FADD/MORT1 regulates the pre-TCR checkpoint and can function as a tumour suppressor

Abstract

Productive rearrangement of the T-cell receptor (TCR) beta gene and signalling through the pre-TCR-CD3 complex are required for survival, proliferation and differentiation of T-cell progenitors (pro-T cells). Here we identify a role for death receptor signalling in early T-cell development using a dominant-negative mutant of the death receptor signal transducer FADD/MORT1 (FADD-DN). In rag-1(-/-) thymocytes, which are defective in antigen receptor gene rearrangement, FADD-DN bypassed the requirement for pre-TCR signalling, promoting pro-T-cell survival and differentiation to the more mature pre-T stage. Surprisingly, differentiation was not accompanied by the proliferation that occurs normally during transition to the pre-T stage. Consistent with a role for FADD/MORT1 in this cell division, FADD-DN rag-1(-/-) pro-T cells failed to proliferate in response to CD3epsilon ligation. Concomitant signalling through the pre-TCR and death receptors appears to trigger pro-T cell survival, proliferation and differentiation, whereas death receptor signalling in thymocytes that lack a pre-TCR induces apoptosis. Later in life all FADD-DN rag-1(-/-) mice developed thymic lymphoma, indicating that FADD/MORT1 can act as a tumour suppressor.

Fig. 1. Expression of death receptors, death ligands and their signalling molecules in thymocyte subsets. (A) Pro–T3, pro–T4, pre–T (divided into large and small cells on the basis of their forward light scatter) and thymic stromal cells from 6- to 10-week-old rag–1^–/– and FADD–DN rag–1^–/– mice were analysed for the presence of specific RNA transcripts by RT–PCR. PCR was performed on 5–fold serial dilutions of cDNA and the amplification products were probed with a ³²P–labelled internal oligonucleotide. (B and C) Pro–T3, pro–T4 and pre–T cells from FADD–DN rag–1^+/+ mice (B) and pro–T cells from FADD–DN rag–1^–/– mice (C) were analysed for FADD–DN expression by cytoplasmic immunofluorescence and flow cytometry using anti-FLAG antibody. Solid lines represent FADD–DN transgenic cells and dotted lines represent wild-type (B) or rag–1^–/– (C) cells. Profiles are representative of analyses of three mice of each genotype.

Fig. 2. FADD–DN promotes development of pro–T4 and pre–T cells in rag–1^–/– mice. (A and B) Flow cytometric analysis of thymocytes from 5- to 13-week-old rag–1^+/+, rag–1^–/–, lpr rag–1^–/– and FADD–DN rag–1^–/– mice. Profiles in (A) show expression of CD4 and CD8. Profiles in (B) show expression of CD25 and CD44 after gating on Thy–1⁺CD3^–4^–8^– cells. CD25 staining varies due to different instrument settings. Total thymic cellularity (C) and CD4⁺8⁺ pre–T–cell content (D) were determined by cell counting and flow cytometric analysis of thymocytes stained with antibodies to CD4 and CD8. Each symbol represents one mouse. (E) Purified pro–T3 and pro–T4 cells from rag–1^+/+, rag–1^–/– and FADD–DN rag–1^–/– mice were analysed for their cell cycle distribution.

Fig. 3. FADD–DN diminishes CD3ɛ ligation-induced production of CD4⁺8⁺ pre–T cells in rag–1^–/– mice. (A) Flow cytometric analysis of thymocytes from 5- to 13–week-old rag–1^–/–, lpr rag–1^–/– and FADD–DN rag–1^–/– mice after intraperitoneal injection with 100 μg of 145-2C11 hamster anti-mouse CD3ɛ monoclonal antibody. Total thymic cellularity (B) and CD4⁺8⁺ pre–T–cell content (C) were determined by cell counting and flow cytometric analysis of thymocytes stained with antibodies to CD4 and CD8. Each symbol represents one mouse.

Fig. 4. FADD–DN does not affect γ-irradiation-induced production of CD4⁺8⁺ pre–T cells in rag–1^–/– mice. (A) Flow cytometric analysis of thymocytes from 5- to 10-week-old rag–1^–/–, lpr rag–1^–/– and FADD–DN rag–1^–/– mice exposed to 5 Gy of γ-irradiation. Total thymic cellularity (B) and CD4⁺8⁺ pre–T–cell content (C) were determined by cell counting and flow cytometric analysis of thymocytes stained with antibodies to CD4 and CD8. Each symbol represents one mouse.

Fig. 5. FADD–DN rag–1^–/– mice develop thymic lymphoma. (A) The incidence of thymic lymphoma in rag–1^–/–, lpr rag–1^–/–, FADD–DN rag–1^–/+, FADD–DN rag–1^+/+ and FADD–DN rag–1^–/– mice that did not develop heart disease. (B) A 19-week-old FADD–DN rag–1^–/– mouse with thymic lymphoma (left) and a healthy rag–1^–/– littermate (right). (C) Thymus from a FADD–DN rag–1^–/– mouse with thymic lymphoma (right) and from a healthy rag–1^–/– littermate (left). (D–G) Histological sections of the thymus (D), spleen (E), liver (F) and kidney (G) from a FADD–DN rag–1^–/– mouse with thymic lymphoma were stained with haematoxylin and eosin. Photographs were taken at 200× (D) or 100× (E–G) magnification. (H) Flow cytometric analysis of cells from a typical FADD–DN rag–1^–/– thymoma.

Fig. 6. The response of FADD–DN rag–1^–/– thymoma cell lines to FasL and γ–irradiation. (A–C) FADD–DN rag–1^–/– thymoma cell lines were cultured with 20 ng/ml FLAG-tagged FasL cross-linked with 1 μg/ml anti-FLAG monoclonal antibody (A), 1 μM dexamethasone (B), or were exposed to 2.5 Gy γ-radiation (C). Cell survival was determined by flow cytometric analysis of PI-stained cells. Survival is shown as a percentage of that in culture medium without treatment. Cells cultured with anti-FLAG antibody alone were indistinguishable from cells cultured in plain medium. (D) FR D71 cells were examined for surface expression of Fas. The solid line represents cells stained with Jo2 anti-Fas monoclonal antibody, and the dotted line represents cells stained with an isotype-matched control antibody. Similar results were obtained with cell lines derived from five other FADD–DN rag–1^–/– mice. (E) Western blot analysis of p53 expression in FR C37 cells after a 5 Gy dose of γ-radiation (2.5 × 10⁶ cell equivalents/well). The blot was probed with a cocktail of monoclonal antibodies to wild-type p53, or as a loading control, with a monoclonal antibody that recognizes mouse HSP-70. Similar results were obtained with the cell line FR C24.

Fig. 7. Model for death receptor-controlled apoptosis and cell production at the pre–TCR checkpoint. Death receptors on CD4^–8^–25⁺44^– pro–T3 cells can signal apoptosis or proliferation depending on the presence or absence of signals from the pre–TCR. (A) In the absence of a signal from the pre–TCR, such as in cells with non-productive TCRβ gene rearrangements, pro–T3 cells die by apoptosis. (B) Expression of a pre–TCR blocks the apoptotic signal from death receptors, thereby allowing cell proliferation and differentiation. Efficient pro–T4 and pre–T–cell generation probably requires signals from death receptors and the pre–TCR.