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*
Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba, Japan; and
Departments of Medical Biophysics and Immunology, Ontario Cancer Institute, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient (DO-Tg/KO) mice in which T cells
have a reduced TCR on the cell surface. In DO-Tg/KO mice, very few
CD4 single positive (SP) thymocytes developed, indicating that the
decrease in TCR signaling resulted in a failure of positive selection
of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/KO
mice resulted in the generation of functional CD4 SP mature thymocytes
in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells
emerged at lower doses of Ag. TCR signal-dependent, sequential
commitment from CD8+ SP to CD4+ SP was also
shown in a class I-restricted TCR-Tg system. These in vivo analyses
demonstrate that the quantity of TCR signal directly determines
positive and negative selection, and further suggest that weak signal
directs positively selected T cells to CD8 lineage and stronger signal
to CD4 lineage. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a consequence of positive selection, two major lineages of T cells are generated: CD4 T cells that recognize peptide Ags complexed with class II MHC and possess helper function, and CD8 T cells that recognize peptides in the context of class I MHC and possess cytotoxic function. This lineage commitment correlates with the specificity of the TCR and the selecting MHC molecules. Two models had proposed to explain the development of T cells bearing matched TCR and coreceptor specificities. An instructive model proposes that MHC restriction of the TCR determines lineage commitment (8, 9). T cells expressing MHC class I- or class II-restricted TCR down-regulate CD4 or CD8, respectively. Alternatively, according to a stochastic model, coreceptor down-regulation may be an arbitrary event, and the initial TCR stimulation leads to random coreceptor down-regulation (10, 11, 12). Continued maturation is dependent on a properly matched MHC-restricted TCR and coreceptor. These studies were interpreted as suggesting that signaling by CD4 or CD8 coreceptor is indispensable for survival and complete differentiation of immature thymocytes via corecognition of the peptide/MHC. However, recently, this idea has been challenged by analyzing the coreceptor re-expression patterns of immature thymocyte subsets. These studies indicated that thymocyte commitment to the CD8+ lineage requires MHC class I-dependent instructive signals, whereas thymocyte commitment to the CD4+ lineage is MHC-independent and may occur by default (13). Furthermore, several new studies analyzing the development of the thymocytes of TCR-transgenic (Tg) coreceptor-deficient mice showed that coreceptors do not possess essential signaling function required for T cell survival and development (14, 15, 16). These studies suggest that signals through the TCR but not coreceptors influence the lineage decision.
At the DP stage, thymocytes express TCR
dimers associated with
the CD3 complex, which is composed of three dimers of 
, 
,
and -. Ag recognition by the TCR dimer initiates
intracellular activation signals mainly through immunoreceptor
tyrosine-based activation motif (ITAM) within the cytoplasmic region of
the CD3 chains. Particularly, the -chain with three ITAMs initially
has been suggested to play a unique and indispensable role in the
signaling function of TCR. However, recent analysis revealed that ITAMs
of are dispensable for T cell activation (17).
Particularly for thymocyte development, previous experiments using Tg
mice expressing lacking the cytoplasmic region have suggested that
the -chain plays a crucial role, not because of its signaling
function during positive selection but rather due to its function to
induce a high level of stable TCRs on the cell surface of immature
thymocytes (18).
Therefore, we addressed the question of how the level of TCR influences
to the selection of T cells by analyzing in vivo T cell development in
mice expressing a low level of surface TCR complexes. We took advantage
of our CD3 knockout
(-
+, KO) mice in
which T cells have a reduced surface TCR complex and impaired signal
transduction through the TCR complex (19). We crossed
KO mice with DO11.10-TCR transgenic (DO-Tg) mice and analyzed T cell
development in DO-Tg/KO mice by administering various amounts of OVA
peptide in vivo. Using this system, we were able to analyze the
contribution of both the level of TCR and the dose of Ag to T cell
development simultaneously without altering the expression of MHC and
CD4/CD8 coreceptors. The data demonstrate that the magnitude of TCR
signaling directly determines both positive and negative selection.
Furthermore, unexpectedly, DO-Tg+
CD8+ single positive (SP) cells emerged by
injection of low doses of OVA peptide, whereas CD4 SP mature thymocytes
were generated by injection of higher doses. These results demonstrate
that the quantity of TCR signal directly regulates not only
positive/negative selection but also lineage commitment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice transgenic for DO11.10 TCR (DO-Tg) on a BALB/c
background (20) were kindly provided by D. Y. Loh,
and were maintained by interbreeding. CD3-deficient (KO) mice
have been previously characterized (19). These mice were
deficient specifically in the CD3 gene but remained normal splicing
and expression of the CD3 gene. Whereas mice deficient for both
and do not develop SP thymocytes expressing surface TCR, our KO
mice possess small numbers of SP thymocytes and express a low level of
the TCR complex on the cell surface. DO-Tg mice were bred with KO
mice that had been back-crossed at least seven times with BALB/c mice
purchased from the Shizuoka Laboratory Animal Corporation (Hamamatsu,
Japan). The offspring were intercrossed to obtain
CD3+/-, CD3-/-
mice, which were DO-Tg. The transgene was analyzed with tail DNA by PCR
using primers (5'-CAGGAGGGATCCAG TGCCAGC-3', and
5'-TGGCTCTACAGTGAGTTTGGT-3'). The CD3 alleles were analyzed with
tail DNA by PCR using primers (5'-TCCTGTCAGCATTCTCAGGCAAG-3', and
5'-GTGTCACCTTGAATCTCGAGCACCG-3'). Lymphocytic
choriomeningitis virus (LCMV)-Tg/2-microglobulin
(2m)-KO mice with C57BL/6 background
were crossed with KO mice, and the offspring were intercrossed to
obtain CD3+/-,
CD3-/- mice, which were
LCMV-Tg/2m-KO mice. Mice were bred and
maintained in our own animal facility.
Abs and peptides
The following Abs were purchased from PharMingen (San Diego,
CA): anti-CD3 (145-2C11)-biotin, anti-TCR
(H57-597)-biotin, anti-heat-stable Ag (HSA) (J11d)-biotin,
anti-CD69 (H1.2F3)-biotin, anti-CD8 (53-6.7)-PE, and
anti-CD4 (RM4-5)-FITC. Biotinylated KJ1-26 (anticlonotypic mAb that
recognizes the TCR of DO11.10) were kindly provided by P. Marrack
(National Jewish Center, Denver, CO). Biotinylated B20.1 (PharMingen)
and F23.1 that recognize V2 and V8.1, respectively, were used for
analysis of FTOC of LCMV-Tg/2m-KO
mice.
Two different OVA peptides (OVA323–339 and
OVA324–334; synthesized by Sawady Technology,
Tokyo, Japan) were used for stimulation of DO-Tg thymocytes. DO-Tg T
cells respond to OVA323–339 when presented
in the context of I-Ad, whereas
OVA324–334, which also binds to
I-Ad, cannot stimulate the Tg TCR. Peptide
sequences are as follows: OVA323–339,
ISQAVHAAHAEINEAGR; and OVA324–334,
SQAVHAAHAEI. A LCMV peptide (p33, KAVYNFATM) (16) was
used for cocultivation with LCMV-Tg/2m-KO
thymic lobes in FTOC.
Induction of positive selection of adult thymocytes in in vitro culture
A total of 1 x 105 nonadherent APCs (irradiated (30 gray) BALB/c splenocytes) and 1 x 106 thymocytes were cultured in a 96-well round-bottom tissue culture plate (Becton Dickinson, Mountain View, CA) in 0.2 ml of RPMI 1640 medium/10% FCS containing Ag peptide for 72 h. Cells were harvested by pipetting, and harvested cells were counted, stained, and analyzed by FACScan as below.
Induction of positive and negative selection in vivo
OVA323–339 or PBS (no peptide) was
administrated to DO-Tg and DO-Tg/KO mice as 250 µl of sterile
solution at various concentrations by i.p. injection every 4 days.
Seven days after the first administration, mice were sacrificed and
thymocytes were counted, stained, and analyzed by FACScan as below.
FTOC
Timed breedings were established between
LCMV-Tg/2m-/-
-/- females with
LCMV-Tg/2m-/-
-/- males or
LCMV-Tg/2m-/-
+/+ males to obtain
LCMV-Tg/2m-/-
-/- or
LCMV-Tg/2m-/-
+/- fetuses, respectively. At day 15 of
gestation, females were sacrificed and thymic lobes were removed from
the fetuses. DNA was extracted from embryonic tails so that Tg fetuses
could be determined using primers specific for the V2 TCR transgene
(21). The fetal thymic lobes were submerged in 200 µl of
IMDM, 1x Nutridoma SP (Boehringer Mannheim, Indianapolis, IN), 50 µM
2-ME, penicillin, streptomycin, 4 mM glutamine, 5 µg/ml
2-microglobulin (2m)
and with or without LCMV peptide. The plates were put into a plastic
bag (Ohmi Odor Air Service, Hikone, Japan), and the air was exchanged
with a mixed gas containing 80% O2, 5%
CO2, and 15% N2
(22). These lobes were then cultured for 7 days at 37°C,
during which time the medium and the mixed gas was exchanged every
other day. After this incubation period, the thymic lobes were teased
apart and viable cells were enumerated by trypan blue exclusion.
Thymocytes were stained and analyzed by FACScan as follows.
Flow cytometric analysis
T cells were stained biotinylated mAb for 40 min, followed by PE-anti-CD4, FITC-anti-CD8, streptavidin-Quantum Red (Sigma, St. Louis, MO) for 30 min. After washing, cells were analyzed on a FACScan (Becton Dickinson) using CellQuest software. Dead cells were gated out by staining with propidium iodide at 4 µg/ml (Sigma) in the FL-3 channel.
Proliferation assay
Thymocytes from OVA-treated DO-Tg/KO mice or nontreated
DO-Tg, BALB/c mice were incubated with anti-HSA mAb (J11D) for 30
min on ice, followed by addition of complement (normal rabbit serum) at
37°C for 30 min to enrich for CD4+ or
CD8+ mature SP thymocytes. Thereafter, the T
cells were stained with anti-CD4-PE and anti-CD8-FITC mAb, and
CD4+ CD8- SP or
CD4- CD8+ SP T cells were
sorted by FACStarPlus (Becton Dickinson). The
purity of the sorted cells was always >99%. SP T cells were
stimulated at a concentration of 2 x 104
cells per well in a 96-well round-bottom tissue culture plate (FALCON)
in 0.2 ml culture medium with 2 x 105
irradiated (30 gray) BALB/c splenocytes and 3 µM
OVA323–339. A total of 2 x
104 SP T cells were stimulated with immobilized
anti-CD3 (20 µg/ml) in a 96-well flat-bottom tissue culture
plate (FALCON) in 0.2 ml of medium containing10% mouse IL-2 culture
supernatant with 2 x 105 irradiated (30
gray) BALB/c splenocytes as a feeder cells. After 3 days of incubation
at 37°C, cells were pulsed with 37 kBq of
[3H]thymidine (37 Mbq/ml; Amersham Pharmacia
International, Aylesbury, U.K.). At the end of another 8-h incubation,
cells were harvested, and incorporated radioactivity was measured with
MicroBeta (Amersham Pharmacia Biotech, Uppsala, Sweden).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thymocyte maturation in our
CD3-+ (KO) mice
has been previously characterized by a reduction of
CD4+CD8+ DP cells as well
as CD4+CD8- and
CD4-CD8+ SP thymocytes,
whereas the CD4-CD8-
double negative (DN) population was not affected (19)
(Fig. 1
, and Table I). This impairment of T cell development
is due to the low expression of TCR on DN thymocytes and the signal to
induce the transition to DP was insufficient. To assess whether the
magnitude of TCR signals affects the positive selection of DO-Tg
thymocytes, we crossed DO-Tg mice with KO mice (DO-Tg/KO mice)
and analyzed T cell development in the thymus (Fig. 1). DO-Tg mice bear
a TCR that recognizes an OVA peptide bound to
I-Ad (20). As reported previously,
the majority of thymocytes bearing this receptor develop into
CD4+ SP cells (Fig. 1c). In contrast,
very few CD4+ SP thymocytes were detected in
DO-Tg/KO mice (Fig. 1d). However, the expression of the
DO TCR transgene restored the cellularity of DP thymocytes in
DO-Tg/KO mice to normal levels (Fig. 2, and Table I). These results indicate
that positive selection of OVA-specific T cells was abrogated by the
deficiency of CD3.
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, and Table II). In DO-Tg mice, the expression level
of the DO-TCR decreases during thymocyte maturation from the DN to DP
stage, whereas mature CD4+ thymocytes up-regulate
the TCR density. The levels of the DO TCR on DN thymocytes from
DO-Tg/KO mice were lower than those of DO-Tg mice but higher than
the endogenous TCR expression on thymocytes from KO mice. This
increased expression of the Tg TCR may explain the normal development
of DP thymocytes in DO-Tg/KO mice. The low level expression of the
TCR-chains on DN cells instead of the pre-TCR may deliver
sufficient signals to differentiate into DP cells. In contrast, TCR
levels on DP thymocytes of DO-Tg/KO mice, which were lower than
those of DO-Tg mice, were insufficient to induce positive
selection.
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KO mice
Because the DO-Tg/KO thymocytes expressed lower levels of TCRs,
it was possible that the reduced avidity was responsible for the lack
of positive selection. We reasoned that the addition of high affinity
peptides for the TCR might be able to increase the avidity between TCR
and MHC/peptide and restore the sufficient TCR signaling required for
positive selection. To test this idea, we administered various
concentrations of OVA peptide (OVA323–339) into
DO-Tg and DO-Tg/KO mice (Fig. 3). In
DO-Tg mice, negative selection was observed with high doses of peptide
in a dose-dependent manner as previously described (23),
whereas the lower doses did not have any effect on the selection. In
contrast to DO-Tg mice, as expected, CD4+ SP
thymocytes in DO-Tg/KO mice were increased in a dose-dependent
manner at the concentration between 75 and 300 µM of peptide and then
were deleted at 1000 µM. It was noted that negative selection took
place at the Ag dose for maximum selection of
CD4+ SP. This suggests that positive selection of
CD4+ cells and negative selection occur at very
close Ag doses. These results demonstrate that in vivo administration
of OVA peptide compensates the avidity to provide sufficient signals
for positive and negative selection in DO-Tg/KO mice. In this
system, we were able to induce three different fates of thymocytes,
neglect, positive selection, and negative selection, by changing the
concentration of a single peptide without manipulating clonotypic
TCR-chains, MHC, and CD4/CD8 coreceptors.
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In the mice administered with OVA peptide,
CD8+ SP cells emerged unexpectedly at low doses
(75–150 µM) of peptide (Fig. 3). However, these
CD8+ SP thymocytes were deleted at intermediate
concentration (300 µM) in which positive selection of
CD4+ SP cells was still induced. Both
CD4+ and CD8+ SP cells that
developed in OVA-administrated DO-Tg/KO mice expressed the DO Tg TCR
(see below). These results suggest that the quantity of TCR signal
directly regulates positive and negative selection, and further
suggests that weaker signals direct positively selected T cells to the
CD8 lineage and stronger signals to the CD4 lineage. To ensure that
these CD4+ or CD8+ SP
thymocytes had undergone positive selection, thymocytes from
OVA-administered DO-Tg/KO mice (150 µM) were stained with the
maturity markers, HSA and CD69 (24, 25). Onsets of HSA
down-regulation and CD69 up-regulation are closely correlated with
positive selection. CD4+ and
CD8+ SP thymocytes from OVA-administered
DO-Tg/KO mice expressed low level of HSA and high level of CD69 as
compared with DP thymocytes (Fig. 4).
Higher levels of expression on SP cells were also shown for Tg TCR
(KJ1-26 staining) and CD3 expression (CD3 staining) despite low
level of TCR expression due to -deficiency. These data suggest that
both CD4+ and CD8+ SP
thymocytes from OVA-administered DO-Tg/KO mice were positively
selected mature thymocytes.
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Proliferation assays were conducted to determine whether the
CD4+ and CD8+ SP DO-Tg
thymocytes selected in the presence of
OVA323–339 peptide were functional.
CD4+ and CD8+ SP thymocytes
from OVA-administered DO-Tg/KO, DO-Tg, and BALB/c mice were
stimulated with irradiated BALB/c splenocytes and OVA peptide or with
immobilized anti-CD3 mAb (2C11) plus IL-2 (Fig. 5). Positively selected
CD4+ and CD8+ SP thymocytes
from DO-Tg/KO proliferated in response to immobilized 2C11 plus IL-2
at about one-third the level of those of the CD4+
and CD8+ DO-Tg thymocytes, consistent with the
impaired function of T cells from KO mice due to the reduction of
TCR expression (19). CD4+
DO-Tg/KO thymocytes proliferated upon stimulation with peptide plus
APCs but not with APC alone, demonstrating that the proliferation was
OVA specific. In contrast, CD8+ DO-Tg/KO
thymocytes, although positively selected with this peptide, failed to
respond to OVA323–339/APC. It is likely that
CD8+ DO-Tg/KO thymocytes did not respond to
OVA/APCs in the absence of CD4. These data demonstrated that
CD8+ DO-Tg/KO thymocytes are not functional
for specific Ags presented by class II molecules, but are capable of
responding to a strong signal by TCR aggregation. We concluded that
OVA323–339 induced functional SP thymocytes in
DO-Tg/KO mice.
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KO thymocytes in
vitro
In vivo administration of OVA323–339 to
DO-Tg/KO mice resulted in the generation of
CD4+ and CD8+ SP cells
expressing Tg-TCR. It is difficult to determine the exact local
concentration of peptide in thymus in the in vivo system. Therefore, we
also tested whether positive selection of OVA-specific T cells could be
induced in vitro. Thymocytes from DO-Tg, DO-Tg/KO, and BALB/c mice
were cultured in vitro with irradiated splenocytes from BALB/c
(H-2d) mice with
OVA323–339 for 72 h (Fig. 6). Although thymocyte development of
DO-Tg mice was not affected at low doses of
OVA323–339, addition of high dose of the peptide
induced the reduction of DP population and relative increase of other
populations, demonstrating that OVA323–339
mediates negative selection of DO-Tg thymocytes in a dose-dependent
manner (23). In contrast, addition of
OVA323–339 induced the appearance of
CD4+ SP cells in DO-Tg/KO thymocytes in a
dose-dependent manner. Furthermore, CD8+ SP cells
also emerged with high doses of OVA323–339.
Because OVA peptide did not modulate the development of BALB/C
thymocytes (Fig. 6), and a control peptide
(OVA324–334, SQAVHAAHAEI) did not affect at all
on the thymocyte development of all of these mice (data not shown), the
induction of positive and negative selection was OVA specific. The SP
cells from DO-Tg/KO cultures treated with
OVA323–339 were KJ1-26 positive and HSA-negative
(data not shown), suggesting that these SP cells had undergone positive
selection and became mature thymocytes.
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KO thymocytes and
cultured with OVA/APC, mature CD4 and CD8 SP thymocytes emerged (data
not shown). Because both CD4 and CD8 SP cells were also
KJ1-26-positive, these in vitro results confirmed in vivo results that
the addition of OVA compensated for the decrease in TCR density and
provided adequate stimulation to positively select DO-Tg/KO
thymocytes. These data also confirm that lineage commitment was
affected by quantity of TCR signals, namely, relatively weak signals
induce CD8 fate, whereas strong signals induce CD4 fate. MHC class I-independent CD8 lineage commitment
Several studies have indicated that CD8 lineage commitment is
dependent on the interaction of the TCR with MHC class I molecules and
may also involve CD8-dependent signals (8, 13). In
2m-deficient mice, the expression of MHC class
I was extremely reduced and positive selection of CD8 T cells was
impaired (26). To determine whether
CD8+ SP cells selected in the presence of OVA are
generated by the MHC class I-CD8 interaction, DO-Tg/KO thymocytes
were cultured with OVA323–339 and APCs from
2m-KO mice (Fig. 7). In culture with
2m-KO APCs, DO-Tg/KO thymocytes exhibit
positive selection and the appearance of both
CD4+ and CD8+ SP cells
similar to those with BALB/c APCs. The culture with OVA in the absence
of APC did not induce SP cells (data not shown), indicating that
positive selection did not occur by the interaction between TCR and MHC
class I on thymocytes. Because 2m-KO APCs did
not express MHC class I even after the culture with
OVA323–339 (data not shown), it is unlikely that
MHC class I on the 2m-KO APCs was induced by
contaminating 2m. Collectively, these data
indicate that positive selection and CD4/8 lineage commitment of
thymocytes bearing DO-Tg TCR is independent of the interaction between
MHC class I and CD8, but depends on TCR recognition of the OVA peptide
in the context of MHC class II on APCs.
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Experiments with DO-Tg/KO mice have hitherto demonstrated that
quantity of TCR signal determines the fate of thymocytes bearing DO TCR
on both selection and commitment. We intended to determine whether this
rule can be applied to other lines of TCR Tg mice, especially the one
expressing MHC class I-restricted TCR. Because class I-restricted
TCR-Tg mice exhibit positive selection of CD8+ T
cells, we could address the particular question whether
increasing TCR signals induce the appearance of
CD4+ T cells. Therefore, we examined the T cell
development of Tg mice expressing TCR (V2/V8.1) specific for the
LCMV peptide presented by H-2Db. We crossed
LCMV-Tg/2m-KO mice with KO mice to obtain
LCMV-Tg mice, which are deficient for 2m and
express low TCR levels. Day 15 fetal thymic lobes from
LCMV-Tg/2m-/-
+/- or -/- mice
were cultured with 2m and various
concentrations of LCMV peptide (p33, KAVYNFATM) in the submersion organ
culture (22) and T cell development in the thymic lobes
was analyzed (Fig. 8). In the thymus from
LCMV-Tg/2m-/-
+/- mice, a low dose of the LCMV peptide (1
nM) induced positive selection of CD8+ SP cells,
and the highest dose of the LCMV peptide (1 µM) induced negative
selection as previously described (7). Furthermore, the
proportion of CD4+ cells increased in a
dose-dependent manner at the concentration between 1 and 100 nM. In
LCMV-Tg/2m-/-
-/- mice, CD8+ SP
cells also developed in the presence of the LCMV peptide. However, a
10- to 100-fold higher concentration of the peptide was required for
positive selection of CD8+ SP cells. Similar to
DO-Tg/KO mice, positive selection of CD4+ SP
cells was observed at higher concentration of peptide in which
CD8+ SP cells were already decreased (Fig. 8).
Peptide-induced CD4+ and
CD8+ SP cells from
LCMV-Tg/-/- (Fig. 9) as well as
LCMV-Tg/+/+ mice (Ref. 7 , and
data not shown) were stained with clonotype-specific anti-V2
mAb, B20.1 (Fig. 9), and anti-V8 mAb, F23.1 (data not shown).
The staining and calculated results of V2+
thymocytes indicate that both SP lineages express the Tg TCR.
Collectively, the data indicate that LCMV-Tg T cells were neglected
without the LCMV peptide, positively selected to the CD8 lineage with a
low dose, positively selected to CD4 lineage with intermediate dose,
and deleted with a high dose of peptide. The fate of LCMV-Tg/KO T
cells shifted to the one of the LCMV-Tg T cells cultured with a low
dose of peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-KO mice express a low level of the
surface TCR and positive selection of SP thymocytes were diminished. In
vivo administration of an appropriate dose of OVA restored efficient
positive selection and an excessive dose promoted negative selection.
Low level of HSA and high level of CD69 confirmed that these SP cells
had been positively selected. In addition, we found that, within the
range of positive selection, low dose of peptide induced commitment to
the CD8 lineage and high dose resulted in commitment to the CD4
lineage. We showed that both positive selection and lineage commitment
were dependent on the interaction of class II-restricted TCR with MHC
class II but not class I. These results demonstrate that the quantity
of TCR signal directly regulates positive and negative selection, and
further indicate that weaker signals direct positively selected T cells
to the CD8 lineage and stronger signals to the CD4 lineage.
In this study, we demonstrated that the decrease in TCR signaling
caused by the CD3 deficiency resulted in the failure of positive
selection in DO-Tg/KO mice. Although T cells in DO-Tg mice use both
CD3 and CD3 in the CD3 complex, those in DO-Tg/KO mice lack
only CD3 and retain CD3. It has been demonstrated that there is
not any significant difference in quality between signals through
CD3 and CD3, especially on T cell selection and development, as
revealed by studies using CD3KO mice (27, 28) and
CD3-transfected hybridomas (29, 30), although the
functional efficiency of the -chain for cell surface expression of
TCR complex and signal transduction appears to be lower than .
Because it has been shown that the number of ITAMs of affects
thymocyte selection in quantitative fashion (48), the
difference may depend on less number of ITAM in . Therefore, it is
unlikely that a distinct signal through CD3 induced positive
selection in DO-Tg mice compared with neglect in DO-Tg/KO mice,
although we cannot exclude the possibility that quantitative difference
between and might induce some qualitative difference in
signaling. Furthermore, previous studies have demonstrated that the
crucial role of the -chain on thymocyte development is to express
the cell surface TCR at high levels, rather than its signaling property
(18). This was shown by introduction of a transgene
with a truncated cytoplasmic tail into KO mice. The "tailless"
transgene restored normal TCR level and thymocyte maturation.
Therefore, it is most likely that T cell selection in DO-Tg/KO mice
reflects a quantitative change in TCR signaling by the reduced TCR
expression but not a qualitative difference.
Previously, we have reported a similar shift in selection from
neglect to positive selection in HY-specific TCR-Tg mice crossed with
KO mice (HY-Tg/KO) (31). In female HY-Tg/KO mice,
HY-specific T cells were neglected, whereas those of female HY-Tg mice
were positively selected. In male HY-Tg/KO mice, HY-specific T
cells, which are autoreactive, were positively selected. In this
system, because the amount of endogenous HY-Ag and MHC were fixed, and
only TCR signals were reduced, these results imply that T cell
selection is controlled by the magnitude of TCR signals. The present
studies using DO-Tg/KO mice extended the analysis to another TCR-Tg
system in which Ag concentration and the surface TCR level could be
manipulated simultaneously. We administered OVA peptide into
DO-Tg/KO mice, which is a high affinity agonist for DO-TCR. In DO-Tg
mice, OVA323–339 induced negative selection of
DO-Tg thymocytes in vivo (20) and in vitro
(23). Our finding that administration of
OVA323–339 into DO-Tg/KO mice resulted in
positive selection of CD4+ SP DO-Tg thymocytes in
a dose-dependent manner, and in negative selection at the higher
concentrations, implies that selection depends upon the quantity of TCR
signal. Although TCR ligation by a very low number of peptide-MHC
complexes results in neglect, the same peptide-MHC complexes result in
positive selection and then negative selection as the peptide
concentration increases. These results support the avidity model in the
light of the quantity of TCR signals. Similar results obtained from
LCMV-Tg/KO thymocytes suggest that, regardless of the
MHC-restriction specificity of TCR, differentiation of mature
CD8+ and CD4+ thymocytes
can be induced by the appropriate avidity of stimulation.
Various experiments have demonstrated that CD4 and CD8 coreceptors play critical roles for lineage commitment through its functional association with p56lck (32, 33), which is involved in early thymocyte development (34, 35). Models have proposed that coengagement of TCR and coreceptor to MHC conveys lck to activate downstream signaling. The developmental link between MHC specificity and coreceptor expression as shown by the analysis of TCR-Tg mice has been explained by an instructional model (8, 9). However, the recent findings demonstrating the existence of coreceptor transitional cells, CD4+CD8low in class II-deficient (11) and CD4lowCD8+ in class I-deficient mice (12), is inconsistent with the instructional model and has been offered as an evidence for a stochastic model, and these populations are thought to be lineage-committed intermediates in positive selection.
Recently, studies have challenged the validity of the stochastic model. Analysis of coreceptor transitional cells in vivo (36) and in vitro (37) revealed that CD4+CD8low population is composed of a mixture of CD4-committed and CD8-committed thymocytes, whereas the CD4lowCD8+ population exclusively consists of CD8-committed cells. Similar conclusions were reached using a coreceptor re-expression assay (13). Appearance of CD8-committed cells among CD4+CD8low or CD4lowCD8+ thymocytes was completely dependent on prior instructional TCR-class I MHC interactions, whereas the appearance of CD4-committed cells among CD4+CD8low thymocytes depends on prior signals through the CD3 complex (13, 38). Furthermore, several studies using coreceptor-deficient mice showed that coreceptors do not possess essential signaling properties required for T cell survival and development (14, 15, 16). These studies implied that signals through TCR but not coreceptors influence the lineage decision.
In this study, when OVA peptides were administered in DO-Tg/KO mice,
low to mid-range concentrations of OVA induced the generation of SP
mature thymocytes expressing the Tg-TCR in a dose-dependent manner.
Within the range of Ag-mediating positive selection, lower doses
induced predominantly CD8+T cells, whereas higher
doses induced CD4+ T cell development. To
understand the mechanism of our findings, we first considered whether
our data could be explained in the context of the stochastic model.
Because DO-Tg/KO mice expresses normal levels of coreceptor and MHC
molecules, the stochastic loss of one coreceptor triggered by TCR
engagement should only leads to CD4+ T cells
survival and complete differentiation. This does not explain why a
certain number of CD8+ T cells would be
positively selected with only low doses of peptide. It is also
difficult to explain the data with an instructional model. Because CD8
was not used in the recognition of MHC class II in DO-Tg/KO mice, it
cannot instruct CD8 lineage commitment by coengagement with TCR
complex. In addition, it is unlikely that processed OVA peptide
presented on MHC class I interacted DO TCR and CD8 and then promote
positive selection because the peptide could not be presented on MHC
class I even when processed (39). If the engagement of CD8
and MHC class I could provide an instructive signal for positive
selection, CD8+ T cells would not appear with OVA
plus APCs from 2m-KO mice. Thus, our results
are inconsistent with the original instructional model.
Therefore, we propose a revised instructional model in which the
intensity of signals per se delivers to DP thymocytes through the TCR
complex determines the lineage choice. In this "quantitative
instructional model," the net quantity of TCR signal directs the
CD4/CD8 lineage choice. Moderate intensity of TCR signal leads to
positive selection, and, in this process, signals of a stronger and
weaker intensity promote CD4 and CD8 lineage commitment, respectively.
In this case, CD4 and CD8 coreceptors do not play any obligatory role
in signaling and generally stabilize TCR-peptide/MHC interactions
(40, 41, 42, 43). For individual thymocytes, the quantity of TCR
signal varies to a certain extent upon triggering with a particular
concentration of Ag. Such signaling heterogeneity including duration of
signals may explain the overlapping between positive selection of
CD4+ SP cells and negative selection of DP cells
at higher dose of Ag in -/- mice. However,
the overall fate of thymocytes changes from neglect to CD8 lineage
commitment, CD4 lineage commitment and negative selection in this
order. It is not clear that the signals generated by the TCR complex
directly promote termination of one coreceptor synthesis or render
thymocytes competent to respond to other lineage-specific signals such
as Notch (44). These data are also consistent with the
observation that the maturation of CD4 lineage requires higher
signaling threshold than CD8 maturation (45) and that the
maturation of CD4 SP cells is more sensitive to diminished TCR level
(46).
A similar quantitative instructional model has been proposed to explain
why a few but distinct proportion of DO-Tg CD8+ T
cells exist in DO-Tg mice and increase when CD4 is absent
(14). In some circumstances, the DO-Tg thymocytes received
suboptimal TCR signals and developed into the CD8 lineage, and, when
CD4 was not available, the weaker signals promoted CD8 lineage
commitment. Furthermore, in CD8-deficient class I-restricted TCR Tg
mice, an increase of the selecting ligand can overcome the need for
coreceptor involvement and promote positive selection of CD8 T cells
(15, 16). Similarly, in KO mice,
CD4+ SP cells were predominantly reduced as
compared with CD8+ SP cells (19)
(Fig. 1b). This is also explained by a quantitative
instructional model where development of CD4+T
cells requires a stronger signal and is mainly affected by a reduction
in TCR expression. A similar mechanism has also been proposed from the
analysis of in vitro commitment induced by PMA/ionophore stimulation,
in which weak stimulation induced CD8 SP and stronger stimulus induced
CD4 SP thymocytes (47).
Our results provide a strong evidence that the quantity of TCR signals directly regulates not only positive and negative selection but also lineage commitment. Molecular mechanisms to determine the relationship between signal intensity and distinct selection have to be solved.
Note added in proof.
A recent report by Yasutomo et al. (49) described that CD4/CD8 lineage commitment is determined by the duration of TCR signaling, which is consistent with our conclusion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
3 Abbreviations used in this paper: DP, double positive; 2m, 2-microglogulin; FTOC, fetal thymus organ culture; ITAM, immunoreceptor tyrosine-based activation motif; KO, knockout; LCMV, lymphocytic choriomeningitis virus; SP, single positive; DN, double negative; HSA, heat-stable Ag; Tg, transgenic.
Received for publication March 27, 2000. Accepted for publication September 5, 2000.
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