Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3856-3862
In Vivo T-Lymphocyte Tolerance in the Absence of Thymic Clonal Deletion
Mediated by Hematopoietic Cells
By
Joost P.M. van Meerwijk and
H. Robson MacDonald
From the Ludwig Institute for Cancer Research, Lausanne Branch,
University of Lausanne, Epalinges, Switzerland.
 |
ABSTRACT |
Thymic negative selection renders the developing T-cell repertoire
tolerant to self-major histocompatability complex (MHC)/peptide ligands. The major mechanism of induction of self-tolerance is thought
to be thymic clonal deletion, ie, the induction of apoptotic cell death
in thymocytes expressing a self-reactive T-cell receptor. Consistent
with this hypothesis, in mice deficient in thymic clonal deletion
mediated by cells of hematopoietic origin, a twofold to threefold
increased generation of mature thymocytes has been observed. Here we
describe the analysis of the specificity of T lymphocytes developing in
the absence of clonal deletion mediated by hematopoietic cells. In
vitro, targets expressing syngeneic MHC were readily lysed by activated
CD8+ T cells from deletion-deficient mice. However,
proliferative responses of T cells from these mice on activation with
syngeneic antigen presenting cells were rather poor. In vivo,
deletion-deficient T cells were incapable of induction of lethal
graft-versus-host disease in syngeneic hosts. These data indicate that
in the absence of thymic deletion mediated by hematopoietic cells
functional T-cell tolerance can be induced by nonhematopoietic cells in
the thymus. Moreover, our results emphasize the redundancy in thymic negative selection mechanisms.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
DURING THEIR DEVELOPMENT in the thymus,
T-cell precursors randomly rearrange their genes for the 
and 
chains of the clonotypic T-cell receptor (TCR). Thus, an immature
thymocyte repertoire is generated that can recognize both self and
foreign antigenic peptides presented by major histocompatability
complex (MHC) molecules. TCR expressing immature thymocytes are
subsequently submitted to a variety of selection processes. Thymic
positive selection is thought to select "useful" cells, ie,
thymocytes capable of recognition of self-MHC molecules. In the process
of negative selection, self-specific cells are inactivated, thus creating a T-cell repertoire tolerant to self-MHC/peptide
ligands.1-3 It has been reported that 3% to 5% of
precursors that develop in the thymus will fully mature and populate
peripheral lymphoid organs.4,5 Thymocytes whose
differentiation is aborted either have not successfully rearranged
their TCR or TCR genes,6 do not express a TCR
suitable for positive selection,7,8 are
autoreactive,9-11 or do not receive other unidentified
thymic microenvironmental signals required for full
development.5,12-14
The quantitative impact of thymic negative selection has been the
subject of recent reports. While the majority of thymic deletion is
thought to be mediated by radiosensitive antigen-presenting cells of
bone marrow origin, thymic positive selection is known to depend on MHC
molecules expressed by radioresistant thymic epithelial
cells,1,15 although quantitatively minor exceptions to this
rule have been reported.16-23 Therefore, bone marrow
chimeras can be generated in which positive selection is fully
fuctional, while negative selection is defective. In irradiation bone
marrow chimeras in which radioresistant cells express MHC class I and II ligands while radiosensitive elements lack expression of these molecules (MHC I°II°
wt), we have observed a twofold
to threefold increase in the generation of mature thymocytes as
compared with control chimeras.10 These data indicate that
half to two thirds of positively selectable thymocytes are deleted
during thymic development. Zerrahn et al9 have reported
that approximately 5% of the preselection thymocyte repertoire is
self-reactive. During normal thymocyte development, these cells are
expected to be deleted. Combined with the estimate of 3% to 5% of
thymocytes successfully undergoing thymic selection,4,5
these data indicate that at least half of positively selectable
thymocytes are deleted during development. Using a more sensitive
experimental system, Merkenschlager et al8 reported that
approximately 20% of immature thymocytes are activated on interaction
with MHC molecules. Because only approximately 5% of developing
thymocytes successfully undergo thymic selection,4,5 the
latter result suggests that around three quarters of MHC reactive
immature thymocytes are deleted. Interestingly, the mature T-cell
repertoire in mice expressing MHC class II molecules presenting a
single peptide has been reported to contain 60% of cells reactive to
MHC class II molecules presenting the normal repertoire of
self-peptides.11 These cells would be expected to be
deleted in mice expressing normal MHC class II molecules on elements of
hematopoietic origin.24 Thus, while in some reports
significantly lower frequencies of negatively selected thymocytes have
been suggested,7,25 most experimental data indicate that
approximately half of positively selectable thymocytes are deleted
during development.
In contrast to quantitative aspects, little information is available
concerning the qualitative impact of thymic clonal deletion. We report
here an analysis of the specificity of an otherwise normal T-cell
repertoire that develops in the absence of thymic clonal deletion
mediated by cells of hematopoietic origin in MHC I°II°wt chimeras. On activation, these cells lysed
targets expressing host type MHC, but only poorly proliferated on
stimulation with host type antigen presenting cells (APC). Moreover,
MHC I°II°wt chimera-derived T lymphocytes did not
induce lethal graft-versus-host disease (GVHD) in syngeneic animals.
Therefore, thymic negative selection mechanisms appear to be redundant.
| |
MATERIALS AND METHODS |
Mice.
Wild-type C57BL/6 mice were purchased from Harlan Netherlands, Zeist,
The Netherlands. Mice on C57BL/6 genetic background deficient in MHC
class I expression (MHC I°) because of a targeted disruption of the
2-microglobulin gene26 were obtained from Dr B.J.
Fowlkes, National Institutes of Health (NIH), Bethesda, MD. Mice
deficient in MHC class II expression due to an introduced disruption of
the I-A gene in C57BL/6 (I-E
) embryonic stem
cells27 (MHC II°) were provided by Dr H. Bluethmann (Roche, Basel, Switzerland). MHC I° and MHC II° animals were
interbred in our facilities to obtain MHC I°II° mice.
Hematopoietic chimeras.
Irradiation bone marrow chimeras were prepared essentially as described
previously.28 Briefly, anti-NK1.1 antibody-treated hosts
(100 µg PK136 intraperitoneal [IP] 29)
were lethally irradiated (1,000 rad 
) using a Cs137
source and reconstituted with 5 to 20 × 106 bone
marrow cells that had been depleted of T cells using anti-Thy1 antibody
AT8330 plus complement. Chimeras were kept on antibiotic containing water (0.2% Bactrim; Roche, Basel, Switzerland) for the
complete duration of the experiment, usually 6 weeks.
Cytotoxic T-lymphocyte assays.
Unseparated thymocytes or splenocytes derived from DBA/2 mice or
chimeras 6 weeks posttransfer were cultured for 6 days in the presence
of T-cell-depleted (anti-Thy1 AT8330 plus complement), irradiated (1,000 rad ) splenocytes in the absence or presence of 30 U/mL interleukin-2 (IL-2) (EL4 supernatant31).
For lysis of RMA targets (C57BL/6 origin32),
C57BL/6 APC-stimulated effectors were used, while P815 (DBA/2
origin33) lysis assays were performed using T cells
stimulated with DBA/2 APC. Targets (2,000 cells per well) were labelled
with Cr51, extensively washed and mixed with effector cells
in duplicate at effector (viable cell) to target (E/T) ratios
indicated. Cr51 release in the supernatant was measured 4 hours later. Specific lysis is Cr51 release above background as a percentage of maximum (as determined by acid lysis of
targets). Actual E/T ratios (E/TA) in the P815 and RMA
lysis assays were corrected (E/Tcorr) for anti-CD3
antibody redirected lysis. P815 targets were incubated with cytotoxic T
lymphocytes (CTL) in the presence of 1%
145-2C1134 supernatant at titrated E/T ratios and E/T ratio
required for half maximal lysis determined (E/T50%).
E/Tcorr = E/TA / E/T50%.
Proliferation assays.
A total of 106 thymocytes or 5 × 105
splenocytes derived from DBA/2 mice or chimeras 6 weeks
postreconstitution were cultured in triplicate in the presence of
indicated numbers of T-cell depleted (anti-Thy1[AT83] plus
complement), irradiated (1,000 Rad ) splenocytes, in the presence or
absence of 30 U/mL IL-2 (EL4
supernatant31). 3H-Thymidine was added
at day 3 or 4 and incorporation measured 16 hours later by direct
counting (Top Count; Packard Instruments, Meriden, CT).
GVHD.
Sublethally irradiated (720 rad ), anti-NK1.1 antibody
PK13629-treated hosts were injected intravenously (IV) with
the indicated number of unseparated thymocytes or splenocytes derived
from DBA/2 mice or chimeras 6 weeks posttransfer. Alternatively,
lethally (1,000 rad ) irradiated PK136-treated hosts were injected
IV with a mixture of T-cell-depleted bone marrow cells plus
unseparated thymocytes, as previously described.35 Mice
were kept on antibiotic containing water (0.2% Bactrim) for the
complete duration of the experiment. Mortality was monitored for 3 months posttransfer.
| |
RESULTS |
Targets expressing host type MHC molecules are lysed by activated MHC
I °II °wt chimera derived CD8+
T cells.
Thymocytes and peripheral T cells from experimental (MHC
I°II°wt) and control (wtwt) chimeras, as
well as from allogeneic controls (DBA/2), were stimulated with
irradiated T-cell-depleted splenocytes in the presence of exogenous
IL-2 in vitro. After 6 days of stimulation, lysis of targets expressing
host type (RMA, H-2b) or allogeneic (P815,
H-2d) MHC was analyzed (Fig 1).
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| Fig 1.
Targets expressing host type MHC are lysed by activated T
cells derived from deletion-deficient chimeras. (A) Thymocytes or
splenocytes of indicated origin were stimulated in vitro with
approximately the same number of irradiated DBA/2-derived (top panels)
or C57BL/6-derived (bottom panels) T-cell-depleted splenocytes in the
presence of exogenous IL-2. After 6 days of stimulation, lysis of
indicated targets was assessed. (B) As in (A), but in vitro stimulation
of splenocytes was performed with or without added IL-2, as indicated.
E/T ratios were corrected for anti-CD3 antibody redirected lysis of
P815 targets as described in Materials and Methods.
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|
MHC I°II°wt and wtwt chimera, but not
control DBA/2-derived thymocytes and splenocytes stimulated in vitro
with DBA/2 APC efficiently lysed P815 targets (which are of DBA/2
origin33) (Fig 1A). No reproducible difference in the lysis
of P815 by wtwt and MHC I°II°wt
chimera-derived thymocytes and splenocytes was observed (Fig 1A).
When stimulated with T-cell-depleted, irradiated C57BL/6 splenocytes
in vitro, DBA/2 and MHC I°II°wt, but not
wtwt chimera-derived thymocytes and splenocytes readily lysed
RMA targets (Fig 1A). These targets are of C57BL/6 origin and therefore
express chimera host type MHC molecules.32 RMA targets were
invariably significantly better lysed by allogeneic DBA/2 effectors
than by MHC I°II°wt-derived T cells. Moreover, the
lysis was completely mediated by CD8-dependent effector cells as
evidenced by antibody blocking experiments (data not shown). No
significant difference in lysis by thymocytes as compared with
splenocytes was observed.
In all experiments described so far, exogenous IL-2 was added to
cultures during the activation phase. Interestingly, when exogenous
IL-2 was omitted, lysis of RMA targets by MHC
I°II°wt-derived splenocytes was reproducibly
(approximately fivefold) reduced as compared with effector cells
activated in the presence of IL-2 (Fig 1B). In contrast, lysis of RMA
by DBA/2-derived splenocytes was not enhanced by addition of exogenous
IL-2 (Fig 1B). These data suggest that sufficient levels of IL-2 are
produced in cultures in which DBA/2 splenocytes are stimulated by
C57BL/6 APC, while insufficient IL-2 production is obtained with MHC
I°II°wt-derived responder T cells. All thymic
effectors required addition of exogenous IL-2 during the activation
phase irrespective of the origin of effectors and targets (data not shown).
Poor proliferative response to host type MHC by MHC
I °II ° wt chimera derived T cells.
Thymocytes and splenocytes derived from wtwt and MHC
I°II°wt chimeras and from control DBA/2 mice were
stimulated in vitro with T-cell-depleted, irradiated C57BL/6 or DBA/2
splenocytes in the presence of exogenous IL-2
(Fig 2). While chimera-derived thymocytes
and splenocytes proliferated well in response to allogeneic (DBA/2)
APC, limited, but significant, proliferation was observed with APC
expressing host type (C57BL/6) MHC (Fig 2). In contrast, allogeneic
DBA/2-derived thymocytes and splenocytes proliferated well in response
to C57BL/6 APC (Fig 2). When exogenous IL-2 was omitted,
proliferation of MHC I°II°wt splenocytes in response to C57BL/6, APC was completely abolished (Fig 2). In contrast to the
results obtained in CTL assays, however, proliferative alloresponses
were also significantly reduced in the absence of exogenous IL-2 (Fig
2).
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| Fig 2.
Deletion-deficient chimera-derived T cells proliferate in
response to host type APC. A total of 5 × 105 Splenocytes
or 106 thymocytes were stimulated with titrated numbers of
T-cell-depleted, irradiated splenocytes. Origin of effectors and APC
was as indicated in the figure. Exogenous IL-2 was added to thymocyte
and splenocyte cultures unless indicated otherwise.
3H-thymidine incorporation was assessed 3 to 4 days
later.
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|
T cells derived from deletion-deficient chimeras fail to induce
lethal GVHD in syngeneic hosts.
MHC I°II°wt chimeras survived for prolonged periods
of time (over 6 months, data not shown). Activation of naive T
lymphocytes, however, requires costimulation delivered by professional
antigen presenting cells.36 The latter cells are of bone
marrow origin and therefore do not express MHC molecules in MHC
I°II°wt chimeras. To test if MHC
I°II°wt-derived T cells react to host type MHC in
vivo, chimera-derived thymocytes and splenocytes were injected IV into
sublethally irradiated (720 Rad) syngeneic (C57BL/6) or allogeneic
(DBA/2) hosts (Fig 3A). While allogeneic
DBA/2 hosts were efficiently killed by MHC I°II°wt
and wtwt chimera-derived thymocytes, no lethal GVHD was
observed in syngeneic hosts up to 100 days posttransfer, at which time
the experiment was terminated (Fig 3A). As expected, C57BL/6 hosts were
readily killed on injection of control allogeneic (DBA/2) T cells (Fig
3A).
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| Fig 3.
T cells derived from deletion-deficient chimeras do not
induce lethal GVHD. (A) A total of 1.5 × 108 thymocytes
or 1.5 × 107 splenocytes were injected IV into
sublethally irradiated, anti-NK1.1 antibody-treated hosts. The origin
of the T-cell populations is indicated in italics. Survival of the mice
was monitored up to 3 months posttransfer. (B) Comparison of GVHD
directed to total host (left) or to cotransferred bone marrow cells
only (right). (Left) As in (A), but titrated numbers (indicated) of
thymocytes were injected. (Right) Lethally irradiated hosts were
injected with a mixture of 107 allogeneic bone marrow plus
titrated numbers of syngeneic thymocytes. (C) Lethally irradiated,
anti-NK1.1 antibody-treated C57BL/6 hosts were injected IV with a
mixture of 5 × 106 C57BL/6 bone marrow cells plus 1.5 × 108 thymocytes of indicated origin.
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|
The lack of lethal GVHD mediated by MHC I°II°wt
chimera-derived T cells seems rather unexpected. Bone marrow-derived
cells express tissue-specific ligands that in the absence of MHC
expression on hematopoietic cells in MHC I°II°wt
chimeras presumably cannot have been encountered during the development
of the MHC I°II°wt chimera-derived T cells. These
cells would therefore be expected to respond to such ligands.
Therefore, we wished to investigate whether MHC
I°II°wt chimera-derived T lymphocytes are capable of
efficient lysis of MHC expressing hematopoietic cells in vivo.
To establish a system in which lysis of bone marrow cells only leads to
mortality, we reconstituted lethally irradiated B10.D2 hosts with
C57BL/6 bone marrow cells and coinjected titrated numbers of B10.D2
thymocytes. Mortality of hosts was observed with (at least) 100-fold
fewer effector cells than that required for a classical
H-2d versus H-2b GVHD (Fig 3B).
To investigate if MHC I°II°wt chimera-derived T
lymphocytes can kill host type hematopoietic cells in vivo, we
reconstituted lethally irradiated C57BL/6 hosts with syngeneic bone
marrow and coinjected large numbers of MHC I°II°wt or
wtwt chimera or DBA/2-derived thymocytes (Fig 3C). No
mortality of C57BL/6 hosts injected with MHC I°II°wt
(or wtwt) chimera-derived thymocytes was observed up to 100 days posttransfer, at which time the experiment was terminated. As
expected, C57BL/6 hosts injected with allogeneic (DBA/2-derived)
thymocytes died rapidly (Fig 3C).
| |
DISCUSSION |
Thymic clonal deletion is thought to be the major mechanism responsible
for the tolerization of the T-cell repertoire.37-39 Consistent with this hypothesis, a twofold to threefold increased generation of mature T cells is observed in the absence of clonal deletion by APC of hematopoietic origin in MHC
I°II°wt chimeras as compared with control
chimeras.10 Half to two thirds of the mature T lymphocytes
developing in these chimeras therefore are presumably self-specific.
Consistent with this notion, we find that CD8+ T cells that
develop in clonal deletion-deficient animals are capable of lysis of
targets expressing host type MHC molecules. Moreover, MHC
I°II°wt-derived T cells proliferate in response to
APC expressing host type MHC, although this response is rather poor. In
contrast, we have found no evidence for in vivo reactivity of T cells
from deletion-deficient chimeras.
Our data relate to earlier work on the tolerogenic capacity of thymic
epithelium and peripheral tissues. It has been observed in several
systems that expression of MHC molecules by thymic epithelial cells or
by peripheral tissues leads to tissue-specific tolerance. Thus, T
lymphocytes derived from athymic nude mice or irradiated recipients
reconstituted with thymic anlagen or fetal thymi deprived of bone
marrow-derived cells are tolerant to donor type MHC expressed by
tissues such as heart and skin, but have occasionally been reported to
be reactive to cells of hematopoietic origin expressing the same MHC
molecules.16,40-47 In irradiation bone marrow (and spleen)
chimeras in which F1 hosts were reconstituted with bone marrow derived
from one parent (PF1), a T-cell repertoire has been shown to
develop that in vivo is tolerant to MHC of the other parent, while in
vitro this is not the case ("split
tolerance").20,48-50 Tolerization of TCR transgenic thymocytes has been shown to occur when the tolerizing ligand is
expressed exclusively on thymic epithelial cells.16,18,23 T
cells developing in bone marrow chimeric mice expressing mammary tumor virus-encoded superantigens exclusively on thymic
epithelial cells are tolerant to the same antigens when expressed on
APC.51,52 Finally, transgenic mice expressing MHC molecules
exclusively on thymic epithelial cells or on peripheral tissues have
been shown to be tolerant to transgene type MHC.17,53-58
Thus, thymic epithelial cells and peripheral tissues appear to have
tolerogenic capability.
In the thymus medullary, but not cortical, epithelial cells are capable
of tolerance induction. It has been reported that in MHC transgenic
mice, the T-cell repertoire is (partly) tolerant to ligands exclusively
expressed by thymic medullary epithelial cells.17,19,41,58
However, T cells from transgenic mice expressing MHC class II molecules
exclusively on cortical epithelial cells react vigorously to APC
expressing the same MHC molecules, indicating a lack of tolerance
induction.25 Moreover T cells derived from relB-deficient
mice, which lack thymic medullary epithelium, proliferate in response
to normal APC.59 Thus, while positive selection seems to be
supported by cortical, but not medullary epithelial cells,60-62 epithelial tolerance induction appears to be
limited to the medulla.
Given the tolerogenic capability of thymic epithelium and peripheral
tissues, the T-cell repertoire in MHC I°II°wt
chimeras may be expected to be tolerant to tissues expressing MHC
molecules in the chimeras, ie, radioresistant cells. In contrast,
because MHC molecules were not expressed on radiosensitive tissues in the chimeras, T lymphocytes would be expected to react to hematopoietic cells when they express MHC, eg, in mixed lymphocyte reaction, CTL
assays, or when injected into syngeneic hosts.
In vitro, targets expressing host type MHC molecules were efficiently
lysed by MHC I°II°wt, but not control wtwt
chimera-derived thymocytes and splenocytes. The target used, RMA, is a
thymoma and therefore of hematopoietic origin.32
Proliferative responses on stimulation of MHC
I°II°wt-derived T cells with host type splenic APC
were significant, but rather limited. Both responses, however, were
well below that observed for allogeneic combinations. Because the
frequency of alloreactivity has been estimated to be around 1% to 10%
for a given combination,63,64 the percentage of MHC
I°II°wt chimera-derived T cells reactive to host type MHC presumably is less than 1%. Because twofold to threefold more mature T cells developed in clonal deletion-deficient chimeras as
compared with control chimeras, the frequency of in vitro reactivity to
host type MHC appears unexpectedly low. Alternatively, the low level of
reactivity of MHC I°II°wt chimera-derived T cells to
host type MHC molecules could be explained by an unusually low TCR
affinity. Both hypotheses can be explained by induction of
nondeletional tolerance mediated by thymic epithelium. Moreover, it has
been reported that the threshold for activation of mature T lymphocytes
is significantly higher (100-fold in one report) than that for thymic
clonal deletion.65 Thus, a proportion of mature T cells
that in normal mice would have been deleted, but that survived in
clonal deletion-deficient chimeras may express TCR with affinity for
self ligands that is too low to lead to activation.
Interestingly, in contrast to lysis by allogeneic effectors, lysis of
RMA by MHC I°II°wt-derived T cells partly depends on exogenous IL-2 added to the priming cultures. Moreover, when stimulated with APC expressing host type MHC, the limited proliferative responses by MHC I°II°wt chimera-derived splenic T-cell
populations were practically absent if exogenous IL-2 was omitted.
These data are consistent with earlier reports showing that
cytotoxicity requires a lower activation threshold than IL-2 production
or proliferation.66 Therefore, it appears that only clones
expressing TCR with low affinity for self ligands remain functional in
the absence of clonal deletion and that other (nondeletional) tolerance
mechanisms are responsible for tolerization of clones expressing TCR
with higher affinity. A more detailed comparative analysis of
cytotoxicity, proliferation, and cytokine production will be required
to test this hypothesis.66,67
In vivo, MHC I°II°wt chimera-derived T lymphocytes
did not induce lethal GVHD in sublethally irradiated host type animals. The main immunopathological consequences of GVHD are medullary aplasia,
enteropathy and sclerodermia.68 While the intestinal tract
has been reported to be targeted in GVHD mediated by MHC class II, but
not class I differences, medullary aplasia has been described to result
from MHC class I or class II differences.68 Moreover, it
has been reported that proliferation is not required to cause lethal
GVHD over MHC class I barriers and that CTL or Th1 lymphocyte-mediated
lysis of bone marrow cells is sufficient to cause death of host
animals.35,68,69 Because in vitro lysis of targets that
express host type MHC was observed even in the virtual absence of
proliferation, it is surprising that MHC I°II°wt chimera-derived T cells do not induce mortality due to medullary aplasia in host type animals. To assure that lysis of bone marrow cells
will lead to mortality in our experimental system and to increase its
sensitivity, we have adapted a previously described GVHD system in
which injected T cells react to coinjected bone marrow cells required
to reconstitute lethally irradiated hosts.35 Our results
indicate that at least 100-fold fewer T effector cells are required to
cause mortality in this system than in classical GVHD. While low
numbers of allogeneic thymocytes were sufficient to cause mortality in
this system, 300-fold more MHC I°II°wt chimera-derived thymocytes were not. Therefore, while we formally cannot exclude the possibility that the fact that deletion-deficient T
lymphocytes are incapable of induction of lethal GVHD is due to
quantitative factors, we favor the hypothesis that other mechanisms of
nondeletional tolerance are responsible.70
Whatever the explanation for the low level of reactivity to host type
MHC by MHC I°II°wt chimera-derived T lymphocytes in vitro and the absence of lethal GVHD in vivo, it is clear from our data
that thymic tolerance mechanisms are largely redundant.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge the expert technical help of R. Lees, P. Zaech, and A.-L. Peitrequin. Members of the Ludwig Institute and in
particular T. Bianchi, S. Marguerat, and T. Renno are acknowledged for
discussions. We thank Drs H. Bluethmann and B.-J. Fowlkes for providing
MHC mutant mice.
| |
FOOTNOTES |
Submitted December 16, 1998; accepted January 18, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Joost P.M. van Meerwijk, PhD,
Institut National de la Santé et de la Recherche Médicale
(INSERM) U395, CHR Purpan, BP 3028, 31024 Toulouse Cedex 3, France.
| |
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ABSTRACT
INTRODUCTION