INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The tumor necrosis factor receptor (TNFR) superfamily comprise a growing family of type I membrane bound glycoproteins which interact with the TNF family of soluble mediators and type II transmembrane proteins. At least 23 TNFR superfamily members and 17 known ligands have been identified in mammals (reviewed in references 3, 35, and 44). These receptors trigger pleiotropic responses, ranging from apoptosis and differentiation to proliferation, and have been implicated in immune regulation, host defense and lymphoid organ development.

Members of the TNFR family are characterized by the presence of varying numbers (three to six) of cysteine-rich repeats in their cytoplasmic domains (52). TNFRs are subdivided based on the presence or absence of a 70- to 80-amino-acid region of homology in the cytoplasmic region called the death domain, through which these receptors trigger apoptosis (20, 48). DR3 (also called Ws1, Apo3, TRAMP, LARD, TR3, and TNFRSF12) is one of six death domain-containing TNFR family members (the others are TNFR1, CD95/FAS, DR4, DR5, and DR6) and is the one most closely related to TNFR1. Studies on the TNFR1 crystal structure suggest that ligand binding or receptor overexpression results in receptor trimerization and recruitment of trimeric intracellular signaling molecules (4, 36). DR3, like TNFR1, recruits TNFR1-associated death domain protein (TRADD) and Fas-associated death domain-containing protein (FADD) (5, 6, 11, 12, 24) as downstream effectors of apoptosis. These, in turn, interact with caspase 8 (FLICE/MACH) (7, 31), and a cascade of interleukin-1-converting enzyme-like cysteine proteases which trigger cell death (13, 17, 27). DR3 also recruits TRAF2 via TRADD (18, 24, 29, 39) and thus activates the transcription factor, NF-B, that induces the transcription of a number of immune genes (19). In this respect, DR3 (like TNFR1) is capable of inducing both apoptosis and expression of survival/activation genes and is likely to have multiple functions depending on the context of its expression.

DR3 was first reported as the only death domain-containing TNFR family member with lymphoid organ-restricted expression (11, 24). More recent studies have, however, shown DR3 expression to be less restricted, though not as ubiquitous expression of as its other death domain-containing TNFR relatives (47, 51). DR3 expression patterns may be further complicated by the presence of at least 13 human (24, 42, 53) and 3 mouse (51) splice variants. DR3 splicing may be developmentally regulated since human peripheral blood leukocytes have been shown to express full-length DR3 mRNA only following activation (42). While the ligand for DR3 has been reported as TWEAK (30, 10), it has also been shown that TWEAK can bind and signal in a DR3-negative cell line via a TNF/TNFR1-related mechanism (40). Thus, the problems with identifying a single ligand together with the complex expression of DR3 have contributed to the lack of functional data for this gene. To date, the only ex vivo indication, albeit indirect, that DR3 has an apoptotic role stems from the detection of translocations affecting the DR3 gene in several neuroblastoma cell lines (16).

To determine the in vivo function(s) of DR3, we generated mice that are deficient in the expression of its murine homologue. DR3-deficient mice show no defects in organ development (lymphoid or otherwise), lymphoid proliferation, or apoptosis triggered by glucocorticoids or DNA-damaging agents. There is also no defect in the progression of double-negative (DN) thymocytes through the pre-T-cell receptor (TCR)-mediated checkpoint, a developmental transition at which TNFR signaling has been shown to be important (33). However, an impairment of negative selection, as well as anti-CD3-mediated cell death of thymocytes, is observed in DR3-deficient mice, suggesting a nonredundant role in TCR-induced apoptosis that establishes central tolerance in vivo.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of DR3^/ mice. A human cDNA corresponding to the full-length DR3 gene was used as a probe to isolate clones from an EMBL3A phage library of 129/Sv mouse strain genomic DNA (51). A 6-kb SmaI-MfeI fragment covering the whole of the coding region of the DR3 gene was replaced with a cassette containing an internal ribosome entry site (IRES), lacZ poly(A), and neomycin resistance (neo) gene flanked by loxP sequences (a gift from Andrew Smith, University of Edinburgh, Edinburgh, United Kingdom). The DR3 gene targeting construct was linearized and transfected into GK129 embryonic stem (ES) cells by electroporation. ES cell clones were selected in G418-containing medium on a monolayer of mitotically inactivated STO feeder cells. Clones were picked and screened for homologous recombinants using the probes shown in Fig. 1A. ES clones with correct 5' and 3' recombinations were microinjected into C57BL/6 blastocysts and introduced into pseudopregnant C57BL/6 mice. Male chimeric offspring were bred to obtain germ line mutant mice which were screened either by Southern blotting or by PCR.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Generation and screening of DR3/ mice. (A) Map of the coding region of the DR3 gene (top), the targeting construct (middle), and the targeted locus (bottom). Restriction enzyme sites are indicated by single letters; B, BamHI; E, EcoRI; H, HindIII; M, MfeI; S, SmaI; X, XbaI. The positions of the DR3 exons are shown. An IRES/lacZ/loxP/neo vector was used to replace the whole coding region of the DR3 gene. The 5' arm was generated using a 3-kb HindIII-SmaI fragment, while the 3' arm consisted of a 3-kb MfeI-EcoRI fragment. (B) Southern blot screening for homologous recombination in ES cell clones. Genomic DNA was isolated from transfected ES cell clones, digested with either BamHI or XbaI, and screened with probe A (left) for 5' recombination or probe B (right) for 3' recombination, respectively. Only ES cell clones with correct recombination at both ends were used for injections. Sizes of fragments are indicated. (C) Southern blot analysis and genomic PCR of representative mouse tail DNA. Genomic DNA was isolated from mouse tails, screened for correct 5' recombination by Southern blotting using a BamHI digest and probe A, and screened for correct 3' recombination by genomic PCR using the three primers (filled triangles) F1, F2 (in the neo gene), and R1. Sizes of fragments, recombinant and wild-type bands are indicated. (D) Reverse transcription-PCR analysis of thymus cDNA. cDNA prepared from total thymus RNA was subjected to PCR analysis using specific primers corresponding to sequences within the DR3 death domain exon. Actin primers were used a positive control. Sizes of fragments are indicated.

Maintenance and breeding of mouse strains. All mice were kept under barrier conditions at the Imperial Cancer Research Fund animal unit. H-Y TCR transgenic mice were kindly provided by H. von Boehmer (22). Rag 1^/ mice were acquired from the Jackson Laboratory. For H-Y TCR transgenic studies, DR3^/ × H-Y TCR mice were obtained by crossing DR3^/ mice with DR3^+/ H-Y TCR heterozygote transgenics, thus deriving DR3^+/ and DR3^/ mice with and without the H-Y TCR transgene. All mice analyzed were between 6 and 14 weeks of age. For analysis of each age group (6, 10, and 14 weeks), mice were sacrificed within one day of their weekly age.

Flow cytometric analysis. Fluorescence-activated cell sorting (FACS) analysis was performed on a FACScan (Becton Dickinson) using CellQuest software. Single-cell suspensions were prepared from freshly isolated bone marrow, lymph nodes (inguinal), spleen, and thymus. All samples were gated on standard forward scatter versus-side scatter gates. Thymocytes were stained using combinations of the antibodies from Becton Dickinson (anti-CD4-fluorescein isothiocyanate [FITC], anti-CD4-phycoerythrin [PE], anti-CD8-FITC, anti-CD8-biotin, anti-CD30, anti-CD44-PE, anti-CD25-biotin, anti-V8-FITC [F23.1], anti-CD3-FITC [2C11], anti-TCR-FITC, anti-TCR-FITC, anti-B220-FITC, and anti-5-bromo-2'-deoxyuridine [BrdU]) or Caltag Laboratories (anti-CD8-Tricolor, streptavidin-Tricolor, anti-neutrophil-FITC, and anti-monocyte-FITC) or kindly donated by P. Kieselow and H. von Boehmer for H-Y transgenic analysis (T3.70 and T3.70-FITC specific for V3 [49]). BrdU and unconjugated T3.70 or CD30 were visualized using anti-mouse immunoglobulin G (IgG)-FITC (Dako).

Lymphocyte proliferation assays. In vivo BrdU incorporation assays were carried out as described elsewhere (56). In brief, 1 mg of BrdU (Sigma) was injected twice intraperitoneally, 30 min apart. Mice were then sacrificed after 6 h, thymuses were extracted, and single-cell-suspension thymocytes were isolated. BrdU incorporation was visualized using a standard BrdU-labeling protocol (28) with an anti-BrdU antibody (Becton Dickinson) and anti-mouse Ig-FITC secondary (Dako) and analyzed on a FACScan (Becton Dickinson).

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Apoptosis assays. For anti-CD3-induced apoptosis assays, experiments were carried out as described elsewhere (46). In brief, 96-well flat-bottomed plates were coated with anti-CD3 MAb 2C11 (2, 10, or 50 µg/ml) or anti-CD3 and anti-CD28 MAb (each at 10 µg/ml) in PBS, overnight at 4°C, or with rat Ig as a control. Plates were washed three times with PBS before use. Thymocytes were extracted, resuspended in single-cell suspension in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum, and aliquoted into plates at 5 × 10⁵ cells/well. For PMA (Sigma) stimulation, a final concentration of 1 ng/nl was used. Samples were then incubated at 37°C for the indicated times before analysis for apoptosing cells using annexin V-FITC (Pharmingen) and propidium iodide staining to gate out dead cells. All samples were performed in triplicate. Values for percent anti-CD3-induced apoptosis were calculated using the following formula: (% annexin V⁺ cells after anti-CD3  % annexin V⁺ cells in rat Ig controls)/(100  % annexin V⁺ cells in rat Ig controls) × 100. For other apoptosis assays, thymocytes were cultured in vitro in standard RPMI medium supplemented with 10% heat-inactivated fetal calf serum and 50 µM -mercaptoethanol, and apoptosis was induced by stimulation with the following reagents: cycloheximide (30 µg/ml) and anti-Fas antibody (1 µg/ml); dexamethasone (2 µM); and etoposide (50 µM). Samples were taken at 4, 8, 12, 24, and 48 h poststimulation. Apoptosis was visualized using both annexin V-FITC staining, or pre-G₁ peak DNA staining with propidium iodide.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of DR3^/ mice. The strategy used to inactivate the DR3 gene in ES cells is shown in Fig. 1A. We chose to disrupt exon 1 and replace all of the coding exons of the gene to exclude the possibility of any expression of DR3 splice variants. The entire coding region of the DR3 gene, from exon 1 (56 bp upstream of the ATG initiation codon) to 297 bp downstream of the polyadenylation site, was replaced with a construct containing an IRES, -galactosidase (lacZ) gene, and neo gene flanked by laxP sites. ES cell clones with correct 5' and 3' recombinations were detected by screening using probes outside the recombination arms of the targeting vector. 5' recombination was detected by the reduction of a 6.5-kb wild-type BamHI fragment to 3.9 kb and 3' recombination by the reduction of an 11.5-kb wild-type XbaI band to 10 kb (Fig. 1A and B). The homologous recombination frequency was 4.2%. Four clones were chosen for injection into blastocysts. Mice heterozygous for the targeted allele, as detected by screening for 5' recombination in genomic Southern blots of tail DNA (Fig. 1C), were generated from two ES cell clones with correct 5' and 3' integrations of the targeting construct.

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Normal development of major organs, peripheral lymphoid organs, and lymphocyte subsets in DR3^/ mice. The pattern of expression of mouse DR3 in the brain, heart, and kidney as well as lymphoid organs (51) prompted us to investigate whether there were any general developmental defects in DR3^/ mice. Histological analysis of all major organs (including brain, heart, and kidney) revealed no abnormalities (data not shown). In particular, the peripheral lymphoid organs such as thymus, spleen, lymph nodes, and Peyer's patches all showed normal development. Primary B-cell follicles and follicular dendritic cell networks were present in the peripheral lymphoid organs from DR3^/ mice (data not shown), unlike their TNFR1^/ counterparts (25, 32, 37).

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Increased thymus size but normal turnover in DR3^/ mice. TNFR family members have been shown to be important regulators of developmental processes. The expression of DR3 in developing thymocytes suggested that DR3 may be a regulator of thymocyte development. To determine whether DR3 is required for normal thymocyte development, thymocyte subpopulations were analyzed by flow cytometry. There were no significant differences in the four major thymocyte populations defined by the expression of the CD4 and CD8 markers. However, a small but significant increase in the average total number of thymocytes derived from DR3-deficient mice, compared to heterozygous littermates, was observed (Fig. 2A). At 2 to 5 weeks of age, DR3^/ thymuses were generally about 10% larger than their heterozygote counterparts, and by 29 to 32 weeks this difference had increased to about 30%. A larger study (data not shown), covering mice aged from 26 to 233 days, showed these differences to be significant using a different statistical analysis (Mann-Whitney U test; U = 5,965, P < 0.05). No difference in thymocyte number was observed between wild-type and heterozygous mice (data not shown). This increase in thymus size appeared not to be a consequence of an increase in thymocyte proliferation and/or turnover, since neither the proportion nor the numbers of thymocytes which incorporated BrdU differed significantly between DR3^/ and DR3^+/ mice (Fig. 2B).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Analysis of thymocyte development in DR3+/ and DR3/ mice. (A) Thymus size in DR3/ mice. Total thymocyte numbers were collected for mice at the ages indicated (DR3+/ and DR3/, n  11 and n  10, respectively, for each age group), and means were generated. DR3/ thymuses were generally larger than their DR3/ counterparts, irrespective of age. Significance using t tests is indicated. (B) Turnover and proliferation in DR3/ thymus. The proportion (left) and number (right) of BrdU+ thymocytes were estimated as described in Materials and Methods. Filled bars, DR3/; hatched bars, DR3+/. (C) Early thymocyte development in DR3/ mice. Upper panels depict representative FACS plots showing expression of CD44 and CD25 in early thymocyte subsets from DR3+/ (left) and DR3/ (right) mice. Thymocytes were extracted and depleted of cells expressing CD4 and CD8 using specific antibodies and comple- ment. The remaining thymocytes were stained with anti-CD44-PE, anti-CD25-biotin with streptavidin-Tricolor and anti-CD4-FITC, anti-CD8-FITC, anti-B220-FITC, TCR-FITC, and TCR-FITC. DN thymocytes were analyzed by gating on the CD4, CD8, B220, TCR, and TCR populations. Lower panels depict representative FACS plots showing CD4 and CD8 thymocyte subsets in Rag-1-deficient DR3+/ (left) and DR3/ (right) mice.

Early thymocyte development is unaffected by the absence of DR3. The small but significant increase in total thymocyte numbers in DR3-deficient mice was consistent with a regulatory role of DR3 at one or more stages of thymocyte development. There are two major checkpoints at which thymocyte development is controlled. The DN-to-double-positive (DP) thymocyte transition selects immature thymocytes with in-frame TCR rearrangements and is regulated by the pre-TCR. The DP-to-single-positive (SP) thymocyte transition selects major histocompatibility complex (MHC)-restricted, nonautoreactive thymocytes and is regulated by the mature TCR. To investigate whether DR3 plays an essential role in early thymocyte development, we initially analyzed DN thymocyte subsets, as defined by the absence of CD4, CD8, TCR, TCR, and B220 surface markers and the differential expression of CD44 and CD25, by flow cytometry. As shown in Fig. 2C (upper panels), this analysis revealed no consistent differences in the relative proportions of DN populations in DR3^/ compared to DR3^+/ mice.

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Negative selection is impaired in DR3^/, H-Y transgenic mice. The DP-to-SP thymocyte transition is a checkpoint at which a developmental decision is made between cell death and survival. Thymocytes expressing TCRs that have no detectable affinity for ligand, or which are autoreactive, undergo apoptosis, whereas cells expressing TCRs with intermediate affinities receive survival signals and generate the pool of mature SP thymocytes and T cells. To investigate whether DR3 is essential for efficient negative selection of thymocytes, the H-Y TCR transgenic mouse model was used (22). The H-Y TCR recognizes a male-specific antigen in the context of H-2^b In male mice, thymocytes expressing the transgenic TCR are deleted, whereas in female mice, transgene-expressing cells undergo positive selection, resulting in an increased proportion of CD8 SP cells.

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View larger version (48K): [in this window] [in a new window]   FIG. 3.   Negative selection in male DR3/ × H-Y transgenic mice. (A) Representative FACS plots showing expression of CD8 and T3.70 in 14-week-old male H-Y transgenic mice. Thymocytes were extracted and stained with anti-CD4-PE, anti-CD8-Tricolor, and anti-T3.70-FITC. DR3/ × H-Y mice (right) showed an increase in the proportions of CD8+ T3.70+ thymocytes compared to their DR3+/ × H-Y littermates. (B) Proportions and numbers of CD8+ T3.70+ thymocytes in H-Y TCR transgenic DR3/ and DR3+/ mice with age. Male DR3/ × H-Y mice showed higher proportions (top) and numbers (bottom) of CD8+ T3.70+ thymocytes with age compared to their DR3+/ × H-Y littermates. Differences were statistically significant using parametric (for numbers) or nonparametric (for percentages) t tests from 10 weeks of age and are indicated on the graphs. Means were calculated from n  7 mice. Standard deviations of the means are indicated by bars. Filled bars, DR3/; hatched bars, DR3+/. (C) BrdU uptake of male DR3/ × H-Y transgenic mice, determined as described in Materials and Methods. The mean from n  5 mice for each thymocyte population is shown. All mice were 10 weeks of age (±1 day). Proportions (top) of BrdU+ thymocytes did not differ between H-Y transgenic, DR3/ and DR3+/ mice, but numbers (bottom) of BrdU+ thymocytes were increased in DR3/ × H-Y mice compared to their DR3+/ littermates at 10 weeks of age. Increased numbers of total BrdU+ cells were due to increases in the BrdU+ DN and transgenic V8+ thymocyte populations. Filled bars, DR3/; hatched bars, DR3+/. (D) CD8 and T3.70 expression on DR3/ × H-Y transgenic thymocytes. Thymocytes were extracted from 10-week-old mice and stained with anti-CD4-PE, anti-CD8-Tricolor, and anti-T3.70-FITC. Thymocytes were either gated for CD8 expression and their T3.70 expression (top) or gated for high T3.70 expression and their CD8 expression shown (bottom). Representative overlay histograms are displayed and labeled with the proportion of positive cells and the level of each surface marker, as measured by their mean fluorescence channel (MFC). Male DR3/ × H-Y mice (thick line) showed an increase in the proportion of T3.70hi thymocytes expressing CD8 compared to their DR3+/ × H-Y (shaded) littermates but no difference in the level of CD8 expression (lower panel). There was no significant difference in the proportion of CD8+ thymocytes expressing T3.70 or the level of T370 expression on CD8+ thymocytes compared with male DR3/ and DR3+/ H-Y transgenic mice.

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Endogenous superantigen-mediated deletion of T cells is unaffected in DR3-deficient mice. T cells expressing TCRs that recognize minor lymphocyte-stimulating determinants encoded by endogenous superantigens in the mouse genome together with MHC class II molecules are deleted. Minor lymphocyte-stimulating determinants expressed on the 129 × C57BL/6 background of DR3-deficient mice result in the deletion of several V-expressing CD4⁺ T cells including V5 (1). To determine whether endogenous superantigen-driven deletion is defective in DR3-null mice, lymph node T cells, splenic T cells, and SP thymocytes were analyzed using antibodies to specific V families. As shown in Table 1, V5-bearing CD4⁺ T cells were efficiently deleted in DR3-null mice, whereas the proportion of nondeleting V8 cells was unaffected. These data show that DR3 is not essential for superantigen-mediated deletion.

Anti-CD3-induced, but not non-antigen receptor-mediated, apoptosis is impaired in DR3-deficient mice. Immature DP thymocytes and T cells are susceptible to an array of cell death-inducing stimuli, including anti-CD3 cross-linking, glucocorticoid treatment, Fas ligation, and DNA-damaging agents. To assess whether these forms of apoptosis are also dependent on DR3 expression, thymocytes and peripheral T cells were subjected to treatment with anti-CD3 cross-linking, the steroid dexamethasone, the DNA-damaging agent etoposide, and the protein synthesis inhibitor cycloheximide in conjunction with an anti-Fas signal.

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View larger version (26K): [in this window] [in a new window]   FIG. 4.   Apoptosis of thymocytes following CD3 ligation. (A) Representative FACS histograms showing reduced proportions of annexin V-positive thymocytes from DR3/ mice compared to DR3+/+ age- and sex-matched controls after CD3 cross-linking. A stimulation of 2 µg/ml was used, and thymocytes were stained after 48 h. Dead cells were gated out using propidium iodide. (B) Thymocyte suspensions were stimulated for up to 48 h with plate bound anti-CD3 or rat Ig as a negative control. Percent apoptosis was measured as the proportion of propidium iodide-negative thymocytes, which were positive for annexin V. Anti-CD3 treatment at 2 µg/ml resulted in an increase of 50.7% in apoptosing thymocytes above rat Ig controls by 48 h in DR3+/ thymocytes (filled squares) compared to 38.7% in DR3/ thymocytes (open squares). There were no differences in apoptosis of DR3/ (open) and DR3+/+ (filled) thymocytes following stimulation with PMA (triangles) or anti-CD3 and anti-CD28 (circles). Results are the means of 3 DR3/ and DR3+/ littermates, with duplicate cultures performed on each mouse. Standard deviations are shown as bars. * and ** represent P < 0.0003 and P < 0.004, respectively, using t tests assuming unequal variance.

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View larger version (34K): [in this window] [in a new window]   FIG. 5.   Apoptosis of thymocytes and lymphocytes after exposure to various apoptosis inducing agents. Assays were carried out over 48 h with apoptosis measured using annexin V-FITC staining or sub-G1 peak following staining with propidium iodide as described in Materials and Methods. There were no differences in the induction of apoptosis using cycloheximide and anti-Fas, dexamethasone, or etoposide on thymocytes (A) or lymphocytes (B) derived from inguinal lymph nodes from DR3+/+ (open triangles), DR3+/ (closed circles), or DR3/ (open circles) mice.

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View larger version (23K): [in this window] [in a new window]   FIG. 6.   Proliferation of lymphocytes, thymocytes, and splenocytes to various stimuli Assays were carried out over 96 h with proliferation measured by [3H]thymidine uptake over the final 18 to 24 h as described in Materials and Methods. There were no significant differences in thymidine uptake of lymphocytes (top), splenocytes (middle), or thymocytes (bottom) between DR3+/ and DR3/ cells using anti-CD3, anti-CD3, and anti-CD28, PMA and ionomycin, or concanavalin A as stimuli. Data are presented as a stimulation index over medium-only controls, which were not significantly different between DR3+/ and DR3/ cells. Filled bars, DR3/; hatched bars, DR3+/+.

Positive selection is unimpaired in DR3^/ mice. The TCR is first expressed at the DP stage, resulting in a population of TCR^lo and CD3^lo thymocytes which then undergo positive selection to generate the TCR^hi and CD3^hi SP subsets. Positively selected thymocytes also transiently up-regulate the expression of CD69. Analysis of CD3 and CD69 expression on thymocytes from DR3-deficient mice showed no significant difference in the proportions of CD3^hi or CD69-positive cells between DR3-null mice and their heterozygous littermates, suggesting that DR3 is not absolutely required for positive selection (data not shown). This conclusion was consistent with a flow cytometric analysis of DR3-deficient female mice expressing the H-Y TCR. No difference was observed in the numbers or proportions of H-Y TCR-expressing CD8 SP cells between DR3-expressing or -deficient thymocytes (Table 2). The H-Y TCR is H-2K^b restricted and therefore selects on both C57BL/6 and 129 mouse strain backgrounds. Thus, the mixed strain nature of the knockouts should not affect the efficiency of positive or negative selection. Taken together, these results demonstrate that DR3 is not required for positive selection during thymocyte development.

View this table: [in this window] [in a new window]   TABLE 2.   Positive selection in female H-Y transgenic DR3/ and DR3+/ mice

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

DR3 belongs to a family of receptors that plays an important role in regulating cell survival and proliferation. These processes are tightly regulated during T-cell development. Thus, signals originating from the pre-TCR mediate the survival and proliferation of pre-T cells that have undergone in-frame TCR gene rearrangements. The TCR controls the survival of DP thymocytes, selecting those cells expressing TCR with intermediate affinity for MHC ligand and inducing programmed cell death of high-affinity, autoreactive T cells. DP thymocytes that fail to express an TCR also undergo apoptosis. The expression of DR3 in developing thymocytes prompted the hypothesis that it is a key regulator of life/death decisions during thymocyte development. This notion was tested by generating and analyzing mice deficient in DR3 expression.

TNFR family members containing death domains (TNFR1, Fas, DR3, DR4, DR5, and DR6) transduce death signals by interaction of their death domains with FADD, a death domain-containing cytoplasmic protein. FADD then recruits other cytoplasmic effectors such as caspase 8, TRADD, TRAF2, and RIP that activate the apoptotic machinery (41). Studies of transgenic mice expressing DN-FADD in developing thymocytes have given rise to the hypothesis that stimulation of death domain-containing TNFRs on early pre-T cells induces cell death via FADD only in the absence of pre-TCR-derived signals (33). In the presence of pre-TCR-derived signals, the TNFR signals proliferation. The present study demonstrates that DR3, which is expressed on early and late pre-T cells, is not essential for the transduction of apoptosis or proliferation signals at the pre-TCR-mediated developmental checkpoint. It may, however, participate in the regulation of -selected thymocytes in concert with other death receptors, such as DR5, Fas, and TNFR1. An analysis of mice deficient in the expression of multiple death receptors would resolve this question.

The DP-to-SP thymocyte transition is associated with extensive cell death. The pathway(s) that, in concert with signals derived from high-affinity TCRs, induce apoptosis in DP thymocytes have not been defined. TNFRs have, however, been implicated in negative selection in the thymus. Thus, a role for Fas in negative selection at high doses of antigen has been proposed (23). Also, negative selection has been shown to be either enhanced following CD30 overexpression (9) or partially impaired in mice deficient in the expression of CD30, a non-death domain-containing member of the TNFR superfamily (2). We show here that DR3 is also important for negative selection, which is impaired in the absence of this receptor. Thus, the induction of apoptosis in DP thymocytes by anti-CD3, an agonist that is presumed to mimic a high-affinity TCR interaction, is impaired at least at low concentrations of anti-CD3 (2 and 10 µg/ml) in DR3-null mice. Interestingly, this effect is abrogated by high anti-CD3 concentrations, suggesting a greater contribution of DR3 to apoptosis when the signaling strength is not maximal. Also, the deletion of H-Y TCR transgenic thymocytes in male mice is partially inhibited, an effect which is manifested most clearly in older mice. Although the numbers and proportions of H-Y TCR transgenic thymocytes were indistinguishable between DR3^/ and DR3^+/ backgrounds in 6-week-old male mice, a significant inhibition of negative selection occurred in DR3-null mice at 10 weeks, at 14 weeks, when H-Y TCR-expressing thymocytes were virtually undetectable in most male mice expressing DR3, transgene-positive cells were easily detectable in all DR3-null mice analyzed. However, substantial negative selection had clearly occurred in male DR3-null mice, since at 14 weeks, male thymuses contained only about 1/100 the numbers of cells found in the thymuses of female transgenic mice.

The notion that death domain-containing TNFRs play a role in negative selection is in apparent contradiction to studies of mice expressing DN-FADD in thymocytes. In one such study, negative selection was unaffected by DN-FADD, although in this study negative selection was assayed in 6-week-old H-Y TCR male mice (50). At this age, we observed no difference in negative selection in the presence or absence of DR3. In an independent study, the deletion of autoreactive thymocytes was enhanced in H-Y TCR⁺ DN-FADD⁺ male thymocytes (34). These studies suggest that FADD signaling does not lead exclusively to cell death in thymocytes. This, in turn, suggests that FADD-independent pathways from death domain-containing TNFRs activate caspases in negative selection. Adapter molecules such as RIP, RAIDD, and Daxx have been shown to interact with the CD95 death domain, thereby activating a caspase cascade leading to apoptosis (15, 55). It is possible that these cytoplasmic signaling molecules also transduce DR3-mediated signals in thymocytes. A recent study has, however, failed to support a role for caspase activity in negative selection. In this study, mice expressing an inhibitor (p35) of caspase activity in thymocytes showed unimpaired negative selection (14). However, an independent study, using an identical strategy, revealed a reduction of negative selection upon inhibition of caspase activity (21). Therefore, the role of caspases in thymocyte negative selection remains unresolved.

The observation that DR3-null mice exhibit a partial impairment of negative selection can be interpreted within a model in which several different surface molecules act in concert to control the removal of autoreactive thymocytes (23). According to this model, disruption of a single surface receptor or signal transduction pathway would not be expected to result in a complete block in negative selection, a prediction that is observed in practice. The age dependency of the negative selection defect in DR3-null mice in H-Y TCR-transgenic mice may be due to developmental regulation of the ligand(s) for DR3 or, alternatively, to the developmental stage at which deletion occurs in the H-Y TCR model which is likely to be at the DN stage or in the transition from DN to DP thymocytes. Thus, the relative importance of a particular deletional mechanism may vary with age or with the stage of thymocyte development at which deletion of a particular TCR occurs.

Despite the impairment of negative selection in DR3-null mice, there were no signs of autoimmunity in these mice as indicated by the presence of DNA autoantibodies, nor was development of autoimmunity significantly accelerated on an lpr (Fas mutant) background (unpublished data). Furthermore, several other forms of apoptosis were unaffected. There was no difference in the rate of spontaneous cell death of thymocytes in culture or of the rate of apoptosis of thymocytes or T cells after treatment with glucocorticoids, DNA-damaging agents, or anti-Fas in the presence of protein synthesis inhibitors. Moreover, no difference in endogenous superantigen-mediated deletion of T cells was detected. This observation is consistent with the analysis of other transgenic models with defects in negative selection. For example, CD30-null mice (2) and mice lacking expression of the helix-loop-helix inhibitor protein Id3 (38) have impaired negative selection, but T cells specific for endogenous superantigens are efficiently deleted. Conversely, the antiapoptotic protein Bcl-2 inhibits multiple forms of apoptosis but not endogenous superantigen-induced negative selection in thymocytes (43), although bcl-2 transgenic mice have impaired negative selection when tested on a TCR transgenic background (45, 54). Taken together with the present study, these data suggest that there are qualitatively or quantitatively distinct cell death programs that operate at different stages of T-cell development to ensure effective central tolerance.