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Division of Molecular Immunology, The National Institute for Medical Research, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Acute administration of antigenic peptides in TCR transgenic mice leads to depletion in the thymus of double-positive thymocytes (16, 17, 18). Under these conditions, mature T cells bearing the TCR are activated and differentiate into effector cells (cytotoxic, helper) secreting a variety of cytokines. These factors would have an effect on the process of double-positive thymocyte deletion. To address directly the question of the extent to which single-positive mature T cells contribute to this process, we have examined Ag-specific deletion of immature thymocytes using mice transgenic for a TCR (F5) that recognizes a peptide (NP68) from the A/NT/60/68 influenza virus nucleoprotein in the context of class I MHC (Db) (17, 19, 20). Acute administration of antigenic peptide to these mice leads to deletion of almost all double-positive thymocytes within 4 days (17). These results are consistent with other models in which the transgenic TCR is restricted by class II MHC (16, 18, 21). In this report using Tg/Ntg chimeras and situations where mature T cells are absent, we report that this deletion is due to direct recognition of the antigenic peptide by the immature thymocytes and that although activated mature T cells are not necessary for this process to occur, they potentiate such deletion.
Furthermore, we show that continued administration of antigenic peptide to F5 mice unexpectedly results in the appearance of thymocytes that express the F5 TCR but are not deleted on subsequent exposure to Ag or after in vivo administration of anti-CD3 Ab. Nevertheless, these cells remain sensitive to steroid-induced apoptosis, indicating that the downstream apoptotic pathway is still operational. Thus, we propose that proximal TCR signaling in the double-positive thymocytes, which have developed in the presence of the cognate Ag, is altered such that they become resistant to Ag-induced deletion. We propose that these cells may be comparable with the peripheral anergic T cells that are generated after prolonged exposure to cognate Ag and that they are the result of an adaptability mechanism that alters the threshold of their activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice transgenic for the TCR-
ß from the F5 cytotoxic T cell
clone were generated as described previously (20).
Rag-1-/- mice were obtained from E. Spanopoulou (22)
and crossed to F5 mice. Inbred C57BL/10 mice were maintained in a
colony at the institute. Mice were used for experiments at 4 to 6 wk of
age, unless otherwise stated.
Reagents
NP68 peptide from the nucleoprotein of influenza virus A/NT/60/68 (ASNENMDAM) was synthesized on an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA). The peptide was dissolved in PBS and 50 nmol/20 g of mouse were injected i.p. as indicated in Results. Long term treatment consisted of 50 nmol/mouse every second day for 4 weeks before analysis. Water-soluble dexamethasone (Sigma) was diluted in PBS, and 1 mg was injected i.p. for 2 consecutive days, anti-CD3 mAb (2C11) (23) was diluted in PBS and 50 µg was injected i.p. for 2 days, and the mice were analyzed on the following day.
Flow cytometry
Thymuses were removed, and cells were teased into medium. For three-color analysis, 105 to 106 thymocytes were stained at 4°C, in the presence of 0.02% sodium azide and 1% BSA, with anti-CD8-FITC (YTS169.4) (24), anti-CD4-phycoerythrin (GK 1.5, Becton Dickinson, San Jose, CA) and biotinylated anti-Vß11 (KT11) (25) mAbs, followed by streptavidin red 670 (Life Technologies, Grand Island, NY). Cells were also stained for activation markers with biotinylated anti-CD69 (PharMingen, San Diego, CA), anti-CD44 (IM7) (26), and anti-CD25 (7D4) (27) mAbs. For analysis of DNA content, thymocytes were stained with 5 µg/ml 7-aminoactinomycin D (Sigma-Aldrich, Poole, U.K.) in PBS containing 2% FCS, 0.1% sodium azide, and 0.3% saponin (Sigma-Aldrich).
Bone marrow chimeras
Bone marrow chimeric mice were made by injection of C57BL/10 mice with a mixture of F5 transgenic and nontransgenic (5 x 106 + 5 x 106) bone marrow cells, 24 h after irradiation of the mice with 9.5 G. Reconstituted mice were tested for chimerism 6 wk later by flow cytometric analysis of PBL stained with the anti-Vß11 mAb.
Fetal thymic organ culture
Fetal thymic lobes were isolated from day 15 F5/RAG-1-/- embryos and transferred onto Nuclepore polycarbonate filters (Costar, Cambridge, MA). The thymic lobes were cultured at 37°C, 5% CO2 in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and antibiotics. After 4 days of culture, the filters were transferred to medium containing 100 µM NP68 or medium alone and cultured for 12 h. Thymocytes were then harvested for analysis by gently disrupting the thymic lobes manually in 1.5-ml Eppendorf tubes.
Proliferation assay
Responder cells were taken from F5/Rag-1-/- spleens. The spleens were teased, and splenocytes washed, resuspended in RPMI, 10% FCS, and incubated for 2 h at 37°C to permit adhesion. Nonadherent cells were used as a source of T cells. APCs were spleen suspensions from C57/BL10 mice injected with 50 nmol NP68 on the day before the experiment and long term NP68-treated F5/Rag-1-/- mice. RBC were removed by brief exposure to hypotonic shock. Spleen APCs were used alone or after 1 h of incubation with 0.5 µM, 5 µM, and 50 µM NP68, followed by two washes in RPMI, 10% FCS. The cells were irradiated (3000 R) using a cesium source before addition to cultures. APCs (1 x 106) were added to 1 x 105 responders in 0.2 ml of flat-bottom 96-well plates in quadruplicate. Human rIL-2 was added to a final concentration of 10 IU/ml. The plates were pulsed after 72 h with 1 µCi/well [3H]TdR. Labelled cells were harvested 6 h later and [3H]TdR uptake was determined using a beta scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Deletion of F5 transgenic double-positive thymocytes following
administration of the antigenic peptide (NP68) has been attributed to
negative selection of these immature cells on recognition of the
cognate ligand. However, peptide treatment also causes activation of
mature F5 T cells, their differentiation into cytotoxic effector cells,
and concomitant cytokine release (17). Both of these latter effects
could contribute to the loss of double-positive cells, which may become
targets for the cytotoxic cells and/or nonspecific victims of systemic
or local cytokine production. To distinguish between these
possibilities, we used bone marrow chimeric mice reconstituted with
50% F5 transgenic and 50% nontransgenic T cells, as judged by the
frequency of F5+ CD8+ T cells in the blood
(data not shown). Such chimeras were treated for 3 days with 50 nmol of
NP68 peptide, and on day 4 the mice were killed and thymocytes were
stained with anti-CD4, anti-CD8, and anti-Vß11
(recognizing the F5 transgenic TCR ß-chain) mAbs. The results shown
in Figure 1
indicate that in a thymus
containing exclusively F5 TCR+ thymocytes, 95% all
double-positive cells disappear on treatment with NP68. In contrast,
50% of double-positive cells remain in the thymuses of NP68-treated
bone marrow chimeric mice (Figs. 1 and 2). The remaining thymocytes
were found to be negative for the F5 TCR. The experiment was repeated
with four additional chimeras, and the absolute number of surviving F5
TCR+ and F5 TCR- double-positive thymocytes
from NP68-treated and untreated control mice was determined. The
results, shown in Figure 2, indicate that
peptide treatment causes a 10- to 20-fold reduction in the absolute
numbers of F5 TCR+ double-positive cells, whereas there is
only an average of 3-fold reduction in the number of F5
TCR- double-positive thymocytes. We conclude from these
data that the presence of activated cells may cause a nonspecific
depletion of immature thymocytes, but direct recognition of cognate Ag
leads to the preferential depletion of F5 TCR+
double-positive thymocytes.
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Experiments using fetal thymic organ cultures
(FTOC)3 were performed
to establish whether F5 TCR+ double-positive thymocytes are
susceptible to deletion on encounter with cognate Ag in the absence of
mature thymocytes. Thymic lobes were taken from day 15
F5/Rag-1-/- embryos and cultured in vitro for 4 days. At
this point in the culture, the majority of thymocytes have progressed
to the double-positive stage of development and the FTOCs are devoid of
mature F5 thymocytes (Fig. 3A). The few CD8
single-positive thymocytes present in the FTOCs at day 4 are of the
immature HSAhigh phenotype (data not shown). Treatment of
these FTOC with NP68 induced large scale apoptosis of the
double-positive thymocytes (Fig. 3B). After 12
h, the percentage of apoptotic double-positive thymocytes (containing
subdiploid DNA) rose from <1% in control cultures to 66.8% in FTOC
exposed to the cognate Ag. These data show that immature thymocytes are
deleted on exposure to cognate Ag, even in the absence of mature
thymocytes. These findings are consistent with experiments showing that
double-positive thymocytes from adult F5/Rag-1-/- mice
from the nonselecting H-2q haplotype (which do not contain
mature CD8 single-positive thymocytes) also undergo apoptosis when
exposed to NP68, presented by H-2b APC, in suspension
cultures (O. Williams, unpublished data).
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In contrast to short term treatment which leads to complete
depletion of F5+ double-positive thymocytes (17),
continuous administration of cognate peptide for longer periods results
in restoration of thymic cellularity and the appearance of a
double-positive thymocyte population that is resistant to deletion by
exposure to the antigenic peptide (15). However, it was possible that
these thymocytes that accumulated were resistant to apoptosis because
they expressed endogenous TCR molecules. To exclude this possibility,
we examined this process in F5/RAG-1-/- mice, which are
unable to rearrange endogenous TCR loci. Figure 4, A and C, shows
the kinetics of initial deletion and subsequent reappearance of
double-positive thymocytes in long term peptide-treated
F5/RAG-1-/- mice. Thus, under continuous administration
of peptide, the initial depletion of double-positive cells by day 4
starts to reverse by day 7, and both the percentage and absolute
numbers of double-positive thymocytes increase to reach a plateau by
day 8 to 9 of peptide treatment. Subsequently, the absolute numbers
stabilize to levels that vary but are consistently above 10 to 20% of
pretreatment values. These thymocytes express low levels of CD69, CD44,
and CD25 (data not shown). Furthermore, the levels of TCR on these
double-positive cells remain unchanged when compared with untreated
thymocytes, with the exception of the absence of a population of cells
with up-regulated levels of TCR from the thymuses of long term
peptide-treated mice.
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The experiments described above indicate that the reappearance of
double-positive thymocytes in long term Ag-treated F5 transgenic mice
is not due to altered environmental factors (i.e., absence of APC or
absence of responsive peripheral T cells). It was plausible that this
reappearance was due to an accumulation of cells in which the apoptotic
mechanism had been lost or had not developed yet. To test this
possibility, long term peptide-treated or untreated
F5/RAG-1-/- mice were injected with dexamethasone,
which causes nonspecific apoptosis in immature thymocytes. As shown in
Table I, dexamethasone induced a 70- to
80-fold reduction in the absolute numbers of double-positive thymocytes
in long term peptide-treated F5/RAG-1-/- mice, indicating
that these cells are still capable of undergoing death through
apoptosis.
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It has been proposed that the thymus contains a population of
double-positive thymocytes in which CD3 signaling is uncoupled from
signaling through the TCRß complex (28, 29). Therefore, it is
possible that the double-positive thymocytes that accumulate in long
term peptide-treated mice are unable to signal after recognition of
cognate Ag because of such an uncoupling of TCR from CD3 signaling;
however, the capacity to signal through the CD3 complex may be intact
in these cells. To test this possibility, long term Ag-treated or
untreated F5/Rag-1-/- mice were injected for 2 days
with 50 µg of anti-CD3
Ab (2C11) and thymocytes were analyzed
on day 3. The results, shown in Table I
, indicate that anti-CD3
treatment causes a 240-fold reduction in the absolute number of
double-positive thymocytes in otherwise untreated
F5/Rag-1-/- mice but does not induce apoptosis in long
term peptide-treated mice. Taken together, these data indicate that the
inability of antigenic peptide or anti-CD3 Ab to delete these
double-positive thymocytes is due to a defect proximal to the TCR-CD3
complex signaling cascade rather than within the later steps in the
apoptotic pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data described in this report are consistent with those published recently by Martin and Bevan (42). In this report, it was shown that Ag injection can cause deletion of immature cortical thymocytes by a dual mechanism, Ag presentation in the thymus and stimulation of mature peripheral T cells.
Prolonged treatment of F5 transgenic mice with peptide resulted in the reappearance of a double-positive (CD4+CD8+) thymocyte population (15). In this paper, we provide additional proof that this phenomenon is not due to accumulation of cells with endogenous TCRs. Furthermore, we provide evidence that this is not due to local or systemic conditions, such as absence of cytolytic activity and cytokine production by activated mature T cells or the absence of functional APCs.
The ability of APCs from long term-treated mice to stimulate naive F5 T cells indicates that their costimulatory capacity is not impaired. However, it is not clear at the moment whether the surviving double-positive thymocytes after long term treatment are able to perceive costimulatory signals, in addition to the defect in coupling the TCR signals to the apoptotic machinery.
Using thymic organ cultures, Finkel et al. (28, 29) described a
double-positive subpopulation of thymocytes that were resistant to
deletion after stimulation through the TCR but were deleted when
treated with anti-CD3 Abs. The authors suggested that these results
could be explained by functional uncoupling between the TCR and CD3
complexes, and they discussed its importance in ontogeny (34). The
physical dissociation of the TCR-CD3 complex after stimulation
suggested that the signal transduction pathway triggered by the whole
TCR complex may differ from that induced by CD3 ligation (35). In
addition, recently, Tokoro et al. (36) have reported that CD3-induced
apoptosis of double-positive thymocytes can occur in
TCR-/- mice that do not express a TCRß complex.
In contrast, in our experimental system the thymocytes resistant to
Ag-induced apoptosis from long term-treated F5/RAG-1-/-
mice are also resistant to deletion mediated by anti-CD3 Abs.
However, since dexamethasone does induce apoptosis, the machinery for
deletion is still operational in these thymocytes. We propose that the
defect in Ag-induced apoptosis probably occurs proximal to the TCR-CD3
complex in the signaling pathway. Further analysis that will allow us
to address the mechanisms underlying the resistance to apoptosis by
examining kinases involved in the signaling cascade is currently
under way.
A T cell population resistant to anti-CD3-induced apoptosis has been described in lpr/lpr mice. In addition, mature T cells in these mice exhibit a normal activation pattern, but once activated they exhibit a qualitative defect in their capacity to undergo Ag-induced apoptosis. It is likely that this resistance to induction of apoptosis is due to the defect in Fas Ag expression in lpr/lpr mice (36). Since double-positive thymocytes show a high constitutive expression of Fas (37), a defect in its expression could explain the resistance to deletion of double-positive cells in long term NP68-treated F5 mice. However, staining with anti-Fas mAb did not reveal significant differences between long term-treated and untreated mice (data not shown). Moreover, other reports have shown that clonal deletion induced by cognate Ag can occur in the absence of Fas or Fas ligand (31, 38, 39). The role of TNF in these processes was not addressed in the present study.
The type of responses generated in double-positive thymocytes described in this report bear many similarities to those seen in peripheral T cells. Both naive mature T cells and immature double-positive thymocytes, which have not been previously exposed to Ag, respond to stimulation by cognate Ag. However, whereas mature T cells become activated as a result of such stimulation, immature double-positive thymocytes die by apoptosis. Continuous stimulation of mature T cells by Ag induces a state of unresponsiveness (15), sometimes known as anergy. In this report, we show that a similar state of unresponsiveness can be generated in double-positive thymocytes after chronic exposure to Ag. Such unresponsive double-positive thymocytes are also seen in the thymus of mice doubly transgenic for the F5 TCR and the cognate Ag influenza nucleoprotein. In these mice, some thymocytes escape clonal deletion, although expression of activation markers suggests that they have interacted with the antigenic peptide. These cells are exported into the periphery but are unresponsive to Ag in vitro, due to the low levels of TCR and CD8 coreceptor expressed on their cell surfaces, and are capable only of differentiating into effector CTL in the presence of exogenous IL-2 (40). Such peripheral T cells are also present in F5 mice treated chronically with Ag (15). It is possible, therefore, that autoreactive cells can escape negative selection at the double-positive stage due to changes in their signaling machinery. These changes may be induced by chronic and/or inappropriate presentation of the cognate ligand, given that in both experimental systems (peptide injections or transgenic Ag expression as a self protein) the peptide ligand is potentially presented by all cells expressing class I MHC molecules. We propose that the unresponsive double-positive thymocytes described in this report are the functional corollary of what is known in the periphery as anergic T cells. This suggests that the unresponsiveness of thymocytes resistant to apoptosis and of peripheral T cells after chronic exposure to Ag are likely to reflect an inherent adaptability of lymphocytes, a notion described at length in an article by Grossman (41). The validity of this model and how it applies in our system is under examination.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dimitris Kioussis, Division of Molecular Immunology, The National Institute for Medical Research, The Ridgeway, London NW7 1AA, U.K. E-mail address:
3 Abbreviations used in this paper: FTOC, fetal thymic organ culture.
Received for publication July 28, 1997. Accepted for publication February 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß versus ß T cell receptor transgenic mice undergoing negative selection. Nature 340:559.[Medline]
ß+ T cells: one population is deleted by ligation of ßTCR. Cell 58:1047.[Medline]
ßT cell receptor and CD3 transduce different signals in immature T cells. J. Immunol. 142:3006.[Abstract]
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