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*
Department of Immunology, University of Washington, Seattle, WA 98195; and
Merck Research Laboratories, Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although TCRs, coreceptors, and MHC molecules clearly direct the transition from the DP to SP stage, it is unlikely that a "correct" combination of these components sends a strictly instructive signal which simultaneously dictates survival and lineage commitment. Several studies support the notion that lineage commitment occurs first and randomly but that survival depends on sustained, optimal coreceptor/TCR interactions with the MHC molecules (reviewed in Ref. 15). For example, constitutive expression of the CD8 molecule permits maturation of CD4+ helper T cells (CD4+CD8Tg+) bearing an MHC class I-restricted TCR (16, 17). Conversely, constitutive expression of CD4 molecules rescues CD8+ cytotoxic T cells bearing a class II-specific TCR (18). Thus, thymocytes expressing class I-restricted TCRs were shown to be positively selected in the absence of CD8 when cultured with peptide variants that artificially improved TCR-MHC interactions (19, 20). These studies indicate that manipulations that change interactions among the TCR, coreceptor, and MHC invoke differential intracellular signals to direct T cell maturation.
Following the TCR 
() chain gene rearrangement, the DP thymocytes
undergoing selection express clonotypic - and the 
-polypeptide
chains which comprise a structure that can bind the peptide/MHC
complex. This heterodimeric receptor also associates noncovalently with
the invariant CD3 subunits and the 
chains, which are required for
transducing intracellular signals (21, 22). CD4 and CD8
coreceptors stabilize TCR-MHC interactions and enhance intracellular
signaling events triggered by the TCR. Thus, co-cross-linking CD4 and
CD3 has been shown to enhance the ability of T cells to flux
Ca2+ relative to CD3 cross-linking alone
(23). In contrast, independent cross-linking of CD4 and
CD3 decreased the Ca2+ response. These results
indicate that coreceptors can contribute to the overall amplitude
and/or quality of signals transmitted by the TCR. Furthermore,
expression of a chimeric coreceptor molecule containing the
extracellular and transmembrane domains of CD8 and the cytoplasmic
domain of the CD4 molecule in F5 TCR (specific for a viral
nucleoprotein peptide presented by H-2Db)
transgenic mice promoted the maturation of CD4+ T
cells expressing this class I-restricted TCR (24). These
results suggest that the cytoplasmic tail of the CD4 coreceptor can
influence the outcome of the thymocyte cell fate decision.
The src family protein tyrosine kinase
p56lck is expressed mainly in T-lineage cells
throughout T cell development (review in Ref. 25). It
associates noncovalently with the cytoplasmic tails of the CD4 and CD8
coreceptor molecules and becomes catalytically activated when the
coreceptors are cross-linked (26, 27, 28, 29, 30). Biochemical
evidence suggests that p56lck can also enhance
signals mediated through the TCR/CD3 complex (31, 32, 33),
although a direct physical association between
p56lck and TCR/CD3 complex has not been
established. More importantly, lack of functional
p56lck abrogated mature T cell activation in a
mutant Jurkat subclone (JCaM1), providing genetic evidence for the role
of p56lck in TCR signaling (34). In
mice, targeted disruption of the lck gene, or transgenic
expression of catalytically inactive p56lck
under the control of the lck proximal promoter (which drives
transgene expression in the
CD4-CD8- double-negative
(DN) and DP thymocytes), interferes with both cellular expansion and
allelic exclusion at the TCR -chain gene locus during the transition
from the DN to the DP stage (35, 36). In addition,
expression of an activated form of p56lck
(p56lckF505) under the control of the
lck proximal promoter arrests thymocyte differentiation
before the DP stage and at high levels causes transformation and
accumulation of immature thymocytes (37, 38).
Data obtained from these studies document the importance of p56lck in delivering signals from the pre-TCR complex, but they do not provide direct evidence for the role of p56lck in later stages of T cell development. To study the role of p56lck in the maturation of SP thymocytes, we have previously used the lck distal promoter, which is active mainly in mature thymocytes and peripheral T cells, to drive the expression of a dominant-negative form of p56lck in mice (39). Using the lck distal promoter circumvents the early developmental block that occurs after expression of a dominant-negative lck transgene under the control of the proximal promoter. These mice possess reduced numbers of SP thymocytes, indicating that dominant-negative p56lck inhibits positive selection. However, these results do not resolve whether activation of p56lck is sufficient to direct survival and/or lineage commitment during the transition from the DP to SP stage. In the present study, we used the lck distal promoter to drive expression of an activated form of p56lck (p56lckF505) in mice. Our data suggest that activation of p56lck can direct survival and lineage commitment during the final stages of thymocyte maturation. Furthermore, we show that CD4:CD8 ratios of developing thymocytes are sensitive to relative levels of p56lckF505, consistent with the idea that signal "strength" assists in determining the CD4+ vs CD8+ lineage choice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lck gene containing the F505 mutation and a 0.6-kb fragment of the 3' human growth hormone gene was isolated from a previously reported construct, pLGF (37), by restriction digest with NotI and StuI. The vector containing the lck distal promoter, pW120 (40), was linearized with BamHI digest, filled in, and was subject to partial digestion with NotI to remove the human growth hormone gene. Finally, we inserted the lckF505 (including the 3'-human growth hormone gene) gene into the lck distal promoter construct by directional ligation. The A-to-T point mutation converting the tyrosine to a phenylalanine, and the joining region of the distal promoter and the lck gene was verified by nucleotide sequencing.
The transgene DNA was microinjected into fertilized (C57BL/6 x DBA/2)F2 embryos to generate transgenic founders. We determined transgene integration by Southern blot analysis of tail DNA and backcrossed four founders with C57BL/6 animals to establish transgenic lines.
Generation and screening of MHC-deficient dLGF animals
To generate MHC class I-deficient and MHC class II-deficient
dLGF animals, we crossed dLGF animals from A16912 line with
commercially available 2-microglobulin
(2m)-/- or
I-Ab-/- mice (Taconic Farms, Germantown,
NY). To generate MHC double-deficient dLGF animals, we crossed the dLGF
A16809 line animals with
2m-/-/I-Ab-/-
mice, also purchased from Taconic Farms.
The genotype at the 2m and
I-Ab loci was determined by PCR. The primer
sequences are as follows. For detection of wild-type
2m locus, the forward primer sequence is
5'-AAA CTG CAG TCA TCT TCC CCT GTG GCC CTC AGA-3', and the reverse
primer sequence is 5'-GTA AGG AAG AAC TTG AGG CTT ACC-3'. For detection
of wild-type I-Ab locus, the forward primer sequence is
5'-AGC ACC GCG CGG TGA CCG AG-3' and the reverse primer sequence is
5'-CAG AGG GCA GAG GTG AGA CAG-3'. The absence of amplification product
was scored as homozygous disruption in each case.
Analyses of transgene expression by immunoblotting
Transgene expression was determined by immunoblotting total lysates from thymocytes and splenocytes. Briefly, 5 x 105 cells were lysed in buffer containing 1% Triton X-100 (50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM Na3VO4), and the resulting lysates were resolved in 10% SDS-PAGE, transferred onto nitrocellulose, blotted with affinity-purified anti-p56lck monoclonal reagent (3A5; Santa Cruz Biotechnology, Santa Cruz, CA) at 0.25 µg/ml followed by incubation with HRP-conjugated sheep anti-mouse secondary Ab (Amersham Life Sciences, Arlington Heights, IL) at 1/3000 dilution, and visualized by chemiluminescence. Relative intensity of the signals was determined by densitometric analyses using Molecular Imager system (Bio-Rad Laboratories, Hercules, CA).
Flow cytometry
Thymocytes and splenocytes were stained and analyzed by flow
cytometry according to standard protocols. After RBC lysis treatment,
106 cells were incubated in staining media
(HBSS-BSA at 10 mg/ml) containing appropriate concentrations of
fluorochrome-conjugated mAbs on ice, washed, incubated with secondary
reagent where required, and fixed in 1% paraformaldehyde (PBS, pH
7.4). Flow cytometry was performed on FACScan (Becton Dickinson, San
Jose, CA) and analyzed using ReproMac software (TrueFacts Software,
Seattle, WA). The following monoclonal reagents were purchased from
PharMingen (San Diego, CA): biotin-anti-CD3
(145-2C11);
biotin-anti-CD69 (H1.2F3); biotin-anti-CD4 (H129.19);
biotin-anti-heat-stable Ag (HSA); PE-anti CD8 (53-6.7); FITC-anti
TCR (H57-597), FITC-anti-Qa-2 (1-1-2). PE-anti-CD4 (CT-CD4),
FITC-anti-CD8 (CT-CD8a), and Tri-Color-conjugated streptavidin were
purchased from Caltag Laboratories (Burlingame, CA).
For intracellular staining of TCR , cells were first stained for
surface markers using biotinylated and PE-conjugated monoclonal
reagents, respectively, followed by incubation with
Tri-Color-streptavidin, fixed in 4% paraformaldehyde (PBS, pH 7.4),
and permeabilized in 0.1% saponin solution (PBS (pH 7.4)-1% FCS-0.1%
NaN3). The cells were then incubated with
FITC-anti- TCR diluted in permeabilization buffer and analyzed on
FACScan.
Isolation of thymocyte subpopulations
To isolate DP and CD4+ SP thymocytes for
cell cycle analysis, total thymocytes were stained with
fluorochrome-conjugated reagents specific for CD4 and CD8 and sorted on
FACStar (Becton Dickinson, San Jose, CA). For proliferation assay, the
CD4+ and CD8+ T cells were
purified from splenocytes by negative selection using magnetic bead
separation methods. Briefly, splenocytes were incubated with Ab
cocktail containing biotinylated anti-B220,
anti-I-Ab, anti-CD11b, anti-NK1.1, and
anti-CD4, or anti-CD8 monoclonal reagents (PharMingen, San
Diego, CA) for 30 min and washed. Then magnetic beads preconjugated
with streptavidin (Dynal, Lake Success, NY) were added at an 
10:1
bead-target ratio and mixed for 45 min at 4°C. Cells that did not
bind the beads were collected after the magnetic separation for
experiments described in the text.
Cell cycle analysis
Purified DP and SP thymocytes were stained with propidium iodide as described by Hardy et al. (41) and analyzed by flow cytometry to determine nuclear DNA content. Briefly, sorted cells were washed in PBS, fixed in 95% ethanol, and incubated in staining buffer containing propidium iodide (Calbiochem, San Diego, CA) at 20 mg/ml, RNase A at 1 mg/ml, and 0.01% Nonidet P-40, followed by analysis on FACScan.
Proliferation assay
Total or purified splenocytes were stimulated in RPMI (supplemented with 10% FCS, L-glutamine, nonessential amino acid, penicillin-streptomycin, and 2-ME) containing various combinations of 145-2C11 ascites, PMA (Calbiochem), recombinant murine IL-2 (Boehringer Mannheim, Indianapolis, IN), and ionomycin (Calbiochem) for 2 days, pulsed with [3H]thymidine, and harvested on day 3. Proliferation was measured by the cpm incorporated and normalized to the number of mature T cells in each well. For allo-specific responses, indicated numbers of total splenocytes were cultured with irradiated splenocytes from C3H/HeJ animals in the presence or absence of exogenous IL-2. The culture was pulsed on day 4 and harvested on day 5.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the role of p56lck during late
stages of thymocyte ontogeny, we generated mice that express an
activated form of p56lck
(p56lckF505) under the control of the
lck distal promoter (Fig. 1
A). These transgenic mice are
herein referred to as dLGF mice. We generated four independent dLGF
lines by backcrossing founders onto a C57BL/6 background. As shown in
Fig. 1B, immunoblot analyses indicate that the total
abundance of p56lck is increased in thymocytes
from transgene-positive mice relative to littermate controls.
Densitometric scanning of a representative blot showed that transgene
expression increased the total abundance of
p56lck 1.4- to 2.5-fold (lanes
2, 4, 6, and 8) compared with littermate controls
(lanes 1, 3, 5, and 7). The overall levels
of transgene expression declined in the periphery (Fig. 1B,
lanes 9–16), a result different from the known pattern of
lck distal promoter activity (40). This
discrepancy may be explained by the fact that the proportion of T cells
among total splenocytes decreases in the periphery of dLGF mice, as
shown in Fig. 2A.
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dLGF animals appeared healthy and did not develop tumors of the
thymi or peripheral lymphoid organs (up to 5 mo of age). We assessed
the effects of transgene expression on thymocyte development by flow
cytometric analyses of CD4, CD8, and CD3 expression. As shown in Fig. 2
A, thymi from dLGF animals contained substantially
increased proportions of cells with mature
CD4+CD8- and
CD4-CD8+ phenotypes
(upper panels). The increase in the percentage of such SP
thymocytes was accompanied by a concomitant decrease in the percentage
of CD4+CD8+ DP thymocytes
(from 87.0% to 34.7%). The increased proportion of SP thymocytes in
dLGF animals reflected an increased production of mature T cells as
indicated by the increased absolute number of the SP thymocytes.
Moreover, the number of SP thymocytes increased as the abundance of
p56lckF505 increased (Fig. 2B).
Importantly, the total number of thymocytes remained normal in all dLGF
lines, indicating that the expansion was selective for the SP
compartment. At a 0.7:1 ratio
(p56lckF505:wild-type
p56lck in A16924 line),
p56lckF505 increased the number of SP thymocytes
at least 3-fold relative to that of the control mice. Thus, production
of SP thymocytes is extremely sensitive to the level of
p56lck activity.
In contrast to thymocytes, the representation and the number of mature
T cells in the periphery of dLGF mice are lower compared with the
littermate control (Fig. 2A, lower panels). These
data suggest either that p56lckF505 inhibits
migration of mature thymocytes to the periphery or that the presence of
p56lckF505 is detrimental to the survival of
peripheral T cells.
In addition to the altered representation of thymocyte subsets, we
observed that a large number of both the DP and the SP thymocytes in
dLGF animals displayed reduced CD4 and CD8 coreceptor expression on
their cell surfaces (Fig. 2A, upper panels). The
mechanisms responsible for regulation of coreceptor expression on T
cell surfaces are presently unknown. Thymocytes from dLGF animals also
display reduced levels of surface CD3 proteins. In control mice,
10% of thymocytes express high levels of surface CD3 and correspond
to SP mature cells (Fig. 2C, left). In contrast,
only 2% of thymocytes from dLGF animals express high levels of surface
CD3 (right), even though the percentage of SP thymocytes is
increased in these animals (10% in control vs 58% in transgenic
mice). Moreover, the percentage of cells arbitrarily defined as
CD3mid is reduced (48.5% in control vs 23.7% in
Tg animals) with a concomitant increase in the percentage of
CD3- cells in dLGF animals, suggesting that a
large proportion of DP thymocytes fail to up-regulate surface TCR-CD3
expression. Activation of p56lck has been
previously correlated with decreased TCR-CD3 surface expression in
immature and mature T cells. In DN thymocytes, activated
p56lck, expressed under the control of the
lck proximal promoter, inhibits TCR -chain gene
rearrangement and thus prevents production and surface expression of
functionally rearranged TCR -chains (42). In mature T
cells, p56lckF505 down-regulates surface
accumulation of TCR by directing TCR-CD3 complexes to lysosomal
compartments for degradation (43). To determine which of
these mechanisms may be responsible for the reduced accumulation of
surface TCR/CD3 in dLGF animals, we permeabilized thymocytes from
control, pLGF, and dLGF animals and stained them for cytoplasmic TCR
-chain (Fig. 2D). The percentage of cells containing
cytoplasmic TCR protein is reduced in the DN thymocytes (Fig. 2D, center) from mice that express high levels of
p56lckF505 under the control of the
lck proximal promoter (pLGF (37)). These
results are consistent with the known effects of
p56lckF505 on TCR -chain gene rearrangement.
In contrast, the percentage of DN cells containing cytoplasmic
-chains remains normal in dLGF animals (Fig. 2D,
right), indicating that TCR gene rearrangement is not
inhibited. Production of TCRs, judged by the content of intracellular
-chain proteins, is also normal in DP thymocytes from these mice
(data not shown). This finding, together with the fact that
p56lckF505 is not expressed in DN thymocytes
(data not shown), is consistent with the idea that TCR-CD3 levels
decrease in dLGF animals due to posttranslational degradation rather
than diminished synthesis of functional TCR chains.
p56lckF505 does not promote cellular proliferation in dLGF animals
The finding that p56lckF505 increases the
proportion and the number of SP thymocytes suggests either that the
transition from the DP to the SP stage is enhanced or that thymocytes
in the SP compartment undergo proliferation. To address these
possibilities, we purified DP and CD4+ SP
thymocytes from control and dLGF animals and measured
their DNA content. As shown in Fig. 3, the percentages of cells that are in S + G2-M
phase are comparable between Tg and control animals in both the DP and
the SP compartments, and the percentage distribution of cells in
G1 and in S + G2-M
phases in our experiments is in agreement with previous reports
(44). These results indicate that
p56lckF505, at expression levels achieved in our
system, does not induce spontaneous thymocyte proliferation, arguing
against the hypothesis that the increase in SP thymocyte number results
from postselection proliferation.
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The observation that p56lckF505 increases
the proportion and the number of SP thymocytes, without inducing their
proliferation, suggests that activated p56lck
accelerates the maturation of thymocytes from the DP to the SP stage.
In normal animals, maturation through this differentiation step
requires the interaction of functional TCRs with appropriate MHC
molecules. However, we observed that SP thymocytes in dLGF animals
develop efficiently despite the reduced surface levels of CD3,
suggesting that p56lckF505 might compensate for
any loss or attenuation of signals normally triggered by TCR-MHC
interaction. To test this possibility, we investigated whether SP
thymocytes can develop in dLGF mice bred to MHC class I
(2m-/-)-, MHC class II
(I-Ab-/-)-, or MHC double-deficient
backgrounds. Fig. 4A shows CD4
and CD8 staining profiles of thymocytes from dLGF mice crossed onto an
MHC class I-/- background. As expected,
nontransgenic (non-Tg) MHC class I-/- mice
(lower left) exhibited a profound deficiency of
CD8+ SP thymocytes when compared with MHC class
I+/- mice (upper left). In contrast,
expression of p56lckF505 in MHC class
I-/- mice (lower right)
reconstitutes the CD8 SP compartments to levels comparable with that of
its MHC class I+/-/dLGF counterpart. These
results indicate that p56lckF505 can drive the
maturation of CD8+ SP thymocytes in the absence
of MHC class I molecules. As in a wild-type background (Fig. 2C), the presence of the dLGF transgene caused decreased
levels of surface TCR-CD3 on DP as well as on SP thymocytes in MHC
class I-/-/dLGF mice (Fig. 4B,
bottom). Thus, in dLGF mice (center and
bottom), <10% of total CD8+ SP
thymocytes express appreciable levels of TCR-CD3, and the level of
TCR-CD3 expression in these cells is significantly reduced compared
with MHC class I+/-/non-Tg controls
(top). Nonetheless, CD8+ SP
thymocytes in MHC class I-/-/dLGF mice
up-regulate CD69 as well as do their MHC class
I+/-/nontransgenic and MHC class
I+/-/dLGF counterparts (Fig. 4C).
This suggests that dLGF thymocytes have received an activating signal
during the DP-to-SP transition, even in the absence of MHC class I
molecules.
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A, MHC class
II-/-/dLGF mice possess a large number of
CD4+ SP thymocytes (lower
right), in contrast to the MHC class
II-/-/nontransgenic control (lower
left). We also noted that the percent of
CD4+ SP thymocytes in MHC class
II-/-/dLGF mice was slightly reduced compared
with the MHC class II+/-/dLGF control. This
result suggests that MHC class II molecules can enhance but are not
required for maturation of SP thymocytes directed by
p56lckF505. The level of surface TCR/CD3 is
again reduced on thymocytes from MHC class
II-/-/dLGF mice (data not shown), although
CD4+ SP thymocytes clearly up-regulate surface
expression of CD69 (data not shown) and the mature T cell marker Qa-2
(Fig. 5B). To test whether CD4+ T
cells acquire the ability to respond to activating signals, we measured
the ability of CD4+ T cells to proliferate. We
purified CD4+ splenic T cells from MHC class
II-positive (MHC class II+/-) or -negative (MHC
class II-/-) and nontransgenic or dLGF animals
and cultured them with H-2k allogeneic
stimulators in the absence or presence of exogenous IL-2. The
CD4+ cells from MHC class
II+/-/nontransgenic control animals
proliferate in response to increasing numbers of stimulators in the
absence of exogenous IL-2, but those from MHC class
II-/-/non-Tg, MHC class
II+/-/dLGF, and MHC class
II-/-/dLGF animals do not proliferate
(Fig. 5C, left). Addition of exogenous IL-2 enhances the
overall response but still fails to restore proliferation of T cells
from MHC class II+/-/dLGF and MHC class
II-/-/dLGF animals to the control level (Fig. 5C, right). The apparent deficit in proliferative
responses by MHC class II+/-/dLGF and MHC class
II-/-/dLGF splenocytes may reflect the reduced
levels of surface TCR on these cells (data not shown). This hypothesis
is supported by the fact that stimulation with ionomycin and PMA, which
bypasses TCR ligation, restores proliferation to near normal levels
(Fig. 5D).
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A, lower right).
Importantly, the overall representation of thymocyte subsets remains
unchanged compared with the MHC-positive dLGF control (upper
right). Moreover, CD4 and CD8 SP thymocytes from MHC-deficient
dLGF mice down-regulate HSA as efficiently as do their MHC-positive
counterparts and better than the MHC-positive non-Tg control (Fig. 6B). These results further support the hypothesis that
p56lckF505 substitutes for a TCR-derived signal
for positive selection and drives thymocyte differentiation to the
mature SP stage.
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We observed that the ratio of CD4:CD8 SP thymocytes varied among
the four dLGF lines. As depicted in Fig. 7A, the mean CD4:CD8 ratio of
individual transgenic lines increases with increasing total
p56lck abundance (A16924 and A16912 lines) but
abruptly returns to the normal ratio at the highest level of transgene
expression (A16809 line). The ratios in individual animals from the two
intermediate lines (A16912 and A16924) varied significantly but were
consistently higher than those in their littermate controls. We
hypothesized that quantitative changes in total
p56lck activity may directly influence the
CD4:CD8 ratio of developing thymocytes or that the site of transgene
integration indirectly affects the balance of lineage commitment. To
test whether alterations in p56lck abundance
affect the CD4:CD8 ratio, we intercrossed transgenic mice from the
A16912 line expressing an intermediate level of
p56lckF505 (and exhibiting increased CD4:CD8
ratios), thus generating animals hemizygous or homozygous for the dLGF
transgene. We analyzed three litters of animals from this cross to
individually assess the total p56lck abundance
in thymocytes and to document the CD4:CD8 SP thymocyte ratio. The
results, summarized in Fig. 7B, demonstrate that augmented
p56lckF505 levels initially increase the
representation of CD4+ cells in the SP
compartment to a ratio of 10:1. Thereafter, further increases in
p56lck abundance results in gradual
normalization of the CD4:CD8 ratio.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2.5- to 3-fold over endogenous wild-type
p56lck levels causes a 5-fold increase in the
number of SP thymocytes (Fig. 1D). This result is in marked
contrast to the effects of p56lck F505
expression under the control of the lck proximal promoter
(pLGF), which blocks generation of mature SP thymocytes
(38). Several lines of evidence strongly suggest that
activated p56lck drives differentiation of
mature SP thymocytes in dLGF mice. First, cell cycle analyses indicate
that the proportion of proliferating cells in the DP and SP
compartments did not increase in dLGF mice (Fig. 3). Thus, massive
expansion of SP thymocytes cannot easily explain the increased number
and representation of these cells. Second, SP thymocytes from dLGF
animals express mature T cell markers: CD69 and Qa-2 are both induced,
HSA is down-regulated, and mutually exclusive expression of the CD4 and
CD8 coreceptors takes place. These results suggest that the presence of
p56lckF505 permits an increased number of DP
thymocytes to survive and undergo final maturation in the thymus.
Interestingly, surface TCR-CD3 and coreceptor expression is notably
diminished in the DP and particularly in the SP compartment in dLGF
animals. Our data show that production of TCR -chains is normal in
these mice. We further considered the possibility that
p56lckF505, acting as an agent that delivers all
aspects of positively selecting signal, may cause premature cessation
of the -chain gene rearrangement in dLGF mice by shutting off the
recombinase-activating gene 1 (RAG1) and RAG2
genes (45, 46, 47, 48). However, TCR -chain production occurs
normally in dLGF mice, as judged by the level of endogenous -chains
expressed in dLGF-TCR double-transgenic mice (J. Alberola-Ila, personal
communication). Previous studies indicate that activated
p56lck can down-regulate surface TCR by a
posttranslational mechanism. Ab-mediated ligation of coreceptors, which
presumably activates p56lck, decreases surface
TCR expression in DP thymocytes (49, 50), whereas
transgenic expression of dominant-negative
p56lck increases surface TCR expression
(39). Subsequently, expression of activated
p56lck (p56lckF505) was
correlated with trafficking of TCR-CD3 complexes to lysosomal
compartment for degradation (43). Given that TCR-
protein levels are normal in DN thymocytes and that TCR -chain
production is normal, posttranslational degradation is almost certainly
responsible for the reduced surface expression of TCRs in dLGF animals.
It is also possible that, in the presence of activated
p56lck, cells expressing fewer TCRs on their
surfaces are preferentially selected and survive. However,
down-regulation of the TCR clearly takes place in the DP compartment
(before selection), indicating that the catalytically active
p56lck can directly down-modulate surface
expression of this receptor. The effects of
p56lck activation on coreceptor expression
remain unclear. However, in vivo administration of anti-CD4 mAbs
has been shown to diminish CD4 expression in addition to its effect on
surface TCR expression (49), suggesting that
p56lck may play a role in regulation of
coreceptor expression or that expression of coreceptors is in some way
coordinately regulated with that of TCR-CD3.
Current models of thymocyte selection posit that the avidity of the
combined interactions among the TCR, coreceptor, and the MHC influences
the cell fate choice in one of three ways. If the combined
interaction is too weak (or does not occur), cells die by neglect. If
the interaction is too strong, cells die by negative selection. In
contrast, those cells that interact with intermediate affinity receive
the appropriate signal for survival and differentiation and become
mature thymocytes. Manipulations that alter these surface interactions
directly affect the efficiency with which mature T cells are generated.
The fact that p56lckF505 increases the number of
SP thymocytes suggests that cells interpret the presence of
p56lckF505 as having received an optimal signal
for survival/differentiation. Quantitatively, this may mean that
p56lckF505 increases signal strength to permit
survival of cells that would normally die by neglect. Whether
p56lckF505 also increases the number of cells
that die in negative selection was not addressed in this study.
Nonetheless, the augmentation of signal strength achieved by modest
expression of the dLGF transgene (encoding a 7-fold more active kinase
protein (51) clearly biases the tendency toward survival
than death, because the number of SP thymocytes increases as a direct
function of the relative level of transgene expression (Fig. 2B). Alternatively, threshold activation of
p56lck may be the qualitative requirement for
survival and/or differentiation of thymocytes undergoing selection. If
this were true, all cells that possess
p56lckF505 may potentially survive and
mature.
The tyrosine-to-phenylalanine mutation renders p56lck constitutively active, which has been shown to greatly potentiate Ag receptor-mediated signals in T cells (32). Thus, we tested whether the p56lckF505-driven differentiation process required TCR ligation at all, by breeding dLGF animals directly onto MHC class I-, class II-, or double-deficient background. Our data show that p56lckF505 drives both maturation of CD8+ SP thymocytes in the absence of MHC class I and maturation of CD4+ SP thymocytes in the absence of MHC class II, implying that constitutively activated p56lckF505 can direct thymocyte maturation in the absence of appropriate MHC engagement. It is unlikely that the effect of p56lckF505 expression is to promote aberrant selection of SP cells on mismatched MHC molecule (52) because CD4+ and CD8+ SP thymocytes emerge even in MHC double-deficient dLGF animals. These results strengthen our view that p56lckF505 drives survival and maturation of thymocytes independent of TCR ligation by MHC. The ability of activated p56lck to substitute for pre-TCR-dependent signals has been previously demonstrated. In RAG-1-deficient mice crossed with pLGF-transgenic animals, expression of p56lckF505 (under the control of lck proximal promoter) was sufficient to overcome the block at the DN-to-DP transition (53). Our results demonstrate that the presence of an activated form of p56lck can bypass the requirement for TCR ligation during the DP-to-SP transition. The phenotype of dLGF mice is also similar to the phenotype of mice lacking Csk, a negative regulator of p56lck (54). In these mice, the absence of Csk in immature thymocytes drove thymocyte differentiation through the DP and SP stages, independent of TCR ligation. The data from our study imply that Csk deficiency leads to hyperactivation (or prolonged activation) of p56lck, which in turn drives thymocyte maturation.
Mature T cells that develop in dLGF animals exit the thymus and
circulate in the periphery but exhibit limited functional capacity.
Purified CD4+ T cells from MHC class
II-/-/dLGF animals proliferated only weakly in
response to allogeneic stimulators (Fig. 5C), although
stimulation with ionomycin and phorbol ester restored the response
significantly (Fig. 5D). These results are perhaps not
surprising, given that T cells from dLGF mice possess substantially
decreased levels of surface TCR, and these cells may remain
refractory to TCR engagement. Alternatively, the presence of
p56lckF505 in mature T cells may be interpreted
as TCR engagement and, in the absence of appropriate costimulatory
signal, may result in anergy. The lack of IL-2 production during T cell
activation has been previously correlated with anergy
(55). IL-2 production by purified
CD4+ T cells from MHC class
II-/-/dLGF mice was indeed greatly diminished
in our experiments (data not shown), suggesting that anergy in vivo may
at least partially account for the observed unresponsiveness of these
cells in vitro. Finally, the TCR repertoire in dLGF mice may be less
sensitive to distinguishing between self and nonself because the
constitutively activated p56lck drives thymocyte
differentiation regardless of individual TCR specificity. In this
scenario, relative frequencies of self-restricted T cells would be
reduced, accompanied by an increased production of potentially
self-reactive T cells. In support of this hypothesis,
CD8+ cytotoxic T cells from dLGF mice exhibited
elevated levels of cross-reactivity toward syngeneic targets in vitro,
relative to non-Tg controls (data not shown).
We also present data that suggest that total
p56lck activity can influence the relative
representation of CD4+ vs
CD8+ SP thymocyte population. Expression of
p56lckF505 increased the representation of
CD4+ SP thymocytes (Fig. 7A), a trend
that was reverted by intercrossing the dLGF mice to further increase in
the abundance of transgene protein (Fig. 7B). We speculate
that at low levels, p56lckF505 promotes
differentiation and/or survival of CD4 lineage cells as a result of its
preferred interaction with CD4 (28). At higher levels of
p56lckF505, the CD4:CD8 ratio returns to normal
as the p56lckF505-CD4 interactions become
saturated and p56lckF505-CD8 interactions
increase. Whether p56lckF505 sends an
instructive signal to direct lineage commitment or rather promotes
survival of precommitted thymocytes remains unclear. Previously, a
model was proposed in which the relative strength of the signal
generated by coreceptor-associated p56lck
determines CD4 and CD8 lineage preference (24). The
quantitative interpretation of this model and our current data is
consistent with the observation that p56lck
interacts with the cytoplasmic tail of the CD4 molecule with higher
affinity than it does with the CD8 molecule (28). Other
studies have indicated that thymocyte lineage commitment occurs
independent of TCR-MHC specificity and even independent of coreceptors,
before selection (17, 19, 20). In our system, the
deficiency of MHC exerted little effect on the ability of
p56lckF505 to influence CD4:CD8 ratios (Figs. 4A, 5A, and 6A), suggesting that the
total activity of p56lck, rather than coreceptor
ligation (by MHC), determines lineage choice and/or survival. Two
recently published studies further strengthen our view that total
p56lck activity determines the CD4:CD8 ratios.
In one study, the level of total p56lck activity
was shown to override MHC restriction of a given TCR in driving the
lineage commitment decisions (56). In a second study,
reconstitution of wild-type p56lck in
lck-/- mice resulted in maturation
predominantly of the CD4+ SP population
(57). The observed skewing toward the CD4 lineage in this
system is consistent with our data that increased
p56lck activity initially favors differentiation
of CD4+ cells (Fig. 2B) and suggests
that the level of activity achieved by the expression of the wild-type
p56lck must fall within this range. A
comprehensive series of experiments now supports the view that
p56lck is a pivotal regulator of early thymocyte
development entrained by the pre-TCR. Our studies make plain that the
selection processes ordinarily attributed to TCR signaling later in
thymocyte maturation can also be mimicked by simple activation of
p56lck. This observation encompasses not only
the emergence of SP cells with "mature" cell surface phenotypes but
also the relative proportion of thymocytes that display
CD4+ vs CD8+
characteristics. It is plausible that the strength of the
p56lck-derived signal can by itself direct all
aspects of lineage commitment during thymocyte maturation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sue J. Sohn, Department of Molecular and Cell Biology, 465 LSA, University of California, Berkeley, CA 94720.
3 Abbreviations used in this paper: DP, double-positive; SP, single-positive; DN, double-negative; 2m, 2-microglobulin; HSA, heat-stable Ag; dLGF, distal promoter driving the lck gene bearing the F505 mutation; RAG, recombinase-activating gene.
Received for publication August 7, 2000. Accepted for publication November 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and SP
thymocytes (•, CD4+ and CD8+ combined) are
shown with the SEM. Solid line, control value (NLC, normal
littermate control), arbitrarily designated as 1. C,
Transgene expression diminishes surface CD3 levels on thymocytes. CD3
profiles of live cell-gated thymocytes are shown as single-parameter
histograms. Percentages of CD3mid and CD3high
populations are also indicated. D, Expression of dLGF
transgene does not inhibit early steps of TCR production. Thymocytes
from control, pLGF, and dLGF animals were stained for surface CD4 and
CD8, followed by permeabilization and staining for cytoplasmic TCR, and
analyzed by three-color flow cytometry. Cytoplasmic TCR profiles of
gated CD4-CD8- DN thymocytes are shown.
Numbers indicate the percentages of cells that stain positive for
cytoplasmic TCR.
) and
control (
) animals were determined by propidium iodide staining and
flow cytometric analysis. The percentages of cells in G1 or
S + G2-M phase among sorted DP and CD4+ SP
populations are shown. The results are representative of three
independent experiments.
), one
MHC class II+/-/dLGF animal (
), and three MHC class
II-/-/dLGF animals (
) are shown. D,
Purified CD4+ T cells were stimulated in culture with media
only or with ionomycin and PMA. The culture was pulsed with
[3H]thymidine on day 2 and harvested and counted on day
3. The mean of triplicate wells and the SEM are also shown.
