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Reveals the Opposite Effects of Pre-TCR at Successive Stages of T Cell Development1
ugich3,*,
*
Laboratory of T Cell Development, Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and
Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-pre-TCR
chain (pT)), first
expressed in a fraction of
CD8-4-CD44-25+ (DN3)
cells, is believed to facilitate or enable an efficient transition from
the CD8-4- double-negative (DN) to the
CD8+4+ double-positive (DP) developmental
stage. Subsequent to pre-TCR expression, DN3 thymocytes receive
survival, proliferation, and differentiation signals, although it is
still unclear which of these outcomes are directly induced by the
pre-TCR. To address this issue, we generated mice bearing a range of
pT transgene copy number under the transcriptional control of the
p56lck proximal promoter. All lines exhibited
increased DN3 cycling, accelerated DN3/4 transition, and improved DN4
survival. However, the high copy number lines also showed a selective
reduction in thymic cellularity due to increased apoptosis of DP
thymocytes, which could be reversed by the ectopic expression of Bcl-2.
Our results suggest that transgenic pT likely caused apoptosis of DP
thymocytes due to competitive decrease in surface TCR formation.
These results highlight the critical importance of precise temporal and
stoichiometric regulation of pre-TCR and TCR component
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or 
lineage commitment and, if the
TCR lineage is chosen, extensive proliferation and subsequent
expression of CD4 and CD8.
To facilitate studies of early T cell development, DN thymocytes
were further subdivided based on the cell surface expression of the
Pgp-1 glycoprotein (CD44) and IL-2R chain (CD25) into four
sequential stages of development:
CD44+CD25-
(DN1)
CD44+CD25+
(DN2)CD44-/lowCD25+
(DN3)CD44-/lowCD25-(DN4)
(reviewed in Refs. 3, 4 , and 6). The major
changes associated with TCR gene rearrangement and cellular expansion
occur at the transition from DN3 to DN4 cells. Some or all of these
changes are mediated by the pre-TCR, formed by association of the
TCR chain with the surrogate TCR (pre-TCR chain (pT)) at
the DN3 stage (7, 8). As a consequence, DN4 cells progress
to the DP stage, where pT expression is down-regulated as the TCR
chain becomes available to pair with TCR (9, 10).
Subsequent expression of the TCR complex thus enables appropriate
selection of thymocytes depending on their TCR specificity and its
proper interaction with self-MHC molecules (11).
The importance of the pre-TCR was highlighted by experiments with
knockout mice where the deletion of either of the pre-TCR components
resulted in a severe decrease of thymic cellularity, apparently caused
by an inefficient transition from DN3 to DN4 stages and/or inefficient
thymocyte expansion (12, 13). Despite these clear-cut
experiments, the exact mechanistic role of the pre-TCR is rather
obscure. Numerous functions have been attributed to the pre-TCR,
including rescue from programmed cell death, proliferation, induction
of CD4 and CD8 expression, induction of TCR locus rearrangement, and
induction of TCR lineage commitment (reviewed in Refs.
8 and 14). However, while the pre-TCR could
directly mediate all of the above functions, it is equally plausible
that it may directly induce one (albeit critical) outcome, such as
rescue from apoptosis, that enables other events to occur in a
pre-TCR-independent manner. In fact, evidence exists to support this
view (15, 16, 17).
To revisit this puzzle, we evaluated T cell development in a
model where the limiting pre-TCR complex component, pT, was
constitutively overexpressed. We generated transgenic mouse lines that
expressed different levels of the pT transgene under the
transcriptional control of the p56lck proximal
promoter. Results of the characterization of these mice, presented
below, highlight the importance of correct developmental regulation of
TCR and pre-TCR component expression and stoichiometry for optimal T
cell differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were bred and maintained under specific pathogen-free
conditions in the Memorial Sloan-Kettering Cancer Center Research
Animal Resource Center (New York, NY). Full-length pT cDNA was
inserted into the BamHI site of the expression cassette
p1017 (18). The transgene was released from the vector by
NotI digestion, purified, and injected into (C57BL/6J
x CBA/CaJ) F2 fertilized eggs. Transgenic
founders were identified by Southern blot analysis of
BamHI-digested genomic tail DNA using the pre-T cDNA as a
probe. Relative copy number was determined by comparison with the
construct loaded at a known copy number. The transgenic lines were
backcrossed to C57BL/6J background for more than nine generations at
the time of analysis, which was conducted on separate mice between 8
and 13 wk of age.
RT-PCR
Total RNA was isolated from mouse thymocytes using TRIzol
reagent (Life Technologies, Rockville, MD). RNA was reverse
transcribed using random primers following the instructions of the
manufacturer (Stratagene, La Jolla, CA). PCR was performed using
primers complementary to sequences in the 5' and 3' ends of pT
(5'-CTGCAACTGGGTCATGCTTC-3' and 5'-TCAGACGGGTGGGTAAGATC-3') and
-actin (Stratagene) as a housekeeping gene. Amplification was
performed for indicated cycles at an annealing temperature of 55°C
using a thermal cycling machine (PerkinElmer/Cetus, Norwalk, CT). After
amplification, 10 µl of the reaction mixture was resolved on a 1.3%
agarose gel, blotted to a nylon membrane, and hybridized with a
purified fragment specific for each cDNA.
Flow cytometric analysis
Single cell suspensions were obtained from thymi, stained with
the indicated Abs, and analyzed using a FACScan instrument and
CellQuest 3.1 software (BD Biosciences, Mountain View, CA). DN cells
were prepared from total thymocytes by two cycles of anti-CD4 and
anti-CD8 mAb plus complement-mediated depletion as previously
described (19). Cell surface expression of CD markers was
determined using anti-CD4-tricolor, anti-CD8 FITC, anti-CD8
PE, anti-CD25 PE, anti-CD25 biotin, anti-CD44 PE (Caltag
Laboratories, San Francisco, CA), and anti-CD44 CyChrome (BD
PharMingen, San Diego, CA) mAbs. T3.70 (H-Y-specific TCR
chain) was purified from ascites and conjugated to biotin in our
laboratory. PE-labeled streptavidin was purchased from Caltag
Laboratories. Hybridoma (2F5) producing mAb specific for the
extracellular domain of pT was kindly provided by Dr. H. von Boehmer
(Dana-Farber Cancer Institute, Boston, MA). The 2F5 mAb was purified
and conjugated to biotin in our laboratory. Signal observed with this
Ab was amplified using the secondary reagent streptavidin-PBXL3 (Martek
Biosciences, Columbia, MD).
Apoptosis assay
Thymocytes were suspended in ice-cold PBS containing 10% FCS.
Where required, DN cells were prepared as above. Cells were cultured in
10% FCS-RPMI 1640 at a concentration of 1 x
106 cells/ml (37°C) and aliquots were removed
at 0, 12, 18, or 24 h to assess apoptosis. Surface staining was
conducted with anti-CD4 tri-color (TC) plus anti-CD8 PE
for total thymocytes, or with anti-CD25 PE plus anti-CD44
CyChrome for DN thymocytes. Cells were washed twice with PBS then
incubated in 250 µl of binding buffer (10 mM HEPES/NaOH (pH 7.4), 140
mM NaCl, and 2.5 mM CaCl2) containing 2.5 µl
annexin V-FITC (BD PharMingen) for 15 min in the dark. Samples were
analyzed by flow cytofluorometry (FCM) within 1 h.
Annexin+ cells were scored as dead (apoptotic and
necrotic) in some assays where the propidium iodide (PI)
cytofluorometer channel was required to detect other fluorophores used
to gate on a selective population of cells. In other assays, cells were
purified and annexin staining was used in conjunction with PI to
discriminate between the early apoptotic and late apoptotic/necrotic
cells. Results of both types of assays were concordant, revealing a
significant early apoptotic component in each case. Figure
legends
denote the use of each of the assays in the figures and comment on the
result of the other assay, which is not shown.
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Procedure used was modified from Refs. 20, 21, 22 . Briefly, one milligram of 5-bromo-2'-deoxyuridine (BrdU) was injected i.p. three times at 4-h intervals. The thymus was taken 12 h after the third injection. Thymocytes were immediately suspended in ice-cold PBS containing 10% FCS. Total thymocytes were distributed in Eppendorf tubes (5 x 106 cells/tube) and incubated with 50 µl of a mixture of anti-CD4 TC and anti-CD8 PE (both from Caltag Laboratories) at optimal dilutions for 20 min at 4°C. DN thymocytes were prepared as above and distributed in Eppendorf tubes (5 x 106 cells/tube) and incubated with 50 µl of a mixture containing anti-CD4 allophycocyanin, anti-CD8 allophycocyanin, anti-CD44 PE, and anti-CD25 biotin (all from Caltag Laboratories) at optimal dilutions for 20 min at 4°C. The cells were then washed and biotinylated Ab was revealed with streptavidin-CyChrome (BD PharMingen). After surface staining, cells were fixed in 200 µl of 1% paraformaldehyde containing 0.01% Tween 20 for 48 h at 4°C in the dark. Cells were washed in PBS and then in 40 mM Tris-HCl, pH 8.0, containing 10 mM NaCl and 6 mM MgCl2, and thereafter incubated for 30 min at 37°C in the same buffer containing 50 U/ml dRNase I (Amersham Pharmacia Biotech, Piscataway, NJ). After washing in PBS containing 0.5% Tween 20, thymocytes were incubated with 5 µl of anti-BrdU FITC (BD Biosciences) for 30 min at room temperature in the dark.
Metabolic labeling and immunoprecipitation
Thymocytes (30 x 106) were labeled
with 2 mCi 35S-methionine (Amersham Pharmacia
Biotech) for 1 h at 37°C. Cells were washed with ice-cold PBS
and the pellet was lysed with 1% Triton X-100, 1% BSA, 0.5 mM PMSF,
0.1 mM
N--tosyl-L-lysine-chloromethyl-ketone
and 5 mM iodoacetamide in TBS (10 mM Tris (pH 7.4) and 150 mM NaCl) for
30 min on ice. Lysates were centrifuged (2000 rpm for 5 min) and
precleared overnight with 2 µl of normal rabbit serum, 75 µl of
Zysorbin (Zymed Laboratories, San Francisco, CA) and 25 µl of a 50%
slurry of protein A-Sepharose (Sigma-Aldrich, St. Louis, MO). After
centrifugation, precleared lysates were incubated with 10 µg of Ab
against the C-terminal (kindly provided by Dr. D. Wiest, Fox Chase
Cancer Center, Philadelphia, PA) for 2 h and then protein
A-Sepharose was added to bring down the immunocomplexes. Samples were
resolved by SDS-PAGE and the polyacrylamide gel was fixed, soaked in
the fluorographic reagent Amplify (Amersham Pharmacia Biotech) for 15
min, dried, and finally exposed to Kodak (Kodak, Rochester, NY) film at
-80°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-transgenic mice
To generate mice with constitutive expression of pre-TCR, we
inserted murine pT cDNA (kindly provided by Dr. H.-J. Fehling,
University of Basel, Basel, Switzerland) into the p1017 cassette
(obtained from Dr. R. Perlmutter, Amgen, Thousand Oaks, CA) under the
transcriptional control of the p56lck proximal
promoter (Fig. 1
A). A
NotI fragment of this construct (Fig. 1A) was
microinjected into fertilized oocytes of (C57BL/6 x CBA)
F2 mice according to standard procedures, and
four transgene (Tg)+ founders (F11, F23,
F29, and F32) were obtained. Founders pT Tg 11 and 32 expressed low
Tg copy number (between two and four copies per genome), with F11
expressing less than F32 (data not shown). F11 mice are therefore
referred to as pT Tglow. Founders 23 and 29
exhibited much higher Tg copy numbers (>20 copies per genome) and are
together referred to as pT Tghigh. All the
transgenic lines were backcrossed to C57BL/6 mice for over nine
generations at the time of the analysis, giving rise to mouse lines 11,
23, 29, and 32. The expression of pT mRNA was detected by RT-PCR and
of the cell surface or cytosolic protein using a mAb specific for
surface pT (kindly provided by Dr. H. von Boehmer, Dana-Farber
Cancer Institute), or a mAb against the C-terminal pT peptide
(generously supplied by Dr. D. Wiest, Fox Chase Cancer Center), as
shown in Fig. 1, C and D. Semiquantitative RT-PCR
analysis indicated that the pT message was present at a higher level
in pT-Tg thymocytes compared with the wild type (wt);
moreover, the level of mRNA expression appeared to be Tg copy dependent
(Fig. 1B). As previously reported, the low cell surface
expression of pT makes its detection by flow cytometry extremely
difficult. Aifantis et al. (23) have shown that a
combination of biotinylated mAb 2F5 and the enhancing fluorochrome
reagent streptavidin-PBXL3 allows pT detection in SCB29 cells. Using
the same approach, we could detect a discrete but reproducible increase
of pT surface expression on pT-Tg DN cells (Fig. 1C). This was independently corroborated by
immunoprecipitation of metabolically labeled protein lysates using a
mAb directed to the pT C terminus. As shown in Fig. 1D,
pT-Tg mice express higher levels of pT protein compared with the
wild type. Moreover, high copy number cell lines expressed more protein
than the low copy number lines. We could not detect pT expression by
either method on single-positive (SP) thymocytes, nor on any of
the peripheral T cell subsets (data not shown).
Thymic cellularity is reduced in mice overexpressing pT
Tg lines 23 and 29 (pT Tghigh) exhibited
a severe decrease in thymic cellularity. Of the two low copy number
cell lines, line 32 had a moderate cellularity reduction, whereas line
11 had no reduction at all (Fig. 2A). This result suggests that
constitutively overexpressed pT unexpectedly perturbs normal T cell
development in a copy number-dependent manner. Reduced thymocyte
numbers could be due to reduced production or to increased elimination.
The CD4/CD8 profiles in pT-Tg mice were not grossly affected,
although an increase in the percentages of DN cells compared with
littermate controls could be observed (Fig. 2D). More
importantly, in lines 23, 29, and 32, the absolute number of pT-Tg
DN cells was not appreciably different in comparison with wild-type
mice (Fig. 2B), but the absolute number of DP cells was
reduced (Fig. 2C), indicating that thymic hypocellularity in
these mice occurs due to a disturbance at the DP level.
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-Tg overexpression potentiates DN3 to DN4 transition and
thymocyte proliferation
The pre-TCR complex is first expressed and is believed to exert
its function in a subset of DN3 cells (8, 24). We
therefore investigated in wild-type and Tg mice the biology of the
DN3/4 transition, focusing on the Tg lines exhibiting low (line 11) or
high Tg copy number (lines 23 and 29, which were phenotypically
indistinguishable from one another). We found that the DN3/4 transition
was potentiated in pT-Tg mice compared with corresponding littermate
controls (Fig. 2D, bottom, and E and
F). The pT transgene caused a copy number-dependent shift
in DN4/DN3 ratios from 0.6 ± 0.4 in wild-type animals to 3.0
± 0.8 in Tglow or Tghigh
lines (Fig. 2, D and F, and data not shown).
To determine the cause of these changes in population dynamics, we
assessed the proliferative potential and susceptibility to apoptosis of
pT-Tg early thymocytes. This was particularly pertinent in light of
certain models of pre-TCR function, which postulate that rescue from
apoptosis alone (15, 16) or rescue from apoptosis and
induction of proliferation (8, 24) are induced by this
receptor. Mice were injected with BrdU i.p., and the percentage of
incorporation of this nucleotide analog into newly synthesized DNA in
each thymocyte subset was revealed by FCM (Fig. 3). The pT-Tg thymocytes exhibited a
transgene copy number-dependent increase in BrdU incorporation among
DN, DP, and SP thymocytes, as compared with non-Tg counterparts (Fig. 3A). Not surprisingly, the increase in proliferation was
more evident in the DN and CD8 SP subsets, which in wild-type mice are
known to be mitotically the most active (3).
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overexpression
induced a significant (p 
0.09) increase in
BrdU incorporation in DN3 cells and a somewhat decreased proliferation
of DN4 cells (Fig. 3B). This decrease was significant only
in pT Tghigh DN4 cells. These data show that
overexpression of pT pronounces proliferation of the DN3 cells. It
remains to be seen whether those Tg DN3 cells that proliferate are
enriched in cells successfully rearranging the TCR locus.
The pT transgene induces increased survival of DN4 yet
apoptosis of DP cells
DP cells of pT-Tghigh mice exhibit a
marked reduction in cell numbers, decisively contributing to the
overall reduction of thymic cellularity. In normal thymopoiesis, the
expression of pT is highest at DN3 and DN4 stages and is
subsequently down-regulated to allow a proper interaction of
successfully rearranged TCR chains with TCR in DP cells. In the
transgenic system using the p56lck proximal
promoter, pT expression is artificially sustained throughout
intrathymic development (18). We therefore considered the
possibility that this constitutive expression may interfere with DP
survival and final maturation. Indeed, freshly isolated thymocytes from
pT-Tghigh mice, where the hypoplasia of the
thymus is more evident, exhibited a 3- to 4-fold higher proportion of
apoptotic cells compared with the wild type (Fig. 4A and results not shown).
Moreover, when Tg and non-Tg thymocytes were cultured overnight in
single cell suspension culture without additional stimulation, both low
and high copy number DP cells showed an increase in annexin
V+ cells (Fig. 4B), indicating that
these thymocytes exhibit a tendency to spontaneously die by a mechanism
most likely triggered before disruption of the tissue for cell
isolation. Again, the effect was copy number-dependent as
Tghigh DP thymocytes exhibited higher levels of
annexin V+ cells than Tglow
cells. By contrast, the Tg protected the immediate precursors of DP
cells, the DN4 thymocytes, from apoptosis in a copy number-dependent
manner (Fig. 4C). This effect was not seen in DN1, DN2, DN3,
or SP thymocytes (data not shown). We conclude that the effect of pT
overexpression is stage-specific, involving increased proliferation of
DN3 and increased survival of DN4 cells as well as increased apoptosis
of DP cells.
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-Tg mice are dying at
an increased rate in vivo, we crossed them to mice overexpressing Bcl-2
(generously provided by Dr. H. Petrie, Memorial Sloan-Kettering Cancer
Center, New York, NY).
pT-Tghigh/bcl-2 double-Tg mice
showed full correction of thymic cellularity (Fig. 5A), DP cell numbers (Fig. 5B), and thymic volume (data not shown) to the levels
observed in wild-type mice (data not shown) or mice single-transgenic
for Bcl-2 (Fig. 5). Surprisingly, in the case of
pT-Tglow mice, where the apoptosis of DP cells
is less pronounced due to lower Tg copy number, the presence of Bcl-2
resulted in larger thymic size and greater absolute numbers of DP
cells (Fig. 5). These results can be explained by the cooperative
effects of increased proliferation of DN3 cells and reduced
p53-mediated apoptosis caused by the pT overexpression (Ref.
15 and this paper), and the apoptosis-blocking effects of
Bcl-2 which neutralize the effects of the ectopic expression of pT
in DP cells.
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, except for pTlow x Bcl-2 mice,
which had significantly more, and pThigh mice,
which had significantly fewer SP cells (cell numbers x
106 were, for CD8 SP and CD4 SP cells,
respectively: 3.1 ± 06 and 6.7 ± 2.1 for Bcl-2
Tg; 3.6 ± 1.0 and 8.2 ± 3.3 for
pTlow; 7.8 ± 1.9 and 22.2 ± 8.9
for Bcl-2 x pTlow; 1.2
± 0.9 and 2.5 ± 1.1 for pThigh, and
2.9 ± 0.4 and 6.4 ± 0.5 for Bcl-2 x
pThigh mice). Two possible models can account
for the observed results. Enforced expression of pTCR could provide
strong signals to the DP cells, thus leading to their apoptosis by a
process akin to negative selection. Alternatively, deregulated
expression of pT could interfere with TCR assembly and thus lead to
less efficient positive selection. In that regard, previous results
(26, 27) suggest that the bcl-2 transgene
delays DP thymocyte death but does not interfere with negative
selection.
Dysregulated pT may interfere with TCR formation and
function
The enforced presence of overexpressed pT in DP cells at the
moment where the formation of the TCR is indispensable for
survival and further development is likely to affect TCR chain
stoichiometry. This, in turn, has the potential to decrease the
efficacy of TCR formation and its interaction with the selecting
self-MHC molecules. To test whether constitutive expression of pT
may be blocking the delivery of survival and/or selection signals, we
crossed pT-Tghigh mice with TCR H-Y-Tg
mice, where the TCR specificity and selection requirements are well
established. Double-Tg female mice showed a 3-fold decrease in the
proportion of CD8 SP cells (Fig. 6A), with a reduction in the
surface TCR-Tg -chain (T3.70) levels within CD8 SP cells (mean
fluorescence intensity for
T3.70+CD8+ cells in H-Y Tg
was 1344, compared with 1156 for H-Y/pT Tg; and 190 for
T3.70-CD8+ cells in H-Y
Tg, compared with 88 in H-Y/pT Tg). The effect was less
pronounced in the case of Tglow mice (data not
shown). This result indicates that while the pre-TCR plays an important
role at earlier stages of development, at the DP stage the presence of
pT interferes with further development.
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could interfere with this process not only by competing with
TCR and thereby reducing the rate of assembly of TCR, but the
presence of pTCR and TCR on the same cell could also lead to strong,
deleting signals that kill DP cells. Currently we have no direct
evidence to discriminate between these two alternatives. However, pT
Tg, H-Y Tg and pT x H-Y Tg thymi all have similar numbers of
DP cells (between 18 and 22 x 106 with
overlapping SD; see Fig. 6B), suggesting that breeding of a
TCR-Tg onto pThigh background does not lead to
further depletion of DP cells by negative selection. By contrast,
absolute numbers of CD8 SP cells are higher in
pThigh x H-Y TCR-Tg double-Tg than in
pThigh thymi but lower than in H-Y TCR-Tg mice
(Fig. 6B), suggesting that excess of TCR helps positive
selection in double-Tg mice. This finding indirectly favors the
"interference with TCR assembly" alternative, a possibility also
consistent with previous observations that premature TCR
expression competes with the pre-TCR in DN cells, leading to curtailed
development toward DP cells (28, 29). Both observations
suggest that pre-TCR and TCR stoichiometry is crucial for proper
developmental progression—first for the assembly of the pre-TCR in DN
thymocytes (in the absence of TCR) and then for the assembly of
the TCR (in the absence of pT-TCR).
Because the stoichiometry of TCR molecules apparently plays an
important role in development, we evaluated whether supplying more
functional TCR chains to the pT Tg may further enhance the
transition regulated by the pre-TCR. The presence of a rearranged
TCR chain (V5 transgenic from the OT-1 TCR), together with pT
Tghigh, did not result in appreciable correction
of thymic cellularity (average cellularity of two mice in this
experiment: wild type, 119 x 106; pT Tg,
57 x 106; V5, 108 x
106; V5/pT double-Tg, 50 x
106 cells), but rather in an even more efficient
transition from the DN3 to DN4 stage, as judged by an overwhelming
dominance of DN4 cells in the DN compartment (Fig. 7). That provision of additional TCR
chains did not correct thymic cellularity is consistent with the
conclusion that the primary defect in
pT-Tghigh DP thymocytes must be in competitive
disruption of TCR formation by the transgene. Based upon these
findings, we conclude that the levels of pT, rather than of the
TCR chain, are limiting for the DN3 and DN4 transition in vivo. This
notion is further supported by the more efficient DN3 to DN4 transition
in pT-Tghigh mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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lacking the cytosolic domain (tailless
pT), controlled by the p56lck promoter
(30), was shown to function nearly as efficiently as the
whole molecule. Another group showed that the intracellular domain,
devoid of most of the extracellular domain, also apparently functions
comparably to the full molecule (31). Although both groups
studied heavily overexpressed molecules (possibly subject to caveats of
spontaneous aggregation and signaling), their conclusions led to the
hypothesis that recognition of an extracellular ligand may not be
required for the pre-TCR function. Consistent with this hypothesis, it
was found that pre-TCR localizes to the liposoluble membrane
microdomains ("rafts") in the absence of signals from the thymic
microenvironment (32), and that ligation of the
pre-TCR-associated CD3
chain leads to calcium signaling
(33). These findings strongly suggest that either there is
no extracellular ligand for pre-TCR or this ligand is provided in a
cell-autonomous or homotypic fashion. An even more difficult question,
not decisively answered so far, is which of the changes occurring in
the aftermath of pre-TCR expression can be directly ascribed to the
pre-TCR itself and which, if any, are produced by concurrent or
immediately subsequent signaling via pre-TCR-independent pathways.
Experiments presented here provide a new angle to these issues owing to
the presence of graded and different amounts of the transgene in
different mouse lines. Our transgene was expressed under the control of
the proximal lck promoter, known to be activated in early T
cell development, between the DN2 and DN4 stages (18, 34).
Our results demonstrate that qualitatively and quantitatively
constitutive expression of pT potentiates many of the features of
early T cell development at the DN/DP interface in a strictly
developmentally regulated manner. We conclude that pT must be the
limiting chain of the pre-TCR complex, because providing an excess of
this chain accelerated the DN3-to-DN4 transition. It is interesting
that the effects of the transgene were strictly stage specific, with
three thymocyte populations exhibiting three distinct effects. The
effects of the Tg were first observed in DN3 thymocytes (in which the
proximal lck promoter first becomes activated; Ref.
34) in the form of increased proliferation, the extent of
which correlated with the transgene dosage. Perhaps somewhat
surprisingly, Tg DN4 cells, known to undergo major expansion in normal
mice, survived better, but actually proliferated slightly less, than
non-Tg cells. Again, pT Tg levels quantitatively correlated with
increased survival and decreased proliferation. Finally, at the DP
stage, the Tg caused increased apoptosis in a copy number-dependent
fashion. Although this could be caused by Tg toxicity, we believe that
this is not the case, because even the low (barely detectable; 
1–2
copies of Tg) levels of Tg caused increased DP apoptosis. Moreover,
these same Tg doses did not cause apoptosis at the two preceding stages
of development (DN3 and DN4), and it is difficult to envision why
nonspecific toxicity would be selectively developmentally regulated. It
is also possible that pT provides a strong signal to DP cells that
alone, or synergistically with TCR, causes their apoptosis
or that increased apoptosis is due to pT competition with
TCR for TCR, leading to decreased TCR assembly and a
consequent lack of efficient positive selection. This explanation was
supported by suggestive, but not definitive, findings of reduced TCR
expression and inefficient positive selection in pT/H-Y TCR
double-Tg mice.
Overall, the results presented in this study underline the importance
of correct temporal regulation of pre-TCR. Moreover, our data also
point to an important role of chain stoichiometry and TCR assembly.
Thus, we previously showed (see Fig. 8, middle) premature expression of TCR (as is the case in
TCR-Tg mice) takes away TCR chains from the pre-TCR complex,
curtailing expansion and imprinting the mature DN developmental fate
upon many of the cells that express this complex (29). By
contrast, providing excess of pT increases proliferation of DN3
cells and survival of DN4 cells, but the failure to extinguish this
expression at the DP stage reduces formation of TCR and increases
DP cell apoptosis that can be blocked by Bcl-2 overexpression. It will
be of interest to further dissect the developmental effects of
overexpressed pT upon the regulation of cell cycle in the course of
the DN/DP transition.
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Acknowledgments |
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peptide, murine pT cDNA, and the p1017
insertion cassette, respectively. We also thank Dr. I.
Aifantis (Dana-Farber Cancer Institute) for useful discussions and
Dr. H. Petrie (Memorial Sloan-Kettering Cancer Center) for
critical reading of the manuscript. |
Footnotes |
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.) and CA-02583 (Memorial Sloan-Kettering Cancer Center Core Cancer Center Award) from the National Institutes of Health, and the DeWitt Wallace Fund (to J.N.-.). 
2 Current address: Laboratory of Molecular Aspects of Hematopoiesis, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
3 Address correspondence and reprint requests to Dr. Janko Nikolich-ugich at the current address: Vaccine and Gene Therapy Institute, Oregon Health and Science University West Campus, 505 NW 185th Avenue, Beaverton, OR 97006. E-mail address: nikolich{at}ohsu.edu
4 Abbreviations used in this paper: DN, double negative; pT, pre-TCR chain; Tg, transgene/transgenic; wt, wild type; DP, double positive; SP, single-positive; FCM, flow cytofluorometry; BrdU, 5-bromo-2'-deoxyuridine; TC, tri-color; PI, propidium iodide.
Received for publication July 6, 2001. Accepted for publication October 1, 2001.
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