Conditional Fas-associated death domain protein (FADD): GFP knockout mice reveal FADD is dispensable in thymic development but essential in peripheral T cell homeostasis

Abstract

Fas-associated death domain protein (FADD)/mediator of receptor-induced toxicity-1 is required for signaling induced by death receptors such as Fas. In earlier studies, FADD-deficient mice died in utero, and a FADD deficiency in embryonic stem cells inhibited T cell production in viable FADD-/- -->RAG-1-/- chimeras. To analyze the temporal requirement of FADD in the development and function in the T lineage, it is necessary to establish viable mutant mice producing detectable FADD-deficient T cells. We generated mice that express a functional FADD:GFP fusion gene reconstituting normal embryogenesis and lymphopoiesis in the absence of the endogenous FADD. Efficient T cell-specific deletion of FADD:GFP was achieved, as indicated by the presence of a high percentage of GFP-negative thymocytes and peripheral T cells in mice expressing Lck-Cre or CD4-Cre. Sorted GFP-negative thymocytes and peripheral T cells contained undetectable levels of FADD and were resistant to apoptosis induced by Fas, TNF, and TCR restimulation. These T cell-specific FADD-deficient mice contain normal thymocyte numbers, but fewer peripheral T cells. Purified peripheral FADD-deficient T cells failed to undergo extensive homeostatic expansion after adoptive transfer into lymphocyte-deficient hosts, and responded poorly to proliferation induced by ex vivo TCR stimulation. Furthermore, deletion of FADD in preactivated mature T cells using retrovirus-Cre resulted in no proliferation. These results demonstrate that FADD plays a dispensable role during thymocyte development, but is essential in maintaining peripheral T cell homeostasis and regulating both apoptotic and proliferation signals.

FIGURE 1

FADD:GFP reconstitutes normal development in FADD^-/- mice. (A) Diagrams of the wild-type (WT) FADD gene consisting of two exons (boxes 1 and 2); the knockout allele (K/O); and the FADD:GFP construct. LoxP sites and the 0.4-kb probe used for genotyping by Southern blots are indicated. RI: EcoR I. RV: EcoR V. (B) Genotyping by Southern blot analysis using mouse tail DNA identified various genotypes present in the offspring of crosses between FADD^+/- and FADD^+/- FADD:GFP⁺ mice. Only when FADD:GFP is present, can FADD^-/- mice become viable and develop normally. (C) Anti-FADD antibodies detected the FADD and FADD:GFP proteins in thymocytes, spenocytes and lymph node cells from mice of various genotypes by Western blot analyses. FADD:GFP differs in size from the endogenous FADD which is absent in FADD^-/- FADD:GFP⁺ mice.

FIGURE 2

Detection of the expression and deletion of FADD:GFP by flow cytometric analysis. Gene deletion efficiency in various lymphoid organs was indicated by the percentage of GFP^- cells detected in FADD^-/- FADD:GFP⁺ mice containing either Lck-Cre or CD4-Cre. Cells from FADD^-/- FADD:GFP⁺ mice (top) were used as GFP⁺ controls, which uniformly express FADD:GFP as indicated by a single right histogram peak (shaded). Cells from wild type FADD^+/+ mice were used as GFP^- controls (bottom). Using these two different controls, GFP⁺ cells in mice expressing either Lck-Cre or CD4-Cre were determined to be those represented by areas shaded in histograms (two middle rows).

FIGURE 3

Detection of FADD:GFP deletion by Western blots analysis using anti-FADD antibodies. (A) Efficient T cell-specific deletion of FADD:GFP in FADD^+/- or FADD^-/- mice was induced by the presence of Lck-Cre (+), as indicated by the dramatic reduction of the FADD:GFP protein in the thymus (top panel). FADD^-/- FADD:GFP mice lacking Lck-Cre (-) were used as non-deletion controls. Deletion of FADD:GFP was not readily detectable in the spleen and lymph nodes due to the presence of the non-T cell population which express FADD:GFP in these secondary lymphoid organs. (B) Absence of FADD:GFP in purified GFP^- thymocytes and mature resting and activated T cells was confirmed by anti-FADD Western blots. GFP⁺ T cells isolated from FADD^-/- FADD:GFP⁺ mice were used as controls. Ponceau S staining shown at the bottom in panels (A) and (B) indicates equal loading and transfer of proteins.

FIGURE 4

FADD-deficient T cells are resistant to cell death induction. FADD^-/- cells were the GFP^- population isolated from FADD^-/- FADD:GFP⁺ Lck-Cre⁺ mice. Littermate FADD^-/- FADD:GFP⁺ mice were used to isolate the control GFP⁺ T cell population (FADD:GFP⁺). Error bars represent ± standard deviation of the means from 3-5 mice of each genotype in each treatment. (A) Thymocytes were stimulated with various concentrations of anti-Fas antibodies to induce cell death. (B) Cell death was induced in resting mature T cells by treatment with various concentration of sFasL. Proliferating T cells, activated by stimulation with anti-CD3 and anti-CD28 antibodies, were incubated with various concentrations of anti-Fas antibodies (C), TNF (50 ng/ml) or anti-CD3 antibodies (D) to induce cell death. Percent cell death is indicated on Y-axis.

FIGURE 5

Flow cytometric analysis of T cell sub-populations. Cells isolated from the thymus, spleen, and lymph nodes were stained for the CD4 and CD8 co-receptors. T cell-specific gene deletion in FADD^-/- FADD:GFP⁺ mice induced by either Lck-Cre or CD4-Cre did not alter thymocyte populations (top), but resulted in reduced T cell populations in the spleen and lymph nodes (middle and bottom). Littermate FADD^+/- FADD:GFP⁺ mice containing Lck-Cre or CD4-Cre were used as controls.

FIGURE 6

Defective homeostatic responses in FADD-deficient T cells. Adoptively transferred FADD-deficient (FADD^-/-) T cells were the GFP^- population isolated from FADD^-/- FADD:GFP⁺ Lck-Cre⁺ mice, which divided at largely reduced rates. Control FADD^+/- T cells were isolated from FADD^+/- FADD:GFP⁺ Lck-Cre⁺ littermates. The percentage of cells that underwent various divisions are indicated in brackets on the graph. CFSE-labeled T cells, adoptively transferred into wild-type B6 hosts, were used as non-proliferation controls (top).

FIGURE 7

FADD is required for TCR-induced proliferation responses. (A) Peripheral T cells were stimulated with anti-CD3 antibodies in the presence (+) or absence (-) of anti-CD28 antibodies for two days and then pulsed with [³H] thymidine for 12 h. Levels of proliferation were indicated by the amount of radioactivity incorporated into T cells. cpm: count-per-minute. Mature FADD^-/- T cells are the peripheral GFP^- population isolated from FADD^-/- FADD:GFP⁺ Lck-Cre⁺ mice. Peripheral T cells from wild-type mice (FADD^+/+) were used as control. (B) Peripheral T cells were stimulated for two days with anti-CD3 and anti-CD28 antibodies (open and filled triangles) or with Con A (open and filled circles). The resulting activated T cells were cultured in IL-2-containing media. Growth curves were generated by counting the cell number in T cell cultures at various times after activation. Peripheral FADD^-/- T cells were the GFP^- population isolated from FADD^-/- FADD:GFP⁺ Lck-Cre⁺ mice. The control peripheral FAGG:GFP⁺ T cells were isolated from FADD^-/- FADD:GFP⁺ littermates. (C) Deletion of FADD:GFP in activated mature T cells also resulted in a reduced growth rate. FADD:GFP⁺ T cells were activated by stimulation with anti-CD3 and anti-CD28 antibodies for 16 h to induce cell cycle entry. The resulting activated, dividing T cells were infected with MSCV-Cre or control MSCV viruses. Two days after infection, cells were cultured in fresh IL-2-containing media and cell number is determined at indicated times. Error bars represent ± standard deviation of the arithmetic means from 3-5 mice of each genotype in each treatment.

FIGURE 8

CFSE-labeling assays. FADD-deficient (FADD^-/-) T cells were the GFP^- population isolated from FADD^-/- FADD:GFP⁺ CD4-Cre⁺ mice and the control FADD^+/- T cells were the GFP^- population isolated from FADD^+/- FADD:GFP⁺ CD4-Cre⁺ littermates. CFSE-labeled T cells were activated by stimulation with anti-CD3 and anti-CD28 antibodies, and analyzed by flow cytometry at 72, 96 and 120 h post stimulation. The percentages of cells that underwent various divisions are indicated in brackets on the graph.