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 TCRβ gene rearrangements. The αβTCR 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 αβTCR 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 β-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−/−
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+
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
). 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
). 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.