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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4248-4254
Viral Superantigen-Induced Negative Selection of TCR
Transgenic CD4+ CD8+ Thymocytes Depends on
Activation, but not Proliferation
By
Isabel Ferrero,
Fabienne Anjuère,
Iñigo Azcoitia,
Toufic Renno,
H. Robson MacDonald, and
Carlos Ardavín
From the Department of Cell Biology, Faculty of Biology, Complutense
University, Madrid, Spain; and the Ludwig Institute for Cancer
Research, Lausanne Branch, University of Lausanne, Epalinges,
Switzerland.
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ABSTRACT |
T-cell negative selection, a process by which intrathymic
immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different
experimental approaches suggest that this process is triggered as the
result of activation-mediated cell death, the signal transduction
pathways underlying this process is not fully understood. In the
present report we have used an in vitro system to analyze the cell
activation and proliferation requirements for the deletion of viral
superantigen (SAg)-reactive V 8.1 T-cell receptor (TCR)
transgenic (TG) thymocytes. Our results indicate that in vitro negative
selection of viral SAg-reactive CD4+ CD8+
thymocytes is dependent on thymocyte activation but does not require
the proliferation of the negatively signaled thymocytes.
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INTRODUCTION |
TCR ENGAGEMENT by either a conventional
antigen or a superantigen (SAg) provokes a T-cell response that could
have opposite effects on the responding T cell depending on its stage
of differentiation.1 Mature peripheral T cells undergo a
cell activation-mediated proliferative response upon T-cell receptor
(TCR) engagement, which is followed by an apoptotic phase
involved in the regulation of T-cell homeostasis. However, the ultimate
consequence of this response may differ depending on the nature of the
antigen: whereas the T-cell response to a conventional antigen
generally leads to the generation of an antigen-specific highly
responsive memory T-cell pool, upon SAg recognition the initial
proliferation response is followed by a massive deletion of the
responding T-cell clones.2,3 Immature thymocytes, on the
other hand, will be eliminated in the thymus upon recognition of either
a specific antigen or a SAg encoded by mouse mammary tumor viruses
(MMTV), by a process termed negative selection, whereby intrathymic
T-cell tolerance is generated through apoptosis-mediated clonal
deletion of potentially autoreactive T cells.4
Although the data generated over the last few years have greatly
improved our knowledge of thymocyte negative selection, the molecular
mechanisms governing this process are not fully understood. Molecules
that play an essential role in signal transduction during T-cell
peripheral activation or positive selection such as p56lck,
p21ras, and MAPK do not appear to participate in negative
selection.5-7 Interestingly, the analysis of negative
selection in ovalbumin-specific TCR transgenic (TG) mice crossed with
ZAP-70-deficient mice8 has shown the participation of this
protein tyrosine kinase which has a crucial role in T-cell activation.
In addition, in vitro induction of thymocyte apoptosis through the
CD3/TCR complex has been reported to involve a series of signaling
events that include activation of protein tyrosine kinases, hydrolysis
of phosphatidylinositol, elevation of cytoplasmic-free
Ca2+, and activation of protein kinase C
(PKC).4 These data suggest that deletion of autoreactive
thymocytes is the result of activation-mediated cell death. However,
the signaling mechanisms underlying the induction of thymic deletion
remain largely unknown. In pioneer studies, treatment with cyclosporin
A (CsA) in vivo was shown to interfere with this
process.9,10 In addition, it has been recently shown that
cell proliferation is required for in vivo bacterial SAg-induced peripheral T-cell apoptosis.11
To get new insights into SAg-mediated negative selection, we have
recently analyzed the capacity of different antigen-presenting cells
(APCs) to induce the deletion of viral SAg-reactive double-positive (DP) thymocytes in an in vitro system based on the coculture of APCs
expressing the Mtv-7 MMTV-encoded SAg with
Mtv-7-reactive V8.1 TCR TG
thymocytes.12,13 In the present report we have used this
experimental model to study the cell activation and proliferation
dependence of negative selection induced by MMTV-encoded SAgs.
Our results indicate that in vitro negative selection of viral
SAg-reactive DP thymocytes is dependent on thymocyte activation but
does not require the proliferation of the negatively signaled
thymocytes.
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MATERIALS AND METHODS |
Animals.
BALB/c mice were purchased from IFFA Credo (L'Arbresle, France).
Mtv-7+ (Mls-1a) congenic BALB/c
(BALB.D2) (H-2d, Mtv-7+) mice were
bred from breeding pairs originally obtained from Dr H. Festenstein
(London Hospital Medical College, London, UK). TCR V 8.1
and TCR V8.2 TG mice were obtained from H.P. Pircher (Department of
Experimental Pathology, University Hospital, Zürich, Switzerland)
and H. Bluethman (Hoffmann-La Roche, Basel, Switzerland), respectively.
Both TCR TG mice were crossed on a BALB/c background. In all
experiments 4- to 6-week-old female mice were used.
Antibodies.
The following antibodies were used: KT3-1.1, anti-CD3; 172.4, H129.19,
and CT-CD4, anti-CD4; CT-CD8a, 53-6.7, and 31M, anti-CD8; AT83,
anti-Thy-1.2; H1-2F3, anti-CD69; RA3-6B2, anti-B220; M5-114, anti-MHC
class II; C1.A3-1, anti-macrophage antigen F4/80; N418, anti-CD11c;
B44, anti-bromodeoxyuridine (BrdU).
Isolation of splenic B cells.
Splenic B cells were purified by depletion of T cells by
complement-mediated cytotoxicity. Splenocytes were incubated with anti-Thy-1 (clone AT83), anti-CD4 (clone 172.4), and CD8 (clone 31M)
monoclonal antibodies for 60 minutes at 4°C, followed by 30 minutes
at 37°C after the addition of rabbit complement (Saxon Europe,
Suffolk, UK) and DNAse I (0.5 mg/mL; Boehringer-Mannheim, Mannheim,
Germany) to prevent excessive clumping of dead cells and debris. Dead
cells were removed by centrifugation on Ficoll-Paque (Pharmacia,
Uppsala, Sweden). Analysis of B220, CD4, and CD8 expression confirmed
that the B-cell preparation obtained had a purity greater than 95%
(data not shown). Contaminating dendritic cells and macrophages represent less than 2% as assessed by the expression of CD11c and
F4/80 (data not shown).
In vitro deletion assay.
In vitro deletion assays were performed as described
previously.13 In brief, thymocytes
(2 × 105/well) from either TCR V8.1 (Mtv-7
SAg-reactive) or TCR V8.2 (nonreactive) TG mice were cultured with
splenic B cells (105/well), from either BALB/c
(H-2d, Mtv-7 ) or BALB.D2
(H-2d, Mtv-7+), in RPMI 1640 medium
(Sigma, St Louis, MO) supplemented with 10% fetal calf serum (FCS,
Sera-Lab, Sussex, UK), 10 mmol/L HEPES (Sigma), and 100 U/mL
penicillin-streptomycin (GIBCO, Grand Island, NY), in round-bottom
96-well plates (Falcon, Plymouth, UK).
Inhibitors.
To study the activation/proliferation dependence of the SAg-mediated
deletion of DP thymocytes, in vitro deletion cultures were performed in
the presence of 6 µmol/L PKC-specific inhibitor bisindolylmaleimide I
(BIM) (Calbiochem, San Diego, CA), 80 nmol/L calcineurin inhibitor CsA
(Calbiochem), or 120 µmol/L G1 cell-cycle blocker mimosine (Sigma).
Due to the autofluorescence of B cells in the FL2 and FL3 channels of
the FACScan when cultured in the presence of BIM, analysis of DP
thymocytes was performed after removing magnetically the B cells using
anti-mouse Ig-coated magnetic beads (Dynabeads; Dynal, Oslo, Norway).
Therefore, to allow the comparison of the results in the different
experimental conditions, B cells were also removed in the cultures with
CsA or mimosine.
Flow cytometry.
Purity of the B-cell preparation was assessed after double staining
with fluorescein isothiocyanate (FITC)-conjugated anti-B220 (clone
RA3-6B2; Caltag, San Francisco, CA) and phycoerythrin
(PE)-conjugated anti-CD4 and anti-CD8 (clone H129.19,
Boehringer-Mannheim; and clone CT-CD8a, Caltag). Deletion of DP
thymocytes was determined after double-staining with PE-conjugated
anti-CD4 and FITC-conjugated anti-CD8 (clone 53-6.7). Expression of the
activation marker CD69 by thymocytes was analyzed after triple staining
with tricolor (TC)-conjugated anti-CD4 (clone CT-CD4; Caltag),
FITC-conjugated anti-CD8, and biotin-conjugated anti-CD69 (clone
H1-2F3) followed by streptavidin-PE (Caltag). FACS data presented
correspond to the cells within the viable cell gate, defined on the
basis of the light forward scatter (FSC) versus light side scatter
(SSC) profiles as previously described.13 Blast formation
was estimated by mean of the FSC profile. All the staining steps were
performed at 0 to 4°C in phosphate-buffered saline (PBS) containing 5 mmol/L EDTA and 2% FCS. Analysis was performed on a FACScan flow
cytometer (Becton Dickinson & Co, Mountain View, CA) at the Flow
Cytometry Laboratories of the Faculty of Biology and the
Fundación Jiménez Díaz (Madrid, Spain) and the
Ludwig Institute (Lausanne, Switzerland), using Lysys II and PC-Lysys
softwares (Becton Dickinson).
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick
end-labeling (TUNEL) staining.
At the indicated times, the cells were stained with TC-conjugated
anti-CD4 and PE-conjugated anti-CD8, washed in PBS, and incubated with
0.15 mol/L NaCl and 95% ethanol for 30 minutes at 4°C, and then
fixed in 1% paraformaldehyde in PBS containing 0.05% Tween-20, for 30 minutes at room temperature followed by 30 minutes at 4°C. The cells
were subsequently washed once in PBS, once in TdT buffer (200 mmol/L
potassium cacodilate, 25 mmol/L cobalt chloride, 125 mmol/L Tris-HCl,
250 µg/mL BSA, pH 6.6 at 25°C), and incubated in TdT buffer
supplemented with 0.7 µmol/L FITC-conjugated dUTP and 25 U/mL TdT
(both from Boehringer-Mannheim) for 30 minutes at 37°C.
Bromodeoxyuridine (BrdU) detection.
For this assay, in vitro deletion cultures were performed in RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin-streptomycin, and 10 mmol/L HEPES, and containing 50 µmol/L 2-mercaptoethanol (GIBCO), and 6.5 µmol/L BrdU (Sigma). Deoxycytidine 9 µmol/L
(Sigma) and thymidine 8 µmol/L were added to the culture medium to
neutralize BrdU toxicity in culture14 and to improve BrdU
incorporation by thymocytes, respectively. After 24, 36, and 60 hours
in culture, the cells were stained with TC-conjugated anti-CD4 and
PE-conjugated anti-CD8, washed in PBS, and incubated with 0.15 mol/L
NaCl and 95% ethanol for 30 minutes at 4°C, and subsequently fixed
in 1% paraformaldehyde in PBS containing 0.01% Tween-20, for 30 minutes at room temperature followed by 30 minutes at 4°C. The cells
were then washed in DNAse buffer (0.15 mol/L NaCl, 4.2 mmol/L
MgCl2, 10 mmol/L HCl, pH 8.0) and incubated
for 1 hour at 37°C in the same buffer containing 50 U/mL DNAse I
(Boehringer-Mannheim). After washing in PBS, cells were incubated in
PBS containing 0.5% Tween-20, 5% FCS, and FITC-conjugated anti-BrdU
(Becton Dickinson) for 45 minutes at room temperature.
Quantitation method.
The absolute number of DP and single-positive (SP) thymocytes has been
obtained by multiplying the total number of cells per well by the
percentage of viable cells, and by the percentage of each thymocyte
subset calculated within the total viable cell population. To allow the
direct comparison of the results obtained in different experimental
conditions, the percentages of DP and SP shown in Fig
1 correspond to corrected values, which
have been calculated considering DP + CD4+SP + CD8+SP = 100%, ie, excluding B cells and double-negative
thymocytes. Percent deletion of DP thymocytes was calculated using the
formula:
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| Fig 1.
Specificity of the in vitro deletion of TCR V8.1 TG
thymocytes. Dot plots show the CD4 versus CD8 profiles (CD4-TC/CD8-FITC staining) of Mtv-7-reactive TCR V8.1 or nonreactive TCR
V8.2 thymocytes cultured for 60 hours alone or with B cells from
Mtv-7+ BALB.D2 or Mtv-7
BALB/c mice. Percent deletion values of V8.1 DP thymocytes were 2%
and 68% when cultured with BALB/c and BALB.D2 B cells, respectively. For V8.2 DP thymocytes percent deletion value was 0% when either BALB/c or BALB.D2 B cells were used. The absolute cell number per well
and the percentage (in boxes) are indicated for each thymocyte subset.
The percentages correspond to corrected values, which have been
calculated considering DP + CD4+SP + CD8+SP = 100%. Histograms represent the CD69
expression and FSC profile of DP thymocytes for each culture condition.
The percentage of CD69+ (FL2 > 20) and blast
(FSC > 80) DP thymocytes are shown.
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RESULTS AND DISCUSSION |
Specificity of the in vitro deletion of TCR V8.1 TG
thymocytes by Mtv-7+ B cells.
The experimental model of in vitro deletion used in this study is based
on the coculture of splenic B cells expressing the Mtv-7 SAg,
obtained from BALB.D2 mice, with Mtv-7-reactive TCR V8.1 TG
thymocytes. Details concerning the potential of splenic B cells to
induce the in vitro deletion of SAg-reactive thymocytes in a similar
culture system, the kinetics of the process, as well as the optimal
APC:thymocyte ratio have been considered in a recent report.13 The specificity of the deletion system is
illustrated in Fig 1, which shows the CD4 versus CD8 profile of
Mtv-7-reactive TCR V8.1 or control nonreactive TCR V8.2
thymocytes, cultured for 60 hours with Mtv-7+ or
Mtv-7 splenic B cells, obtained from BALB.D2 and
control BALB/c mice, respectively. For each thymocyte subpopulation,
the absolute cell number per well and the percentage are shown.
Culture of TCR V8.1 TCR TG thymocytes with BALB.D2 B cells induced a
strong deletion of DP thymocytes (68% in the experiment shown in Fig
1), as indicated by the decrease in their percentage (20% v
63%) as well as in their absolute number (9.400 v 29.200), confirming our previous results using a similar experimental
method.13 In the in vitro deletion assay described in the
present report, thymocyte viability in culture has been improved by
adding 10 mmol/L HEPES to the culture medium, and by using an FCS batch more adequate for these cultures. Percent deletion has been calculated following the formula described in Materials and Methods on the basis
of the absolute DP cell number, according to Vasquez et al,15 instead of using the parameter "percentage of DP
in the culture" used by Pircher et al,16 because a
decrease in the percentage of DP thymocytes in this culture system may
reflect not only a deletion process, but also the proliferation of SP thymocytes. In this sense, as previously described,13
deletion of SAg-reactive DP thymocytes is paralleled by cell
activation, as assessed by the expression of CD69 (80%
CD69+ DP cells) and the FSC profile (89% DP with
FSC > 80), and by an increase in the absolute number of SP
thymocytes per well, particularly of the CD4+ SP subset
(23.500 v 10.400). Upregulation of CD69 has also been reported
during the in vitro deletion of TCR TG DP thymocytes specific for the
lymphocytic choriomeningitis virus,17 or the 2 oxoglutarate
dehydrogenase (2C TCR TG mice).18
In contrast, when splenic B cells from Mtv-7
BALB/c mice were used as APCs, no significative deletion (2% deletion)
or activation (10% CD69+ DP cells) of TCR V8.1 DP
thymocytes were observed. Similar results were obtained when control
nonreactive TCR V8.2 thymocytes were cultured with either
Mtv-7+ or Mtv-7 B cells from
BALB.D2 or BALB/c mice, respectively. These results indicate that
deletion of TCR V8.1 TG DP thymocytes induced by BALB.D2 B cells is
Mtv-7 SAg specific.
Analysis of cell activation, cell proliferation, and apoptosis during
in vitro SAg-mediated deletion of TCR V8.1 DP
thymocytes.
Figure 2 compares the phenotype of TCR
V8.1 DP thymocytes cultured for 36 or 60 hours alone or with BALB.D2
B cells. A considerable decrease in the absolute cell number per well
of DP thymocytes was already observed after 36 hours (77% deletion).
DP deletion was accompanied by cell activation, as assessed by CD69
expression and the FSC profile. Analysis of DNA fragmentation by the
TUNEL method indicated that around 40% (37% in the experiment shown in Fig 2) of DP thymocytes were apoptotic. However, no BrdU
incorporation was detected, indicating that after 36 hours DP
thymocytes undergoing in vitro SAg-mediated negative selection did not
enter into cell cycle.
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| Fig 2.
Phenotype of TCR V8.1 DP TG thymocytes during in vitro
Mtv-7 SAg-mediated negative selection. Dot plots show the CD4
versus CD8 profiles (CD4-PE/CD8-FITC staining) of TCR V8.1 TG
thymocytes cultured for 36 or 60 hours alone or with BALB.D2 splenic B
cells. The absolute cell number of DP and SP thymocytes is indicated. Circles indicate the DP CD4+CD8int
population. Histograms represent the FSC, CD69 expression, TUNEL staining, and BrdU incorporation of DP thymocytes for each culture condition. Staining details are given in Materials and Methods. The
percentage of CD69+ (FL2 > 20), blast (FSC > 70),
TUNEL+ (FL1 > 300), and BrdU+
(FL1 > 130) DP thymocytes are indicated. The experiment is
representative of four experiments with similar results. (Note that the
CD4-TC/CD8-FITC staining used in Fig 1 dot plots does not allow a clear
definition of the CD4+CD8int DP
population.)
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DP thymocyte deletion was almost complete when TCR V8.1 thymocytes
were cultured for 60 hours with BALB.D2 B cells. After this period in
culture most DP thymocytes were activated blasts, and approximately
50% of them were apoptotic, as shown by the TUNEL method.
Interestingly, around 40% (35% in the experiment shown) DP thymocytes
had entered into cell cycle as assessed by BrdU incorporation.
TCR V8.1 DP thymocytes were not activated and did not incorporate
BrdU when cultured alone for 36 or 60 hours; a proportion of them, most
likely corresponding to apoptotic nonpositively selected thymocytes,
were positive for TUNEL staining, in agreement with the findings of
Surh and Sprent,19 who suggested that the high proportion
of apoptotic cortical thymocytes detected in situ with the TUNEL method
is primarily a reflection of lack of positive selection.
Cell activation and cell proliferation requirements of SAg-mediated
deletion of TCR V8.1 DP thymocytes.
The data shown in Fig 2 indicate that most of the DP thymocytes deleted
after 60 hours in culture with BALB.D2 B cells were activated, as more
than 75% of DP remaining at 36 hours were eliminated at 60 hours, and
up to 70% of DP expressed the CD69 activation molecule at 36 hours.
This suggests that cell activation may be required for SAg-mediated DP
deletion to occur. However, deletion of DP thymocytes does not appear
to be paralleled by cell proliferation, because at 36 hours a strong
deletion occurred but no cycling cells were observed, as demonstrated
by the absence of BrdU+ cells (Fig 2). To exclude the
possibility that DP thymocytes already deleted at 36 hours might have
entered into cell cycle during the early phase of our in vitro assay,
BrdU incorporation was analyzed after 24 hours in culture. As shown in
Fig 3, only 27% deletion of DP thymocytes
was observed after 24 hours, and no BrdU incorporation was detected at
this timepoint when TCR V8.1 thymocytes were cultured alone or with
BALB.D2 B cells, indicating that DP deletion observed at 36 hours was
not preceded by entry into cell cycle. Interestingly, around 50% of DP
thymocytes were CD69+ after 24 hours in culture with
BALB.D2 B cells. Proliferating DP thymocytes detected by BrdU
incorporation after 60 hours in culture probably represent
Mtv-7 SAg-stimulated postpositively selected DP thymocytes,
which have been shown to be less sensitive to negative selection than
mature SP thymocytes.20-22 This is supported by the finding
that most DP thymocytes remaining after 60 hours have a
CD4+CD8int phenotype (see Figs 2 and
4), corresponding to postselected
DP.23 Furthermore, most BrdU+ DP cells are
located within the CD4+CD8int population (data
not shown). In addition, up to 60% CD4+ SP thymocytes were
CD69+ BrdU+ cells (data not shown). These data
indicate that TCR stimulation by viral SAg may result in deletion or
proliferation depending on the thymocyte differentiation stage.
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| Fig 3.
Analysis of cell activation and BrdU incorporation during
the early phase of in vitro SAg-mediated deletion of V8.1 DP TG thymocytes. Dot plots show the CD4 versus CD8 profiles of TCR V8.1
TG thymocytes cultured for 24 hours alone or with BALB.D2 splenic B
cells. The absolute cell number of DP and SP thymocytes is indicated.
Histograms represent the CD69 expression and BrdU incorporation of DP
thymocytes. The percentage of CD69+ (FL2 > 20) and
BrdU+ (FL1 > 80) DP thymocytes are indicated. The
experiment is representative of three experiments with similar
results.
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| Fig 4.
Cell activation and cell proliferation requirements of
SAg-mediated deletion of TCR V8.1 DP TG thymocytes. TCR V8.1
thymocytes were cultured for 60 hours alone or with BALB.D2 splenic B
cells in control conditions (untreated), or in the presence of 6 µmol/L BIM (PKC inhibitor), 80 nmol/L CsA (calcineurin inhibitor), or 120 µmol/L mimosine (cell-cycle blocker). Dot plots show the CD4 versus CD8 profile (CD4-PE/CD8-FITC staining) after removing
magnetically the B cells using anti-mouse Ig-coated magnetic beads (see
Materials and Methods for details). The absolute cell number of DP
thymocytes is indicated. The statistics corresponding to four similar
experiments are given in Table 1. Histograms represent the CD69
expression and BrdU incorporation of DP thymocytes for each culture
condition. The percentage of CD69+ (FL2 > 20) and
BrdU+ (FL1 > 80) DP thymocytes are indicated.
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To investigate whether DP thymocyte deletion depends on cell
activation, but does not require cell proliferation, TCR V8.1 TCR TG
thymocytes were cultured for 60 hours with BALB.D2 B cells in the
presence of inhibitors of cell activation and/or cell
proliferation. BIM and CsA were used as inhibitors of the signaling
molecules PKC and calcineurin, respectively, known to play an essential role in T-cell activation. The G1 cell-cycle blocker mimosine was used
to inhibit cell proliferation. Each inhibitor was used at a
concentration chosen to get the optimum effect with a minimum toxicity,
after testing a broad range of concentrations. The results obtained are
shown in Fig 4 and Table 1. When cultured in the presence of BALB.D2 B cells and 6 µmol/L of the PKC-specific
inhibitor BIM, DP thymocytes were not activated, did not enter into
cell cycle, and SAg-mediated DP deletion was almost completely
inhibited. Inhibition of PKC activation by staurosporine, calphostin,
or H-7 has also been reported to block the in vitro deletion of DP thymocytes from cytochrome c-specific TCR TG mice,15
although it has been shown that these inhibitors are poorly selective
whereas BIM displays a high degree of selectivity.24
Therefore, our results obtained with BIM establish more convincingly
the requirement for PKC in the signal transduction pathway underlying
SAg-mediated T-cell negative selection.
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Table 1.
Effect of Inhibitors of Cell Activation
and/or Cell Proliferation on the In Vitro Deletion of TCR
V8.1 TG DP Thymocytes
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Treatment with the calcineurin inhibitor CsA at 80 nmol/L induced a
partial inhibition of DP thymocyte activation after culture with
BALB.D2 B cells, and no BrdU incorporation was detected. Percent
deletion values were around 40%, indicating a partial inhibition of DP
thymocyte deletion. In contrast with the results obtained with BIM, an
almost complete inhibition of DP activation and deletion was not
reached with CsA, even when used at very high and, therefore, toxic
concentrations. These data suggest a direct correlation between the
level of DP activation and the degree of DP deletion. In support of
this, lower BIM and CsA concentrations induced a lower inhibition of
both DP activation and deletion (data not shown). Data dealing with the
participation of the calcineurin-mediated signaling pathway in negative
selection are controversial. In vivo and in vitro experiments using the
calcineurin inhibitors FK50625 and CsA,21
respectively, in TCR TG thymocyte deletion assays, argue against
calcineurin requirement. However, other in vivo and in vitro
experimental evidences have firmly shown the involvement of this
calcium/calmodulin-dependent phosphatase in negative selection induced
by SAgs,9,10 and by antigenic peptides derived from
cytochrome c and the male H-Y antigen, in TCR TG mice.26,27
Interestingly, in agreement with our data, in these reports it was
shown that after blockage of calcineurin by CsA, negative selection was
partially inhibited or delayed. In addition, experiments of TCR TG
thymocyte deletion mediated by an antigenic peptide or a peptide
analogue have shown that CsA did not interfere with thymocyte deletion
in vitro when the antigenic peptide was used at high concentration, but
partially inhibited deletion when the peptide analogue or lower
concentrations of the antigenic peptide were used.27 These
data support the hypothesis that requirement for the
calcineurin-mediated signal transduction depends on the intensity of
the TCR-coupled signaling triggered by the deleting ligand and may
provide an explanation for the reports by Wang et al25 and
Vasquez et al21 in which no inhibition of negative
selection by CsA or FK506 was observed. Therefore, in contrast to
signaling through PKC which appears to have a crucial function in
negative selection, calcineurin-mediated signaling may be overcome when
the deleting antigen determines a very high-affinity TCR-peptide
interaction, but may be required when this interaction has a lower
affinity, or when the deleting antigen is present at low concentration.
Similar observations have been reported with regard to costimulatory
signal requirements during T-cell negative
selection.28
BrdU incorporation but not activation of DP thymocytes was inhibited
after culture with BALB.D2 B cells in the presence of the cell-cycle
blocker mimosine. Interestingly, in accordance with our previous
hypothesis, no inhibition of DP deletion was obtained under these
culture conditions, demonstrating that in vitro deletion of
SAg-reactive TCR V8.1 TG thymocytes does not depends on cell
proliferation. This is apparently in contrast with recent data
establishing a correlation between proliferation and cell death upon
stimulation of peripheral T cell by bacterial SAgs.11
However, our results are in agreement with Boehme and Lenardo,29 who reported that progression through the cell
cycle was not required for TCR-mediated apoptosis of activated
peripheral T cells. Interestingly, these investigators have shown that
although peripheral T cells blocked in S phase after treatment with
aphidicolin were susceptible to TCR-induced apoptosis, cells blocked in
G1 phase by treatment with mimosine had a diminished susceptibility to
TCR-induced apoptosis. These data suggest that in vitro SAg-mediated deletion of TCR V8.1 TG thymocytes is less sensitive to cell-cycle blockade in G1 than TCR-induced apoptosis of peripheral T cells.
In conclusion, the data shown in the present report indicate that viral
SAg-mediated T-cell negative selection requires cell activation but
does not depend on cell proliferation. Finally, the differential
inhibition effect on SAg-mediated DP deletion, obtained by blocking PKC
or calcineurin, support the hypothesis that synergy between different
TCR-mediated signaling pathways may be required depending on the
intensity of TCR-antigen interaction mediating the negative selection.
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FOOTNOTES |
Submitted November 25, 1997;
accepted January 15, 1998.
Supported in part by grants from the Dirección General de
Investigación Científica y técnica,
Ministerio de Educación y Ciencia, Spain (to C.A.) and from the
Human Frontiers of Science Program (to H.R.M.). T.R. was supported by a
Centennial Fellowship from the Medical Research Council of Canada.
Address reprint requests to Carlos Ardavín, PhD,
Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Drs Hanspeter Pircher and Horst Bluethmann for the TCR TG
mice, and Pilar Martín for help in FACS experiments. We are
grateful to the Flow Cytometry facility of the Fundación Jiménez Díaz for making it possible to perform FACS
analysis after hours.
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Abstract
Introduction
Abstract