By
From the Department of Immunology, IMM4, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
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Abstract |
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To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses
of antigen. In normal mice, injection of 1-100 µg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive V8+ cells at the level of the semi-mature population
of HSAhi CD4+ 8
cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal.
SEB injection also caused marked elimination of V
8+ HSAhi CD4+ 8
thymocytes in lpr/lpr
mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (
1 µg) but not by high doses (100 µg). Similar findings applied when
T cell receptor transgenic mice were injected with specific peptide. These findings suggest that
clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen
but Fas dependent at high doses. Previous reports documenting that negative selection is not
obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level.
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Introduction |
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Self-tolerance induction is largely a reflection of negative selection (clonal deletion) of immature T cells during maturation in the thymus (1). Despite the importance of central (thymic) tolerance, some self-antigens, e.g., tissue-specific antigens, are poorly represented in the thymus. Hence, unresponsiveness of T cells to these antigens is thought to involve peripheral mechanisms. Of the various mechanisms proposed to account for peripheral tolerance, considerable attention has been focused on the role of Fas (CD95) (5). This cell-surface molecule is upregulated after TCR stimulation and results in activation-induced cell death (AICD)1 through interaction with Fas ligand during the late stages of the primary response. In support of this view, the normal elimination of T cells after the primary response (9) is impaired in Fas-deficient lpr/lpr mice (11).
During later life lpr/lpr mice develop massive lymphadenopathy and auto-antibody production (17). This syndrome is considered to reflect a breakdown of peripheral tolerance as the result of defective AICD (8, 11). The possibility that lpr/lpr mice have a defect in central tolerance seems unlikely since most groups have failed to find evidence for impaired negative selection in the thymus of lpr/lpr mice (11, 12, 17). Nevertheless, a recent study found reduced apoptosis of cortical thymocytes in lpr/lpr mice after injection of specific peptides or anti-TCR mAb (26). However, this effect was only apparent within the first 24 h after injection.
Most studies on thymic tolerance have focused on negative selection occurring in the cortex. Recently, we obtained evidence that negative selection can operate at the
level of the semi-mature subset of heat-stable antigen
(HSA)hi CD4+ 8 cells found in the medulla (27). Thus,
combined TCR/CD28 ligation in vitro induced rapid
(<24 h) induction of apoptosis in HSAhi CD4+ 8
cells; by
contrast, for fully mature HSAlo CD4+ 8
thymocytes
TCR/CD28 ligation caused T cell activation rather than
death. An unexpected finding in these experiments was
that Fas played a decisive role in TCR/CD28-mediated
apoptosis, but only when TCR ligation was induced with a
high concentration of anti-TCR mAb. With a low-to-moderate concentration of this mAb, apoptosis induction
was Fas-independent. Therefore, the implication is that Fas
expression might play an important role in negative selection, but only for antigens expressed at a high level.
Since the above data were derived from a highly artificial
in vitro model, the relevance of the data to normal negative
selection in vivo is questionable. To seek direct evidence
on the possible role of Fas in negative selection, we have
now examined the effects of injecting normal versus lpr/lpr
mice with various doses of Staphylococcus enterotoxin B
(SEB), a soluble superantigen (SAg) recognized by V8+
CD4+ T cells (28); previous studies have shown that injection of this antigen induces clonal elimination of T cells in
the thymus (29). We also examined negative selection in
D011 TCR transgenic mice (30) after injection of specific
ovalbumin (ova) peptide. In each situation, injection of antigen caused elimination of HSAhi CD4+ 8
thymocytes.
However, in lpr/lpr mice, negative selection failed to occur
when the dose of antigen was raised to a high level.
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Materials and Methods |
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Mice.
C3H/HeJ (C3H), MRL/Mpr-Faslpr (MRLlpr/lpr), C3H. MRL-Faslpr (C3Hlpr/lpr), C57BL/6J (B6), and B6-Faslpr (B6lpr/lpr) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility. D011 TCR transgenic mice (30) were bred in our facility and backcrossed three times to MRLlpr/lpr; when used in experiments, neonatal mice were typed individually for Fas and TCR clonotype expression using specific mAbs.Antibodies.
Antibodies specific for the following markers have been previously described (30, 31): CD4 (RL172, rat IgM), CD8 (3.163.8, rat IgM), CD25 (7D4, rat IgM), HSA (J11D, rat IgM), Thy 1.2 (J1j, rat IgM), D011 TCR (KJ1-26, mouse IgG), TCR VOva Peptide.
Ova 323-339 peptides (ISQAVHAAHAEINEAGR) were synthesized on a synthesizer (model 431A; Applied Biosystems, Foster City, CA) by standard solid phase peptide synthesis method (tBoc chemistry) and purified with C18 reverse-phase HPLC. The concentration of peptides was determined by quantitative amino acid analysis.Cell Purification.
Purification of HSAhi or HSAlo CD4+ 8In Vivo Treatment for the Deletion of Immature Thymocytes.
Newborn (1-6-d-old) mice and adult (7-8-wk-old) mice were injected intraperitoneally with SEB (Sigma Chemical Co., St. Louis, MO), anti-TCR mAb (H57-597), or ova 323-339 peptide at the dose specified. At 20 (day 1), 44 (day 2), or 68 (day 3) h after injection, the mice were killed and cell surface markers of thymocytes were analyzed.In Vivo BrdU Labeling.
Mice (4-6 d) were intraperitoneally injected with 0.3 mg of BrdU (Sigma Chemical Co.) in PBS twice, at 20 and 12 h before the mice were killed (34, 35).Culture Conditions.
As described previously, purified cells (2-3 × 105) were cultured in 0.2 ml of RPMI medium supplemented with 5 × 10Flow Cytometry Analysis.
For the in vivo studies, thymocytes were incubated with FITC-conjugated anti-HSA mAb, PE-conjugated anti-CD8 (53-6.7), Cy5-conjugated anti-CD4 (GK1.5), and biotinylated anti-V ![]() |
Results |
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To examine the effects of SEB on negative selection, SEB was injected intraperitoneally into normal C3H
versus Fas-deficient MRLlpr/lpr mice. In these immunogenic
I-E+ strains, SEB is recognized by V8+ T cells, especially
by CD4+ cells (29). In a previous study, injection of SEB
was reported to cause a twofold reduction in the proportion of V
8+ cells in unseparated thymocytes 1 d later both
in normal and lpr/lpr mice (16); whether this finding was
V
specific was unclear. The V
-specific effects of SEB injection on subsets of thymocytes in normal C3H mice is
discussed below. In all experiments a single dose of SEB
was injected.
At the level of CD4+ 8+ double-positive (DP) thymocytes, the effects of SEB injection in adult (10 wk) and
neonatal (4 d) C3H mice were quite different (Fig. 1). In
adult mice, injection of a large dose of SEB (200 µg)
caused pronounced destruction of DP cells and reduced the
total cellularity of the thymus by ~80% at 2 d after injection; the elimination of DP cells was associated with a reciprocal increase in single-positive (SP) CD4+ 8 and
CD4
8+ cells. In neonatal mice, by contrast, a large dose
of SEB (50 µg) caused little or no reduction in the proportion of DP cells and led to a smaller (20-50%) reduction in
total cellularity.
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In contrast to DP cells, SEB injection had a clear V-specific effect on CD4+ 8
SP cells, in both adult and neonatal mice (Fig. 1). At 2 d after injection, SEB injection
caused little or no alteration in the total proportion of SEB-reactive V
8+ CD4+ 8
cells. However, when these cells
were typed for HSA expression, the V
8+ CD4+ 8
subset
showed a marked and selective reduction of semi-mature HSAhi cells and a reciprocal increase in fully mature HSAlo
cells. In control PBS-injected mice, by contrast, the majority of the V
8+ CD4+ 8
cells were HSAhi. The effect of
SEB injection appeared to be specific for V
8+ cells since
there was no change in the HSA phenotype of V
6+ cells.
SEB injection thus led to substantial elimination of HSAhi
V
8+ CD4+ 8
cells while causing apparent expansion of
HSAlo cells; these findings applied in both adult and neonatal mice. Unless stated otherwise, the experiments discussed
below refer to neonatal (4-d-old) C3H mice.
The above data apply to mice given SEB 2 d
before. Other time points are shown in Fig. 2. Gating on
unseparated CD4+ 8 cells (Fig. 2 A, column a) revealed
that the proportion of V
8+ cells was slightly reduced on
day 1 (20 h), increased on day 2 (44 h), and greatly reduced
on day 3 (68 h). Examining HSA expression on V
8+
CD4+ 8
cells (Fig. 2 A, column b) showed that HSAhi
cells were present on day 1, greatly reduced on day 2, but
again visible on day 3. When the data were expressed as total numbers of cells/thymus (Fig. 2 B, left), numbers of
V
8+ HSAhi CD4+ 8
cells were slightly reduced on day 1 and substantially reduced on days 2 and 3; levels of V
6+
cells, by contrast, remained largely unchanged (Fig. 2 B,
right). Essentially similar findings applied when V
8 expression was examined on gated HSAhi CD4+ 8
cells (Fig. 2
A, column c). Thus, V
8+ cells in the HSAhi subset were
present at day 1 but markedly reduced on days 2 and 3. This applied to both the proportion of cells (Fig. 2 A, column c) and total cell numbers (Fig. 2 B).
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The data on V8+ HSAlo CD4+ 8
cells were more
complex. Thus, total numbers of these fully mature cells
were moderately reduced on day 1, elevated on day 2, and
greatly reduced on day 3 (Fig. 2 B, left). Similar findings applied to the proportion of V
8+ cells in gated HSAlo CD4+
8
cells (Fig. 2 A, column d).
Since CD4+ 8 thymocytes
arise from CD4+ 8+ precursors through progressive downregulation of CD8, FACS-gated CD4+ 8
thymocytes include a proportion of CD4+ 8lo cells, which are presumably
less mature than CD4+ 8
cells. The tolerance susceptibility of CD4+ 8+ (CD4+ 8hi), CD4+ 8lo, and CD4+ 8
cells
to SEB is shown in Fig. 3 (note the "sharper" FACS® gate
for CD4+ 8
cells in Fig. 3 than in Figs. 1 and 2). At day 2 after SEB injection, it can be seen that the proportion of
V
8+ cells (the data in parentheses refer to mean values)
was considerably reduced in the CD4+ 8lo subset but markedly elevated in the CD4+ 8
subset; proportions of V
6+
cells remained relatively unchanged. For CD4+ 8hi cells,
V
8+ cells consisted mostly of TCRlo cells with very few
TCRhi cells. Significantly, SEB injection failed to deplete
either V
8hi or V
8lo cells from the CD4+ 8hi subset.
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The above data suggest that, for the FACS® gates used,
SEB-induced elimination of thymocytes was restricted to
CD4+ 8lo cells and did not include either CD4+ 8hi or
CD4+ 8 cells. Here two points should be made. First, before injection, nearly all of the V
8+ CD4+ 8lo cells were
HSAhi (90%), attesting to their relative immaturity. However, after SEB injection the depleted population of V
8+
cells found on day 2 was enriched for HSAlo cells (50%),
implying that SEB selectively deleted the HSAhi V
8+ cells
and caused expansion of the small subset of V
8+ HSAlo
cells (data not shown). Second, HSAhi cells also accounted
for a considerable proportion of CD4+ 8
cells (60%).
However, at 2 d after SEB injection the expanded population of V
8+ cells consisted almost entirely of HSAlo cells
(>90%) (data not shown).
Collectively, these data appear to indicate that the V8+
cells eliminated by SEB injection were predominantly
HSAhi CD4+ 8lo cells. However, whether deletion also affected the slightly more mature population of HSAhi cells
found in the CD4+ 8
subset could not be accurately established because of the high levels of fully mature HSAlo
cells in the CD4+ 8
subset. To seek direct information on
this issue, we prepared purified populations of HSAhi
CD4+ 8
cells by positive panning (for HSA+ cells) and
negative selection with anti-CD8 mAb + C. This treatment removed both CD4+ 8hi and CD4+ 8lo cells. The
HSAhi cells surviving this procedure were thus CD4+ 8
rather than CD4+ 8lo. The susceptibility of these purified
HSAhi CD4+ 8
cells to SEB-mediated deletion in vitro is
discussed below. For the in vivo experiments that follow, it
should be pointed out that the FACS® gate used to define
CD4/CD8 expression was the same as in Figs. 1 and 2
(rather than in Fig. 3); with this gate the cells defined as being CD4+ 8
consisted predominantly of CD4+ 8
cells
plus a small proportion of CD4+ 8lo cells (~30%). For convenience, the HSAhi cells in this mixed population will be
referred to hereafter as HSAhi CD4+ 8
cells.
In interpreting the above data, a key issue is whether the disappearance of HSAhi CD4+ 8 cells on day 2-3 after SEB injection (Fig. 2) simply reflected a switch to HSAlo cells. This
possibility is unlikely because previous studies showed that
culturing purified HSAhi CD4+ 8
cells with a combination of cross-linked anti-TCR + anti-CD28 mAb in vitro
caused many of the cells to undergo apoptosis (27) but
failed to downregulate HSA expression on the surviving
cells (our unpublished data). Similar findings applied when
purified HSAhi CD4+ 8
cells (depleted of 8lo cells; see
above) were cultured overnight with SEB plus spleen APC
in vitro (Fig. 4). As manifested by TUNEL staining, this
treatment induced substantial apoptosis of V
8+ HSAhi
cells (but not V
6+ cells) relative to cells cultured with
APCs without SEB (Fig. 4, A and B). However, there was
no reduction of HSA expression on the surviving (TUNEL
)
cells (Fig. 4 C). With mature HSAlo cells, exposure to SEB
failed to cause apoptosis of V
8+ cells (Fig. 4 D); since the
culture period was brief (20 h), expansion of V
8+ HSAlo
cells was not apparent. These findings applied to normal
cells stimulated with SEB in vitro. If the disappearance of
V
8+ HSAhi CD4+ 8
cells from the thymus between day
1 and 2 after SEB injection (Fig. 2) reflected death of these
cells (rather than a switch to HSAlo cells), onset of apoptosis
would be expected if the cells were harvested on day 1 after
in vivo SEB injection and then cultured in vitro without
further stimulus. This was indeed the case. Thus, when HSAhi
versus HSAlo CD4+ 8
thymocytes were purified from
mice given SEB 1 d before and cultured in vitro overnight
without APCs, there was significant apoptosis of V
8+
HSAhi cells but no apoptosis of V
6+ HSAhi cells (relative
to the background apoptosis in total HSAhi CD4+ 8
cells)
(Fig. 5). For the HSAlo cells, there was slight apoptosis of
V
8+ cells and no apoptosis of V
6+ cells.
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Collectively, these data suggest that
the disappearance of V8+ HSAhi CD4+ 8
cells after SEB
injection reflected elimination of these cells rather than a
switch to HSAlo cells. For the HSAlo cells, the transient reduction in V
8+ cells at 1 day after SEB injection (Fig. 2)
suggested that a small subset of these cells was eliminated at
day 1. The minor degree of apoptosis seen when the cells
were cultured in vitro at 1 day after SEB injection (Fig. 5)
is consistent with this possibility. The expansion of V
8+
HSAlo cells at day 2 after SEB injection presumably indicated proliferation of mature T cells to SEB.
To examine this possibility, groups of neonatal C3H
mice were injected with SEB and thymocytes were prepared 1, 2, or 3 d later; BrdU (2 injections 12 h apart) was
administered for the last 20 h before preparation of thymocytes. The data in Fig. 6 make three points. First, in
agreement with the known high turnover of CD4+ 8 cells
in the neonatal thymus (34), 20 h of exposure to BrdU labeled a high proportion of the V
8+ and V
6+ CD4+ 8
thymocytes in PBS-injected control mice; labeling was
higher for HSAhi cells than for HSAlo cells and, for V
6+
HSAhi cells of the SEB-injected groups, labeling declined
progressively as the 4-d-old mice aged over the 3-d period
studied. Second, relative to V
6+ cells, SEB injection
caused no obvious change in BrdU labeling of V
8+ cells
at 1 d after injection; this applied to both HSAhi and HSAlo
V
8+ cells. Third, labeling of V
8+ HSAlo cells increased
markedly at day 2 after SEB injection and then declined
abruptly on day 3. Labeling of V
8+ HSAhi cells, by contrast, was below normal on both day 2 and 3. Collectively, these data indicate that SEB injection caused V
8+ HSAlo
cells to proliferate for a brief period (evident only on day 2)
and then disappear. This transient proliferative response did not affect V
8+ HSAhi CD4+ 8
cells.
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As discussed earlier (see Introduction), apoptosis of purified HSAhi CD4+ 8 thymocytes in response to combined TCR/CD28 ligation in
vitro was found to be Fas independent with a low concentration of cross-linked anti-TCR mAb, but was completely
Fas dependent with a high dose of this mAb (27). This
finding is illustrated in Fig. 7, where it can be seen that
TCR/CD28 ligation of Fas-deficient B6lpr/lpr HSAhi CD4+
8
cells in culture caused significant apoptosis at a low concentration of anti-TCR mAb (0.1 µg/ml) but not at a high
concentration (10 µg/ml). By contrast, apoptosis of HSAhi
CD4+ 8
cells from normal B6 mice was higher with a
high concentration of anti-TCR mAb than with a lower
concentration (Fig. 7). Similar findings applied when Fas
expression on normal B6 thymocytes was blocked with
Fas-Ig fusion protein (27).
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To examine whether this role of Fas in negative selection also applied in vivo, we tested the effects of injecting
normal versus lpr/lpr neonatal mice with graded doses of
SEB. Based on the results considered above, the expectation was that a moderate-to-low dose of SEB would induce negative selection of HSAhi V8+ CD4+ 8
cells via a
Fas-independent pathway and thus cause cell deletion in
both normal and lpr/lpr mice. By contrast, with a high dose of antigen, negative selection would be Fas dependent and
thus occur only in normal and not lpr/lpr mice.
In accordance with this prediction, injecting graded
doses of SEB into normal neonatal C3H or MRL mice induced strong deletion of HSAhi V8+ CD4+ 8
cells at
doses ranging from 1 to 100 µg SEB/mouse (Fig. 8 A and data not shown). When injected into either MRLlpr/lpr or
C3Hlpr/lpr hosts, by contrast, SEB induced strong deletion at
up to 1 µg but caused little or no deletion at higher doses,
e.g., 100 µg. This difference between normal and lpr/lpr
mice was restricted to HSAhi cells and did not apply to control V
6+ cells (Fig. 8). Similar findings applied when SEB
was injected into adult mice (Fig. 8 B) and also when anti-TCR mAb was injected into neonatal mice (Fig. 8 C). In
each situation, a moderate dose of antigen induced strong
deletion of HSAhi CD4+ 8
cells in both normal and lpr/lpr
mice, whereas a high dose of antigen induced deletion only
in normal and not lpr/lpr mice.
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To seek
further information on the role of Fas in negative selection,
we examined the effects of injecting specific peptide into
neonatal (4-d-old) normal versus Fas-deficient D011 TCR
transgenic mice; mature CD4+ 8 cells from this line are
strongly reactive to an ova peptide, ova 323-339, presented
by IAd (30).
The features of thymocytes from uninjected neonatal
D011 mice are shown in Fig. 9 A. Gating on the subset of
CD4+ 8 thymocytes revealed that nearly all of these cells
were clonotype-positive (Id+). In contrast to the discrete
subsets of HSAhi and HSAlo CD4+ 8
thymocytes found in
normal nontransgenic mice (Fig. 9 A, bottom, thick line), the
vast majority of CD4+ 8
thymocytes from D011 mice
were HSAhi (~90%) and therefore are potentially tolerance
susceptible. For this reason, detecting negative selection of
D011 thymocytes after ova peptide injection was relatively
easy and merely entailed examining CD4/CD8 expression
(rather than enumerating HSAhi CD4+ 8
cells).
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The effects of injecting various doses of ova peptide into
neonatal D011 mice are shown in Fig. 9 B; thymocytes
were examined at day 2 after injection. For normal Fas+
D011 mice, injecting either a moderate dose (1 µg) or a
high dose (100 µg) of ova peptide caused a marked depletion of (total) CD4+ 8 thymocytes. With Fas-deficient
D011lpr/lpr mice, by contrast, depletion of CD4+ 8
thymocytes was apparent only with the lower dose of peptide; with a high dose of peptide deletion of CD4+ 8
thymocytes was minimal. Similar findings applied when the
data were expressed as total numbers of HSAhi CD4+ 8
thymocytes (Fig. 9 C, left). These data apply to thymocytes
examined at day 2 after injection. As for SEB, elimination
of HSAhi CD4+ 8
cells at day 1 after ova peptide injection
was quite limited (data not shown).
Injecting D011 mice with ova peptide caused little if any
expansion of the residual population of HSAlo CD4+ 8 thymocytes. Indeed, especially in Fas+ D011 mice, peptide injection appeared to delete both HSAhi and HSAlo CD4+ 8
cells (Fig. 9 C, middle). In interpreting this finding it should be noted that, in contrast to normal thymocytes, D011 thymocytes lacked a discrete subset of HSAlo CD4+ 8
cells
(Fig. 9 A, bottom): HSA expression was relatively homogeneous and the few HSAlo cells detected expressed intermediate levels of HSA and formed a continuum with the
HSAhi cells. Hence it is not surprising that the HSAlo cells
resembled HSAhi cells in being tolerance susceptible.
Unlike CD4+ 8 thymocytes, D011 CD4+ 8+ thymocytes were comparatively resistant to negative selection
(Fig. 9, B and C, right). Thus, even large doses of peptide
caused only minimal (30%) depletion of CD4+ 8+ cells.
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Discussion |
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The data in this paper make two main points. First, injecting mice with antigen (either SEB or ova peptide)
caused marked antigen-specific elimination of the semi-mature population of HSAhi CD4+ 8 thymocytes within
2 d. Second, based on studies with normal versus lpr/lpr
mice, the disappearance of these cells was Fas independent at low doses of antigen but Fas dependent at high doses.
Before discussing the role of Fas, several features of the
elimination of HSAhi CD4+ 8
cells require comment.
The finding that the disappearance of V8+ HSAhi
CD4+ 8
cells after SEB injection was associated with a reciprocal increase in HSAlo cells raised the possibility that
the HSAhi cells were not deleted but simply switched to
HSAlo cells. This possibility is unlikely because exposing
HSAhi cells to SEB plus APCs in vitro caused rapid onset of
apoptosis of V
8+ cells and failed to reduce HSA expression on the surviving cells. In addition, when purified
HSAhi CD4+ 8
cells were prepared from SEB-injected
mice at 1 d after injection, the V
8+ subset of these cells
underwent apoptosis in vitro without further contact with
SEB, implying that the prior in vivo exposure to SEB had
already signaled the cells to die. In light of these findings, the disappearance of V
8+ HSAhi CD4+ 8
cells after SEB
injection appeared to reflect clonal deletion of these cells
rather than differentiation into HSAlo cells. For D011 mice,
the paucity of HSAlo CD4+ 8
thymocytes in these mice
makes it most unlikely that the deletion of HSAhi cells reflected a switch to HSAlo cells. Thus, in this situation, peptide injection caused extensive deletion of total CD4+ 8
thymocytes.
In tissue culture, deletion of HSAhi CD4+ 8 thymocytes occurred quite rapidly. Thus, when HSAhi CD4+ 8
cells were exposed to SEB plus APCs in vitro, apoptosis of
V
8+ cells was clearly apparent after overnight culture;
whether these rapid kinetics also apply to ova peptide was
not tested. However, under in vivo conditions the elimination of HSAhi CD4+ 8
cells was limited on day 1 but
prominent at days 2 and 3 both for SEB and ova peptide.
Why the disappearance of HSAhi CD4+ 8
cells was delayed in vivo is unclear. One possibility is that the intraperitoneal route of injection impeded entry of antigen into
the bloodstream, and thus led to relatively slow accumulation in the thymus. In support of this idea, injecting SEB
intravenously rather than intraperitoneally accelerated the
onset of V
8+ HSAhi cell elimination by ~10 h (our unpublished data). For ova peptide, we have yet to examine
the effects of peptide injection beyond day 2. For SEB, the
maximal deletion of V
8+ HSAhi CD4+ 8
cells after intraperitoneal injection of SEB was at days 2 and 3. These
cells began to reappear in the thymus at about day 5 after
injection and reached normal levels by day 7 (our unpublished data). The implication is that SEB was cleared from
the thymus within a few days, thus allowing rapid replacement of the deleted cells by a new wave of HSAhi CD4+ 8
cells derived from DP precursors in the cortex.
With regard to the site of negative selection, many
workers consider that the deletion of immature T cells occurs largely in the cortex (3). In favor of this idea, it is well
documented that injecting adult mice with antigen causes
massive apoptosis in the cortex (30). However, in this situation cortical apoptosis could be a reflection of stress induced by stimulation of mature T cells in the periphery
(37). Such nonantigen-specific destruction of cortical thymocytes is less of a problem in neonatal mice, which have
few peripheral T cells. Here, it is of interest that, for both
SEB and ova peptide, injecting neonatal hosts with antigen caused marked elimination of HSAhi CD4+ 8 cells but
minimal deletion of CD4+ 8+ cells. We considered the
possibility that injection of antigen caused deletion of a
small subset of CD4+ 8+ cells, e.g., TCRhi cells. However,
gating on typical CD4+ 8+ (8hi) cells after SEB injection
showed no detectable deletion of either V
8lo or V
8hi
cells (Fig. 3). Similarly, we failed to see deletion of either TCRlo or TCRhi CD4+ 8+ cells in D011 mice after ova
peptide injection (our unpublished data). Hence, for neonatal hosts and the two antigens studied here, negative selection of typical cortical CD4+ 8+ cells appeared to be
very limited. It should be emphasized that these data do not
exclude the possibility that the cortex is an important site of
negative selection in other systems. Nevertheless, it is notable that, even in adult mice, SEB-induced deletion of V
8+ thymocytes is reported to be undetectable in mice
expressing MHC class II molecules only in the cortex (on
cortical epithelium) but not in the medulla (38).
In contrast to typical cortical CD4+ 8+ cells, the immediate progeny of these cells, namely TCRhi HSAhi CD4+
8lo cells, were strongly deleted after antigen injection (Fig.
3). Since the anatomical localization of CD4+ 8lo cells in
the thymus is unclear, it is possible that some of these cells
were deleted in the cortex before entering the medulla. However, it is important to emphasize that, for both SEB
and ova peptide, the elimination of SP thymocytes applied
not only to HSAhi CD4+ 8lo cells but also to the larger
population of HSAhi CD4+ 8 cells, which are presumably
slightly more mature than HSAhi CD4+ 8lo cells. In fact
most of the experiments with SEB concerned CD4+ 8
cells rather than CD4+ 8lo cells. Similarly, for D011 mice,
injecting peptide deleted both HSAhi CD4+ 8lo and HSAhi
CD4+ 8
thymocytes (Fig. 9 B).
Since the vast majority of lymphoid cells in the medulla
are typical SP cells (39), most HSAhi CD4+ 8 thymocytes
are probably situated primarily in the medulla rather than
the cortex. In support of this idea, the medulla contains considerable numbers of HSA+ cells in tissue sections (our
unpublished data), suggesting that many medullary T cells
are HSAhi. In addition, TUNEL staining has revealed that
SEB injection leads to increased numbers of apoptotic cells
in the medulla at day 2 after injection (our unpublished
data). For these reasons, it would seem highly likely that
the deletion of HSAhi CD4+ 8
cells takes place mainly in
the medulla (although the possibility that these cells initially
receive a death signal in the cortex cannot be excluded
[40]).
The elimination of HSAhi CD4+ 8 cells after antigen
injection did not appear to involve cell division. Thus, cell
cycle analysis of HSAhi CD4+ 8
cells has shown that the
rapid death of these cells after TCR ligation in vitro does
not involve entry into cell cycle (our unpublished data).
Likewise, the elimination of HSAhi CD4+ 8
cells in vivo
was not preceded by an increase in BrdU incorporation (Fig. 6). This finding contrasts with fully-mature T cells
where TCR-mediated apoptosis (AICD) generally occurs
slowly and is preceded by an overt proliferative response
(41). Hence, for the fully mature subset of HSAlo CD4+ 8
thymocytes, we expected initial contact with SEB to be directly immunogenic for these cells. In line with this prediction, SEB injection caused V
8+ HSAlo CD4+ 8
cells to
proliferate (incorporate BrdU) at 2 days after injection and
undergo considerable expansion. Surprisingly, however,
the expansion of V
8+ HSAlo cells was preceded by a transient decrease in numbers of these cells at day 1 after injection, probably via deletion. Why a proportion of these mature T cells succumbed to early deletion is unclear, although
a similar finding has been reported for peripheral T cells after SEB injection (10, 42). It is of interest that the expansion of V
8+ HSAlo thymocytes was evident only at day 2 after injection and was followed by a marked reduction in
numbers of these cells on day 3; the disappearance of V
8+
HSAlo cells was also prominent on day 5 and levels of these
cells did not return to normal until after day 7, i.e., ~2 d
later than for HSAhi cells (our unpublished data). At face
value the rapid elimination of proliferating V
8+ HSAlo
thymocytes after day 2 could reflect AICD and be the
counterpart of the rapid sequence of expansion followed by
deletion reported for peripheral V
8+ cells after SEB injection (10). However, whether the V
8+ HSAlo cells in the
thymus died in situ or migrated to the periphery is still unclear.
For SEB, it is worth noting that the near-complete elimination of V8+ cells, including mature HSAlo cells, seen at
days 3-5 after a single injection of SEB, is in line with the
original report that multiple injections of SEB caused
marked depletion of V
8+ SP thymocytes 8 d after the last
injection (29). Since earlier time points were not examined, the authors may have missed the prior expansion of
V
8+ cells reported here for HSAlo SP cells.
With regard to Fas, the main finding in this paper is that
in lpr/lpr mice the strong elimination of HSAhi CD4+ 8
thymocytes induced by SEB, ova peptide, and anti-TCR
mAb injection failed to occur when the dose of Ag/mAb
was increased to a high level. The implication therefore is
that negative selection is critically dependent upon Fas, but
only for antigens expressed at a high level. This finding
would seem to disagree with reports that injecting large
doses of specific peptide into TCR transgenic mice reduced the cellularity of the thymus by >10-fold, in both normal and lpr/lpr mice (11, 25). But because adult mice
were used in these experiments, the possibility remains that
thymocytes were not eliminated via negative selection but
destroyed nonspecifically by stress (37). In another study,
giving multiple injections of SEB to neonatal mice over a
2-wk period caused equivalent deletion of V
8+ cells in
both normal and lpr/lpr mice (43). Since the effects of varying the dose of injected SEB was not tested, the relevance of this finding to our data is unclear.
Much of the evidence that Fas is not involved in negative selection has come from the finding that lpr/lpr mice show relatively normal thymocyte deletion in response to endogenous SAgs and the male (HY) antigen (8, 12, 15, 17, 22). A corollary of the present data is that Fas is irrelevant for negative selection to endogenous self-antigens unless the concentration of these antigens in the thymus is unusually high. Hence if endogenous SAgs and the male antigen are expressed at only an "average" level (equivalent to a low-to-moderate dose of SEB or ova peptide), there is no discrepancy with the present data.
Although the range of self-antigens causing negative selection is unknown, one can envisage that some antigens, e.g., peptides derived from histones or other common intracellular proteins, are expressed at a relatively high level. Hence, based on our findings with SEB and ova peptide, the elimination of T cells specific for these common self-proteins might be Fas dependent and thus allow the reactive T cells in lpr/lpr mice to escape central tolerance induction in the thymus. Upon maturation and exit from the thymus, these nontolerant T cells would continue to see these antigens at a high concentration, but now in immunogenic rather than tolerogenic form. Accordingly, the onset of lymphadenopathy and autoimmune disease in lpr/lpr mice (8, 17) might be a reflection of an ongoing immune response of nontolerant T cells directed to ubiquitous self-antigens expressed at a high level.
This scenario rests on two assumptions. First, one has to
argue that the peripheral T cells in lpr/lpr mice are indeed
responding to self-antigens. Such a possibility has yet to be
proved, although the finding that all subsets of T cells in
lpr/lpr mice, including CD4+ 8 and CD4
8+ cells, have
an abnormally high turnover in the periphery is consistent with this idea (24, 44). The second assumption is that if T
cells in lpr/lpr mice are indeed autoreactive, the antigens recognized by these cells are expressed at a high level relative to other antigens. Assessing this idea will hinge on defining the peptide specificity of autoreactive T cells from
lpr/lpr mice.
As a final comment, it is important to emphasize that our data suggest that Fas regulates negative selection at a relatively late stage of thymocyte differentiation. The data are thus in contrast with the report that Fas controls the early onset of cortical apoptosis after peptide injection in TCR transgenic models (26). Since these findings applied to adult mice, cortical apoptosis may have been stress related. If so, it is of interest that, for mature T cells, Fas ligand can act as a costimulatory molecule (45), and that cytokine production by lpr/lpr T cells in vitro is lower than for normal T cells (11). Hence, stress-induced thymic atrophy after antigen injection could be less marked in lpr/lpr mice.
In conclusion, the data in this paper indicate that clonal elimination of antigen-specific T cells in the thymus after antigen injection occurs at a relatively late stage of differentiation and does not involve Fas when the dose of antigen is kept to a low-to-moderate level. However, with a high dose of antigen the presence of Fas becomes crucial for negative selection. In light of these findings, the prior failure to find a clear role for Fas in central tolerance may reflect that the antigens studied were expressed at a relatively low level.
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Footnotes |
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Address correspondence to Jonathan Sprent, Department of Immunology, IMM4, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, California 92037. Phone: 619-784-8619; Fax: 619-784-8839; E-mail: jsprent{at}scripps.edu
Received for publication 11 August 1997 and in revised form 2 March 1998.
This is publication no. 10869-IMM from the Scripps Research Institute.We thank Ms. Barbara Marchand for typing the manuscript and Dr. Z. Cai for preparing ova peptides.
This work was supported by grants CA38355, CA25803, AI21487, AI32068, AI38385 and AG01743 from the United States Public Health Service. Dr. Kishimoto is a recipient of a fellowship from the Cancer Research Institute.
Abbreviations used in this paper AICD, activation-induced cell death; BrdU, bromodeoxyuridine; DP, double positive; guinea pig C, guinea pig complement; HSA, heat-stable antigen; ova, ovalbumin; SEB, staphylococcal enterotoxin B; SP, single positive; TUNEL, TdT-mediated dUTP-biotin nick end labeling.
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