By
From the * Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892; Immune Cell Biology Department, Naval Medical Research Institute, Bethesda, Maryland 20889-5607;
and § Department of Immunology, Scripps Research Institute, La Jolla, California 92037
Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.
Acentral feature of the immune system is its ability to
respond to foreign antigens while tolerating self-antigens. Burnet's model of self-nonself discrimination proposed that receptor engagements that were stimulatory for
mature immune cells would induce immature immune cells
to die, resulting in the removal of autoreactive receptor specificities from the developing repertoire (1). The removal
of autoreactivities from the functional lymphocyte repertoire is known as negative selection. For T cells, negative selection can occur at different developmental stages in the
thymus by a variety of mechanisms, including (a) clonal deletion, (b) developmental or clonal arrest, or (c) clonal inactivation or anergy (3). CD4+CD8+ double positive
(DP)1 thymocytes in particular are susceptible to clonal deletion induced in response to TCR signals (5). Curiously, while clonal deletion of DP thymocytes must involve
TCR engagement, TCR engagement by itself is insufficient to stimulate DP thymocyte apoptosis in vitro (8).
Rather, DP thymocyte death has been found to require signals in addition to TCR, such as those provided by CD28
(9). This requirement for second signals parallels the requirement for second signals in TCR-mediated activation
and proliferation of mature single positive (SP) T cells (13).
Because CD28 is not a unique costimulatory molecule for
mature T cells (14), it is likely that CD28 is not the only
molecule capable of transducing second signals for TCR
dependent DP thymocyte apoptosis (6, 14, 23). Indeed,
other molecules have been implicated in clonal deletion of
DP thymocytes, such as CD30 (21) and fas (25). The role of
fas in negative selection is particularly controversial (26).
TCR-dependent apoptosis of mature T lymphocytes
was not foreseen in Burnet's model of self-nonself discrimination but is now a recognized consequence of mature T
cell activation (reviewed by 30-31). In mature T cells, TCR
signals do not directly induce an apoptotic program, but
rather act indirectly by upregulating surface expression of
members of the TNF family, specifically fas ligand (fasL) and
TNF (32). These proteins bind to fas or TNF receptor
(TNFR) which mediate apoptosis through death domains expressed in their cytosolic regions (35). This mechanism
of apoptosis necessarily results in death of both TCR-stimulated T cells and neighboring (or bystander) cells expressing fas or TNFR. In contrast, clonal deletion in the thymus
is presumed not to involve bystander death since it should
be limited to cells directly stimulated by TCR interactions.
In this study we have focused on identifying mechanisms
of TCR-dependent clonal deletion of rigorously purified
DP thymocytes to avoid confounding signals that can result
from contact with other thymic elements. We identify two
distinct mechanisms for generating second signals leading to
TCR-induced DP thymocyte apoptosis: (a) a CD28-dependent mechanism that requires simultaneous engagement of
TCR and CD28 surface molecules, and (b) a CD28-independent mechanism initiated by TCR signals but mediated
subsequently by APC signals. While we found that fas signals can induce DP thymocyte death, they were not involved in either TCR-specific apoptotic mechanism. We
propose that the two mechanisms of DP apoptosis revealed
in this report represent two distinct TCR-specific clonal deletion strategies in the thymus: (a) a CD28-dependent
mechanism specific for antigens on B7+ cells, i.e., APC and
medullary epithelium, and (b) a CD28-independent mechanism specific for antigens on B7 Mice.
Young adult female C57BL/6 (B6) mice, gld/gld mice
(B6-SMN C3H fasL), and lpr/lpr mice (B6 mrl lpr/lpr) were obtained from The Jackson Laboratory (Bar Harbor, ME) and
young adult female B6 Ly5.2 mice were obtained from the Frederick Cancer Research Center (Frederick, MD). CD28-deficient
mice (CD28 KO) (14) were bred to the C57BL/6 background
and maintained at the Bethesda Naval Medical Research Institute.
Mice transgenic for the human bcl-2 gene (driven by the lck proximal promoter [36] were generously provided by Dr. Stanley
Korsmeyer and bred in our facility at the NIH. Mice deficient in
either the p55 TNFR (p55) (37) or both p55 and p75 TNFR (38 and 38a) were obtained from Immunex (Seattle, WA).
Cell Preparation.
CD4+CD8+ thymocytes were purified from
4-6-wk-old female mice either by panning on anti-CD8 (83-12-5)
coated plates (39) or by Percoll density fractionation (40). Similar
experimental results were obtained with cells isolated by either procedure. In each case >95% of isolated cells were CD4+CD8+.
APC were prepared by treating splenocytes with anti-CD4 (RL-172), anti-CD8 (3-155), anti-Thy-1 (30H12), and rabbit complement as previously described (3). Viable cells were isolated by
centrifugation over Lymphocyte-M (Cedarlane Laboratory, Ltd.,
Ontario, CA). These cell preparations were free of T cells as determined by CD3 staining and FACS® analysis. Lymph node T
cells were prepared from B6 and gld/gld mice by treating single-cell suspensions of cells isolated from a pool of popliteal, inguinal,
axillary, brachial, and submandibular lymph nodes with a combination of anti-class II (M5114), anti-NK1.1 (PK136), anti-HSA
(J11d) culture supernatants, and rabbit complement.
Antibodies.
Anti-CD28 (37.51 [41]) and anti-TCR- Reagents.
Murine recombinant TNF- Culture and Stimulation Conditions.
Purified cell populations
were cultured for 16-20 h in a 7% CO2 humidified incubator in
RPMI 1640 supplemented with 5 × 10 Staining and Flow Cytometry.
Cell death was assayed as previously described (9, 48). In brief, 5 × 105 cultured cells were incubated with saturating concentrations of the FITC conjugated antibodies specified in staining medium (HBSS, 0.5% BSA, 0.5%
NaN3) for 30 min at 4°C, washed, and then incubated with a 1 µg/ml ethidium bromide (EtBr; Sigma) for another 30 minutes at
4°C. Cells were washed again and analyzed using CellQuest software on a FACScan®. In mixed cell culture experiments, DP thymocytes were identified by expression of Ly5.1, Ly5.2, HSA, and
Thy-1, as indicated.
Measures of Cell Death.
Specific cell death (% cell death) was
determined by EtBr staining and was calculated as a normalized
value as follows: (%EtBr+ (stimulated) To identify the molecular requirements for
TCR-dependent DP thymocyte apoptosis, we stimulated
purified DP thymocytes in vitro and assessed them for EtBr
uptake, which identifies cells undergoing apoptosis (9, 48).
We initially examined surface molecules for their ability to
induce apoptosis of DP thymocytes in the absence of TCR
engagement (Fig. 1 a). In fact, we identified two stimuli
that induced DP thymocyte apoptosis in a TCR-independent manner, namely (a) fas engagement by immobilized platebound antibody and (b) TNF
Even though fas and TNFR are members of the TNFR
family of proteins (52), many of which induce cell death,
platebound antibody engagement of three other members
of the family (CD27, CD30, and 41BB) did not affect DP
thymocyte viability (Fig. 1 a, right). In fact, no other molecules examined, including platebound antibodies specific for
CD4, CD5, CD8, CD69, and LFA-1 (9 and data not shown), were capable of mediating TCR-independent DP thymocyte apoptosis (Fig. 1 a).
We next assessed molecules that could induce DP thymocyte apoptosis in the presence of TCR engagement (Fig.
1 b). As expected (9), TCR engagement alone failed to induce significant DP thymocyte apoptosis (Fig. 1 b). We examined three specific sets of surface proteins for their ability
to cooperate with the TCR to kill DP thymocytes (Fig. 1 b
and data not shown), namely (a) molecules thought to be
costimulatory (CD9 [20]), CD28 [reviewed in 53], CD43
[19], and CD81 [54], (b) coactivating molecules that enhance TCR signaling (CD2, CD4, CD5, CD8, CD24,
CD69, and LFA-1), and (c) selected TNFR family members (CD27, CD30, 41BB), which may also exhibit costimulatory activity (15, 17, 21, 52). Importantly,
antibody engagement of each of these molecules cooperated with TCR signaling to upregulate CD5 expression on
DP thymocytes (data not shown). However, only CD28
cooperated with TCR to induce DP thymocyte apoptosis
(Fig. 1 b).
Thus, there exist both TCR-independent and TCR-dependent mechanisms of DP thymocyte apoptosis: TCR-independent mechanisms can be mediated by either fas or
TNFR engagement, whereas the TCR-dependent mechanisms can be mediated by coengagement of TCR and CD28.
The ability of fas and TNF-
While fas and TNFR are not involved in TCR-CD28-
induced apoptosis, additional members of the TNFR family
expressing the "death domain" associated with apoptotic
signaling continue to be identified (56). To determine
if TCR-CD28 coengagement induced apoptosis in trans of
bystander thymocytes via ligands (known and unknown), we used thymocytes from CD28 KO mice as bystander
cells for they do not die in response to TCR-CD28 stimulation (Fig. 3 a). In this mixed culture experiment, we
stimulated cocultures of DP thymocytes from wild-type
(B6-Ly5.2) and CD28 KO mice with both anti-TCR and
anti-CD28 platebound antibodies (Fig. 3 b). We found that
the majority of wild-type DP thymocytes died in cocultures in response to TCR-CD28 coengagement. In contrast, bystander CD28 KO DP thymocytes did not die even
though they were present in the same cocultures and even
though they were stimulated by the immobilized anti-TCR
antibodies (Fig. 3 c). These results demonstrate that TCR-CD28 coengagement only induces death of DP thymocytes that had been stimulated by both TCR and CD28 signals,
and suggests one of two scenarios as illustrated in Fig. 3 d.
Either TCR-CD28 transduces intracellular signals that directly activate an intrinsic death program in DP thymocytes, or TCR-CD28 coengagement stimulates surface expression of an unknown death domain bearing molecule that signals cell death upon interaction with its ligand which must also be expressed on DP thymocytes. In the latter
case, the death receptor-ligand interaction could conceivably operate either on an individual DP thymocyte (i.e., cis)
or between interacting DP thymocytes (trans).
To characterize the molecular basis for
TCR-CD28-induced apoptosis of DP thymocytes, we assessed specific inhibitors for their ability to abrograte DP
thymocyte death (Fig. 4). We found that neither cyclosporine A (reviewed in reference 59) nor wortmannin (60, 61)
inhibited TCR-CD28-mediated death (Fig. 4), indicating that calcineurin and PI 3-kinase activity are not required.
In contrast, DP thymocyte apoptosis induced by TCR-CD28
coengagement was inhibited by (a) GF109203X, a specific
inhibitor of protein kinase C
These data further suggest that the death mechanism initiated by TCR-CD28 coengagement is distinct from that
initiated by fas engagement, for unlike TCR-CD28-mediated death, fas mediated death of thymocytes is not inhibitible by cycloheximide (65, 66) and may not be inhibitable
by bcl-2 (67). To demonstrate this directly, we compared
the effect of bcl-2 on DP thymocyte apoptosis induced by
fas and TCR-CD28 engagement (Fig. 5). In this experiment, we cultured DP thymocytes from wild-type and bcl-2
transgenic mice with three different stimulators of apoptosis: dexamethasone, platebound antibodies against fas, and
platebound antibodies against TCR-CD28. As expected,
dexamethasone killed wild-type DP thymocytes but not DP
thymocytes from bcl-2 transgenic mice (Fig. 5). More importantly, DP thymocytes that expressed bcl-2 were susceptible to fas mediated death but resistant TCR/CD28-induced death (Fig. 5). Thus, the death program initiated
by TCR-CD28 coengagement is distinct from that initiated by fas engagement.
We conclude that apoptotic signals generated by TCR-CD28 coengagement are mediated by PKC Our studies using
an array of antibodies to known molecules on the surface of
DP thymocytes did not identify any proteins other than
CD28 that could cooperate with TCR to induce DP thymocyte apoptosis. To determine if any such molecule existed, we asked whether APC expressed ligands, known or
unknown, for surface molecules that would cooperate with
TCR to induce DP thymocyte apoptosis. In this coculture
experiment, we stimulated DP thymocytes from wild-type
and CD28 KO mice with platebound anti-TCR in the
presence of APC. We confined our assessment of EtBr staining to DP thymocytes by excluding APCs from the analysis
using an allelic marker, Ly5. DP thymocytes from wild-type mice died in response to TCR stimulation in the presence of APC. More importantly, DP thymocytes from CD28 KO mice also died in response to TCR stimulation in the
presence of APC, indicating that APC possess ligands that
engage molecules other than CD28 that can induce death
of TCR-stimulated thymocytes. It is important to note that
this CD28-independent mechanism of DP thymocyte death
strictly requires engagement of TCR on DP thymocytes, as
APCs did not kill unstimulated CD28 KO DP thymocytes
(Fig. 6). From these data we can conclude that APC express or secrete ligands for surface molecules on DP thymocytes that are capable of cooperating with the TCR to
induce TCR-dependent but CD28-independent apoptosis.
The present
results clearly indicate that CD28 is not the only surface
molecule that the TCR can use to kill DP thymocytes. However, it is possible that even though CD28-dependent
and CD28-independent mechanisms of TCR-induced DP
thymocyte apoptosis are mediated by different surface receptor molecules on DP thymocytes, the second signals induced by these different proteins might be identical. That
this is not the case was revealed by experiments in which TCR and second signals were induced sequentially rather
than simultaneously. In this experiment, we prestimulated
DP thymocytes with immobilized anti-TCR for 6 h and
subsequently transferred them to cultures with platebound
anti-CD28. We found that death of prestimulated DP thymocytes was not affected by subsequent stimulation through CD28 (Fig. 7 a). In fact, only subsequent coengagement of
both TCR and CD28 together increased apoptosis of prestimulated DP thymocytes above background (Fig. 7 a). In
contrast, we found in the same experiment that DP thymocytes from CD28 KO mice similarly prestimulated with
immobilized anti-TCR and subsequently transferred to
APCs were killed efficiently (Fig. 7 b). Thus, to stimulate DP thymocyte apoptosis, TCR and second signals induced
by CD28 must be generated simultaneously, whereas TCR
and second signals induced by APC can be generated sequentially, as illustrated in Fig. 7 c. Consequently, CD28-dependent and CD28-independent pathways of cell death
represent two distinct mechanisms by which TCR can induce DP thymocyte apoptosis.
To determine if fas/
fasL interactions were involved in APC-induced cell death,
we prestimulated CD28 KO DP thymocytes with platebound
anti-TCR and then transferred them to cultures containing APCs isolated from fasL-deficient (gld) mice (Fig. 8). FasL-deficient APCs from gld mice mediated the death of TCR-stimulated DP thymocytes as efficiently, if not more efficiently,
than wild-type APCs (Fig. 8). Thus, the CD28-independent mechanism of TCR-mediated DP cell death is also
independent of fas/fasL interactions.
It was conceivable that the CD28-independent mechanism of cell death involved a CD28-like molecule such as
CTLA-4 that could be engaged by B7 ligands on APC
(68). In fact, this is not the case, as blocking antibodies to
B7-1 and B7-2 failed to inhibit the ability of APCs to kill
TCR-prestimulated DP thymocytes from CD28 KO mice
(Fig. 8). Similar results were obtained with soluble CTLA-4 Ig
(data not shown). Furthermore, the apoptosis observed was
not a consequence of the transfer of small amounts of anti-TCR which could bind to Fc receptors (FcR) on APC, for
the anti-FcR antibody 2.4G2 did not inhibit the apoptotic
effects of sequential TCR and APC engagement (Fig. 8).
Finally, these data suggest that TCR prestimulation (in
the absence of CD28 coengagement) "prepares" DP thymocytes to undergo apoptosis upon subsequent interaction
with APC, possibly by inducing expression of surface molecules containing death domains that bind APC-derived
ligands. Consistent with such a possibility, cycloheximide
reduced by over 70% the number of TCR prestimulated DP thymocytes that died upon subsequent exposure to
APC (data not shown), indicating that new protein synthesis is required to make TCR prestimulated DP thymocytes
vulnerable to death. TNFR family members are attractive
candidate molecules whose synthesis could be induced on
DP thymocytes by TCR prestimulation. Indeed, CD30 has
been reported to play a role in thymocyte negative selection (21). Nevertheless, CD30 does not appear to be involved as neither soluble CD30-Ig fusion protein (45) (Fig. 8)
nor antibodies specific for the CD30 ligand (data not shown)
blocked the ability of APCs to mediate CD28-independent
apoptosis of prestimulated DP thymocytes.
The present report identifies two distinct mechanisms by
which DP thymocytes can be induced to undergo apoptosis in response to TCR signals. In the CD28-dependent
mechanism, TCR signals are delivered simultaneously with
CD28 costimulatory signals, resulting in DP thymocyte death.
In the CD28-independent mechanism, TCR signals are
not accompanied by costimulatory signals and death signals
are generated upon subsequent interaction with APCs. In addition the present study demonstrates that purified DP
thymocytes can also be induced to undergo apoptosis in the
absence of TCR signals by fas-fasL and TNF-TNFR interactions, but such TCR-independent mechanisms of apoptosis cannot be the basis for TCR-specific clonal deletion
of DP thymocytes in vivo.
The CD28-dependent mechanism of apoptosis is a consequence of simultaneous TCR and CD28 coengagement
on DP thymocytes and results exclusively in death of DP
thymocytes that have simulataneously received both TCR
and CD28 signals. Such apoptotic signals do not require
the activity of PI 3-kinase, a signaling molecule associated
with CD28 (69), but are resistant to the effects of cyclosporine A, a hallmark of CD28 involvement in mature T cells
(70). Apoptosis induced by TCR-CD28 coengagement requires PKC The mechanism responsible for CD28-independent DP
apoptosis is distinct from that of TCR-CD28-induced apoptosis. CD28-independent DP thymocyte apoptosis does
not require costimulatory signals. Rather, TCR engagement alone on DP thymocytes appears sufficient to induce
surface expression of a molecule which triggers death upon
subsequent engagement by ligands expressed by APCs.
This proposed surface molecule has features of death domain containing receptors, such as some members of the
TNFR family. Thus, TCR signals may stimulate expression of a TNFR family member which is neither fas, nor
CD30, nor 41BB, but which generates apoptotic signals
when engaged by an appropriate ligand expressed or secreted by APC.
It is conceivable that all programmed cell death is initiated by signals transduced by death domains on specialized
surface receptors. From this perspective, surface death receptors would be responsible for death of DP thymocytes
stimulated either by TCR-CD28 costimulatory signals or
by CD28-independent TCR signals alone. However, it is
important to appreciate that the identity and specificity of
the death receptors induced by TCR-CD28 signals must
be different than those induced by TCR signals alone. This conclusion is based on our data showing that TCR-CD28-
stimulated DP thymocytes kill themselves or each other,
but do not kill TCR-stimulated DP bystander cells (Fig. 3 c).
Rather, TCR-stimulated DP thymocytes are induced to
die upon subsequent interaction with APCs (Fig. 7 b).
Therefore, from the perspective that all cell death is mediated by death domain containing receptors, the ligand for
TCR-CD28-induced death receptors must be expressed
on DP thymocytes themselves, whereas the ligand for TCR
induced death receptors is not expressed on DP thymocytes
but is expressed on APCs.
The importance of APCs in inducing clonal deletion in
the thymus has long been appreciated (8, 71). However, the mechanism by which APCs induce DP thymocyte apoptosis has not been fully understood. Here we show
for the first time that APCs can induce death of TCR stimulated DP thymocytes in two distinct ways via two distinct
sets of ligands. Our observation that TCR signals can induce DP thymocyte apoptosis by two different mechanisms is relevant to an understanding of clonal deletion of DP
thymocytes during normal in vivo development. In Fig. 9
we illustrate a model of in vivo clonal deletion that is based
on our present data. We propose that the two mechanisms
of TCR-mediated DP apoptosis identified in this report
represent two distinct strategies to rid the developing DP
thymocyte repertoire of autoreactive specificities during thymocyte development (Fig. 9). Only DP thymocytes whose TCRs bind MHC-peptide complexes on cells expressing
the CD28 ligands B7-1 or B7-2 will be killed as a consequence of TCR and CD28 coengagement. Because B7-1
and B7-2 are not expressed in the thymic cortex but are
only expressed on APCs and medullary epithelial cells, this form of clonal deletion would be confined to DP thymocytes autoreactive to self-antigens encountered either at
the corticomedullary junction, where most APCs are located, or on medullary epithelium. In contrast, high-affinity
interactions of TCR on DP thymocytes with self-antigens in the cortex would induce expression of a surface molecule that "tags" DP thymocytes for future disposal (i.e., a
"death tag"). Such DP thymocytes would not die in response to TCR engagement per se, but would travel
through the cortex and enter the corticomedullary junction
where they would encounter a "screen" of APCs constitutively expressing or secreting a ligand that bind the putative
death tag and induce apoptosis.
This model provides thre main insights. First, apoptosis
of DP thymocytes targeted for deletion occurs either at the
corticomedullary junction (CMJ) or in the thymic medulla,
regardless of whether DP cells encountered antigen in the
cortex. As a result, DP thymocyte deletion requires that
DP cells migrate out of the thymic cortex to the CMJ or
medulla which is considered to be a consequence of positive selection. Thus, our model predicts that DP thymocytes can only be clonally deleted in vivo if they are first
positively selected. Observations that TCR-mediated apoptosis is confined to the thymic medulla are consistent with
this perspective (74). Indeed, only DP thymocytes dying
of neglect appear to undergo apoptosis in the cortex (75).
Second, the model proposes that CD28-dependent and
CD28-independent mechanisms of TCR-mediated DP apoptosis eliminate clones bearing TCR specific for distinct
sets of antigens: the CD28-dependent mechanism disposes
of TCR reactivities to APC and medullary antigens, whereas
the indirect, CD28-independent mechanism of TCR-mediated DP apoptosis disposes of TCR reactivities to cortical
antigens. Recently, experimental mice were generated in
which expression of MHC class II was confined to cortical
epithelium (76). Our model predicts that these experimental mice would lack the CD28-dependent mechanism of
death because medullary cells expressing B7 (thymic APC or
medullary epithelium) would lack MHC class II expression and fail to engage TCR on DP thymocytes. Indeed, T cells
in these animals were not tolerant to self-antigens on Class
II+ APCs. Importantly, while CD28-dependent clonal deletion is absent in these mice, our model predicts that the
CD28-independent mechanism of clonal deletion should
be intact, so that their CD4+ T cells would be tolerant to
self-antigens expressed on class II+ cortical epithelium.
Third, the model predicts that there can be a delay between the receipt of a negative selecting TCR signal and
the death of the cell by the CD28-independent mechanism. Thus, the presence of autoreactive TCR specificities
among DP thymocytes is not necessarily indicative of a failure of negative selection.
Our efforts to define the molecular basis for CD28-dependent mechanisms of TCR-mediated DP apoptosis revealed
that CD28 is surprisingly unique in its capacity to cooperate with TCR to directly produce apoptotic signals. Even
molecules that have been described as augmenting proliferation of mature T cells, such as CD9, CD43, CD81, CD27,
41BB, and CD30, were not able to stimulate TCR-mediated death of DP thymocytes, so DP thymocyte apoptosis is
not the result of simultaneous engagement of TCR with
any costimulatory molecule. Rather, CD28 appears to generate unique second signals whose identities have not yet
been elucidated. Interestingly, TCR-CD28-induced thymocyte apoptosis does not require the activity of PI 3-kinase, one molecule known to associate with CD28.
The identity of the APC-derived signals responsible for
CD28-independent thymocyte apoptosis is not known.
While such signals may well involve molecules containing
death domains, this study has ruled out two attractive candidates, CD30 and 41BB (Fig. 1 b). However, proteins expressing death domains continue to be identified. In fact,
three newly identified molecules containing death domains
(DR-3, DR-4, and TRAMP) are expressed in lymphopoietic tissues (56).
Finally, it is important to draw a distinction between TCR-mediated apoptosis of DP thymocytes and the phenomenon of negative selection. Negative selection refers to any
process that rids a developing T cell repertoire of an autospecificity. Although TCR-mediated clonal deletion of DP
thymocytes is thought to be a major component of negative selection, it is clearly not the only mechanism responsible for negative selection. Indeed, we have previously shown
that thymocytes can be developmentally arrested before the
DP stage in response to TCR signals (3) and others have
shown that negative selection can occur after the DP stage of development (77). In all likelihood the molecular
mechanisms that operate at these other developmental stages
(both pre- and post-DP) are distinct from those responsible
for TCR-dependent apoptosis of DP thymocytes. In support of this possibility, it is interesting to note that (a) transgenic bcl-2 expression does not always affect negative selection (36) even though it abrogates clonal deletion of DP
thymocytes and (b) fas appears to play a role in the TCR-mediated death of semimature T cells in the thymus (79)
but clearly does not play a role in the TCR-mediated death of rigorously purified DP thymocytes.
In conclusion, this study identified both TCR-independent and TCR-dependent mechanisms of DP thymocyte
apoptosis and reveals that TCR-dependent mechanisms of
DP thymocyte death occur by two mechanisms: (a) a CD28-dependent mechanism in which TCR and CD28 costimulatory signals must be received simultaneously to generate apoptotic signals and (b) a CD28-independent mechanism
in which TCR signals are indirectly responsible for apoptosis by upregulating molecules which, when subsequently
engaged by APCs, will induce cell death. We propose that
these two mechanisms represent two distinct strategies used
by the thymus to dispose of autoreactive DP thymocytes
and that the strategy used depends on where the antigen is
encountered. Hence, DP thymocytes autoreactive to cortical antigens will be removed from the T cell repertoire by
the indirect CD28-independent mechanism, whereas DP
thymocytes autoreactive to APC or medullary cell antigens
will be removed from the repertoire by CD28-dependent
mechanisms (Fig. 9). Thus, the present study reveals an unexpected diversity of molecular mechanisms responsible for
TCR-specific clonal deletion.
cells in the thymic cortex.
(H57-597
[42]) were affinity purified in our laboratory from hybridoma culture supernatant on columns of protein G- and protein A-Sepharose
(Pharmacia LKB Nuclear, Gaithersburg, MD), respectively. Anti-CD2 (RM2-5), anti-CD43 (S7), anti-CD27 (LG.3A10), anti-41BB
(1AH2), neutralizing anti-TNF-
(G281-2626), anti-CD80 (1G10),
anti-FcR (2.4G2), anti-fas (Jo2), and FITC-conjugated anti-HSA,
FITC anti-Ly5.2 (CD45.1) and FITC anti-Ly5.1 (CD45.2) were
purchased from Pharmingen laboratories. FITC-conjugated anti-CD5 (Ly-1) was purchased from Beckton Dickinson (San Jose, CA).
Anti-CD81 was the product of hybridoma 2F7 (43). Anti-CD9
(9D3 [20]), anti-CD24 (20C9 [44]), anti-CD30 (mCD30.1 [45]),
anti-CD30L (M15 [46]), and anti-B7-2 (GL1 [47]), were generously provided by Drs. Hiromi Fujiwara (Osaka University, Osaka,
Japan), Charles Janeway (VCI, NIH, Bethesda, MD), Eckhardt
Podack (University of Miami School of Medicine, FL), Phil Morrissey (Immunex, Seattle, WA), and Richard Hodes (NIH, Bethesda, MD), respectively.
(R&D Labs., Minneapolis, MN) was used at a final concentration of 100 ng/ml.
CD30-Ig was generously provided by Dr. Eckhardt Podack. Cyclosporine A was purchased from Calbiochem-Novabiochem (La
Jolla, CA) and cycloheximide, wortmannin, and GF109203X were
purchased from Sigma Chem. Co. (St. Louis, MO). The caspase
inhibitor Cbz-Val-Ala-Asp-(Ome)-fluoromethyl ketone (ZVAD-FMK) was purchased from Enzyme Systems Products (Dublin, CA).
5 M 2-ME and 10% FCS
at 37°C. Single-cell suspensions of DP thymocytes were plated in
24-well tissue culture plates (Corning Glass, Corning, NY) at a
cell density of 2 × 106/ml in a total of 500 µl per well. When
mixed culture experiments were performed, DP cells from CD28-deficient mice were incubated with DP cells from Ly5.2 mice at a
1:1 ratio and with LN T from Ly5.2 mice at a 1:2 or 1:3 ratio.
APC were mixed with DP thymocytes at a 2:1 or 3:1 ratio and
3 × 106 total cells were plated per well. For stimulation, wells in
a 24-well plate were coated with antibody combinations by incubating them overnight at 4°C with 350 µl of a 10 µg/ml (most
antibodies) or 50 µg/ml (anti-CD28) of each affinity-purified antibody specified in PBS.
%EtBr+ (unstimulated
control))/(100
%EtBr+ control). Background EtBr staining of
cultured DP thymocytes ranged between 18 and 30%. To compare thymocyte apoptosis in response to TCR-CD28 signals among
experimental groups with different internal controls (i.e., different mouse strains, different solvents, etc.), individual responses
were normalized and expressed as a killing index. The killing index was calculated as follows: (% DP cell death induced by TCR-CD28 under experimental conditions)/(% DP cell death induced
by TCR-CD28 under control conditions). Killing index = 1.0 means that the indicated condition did not affect TCR-CD28-
induced apoptosis; killing index >1 means that the indicated condition inhibited TCR-CD28-mediated death. All experiments displayed were performed three or more times with similar
results.
Surface Molecules Expressed by CD4+CD8+ Thymocytes
That Kill Cells by Either TCR-dependent or TCR-independent
Mechanisms.
engagement of
TNFR. Antibody engagement of surface fas molecules induced apoptosis of most DP thymocytes, consistent with
others' observations and the high expression of fas on DP thymocytes (49); and soluble TNF-
engagement of surface receptors induced apoptosis of 20% of DP thymocytes,
which may represent a distinct TNF-
responsive subpopulation (50) (Fig. 1 a). Our present finding that immobilized anti-fas antibody efficiently induces DP thymocyte death
differs from that of Ogasawara et al. (51) who found that
metabolic inhibitors were necessary to enhance DP thymocyte death induced by soluble anti-fas antibodies. We think
that our different observations may be due to differences in
the efficiency by which fas is crosslinked by soluble versus immobilized antibody.
Fig. 1.
TCR-independent
and TCR-dependent mechanisms of DP thymocyte apoptosis. Antibodies against cell surface
molecules that possess costimulatory/coactivating activity and
antibodies against TNFR family
members were assessed for their
ability to induce apoptosis of rigorously purified DP thymocytes.
Control EtBr percentages ranged
between 18 and 30% in all experiments presented in this report.
(a) Only TNF- and fas induce
death of DP thymocytes in the
absence of TCR stimulation. DP
thymocytes isolated from young adult female B6 mice were stimulated by platebound antibodies or
by 100 ng/ml recombinant murine TNF-
. Cells were harvested after overnight incubation and
percent cell death was quantitated by EtBr staining (see Materials
and Methods). (b) Only CD28 cooperates with TCR to induce death of DP thymocytes. Cells were stimulated by platebound antibody combinations as
indicated. Anti-TCR-
(H5-597) was plated at a concentration of 10 µg/ml. Even though anti-CD28 antibody was unique in its ability to induce cell
death, every experimental antibody enhanced TCR-mediated upregulation of CD5 (data not shown). In a and b, each experimental antibody (other than
anti-CD28) was plated at both 10 and 50 µg/ml concentrations. Results from the 10 µg/ml plating preparations are shown and were indistinguishable
from results with 50 µg/ml concentrations. Cells were harvested and percent cell death was quantitated by EtBr staining (Materials and Methods).
[View Larger Versions of these Images (20 + 24K GIF file)]
to mediate DP thymocyte apoptosis
raised the possibility that both mechanisms of DP thymocyte apoptosis (TCR-independent and TCR-dependent)
may ultimately result from engagement of fas or TNFR.
To address this possibility, we examined the ability of TCR-CD28 coengagement to kill purified DP thymocytes from
mice deficient in (a) fas (lpr, reviewed in reference 55), (b)
fas ligand (gld), (c) the p55 murine TNFR, or (d) both the
p55 and p75 murine TNFRs (38, 38a) (Fig. 2). None of
the mutations significantly affected the ability of TCR-CD28 coengagement to induce apoptosis of DP thymocytes, indicating that neither fas nor the TNF receptors
p55 or p75 were required (Fig. 2). In addition, neutralizing
anti-TNF-
antibodies had no effect on TCR-CD28-
induced apoptosis of wild-type DP thymocytes (data not
shown). We also found that TCR-CD28-induced death
was not blocked by CD30 Ig, a fusion protein that blocks
CD30-CD30L interactions (data not shown). We conclude
that TCR-CD28 coengagement induces DP thymocyte
apoptosis by a mechanism that is independent of fas/fasL,
TNF-TNFR, and CD30-CD30L interactions.
Fig. 2.
TCR-CD28 killing of DP thymocytes is not mediated by
fas-fasL or TNF-TNFR interactions. DP thymocytes were isolated from
the mice indicated (wild-type B6, gld/gld (fasL deficient), lpr/lpr (fas deficient), TNFR (p55)-deficient, and TNFR (p55 and p75)-deficient mice
strains. Single-cell suspensions were stimulated overnight by platebound
anti-TCR- and anti-CD28 antibodies, and then were harvested and
stained with EtBr. To compare thymocyte apoptosis from different mouse
strains with different internal controls, we have normalized individual responses to their respective controls. The normalized value is referred to as
a killing index.
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Only TCR-CD28-stimulated DP thymocytes die in response to TCR-CD28 coengagement. (a) TCR-CD28 stimulation will
not kill bystander CD28 KO DP thymocytes. Individual populations of
DP thymocytes from wild-type B6 mice and CD28 KO mice were cultured and stimulated independently with platebound antibodies. % Cell
death of each population of DP thymocytes was quantitated and normalized as described in Materials and Methods. (b) Experimental design. DP thymocytes from wild-type mice (CD28+Ly5.2+) were mixed in a 1:1 ratio
with DP thymocytes from CD28-deficient mice (CD28/
Ly 5.1+) and
stimulated by platebound anti-TCR-
and anti-CD28. Thymocytes were harvested after overnight culture and percent cell death in each population was determined. CD28+/+ and CD28
/
DP thymocytes were
distinguished by the presence or absence of Ly5.1 staining. (c) Bystander
CD28 KO DP thymocytes are not killed by TCR-CD28 signals. DP thymocytes were isolated from wild-type and CD28-deficient (CD28 KO)
mice which differed in Ly5 expression such that wild-type DP thymocytes were Ly5.2+ and CD28 KO thymocytes were Ly5.1+. Harvested
cells were stained with both anti-Ly5.1 antibody and EtBr to determine
cell death in each population of cocultured DP thymocytes. Percent cell death was quantitated and normalized as described in Materials and Methods. (d)
Schematic of the mechanism by which TCR-CD28 coengagement kills DP thymocytes. This figure illustrates two possible CD28-dependent mechanisms of TCR-mediated apoptosis of DP thymocytes, both of which result in death exclusively of TCR-CD28-stimulated DP thymocytes. The upper
figure (i) illustrates one scenario in which simultaneous coengagement of TCR and CD28 molecules directly and cell-autonomously induces an apoptotic program. The lower figure (ii) illustrates an alternative scenario in which simultaneous coengagement of TCR and CD28 induces expression of a
death domain containing receptor (Y) that signals apoptosis upon interaction with its ligand (Y-L, Y ligand) that is also expressed on DP thymocytes. In
this latter case, the ligand could conceivably engage the death receptor in either cis or trans.
[View Larger Versions of these Images (16 + 14 + 16 + 12K GIF file)]
(PKC
(61, 62), (b) the protein synthesis inhibitor, cycloheximide, and (c) the caspase
(ICE-family protease) inhibitor, ZVAD-FMK (63, 64) (Fig.
4). Consistent with the participation of caspases, TCR-CD28
coengagement failed to induce significant apoptosis in DP
thymocytes from bcl-2 transgenic mice which constitutively overexpress the anti-apoptotic bcl-2 protein (Fig. 4).
Fig. 4.
Inhibitors of TCR-CD28-mediated death of DP thymocytes. DP thymocytes from wild-type (B6) mice were stimulated by
platebound anti-TCR- and anti-CD28 in the presence or absence of
the following pharmacological agents: the calcineurin inhibitor, cyclosporine A (1 µg/ml); the PI-3-kinase inhibitor, wortmannin (800 ng/ml); the
PKC
inhibitor, GF109203x (800 ng/ml); the protein synthesis inhibitor,
cycloheximide (10 µg/ml); and the caspase inhibitor, ZVAD-FMK (100 µM). To compare the effects of various reagents on TCR-CD28-mediated DP thymocyte apoptosis in experiments performed with different
solvent controls, individual responses were normalized to their respective
controls (killing index). As positive controls for the pharmacologic agents
used: cyclosporine A and GF109203x used in this experiment inhibited
TCR-mediated CD5 upregulation, and wortmannin used in this experiment blocked NK-mediated target cell lysis (data not shown). Also displayed in the same format are the results of anti-TCR-CD28 stimulation
of DP thymocytes isolated from bcl-2 transgenic mice.
[View Larger Version of this Image (18K GIF file)]
Fig. 5.
Distinct signaling mechanisms of DP thymocyte apoptosis as
revealed by differential sensitivity to bcl-2. DP thymocytes isolated from
wild-type (B6) and bcl-2 transgenic (bcl-2 TG) mice were cultured with
three distinct apoptotic stimuli: dexamethasone (106 M), platebound anti-fas antibodies, and platebound anti-TCR and anti-CD28. It can be seen
that transgenic bcl-2 expression abrograted TCR-CD28-mediated apoptosis but not fas-mediated apoptosis of DP thymocytes. It might be
noted that thymocyte death by TCR-CD28 engagement does not involve glucocorticoids as the steroid inhibitor RU486 only blocked death
induced by dexamethasone but not by TCR-CD28 (data not shown).
[View Larger Version of this Image (13K GIF file)]
, require protein synthesis, and result in the activation of intracellular
caspases that are regulated by bcl-2.
Fig. 6.
A CD28-independent mechanism of TCR-mediated death
of DP thymocytes. DP thymocytes from either B6 (Ly 5.1+) or CD28
KO (Ly5.1+) mice were co-cultured with Ly 5.2+ APC in the presence
or absence of platebound anti-TCR. Harvested cells were stained with
both anti-Ly5.1 antibody and EtBr. DP thymocytes were distinguished
from APCs by expression of Ly5.1.
[View Larger Version of this Image (14K GIF file)]
Fig. 7.
CD28-dependent and CD28-independent mechanisms of DP thymocyte apoptosis are distinct. (a) TCR and CD28 signals must be received simultaneously to mediate death of DP thymocytes. DP thymocytes from B6 mice were prestimulated by platebound anti-TCR- for 6 h. They were
then removed from this stimulus and transferred to wells that had been precoated with the antibodies indicated on the x-axis. Cells were harvested and
stained with EtBr. The background cell death observed in TCR pretreated groups is likely due to cell damage inflicted by their physical removal from
platebound anti-TCR antibody. (b) TCR and second signals derived from APCs do not have to be simultaneous to induce CD28-independent DP thymocyte death. DP thymocytes from CD28 KO (Ly 5.11) mice were prestimulated by platebound anti-TCR-
for 6 h. They were then removed from this
stimulus and transferred either into wells that had been precoated with anti-CD28 or into wells containing APCs from B6 Ly5.2 mice (in a 2:1 ratio with
the DP cells). Cells were harvested and stained with both Ly5.1 and EtBr. DP thymocytes were distinguished from APC by expression of Ly5.1. As can
be seen, preengagement of TCR on DP thymocytes made them susceptible to APC-induced cell death. (c) Schematic of the CD28-independent mechanism of DP thymocyte apoptosis. This figure illustrates the proposed mechanism by which TCR and subsequent APC signals induce apoptosis of DP thymocytes in a CD28-independent manner. TCR prestimulation of DP thymocytes (1) induces upregulation of a molecule X which might express death domains (2). Subsequent engagement of molecule X with a ligand expressed by APCs (X ligand or X-L) induces apoptosis of only prestimulated DP thymocytes (3).
[View Larger Versions of these Images (20 + 16 + 13K GIF file)]
Fig. 8.
Characterization of the CD28-independent mechanism of
DP thymocyte apoptosis. DP thymocytes from CD28 KO mice were
presetimulated with platebound anti-TCR antibody for 6 h, then cocultured with APC from either gld mice or B6 Ly5.2 mice in the presence or
absence of the following reagents: anti-FcR antibody (2.4G2, 10 µg/ml); the fusion protein CD30 Ig (10 µg/ml) which blocks CD30-CD30L interactions; or a combination of anti-B7-1 and anti-B7-2 antibodies (10 µg/ml each) which blocks B7 ligand engagement by both CD28 and
CTLA-4. To compare the effects of various reagents on TCR-CD28- mediated DPthymocyte apoptosis in experiments performed over time, individual responses were normalized to their respective controls and the
normalized value is referred to as a killing index.
[View Larger Version of this Image (17K GIF file)]
and employs a common effector pathway involving caspases that is inhibitible by bcl-2. The present
results are consistent with either of two possibilities: simultaneous TCR-CD28 signals induce DP thymocyte death
either by directly initiating a novel intracellular apoptotic
program or by inducing surface expression of an uncharacterized death receptor (e.g., a TNFR family member) that
is engaged by surface ligands. In either case, the CD28-dependent mechanism of death requires that both TCR
and CD28 signals be delivered simultaneously and requires
that the death ligand, if it exists, be expressed on DP thymocytes for these were the only cells present in our cultures.
We favor the possibility that TCR-CD28 directly signals
an apoptotic program because we have found that TCR-CD28-induced apoptosis of DP thymocytes occurs efficiently at very low cell densities at which cell-cell interactions are unlikely to occur (data not shown). However, we
cannot exclude the possibility that TCR/CD28 stimulation
results in lethal death receptor/ligand interactions occurring
in cis on the surface of individual DP thymocytes (Fig. 3 d).
Fig. 9.
Proposed model of two distinct mechanisms of intrathymic
DP clonal deletion. Applying the present data to in vivo thymocyte development, we propose that there are two distinct mechanisms by which
autoreactive DP thymocytes are deleted in the thymus. CD28-dependent
mechanism (right): DP thymocytes recognizing self-antigens on B7+ cells
(i.e., medullary epithelial cells or APCs) are killed by signals generated by
simultaneous engagement of TCR and CD28. TCR-CD28 coengagement may directly initiate an apoptotic program (i) or may upregulate a
death domain containing receptor that signals death upon interaction with
its ligand on DP thymocytes (ii). CD28-independent mechanism (left):
DP thymocyte expressing TCR's recognizing self-antigens on cortical epithelium do not die as a consequence of TCR engagement. Instead, they
are induced to express a surface molecule (i.e., a death tag, X) that signals them to undergo apoptosis upon subsequent interaction with APC's present in the CMJ. It should be noted that in this model, both mechanisms of clonal deletion of autoreactive DP thymocytes are proposed to
occur in the CMJ or medulla even if they encounter self-antigen in the
cortex.
[View Larger Version of this Image (25K GIF file)]
Address correspondence to Alfred Singer, Experimental Immunology Branch, NCI/NIH, Bldg 10, Rm 4B-17, Bethesda, MD 20892. Phone: 301-496-5461; Fax: 301-496-0887; E-mail: singera{at}nih.gov Dr. Punt's current address is Haverford College, Haverford, Pennsylvania, 19041. Dr. Abe's current address is Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda City, Chiba 278, Japan.
Received for publication 21 July 1997 and in revised form 12 September 1997.
This report was supported in part by National Medical Research and Development Command EQ.0095.003.1412. The views expressed in the article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, or the US Government.We are grateful to Drs. Eckhardt Podack, Philip J. Morrissey, Charles Janeway, and Karen Hathcock for generously providing reagents and mice; to Shabnam Shahabadi for excellent technical assistance; to Drs. Barbara Osborne, E. Wendy Shores, Susan Sharrow, and Pierre Henkart for critically reviewing the manuscript.
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