suspensions obtained from C57BL6 (WT), IFN-γ−/−, Stat1−/−, 3A9/+, or 3A9/IL-2−/− mice. These cells were activated with antibodies to CD3 and CD28, with no added cytokine, or with added IFN-γ. Activated cells were collected after 3 d, and mRNA for caspases was assayed by RNase protection. The protected species were resolved on a TBE/Urea gel, and the bands quantified using a PhosphorImager, a representative example is shown in A. Levels of mRNA (B and C) were calculated by averaging the two bands for the housekeeping genes (L32 and GAPDH), and determining the percentage of the value for each of the caspase probes. The data were pooled from two independent experiments. (D) The levels of caspase-8 protein in CD4+ T cells from C57BL-6 (WT), IFN-γ−/−, and Stat1−/− mice activated for 1 or 3 d with no added cytokine or with IFN-γ (10 U/ml) were determined by Western blot analysis.

Figure 3.

Figure 3.

Retrovirally transduced caspase-8 is able to correct the AICD defect observed in Stat1−/− T cells. (A) CD4+ T cells were purified from pooled spleen and lymph node suspensions obtained from either C57BL-6 (wt), or Stat1−/− mice. These cells were activated and infected in vitro with bicistronic retroviral vectors encoding caspase-8 (two different clones, 8–7 and 8–15) or empty vector (pMIG). The infected cells were then incubated on anti-CD3 coated plates for 20 h to induce AICD, and apoptosis was assayed in GFP+ cells by flow cytometry. The data presented here were obtained from averaging the results of triplicate wells, from one experiment representative of three. (B) The specificity of the requirement for caspase-8 was determined by retrovirally transducing CD4+ T cells from either C57BL/6 (wt), or Stat1−/− mice with viruses encoding caspase-3 (Casp3), caspase-8 (Casp8), or caspase-9 (Casp9), and processed as in A. (C) The levels of caspase-8 expression in retrovirus-infected activated C57BL/6 (WT) and Stat1−/− cells were verified by Western blot analysis.

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