T-cell receptor complex is essential for Fas signal transduction

Abstract

The Fas receptor (also known as CD95 and APO-1) is a member of the tumor necrosis factor alpha-family of death receptors that mediate T-cell responses. Here, we show that Fas receptor signaling requires a functional T-cell receptor (TCR) complex. Fas receptor directly binds to and activates TCR components in a stimulus-dependent manner. Fas receptor stimulation does not activate canonical downstream TCR pathways, but instead the TCR complex is required specifically for Fas-mediated calcium release. Importantly, null mutations in Lck, ZAP70, and the TCR alpha- and beta-chains abrogate Fas signaling. Our results reveal a direct role for the TCR complex in mediating Fas-specific signaling events critical for T-cell homeostasis.

Fig. 1.

Lck is required for Fas ligand-mediated calcium release and cell death in Jurkat cells. (A) PLC-γ1, Lck/Fyn (pSrc), and ZAP70 activation in wild-type and Lck-null Jurkat cells after Fas ligand (FasL) vesicle stimulation as determined by phospho-specific antibodies. (B) Fas-dependent calcium release in wild-type, Lck-null, and Lck-null Jurkat cells stably expressing Lck (Lck rescue). Shown are single-cell responses representative of hundreds of determinations. (C) Caspase 3 activity in wild-type, Lck-null, and Lck rescue Jurkat cells 12 and 24 h after Fas ligand stimulation, relative to untreated cells. (D) Time-dependent increases in cytosolic calcium concentration in wild-type, Lck-null, and Lck rescued cells. (E) Cell death (propidium iodide–positive cells as a percentage of the total) in wild-type, Lck-null, and Lck rescued Jurkat cells. (F) Coimmunoprecipitation of PLC-γ1 and Lck with monoclonal anti-Fas receptor antibody CH11. Total lysates (1/5 load) are depicted as input. (G) Sucrose step gradient centrifugation of lysates prepared from Jurkat cells. The lipid raft fractions (9, 10) were confirmed by dot blotting with the GM1 ganglioside binding-protein cholera toxin-B subunit. (H) PLC-γ1, Lck/Fyn, and ZAP70 activation in primary murine CD4⁺ cells after Fas ligand stimulation. (I) Fas-dependent calcium release in primary murine CD4⁺ cells. Approximately 20% of primary CD4⁺ cells responded to Fas ligand stimulation. This variability in response to Fas ligand stimulation is likely the result of multiple subsets of CD4⁺ cells isolated (e.g., naive, memory, and effector cells).

Fig. 2.

ZAP70 is required for Fas ligand-mediated PLC-γ1 activation and calcium release in Jurkat cells. (A) PLC-γ1, Lck/Fyn, and ZAP70 activation in wild-type and ZAP70-null Jurkat cells (B) Fas-dependent calcium release in wild-type, ZAP70-null, and ZAP70 rescued Jurkat cells. Red trace is ZAP70-null, blue trace is ZAP70 rescue. Wild-type Jurkat responses are not shown, but were performed in parallel as a positive control. (C) Caspase 3 activity in wild-type, ZAP70-null, and ZAP70 rescue Jurkat cells 12 and 24 h after Fas ligand stimulation, relative to untreated cells. (D) Time-dependent increases in cytosolic calcium concentration in wild-type, ZAP70-null, and ZAP70 rescue Jurkat cells. (E) Cell death in wild-type, ZAP70-null, and ZAP70 rescue Jurkat cells.

Fig. 3.

The TCR β-chain is required for Fas ligand-mediated PLC-γ1 activation and calcium release in Jurkat cells. (A) PLC-γ1, Lck/Fyn, and ZAP70 activation in wild-type and TCR-β-null Jurkat cells after Fas ligand stimulation. (B) Fas-dependent calcium release in TCR-β-null and TCR-β rescue Jurkat cells. Red trace is TCR-β-null, blue trace is TCR-β rescue. As in Fig. 2, wild-type Jurkat responses were performed in parallel but are not shown for clarity. (C) Caspase 3 activity in wild-type and TCR-β-null Jurkat cells 12 and 24 h after Fas ligand stimulation relative to untreated cells. (D) Time-dependent increases in cytosolic calcium concentration in wild-type and TCR-β-null Jurkat cells. (E) Cell death in wild-type and TCR-β-null Jurkat cells. (F) Erk1/2, Lck/Fyn, and PLC-γ1 activation upon Fas ligand, PMA/ionomycin and anti-CD3 antibody stimulation. (G) Production of IL-2 in Jurkat cells activated with anti-CD3 antibody or Fas ligand. IL-2 was measured by ELISA 12 h following cell stimulation. (H) CD69 expression flow-cytometry profile of Jurkat cells stimulated with Fas ligand or anti-CD3 antibody for 12 h.

Fig. 4.

Rearrangements at the TRA@ locus associated with acute lymphoblastic leukemia (T-ALL) leads to defective Fas ligand-mediated calcium release and cell death. (A) Fas receptor expression by flow cytometry of Jurkat and T-ALL cell line Sup-T1. The majority of Sup-T1 cells express higher levels of Fas receptor, A small population (∼15%) express lower levels of Fas receptor. (B) PLC-γ1, Lck/Fyn, and ZAP70 activation in Jurkat and Sup-T1 cells. (C) Fas ligand-dependent calcium release in Sup-T1 cells and Sup-T1 cells expressing TCR α- and β-chains. (D) Caspase 3 activity in Jurkat and Sup-T1 cells 12 and 24 h after Fas ligand stimulation relative to untreated cells. (E) Cell death in Jurkat, Sup-T1 cells, and Sup-T1 cells expressing TCR α- and β-chains 12 and 24 h after Fas ligand stimulation.