By
From the Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP), Strasbourg, 67404 Illkirch Cedex, France
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Abstract |
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A system to innocuously visualize T cell lineage commitment is described. Using a "knock-in"
approach, we have generated mice expressing a -galactosidase reporter in place of CD4; expression of
-galactosidase in these animals appears to be an accurate and early indicator of
CD4 gene transcription. We have exploited this knock-in line to trace CD4/CD8 lineage
commitment in the thymus, avoiding important pitfalls of past experimental approaches. Our
results argue in favor of a selective model of thymocyte commitment, demonstrating a fundamentally symmetrical process: engagement of either class of major histocompatibility complex
(MHC) molecule by a differentiating CD4+CD8+ cell can give rise to T cell antigen receptor
(TCR)hi thymocytes of either lineage. Key findings include (a) direct demonstration of a substantial number of CD4-committed, receptor/coreceptor-mismatched cells in MHC class II-
deficient mice, a critical prediction of the selective model; (b) highly efficient rescue of such
"mismatched" intermediates by forced expression of CD8 in a TCR transgenic line, and an explanation of why previous experiments of this nature were less successful
a major past criticism of the selective model; (c) direct demonstration of an analogous, though smaller, population of CD8-committed mismatched intermediates in class I-deficient animals. Finally, we
found no evidence of a CD4 default pathway.
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Introduction |
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Most conventional T lymphocytes fall into two classes
MHC class II-restricted, CD4+ helper, and MHC
class I-restricted, CD8+ cytotoxic cells. Precursors of both
classes differentiate in the thymus, according to an elaborate
program classically visualized by following changes in expression of cell surface markers, in particular the CD4 and
CD8 coreceptors (1, 2). The most immature thymocytes
express no (or very little) CD4 or CD8 and are thus termed
double-negative (CD4
CD8
; DN)1 cells; they also display no
/
TCR on the surface. DN thymocytes differentiate into double-positive (CD4+CD8+; DP) cells, most
of which express low levels of surface TCR. Only 3-5% of
DP thymocytes survive the transition to end-stage single-positive (SP) cells (3, 4) committed to either the CD4+ or
CD8+ lineage and expressing high levels of TCR.
Survival and commitment at the DP stage of thymocyte differentiation are dictated by positive selection events that depend critically on specific interaction between the thymocyte's TCR and MHC molecules expressed on thymic stromal cells. Experiments involving TCR transgenic (tg) mice have demonstrated that DP thymocytes expressing class I-reactive TCRs are selected to become CD8+ cells (5), whereas those displaying class II-reactive receptors differentiate into CD4+ cells (8, 9). Thus, lineage commitment is an inseparable by-product of the positive selection process, the specificity of the TCR being matched to a particular class of MHC molecule and thereby to a particular coreceptor.
The mechanism by which this match is achieved and the nature of the signals directing CD4/CD8 lineage commitment remain unclear. For several years, discussions have crystallized around the issue of whether lineage choice is primarily instructive (directed by the TCR-MHC interaction) or selective (decided after an initially noncommittal TCR-MHC interaction), or is some combination of the two. A series of studies on MHC-deficient and TCR tg mice had concluded that DP thymocytes expressing a class I-reactive TCR can commit to either the CD4+ or CD8+ lineage (10); the same appeared true of DP cells with a class II-reactive receptor (10, 15). These conclusions, in line with a selective mechanism of lineage commitment, were based mainly on two kinds of observations. First, intermediate thymocyte populations (CD4+CD8int, CD4intCD8+) were described, apparently composed of cells in transit between the DP and SP compartments and including a fraction with mismatched receptors and coreceptors (10, 15, 16). Second, it was possible to "rescue" a population of mature lymphocytes seemingly derived from these "mistaken" thymocytes by forcing expression of the appropriate coreceptor (14, 15). In most of these studies, lineage commitment was assessed by one of two criteria: reduced surface expression of one of the coreceptors after positive selection, and helper or cytotoxic function of mature lymphocyte descendents in cases where terminal differentiation was (or was made) possible.
Unfortunately, results from more recent experiments have seriously undermined these conclusions, in particular the reliance on surface levels of coreceptor to identify intermediate populations and to serve as a marker of lineage commitment. For example, when the class I-reactive population of CD4+ CD8int thymocytes from MHC class II-deficient mice was injected into wild-type thymi, only CD8+ thymocytes were eventually recovered (18). Furthermore, when the same population was analyzed via an assay based on protease stripping and coreceptor reexpression, it appeared that many CD4+CD8int cells had turned off CD4 but not CD8 transcription (21, 22). These findings argued for greater complexity during the positive selection process, and demonstrated that surface coreceptor levels can be tardy and potentially misleading indicators of gene activity and lineage commitment.
However, intrathymic cell transfers and coreceptor reexpression assays are themselves potentially artifactual, as they entail manipulation of surface molecules (i.e., cross-linking, proteolysis) that could easily alter experimental outcome. Thus, the mechanism of lineage commitment is still a contentious issue, but it remains an important one as a framework for understanding the positive selection decision-making process at the molecular level. Therefore, we sought to develop a system for visualizing T cell commitment ex vivo in an innocuous manner. We searched for a reporter of gene activity that could be detected easily without perturbing manipulations and whose expression would be independent of transport or stability of the coreceptors. We report here the generation of mice with the LacZ gene targeted into the CD4 locus and experiments exploiting them to study CD4/CD8 lineage commitment.
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Materials and Methods |
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Construction of the Recombinant Vector and Production of CD4+/L Mice.
To obtain CD4+/L mice, we constructed a targeting vector as illustrated in Fig. 1. In brief, a 7.8-kb AvrII-SphI genomic fragment from the CD4 locus was subcloned. A 323-bp fragment containing most of exons 2 and 3 (including a 30-bp intron) was deleted, and three cloning sites (XhoI, EcoRI, and XbaI) were inserted by single-strand mutagenesis. The fragment's coding potential was interrupted by insertion of the 4.3-kb
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Antibodies, Cytofluorimetric Analyses, and Cell Sorting.
The following antibodies were used: FITC-labeled anti-CD8Fluorescein Digalactopyranoside Staining.
Fluorescein digalactopyranoside (FDG) staining was adapted from Nolan et al. (29). In brief, 4-6 × 106 cells were stained, washed, and resuspended in 120 µl PBS. FDG (Molecular Probes, Inc., Eugene, OR, or Sigma Chemical Co., St. Louis, MO) at the stock concentration of 100 mM in H2O/DMSO/ethanol, 1:1:1 (vol/vol), was diluted to a working condition of 7.5 mM with H2O. Both cells and diluted FDG were warmed to 37°C for 5 min. 80 µl of warmed FDG was added to cells while gently vortexing. Cells were incubated for 5 min at 37°C. FDG loading was stopped by adding 2 ml of ice-cold PBS. Cells were kept on ice for 5 min, then transferred to a 15°C water bath for 15-30 min to enhanceImmunoprecipitation Experiments.
Thymocyte suspensions were washed three times with methionine-free DME (GIBCO BRL, Gaithersburg, MD) supplemented with 5% FCS. Cells were labeled with [35S]methionine (~1,200 Ci/mmol; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at 107 cells/ml/1 mCi. To begin the chase, thymocytes were washed with DME containing 10× cold methionine, and then recultured in DME (plus 5% FCS, 10× cold methionine) for the different time points. At each time point, aliquots (107) of cells were washed twice with PBS and frozen as pellets in liquid nitrogen. Frozen pellets were lysed with 1 ml lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, plus protease inhibitors) on ice for 30 min. After lysis, supernatants were cleared of cellular debris by centrifugation at 13,000 rpm for 15 min. Cell lysates were precleared twice for 30 min with 25 ml (50%) rat serum-coupled protein G-Sepharose 4FF (Amersham Pharmacia Biotech, Inc.). The first incubation was usually overnight. The precleared lysate was then precipitated for 1 h with protein G coupled to GK1.5 (specific for CD4 [30]) or anti-Reverse Transcriptase PCR.
RNA was isolated from sorted cells by standard techniques. In brief, RNA was prepared by NP-40 lysis from 1-5 × 104 sorted cells to which 106 HeLa cells were added as carrier. cDNA was synthesized by reverse transcriptase. Serial dilutions of the cDNA were then used as a template for PCR amplification with specific primers for CD8 (5'-ATGGACGCCGAACTTGGTCAGAAGGTG-3' and 5'-CCACGTTATCTTGTTGTGGGATGAAGCC-3') and TCR- ![]() |
Results |
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To provide a convenient
and harmless means of monitoring CD4 gene activity, we
set out to confer a protein reporter with an identical pattern of expression. gal seemed a good reporter candidate because it is easy to visualize, is inert, and, being cytoplasmic, possesses no signaling capacity, a disadvantage of most
cell-surface reporters (31, 32). We chose a "knock-in" approach rather than transgenesis of a promoter-reporter
construct in order to mimic as closely as possible the complex transcriptional controls on the native CD4 gene (33).
Thus, mice expressing
gal in place of CD4 were generated by targeting a
gal coding region into the CD4 locus
via homologous recombination in ES cells (Fig. 1). To ensure faithful expression of the
gal reporter (and to inactivate the CD4 gene at the same time), we introduced the
gal coding sequences exactly at the CD4 initiation codon
in exon 2. The inserted sequence directs the translation of a
fusion protein comprised of
gal and the neomycin (neo)
resistance domain (
geo, chosen because it is known to be
expressed in normal T cells [23]); the insert also contains an
expressible neo gene under an independent PGK promoter
for selection of ES cell transfectants (Fig. 1).
The final construct was electroporated into ES cells, and
G418-resistant clones carrying the specific homologous recombination event were selected. Chimeras generated
from the injection of a positive clone into B6 blastocysts
were mated with B6 females to obtain germline transmission
of the knock-in mutation. The resulting mouse line was
generally kept in heterozygous form (CD4+/L; hereafter "+"
designates the wild-type CD4 locus, "L" our targeted CD4-gal insertion, and "0" the original CD4 knockout mutation of Killeen et al. [26] used in some of the crosses). In the
heterozygotes, both
gal and CD4 (at half normal levels;
data not shown) should be simultaneously expressed under
the influence of elements that control the CD4 locus.
It was first necessary to determine just how closely the
expression of gal matched that of CD4. Lymph node cells
and thymocytes were stained with FDG to detect cytoplasmic
gal expression (29) in addition to antibodies against
CD4, CD8, and CD3, and were analyzed by flow cytometry (Fig. 2).
gal expression was essentially superimposable
with that of CD4 in both primary and secondary lymphoid
organs. In the lymph nodes, CD4+ cells expressed high
levels of
gal, whereas CD8+ and non-T cells expressed
low to background levels (Fig. 2 A). Similarly in the thymus, CD4 SP and CD4+CD8+ DP cells were
gal+,
whereas mature CD8 (CD3hi) SP and immature DN cells
were not (Fig. 2 B).
gal did not appear in only a select
subset of CD4+ T cells: for example, the minor (and peculiar) NK1.1+CD4+ subset of
/
T cells (38) was also
gal+ in thymic and spleen cell suspensions of CD4+/L
mice (not shown). Importantly,
gal expression did not detectably alter thymus cellularity or population distributions,
as assessed with a panel of markers including CD3, CD4,
CD8, CD69, CD24, CD5, CD44 and peanut agglutinin-
binding polysaccharides (not shown).
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Upon closer examination, we noticed some interesting
features of the expression of the gal reporter. First, CD3
thymocytes within the CD8 SP population, the immediate
precursors of DP cells, were
gal+, implying that enzymatic activity was detectable earlier than surface expression
of CD4 (Fig. 2 B). Second, a proportion of the CD4+
CD8+CD3hi thymocytes expressed low levels of
gal, suggesting that reporter activity was downregulated earlier
than surface CD4 upon positive selection. These observations argued that
gal levels responded faster to changes in
transcriptional activity at the CD4 locus than the level of
CD4 protein itself. To test this hypothesis, we compared
the turnover of CD4 and
gal proteins (Fig. 2 C). In vitro
metabolic labeling with [35S]methionine and precipitation
with antibodies against CD4 and
gal revealed that the
half-life of
gal (4.1 h) was significantly shorter than that of
CD4 (6.2 h).
Thus, not only was gal a faithful reporter of CD4 gene
expression, it also appeared to be an early indicator.
Since gal appeared to be an early indicator
of CD4 gene transcription, it represented a valuable marker
of lineage commitment during the initial stages of T cell selection in the thymus. Thus, we stained CD4+/L thymocytes simultaneously for CD4, CD8, CD3, and
gal. In
Fig. 3 A, CD3hi thymocytes are displayed in a CD4/CD8
plot, and show the customary boomerang-shaped distribution extending from the mature CD4 SP to the mature
CD8 SP compartments. Several distinct gates were set
within this boomerang to permit us to investigate the activity of the CD4 gene, as reflected by
gal staining, within
different populations. The terminally differentiated populations (gates B and C) showed essentially homogeneous patterns of
gal staining: CD4 SP thymocytes were
gal+,
CD8 SP cells
gal
. In contrast, most of the intermediate
populations (gates E, G, and F) contained cells of both
phenotypes, even the small CD3hi subpopulation which
displayed high surface levels of CD4 and CD8 (gate D).
These profiles confirmed the notion that surface coreceptor
levels do not fully and immediately reflect lineage commitment (18, 21), and encouraged us to exploit the
gal marker for investigating lineage commitment ex vivo under unmanipulated conditions. However, it should be kept
in mind that, although the
gal
phenotype unambiguously denotes a shut-off of CD4 transcriptional activity in
this context, and thus commitment to the CD8 lineage,
gal+ populations cannot be interpreted as readily, potentially including cells that have not yet committed to or
those that are already committed to the CD4 lineage.
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CD4+/L mice were crossed with MHC class II-negative
(II0) animals (25) to allow us to evaluate the commitment
status of MHC class I-reactive CD4+CD8int thymocytes,
cells proposed to be transitory intermediates committed to
the CD4 lineage (10). Thymocytes from II0 CD4+/L mice
were analyzed as above (Fig. 3 B). The CD4+CD8int population (gate E) split clearly into two subsets, gal+ and
gal
; there was a significant increase in the
gal
subset
compared with the same transitional population in the class
II-positive mouse. This result is consistent with the notion that at least some CD8-committed thymocytes transiently
downmodulate surface CD8 levels after positive selection
(18, 21), but also suggests that a sizeable contingent of
CD4-committed cells can be selected in the absence of
MHC class II molecules.
That CD4-committed thymocytes were selected on MHC
class I (in the absence of class II) molecules is substantiated
in Fig. 4, a five-color cytofluorimetric analysis of the expression of CD4, CD8, CD3, gal, and CD69, an early
and transient marker of positive selection (14, 39).
When CD69+ thymocytes from II0 mice were displayed in
CD3/
gal plots, it seemed that CD4- and CD8-committed
thymocytes could be traced by virtue of their very positions
within this plot. The CD8-committed cells appeared to reside chiefly within the CD3hi
gallo and
gal
gates; as they
lost
gal expression, they also began to switch from the transitional CD4+CD8int phenotype to the definitive CD4int
CD8+ and CD8 SP phenotypes (compare the CD4/CD8
profiles, gates A and B of Fig. 4). In contrast, only CD4+
thymocytes were found within the CD3hi
galhi population
(gate C), well separated from cells along the CD8 pathway.
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Although rendered unlikely by the very appearance of
the profiles in Fig. 4, a caveat to these interpretations is that
CD4+CD8int cells with high gal expression may still have
been destined for the CD8 lineage, but just had not yet
downregulated
gal expression. To address this point, we
purified CD4+CD8intCD3hi
galhi and CD4+CD8intCD3hi
gallo thymocytes, and compared their CD8 mRNA content by reverse transcriptase (RT)-PCR (Fig. 5). If the
CD4+CD8int
galhi cells were CD8-committed or even uncommitted, their CD8 mRNA levels should be as high as
those detected in the CD4+CD8int
gallo or DP populations. However, if the CD4+CD8int
galhi thymocytes were
CD4-committed, they should have downregulated CD8
transcription at least to some degree. The latter scenario appears to be the correct one: CD4+CD8int
galhi thymocytes
expressed on average two- to threefold less CD8 mRNA than their
gallo counterparts, suggesting that many, if not
all, of the CD4+CD8int
galhi thymocytes in II0 mice were
CD4 committed.
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Our original results (10) and those of van Meerwijk and
Germain (16) suggested that a mirror-image population,
committed to the CD8 lineage upon selection on MHC
class II molecules, also exists. However, this interpretation
was questioned in subsequent reports (21, 22, 42). Therefore, we analyzed 2-microglobulin (
2m)-negative (I0)
mice (43) in which the CD4-
gal reporter had been introduced, allowing a distinction between true CD8-committed cells and other populations (i.e., DPs, CD4-committed). The cytometric analysis in Fig. 6 A illustrates that
CD4intCD8+CD3hi cells did exist in class I-deficient mice,
albeit at a much lower frequency than their counterparts in
class II-deficient animals, and that they displayed the
gallo
phenotype expected of the CD8 lineage. The low expression of
gal established that these cells were not contaminants from the neighboring CD4intCD8intCD3hi population, the great majority of which were
galhi. Our results
also indicated that the CD4intCD8+CD3hi cells were a homogeneous set, as we did not detect CD4-committed cells
within this population, a result consistent with recent findings from other groups (18, 21, 44, 45).
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In the same series of experiments, MHC double-deficient (I0II0) mice with a CD4+/L genotype were analyzed
(Fig. 6 B): the CD3hi CD4+CD8int and CD4intCD8+ thymocyte populations present in II0 and I0 mice, respectively,
were not observed in MHC-deficient animals, in agreement with our previous findings (10); the rare CD3hi cells
were all galhi and probably represent nonstandard T lineages (46). This result confirms that the intermediate populations discussed above require MHC engagement in order
to be positively selected, indicating that a CD4 lineage default pathway in the absence of MHC recognition does not
exist in vivo.
We wondered whether
all T cells destined for the CD8 lineage transit through the
CD4+CD8int stage, and just how early CD8-committed
cells switch off CD4 gene activity. Therefore, we crossed
the CD4+/L line with two TCR tg lines expressing different MHC class I-restricted receptors: the "HY" TCR (selected on Db) and the "OVA" TCR (selected on Kb) (47,
48). Thymocytes from HY+CD4+/L and OVA+CD4+/L
mice were stained for CD4, CD8, and gal in addition to
the appropriate V regions (Fig. 7; panels are gated on
clonotypehi cells, thereby focusing the analysis only on cells
expressing the transgene-encoded receptor). In terms of
gal expression, the patterns were quite similar in the two
types of animals: downregulation of the
gal marker began
in the DP compartment, and the CD4+CD8int population
contained both
galhi and
gallo cells; from the CD4int
CD8int stage onwards,
gal expression was essentially shut
off. Furthermore, both the timing and levels of TCR expression, as reflected by CD3 expression (not shown), were
also similar for the two tg lines, not surprising since expression of both transgenes are driven by the same TCR V
promoter (Correia-Neves, M., unpublished results). However, the HY and OVA mice were strikingly different in
their numbers of intermediate CD4+CD8int thymocytes.
Consistent with a previous report (18), there were three to
four times more transitional CD4+CD8int thymocytes in
OVA than in HY mice. (Note that the OVA line used here
expresses the same receptor as the OVA-tcr-1 line reported previously [48], but was generated using a different vector
for transgenesis; that the two behave identically in having a
large CD4+CD8int population indicates that this phenotype
is not an artifact of transgene vectors or sites of integration.)
Thus, in the HY system, differentiation towards the CD8
lineage seems to proceed quite directly, from DP to CD4int
CD8int to CD4
CD8+ cells; with the OVA line, it appears
more convoluted, generating large numbers of CD4+
CD8int cells. This also appears to be true when endogenous
TCR gene rearrangements in both transgenic lines are prevented by the SCID, RAG, or TCR C
mutation (10;
Heath, W., personal communication; Correia-Neves, M.,
unpublished results). Such a dichotomy suggests that there
must be at least two modes (or a continuum) of class I-restricted CD8 lineage differentiation.
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With both class I-restricted TCR tg lines, the CD4+
CD8int populations contained gallo and
galhi cells.
gallo
cells were most likely CD8-committed, in keeping with results from cell transfer experiments (18). A key question was
whether the
galhi cells were just precursors of the CD8-committed cells or whether they really included a CD4-committed population as we suspected. If there were CD4-committed cells, destined to die because they bear mismatched
T cell receptors and coreceptors, it should be possible to
rescue them by restoring surface CD8 expression. Furthermore, the efficiency of the rescue should correlate with the
ability of the TCR to generate the transitional CD4+
CD8int
galhi phenotype. Thus, one would predict that such
a rescue experiment should be more successful in the OVA
than the HY animals, which had previously yielded few
rescued cells (49). We tested this prediction by crossing
the HY and OVA lines with a transgenic mouse line expressing near-physiological levels of the CD8
and
chains (CD8
[42]). Representative CD4/CD8 profiles
of thymocytes and lymph node cells from these crosses are
shown in Fig. 8. As observed previously (49), rescue of
CD4+(tgCD8+) T cells was very limited in the HY mouse;
in striking contrast, close to half of the peripheral clonotypehi
lymphocytes in the OVA cross were of the CD4 lineage.
Thus, there was a direct relationship between the number of
CD4+CD8int
galhi thymocytes and the number of rescued
class I-restricted CD4+(tgCD8+) cells. We also observed a
significant decrease in the positive selection of OVAhi
thymocytes into the CD8 SP compartment. As the affinity of
the OVA receptor may be quite high for its selecting ligand
(18), it is possible that the CD8-committed cells were
deleted due to the higher levels of CD8 expression.
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Finally, we made
use of the gal reporter mice to examine the issue of CD4
lineage commitment in the absence of CD4. Previous studies had demonstrated a population of MHC class II-
restricted, CD4
CD8
TCRhi Th cells in CD4-deficient
mice (26, 52). Although it was never formally demonstrated, these cells were quite logically considered to belong
to the CD4 lineage.
We readdressed this issue by analyzing offspring from
crosses of CD4-gal knock-in mice with one of the original CD4 knockout lines (26), comparing profiles of
gal+
cells in heterozygous CD4+/L animals with those in fully
deficient CD40/L animals. In a four-color cytofluorimetric
analysis using FDG and antibodies against CD4, CD8, and
CD3, the usual CD4/CD8 profile can be substituted with a
gal/CD8 profile (Fig. 9). In CD4+ mice, the
gal+CD8
cells in the thymus and lymph nodes were essentially all
CD4+CD3hi, as expected; on average, they amounted
to 4.0 and 15.1% of total thymocytes and lymphocytes,
respectively. When the same gate was applied to
gal+
CD8
cells from CD4-negative CD40/L mice, the same
populations of CD3+ T cells were present (now CD4
).
The thymocyte population was only moderately diminished in number, whereas the reduction was more marked
in the lymph nodes. The
gal+CD8
cells were analyzed
for various surface markers and for their TCR V
region
usage. Preliminary results indicated that these cells were not
particularly different in CD4+ and CD4
mice, implying
that the cells selected in the absence of CD4 were not a
special subset of Th cells (not shown).
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These results formally demonstrate that thymocytes can be selected into the CD4 lineage in the absence of cell surface CD4.
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Discussion |
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The CD4-gal knock-in mouse line provides an accurate, convenient, and harmless means of monitoring CD4
gene activity directly ex vivo. Expression of the
gal reporter faithfully mimicked CD4 expression, was readily
detectable, and had no detectable immunological consequences. The reporter could be used whether or not the
CD4 molecule itself was present. The choice of reporter
also proved advantageous because the half-life of the
geo
chimeric protein was significantly shorter than that of CD4,
meaning that
gal activity more closely approximated CD4
gene transcription than surface display of CD4 itself. In this
report, we have exploited the CD4-
gal line to study
CD4/CD8 lineage commitment.
For some years, debate on the mechanism of
CD4/CD8 lineage commitment has centered around the
issue of whether commitment is essentially instructive or
selective (49, 55, 56). A crucial distinguishing feature of
these two models is the possibility of transitional populations, with mismatched receptors and coreceptors in the
latter but not the former. Evidence for such populations was provided by studies on MHC-deficient and TCR tg
mice and by rescue experiments based on forced expression
of coreceptors (11, 12, 14, 15). However, a key finding in
several of these studies was brought into question because
of results from transfer experiments (18, 19) and coreceptor
reexpression assays (21). In particular, these data were considered to invalidate the support given the selective model
of lineage commitment by the demonstration of transitional intermediates in class II- and class I-deficient mice.
We now exploit the gal marker to establish that the
CD4+CD8int population from class II-deficient mice includes cells committed to both the CD4 and CD8 lineages.
CD4-committed cells maintain active CD4 gene transcription, whereas CD8-committed cells have turned off the
CD4 gene, as reflected by expression of the
gal reporter
(Figs. 3 B and 5).
It might be argued that the CD4+CD8intgalhi thymocytes seen in class II-deficient mice are just precursors
of the
gallo cells, having not yet shut down transcription
of the CD4 gene. Several observations are inconsistent
with this contention: the low level of CD8 mRNA in
CD4+CD8int
galhi cells compared with their
gallo counterparts, as predicted if they are of the CD4 lineage; the very position of the
galhi cells on the
gal/CD3 plot, visibly on a different branch than that of the
gallo cells (Fig.
4); the correlation between the numbers of CD4+CD8int
galhi cells and the ability of the CD8 transgenes to rescue
the CD4-committed cells in OVA and HY transgenic mice
(Figs. 7 and 8). Finally, we have recently found that altering the avidity of TCR signaling by introducing a CD5
knockout mutation (57) increases the numbers of CD4+
CD8int
galhi cells in II0 mice, without affecting CD4+CD8int
gallo numbers, a change in ratio incompatible with a simple precursor/product relationship (Chan, S., manuscript in
preparation).
The data presented here do not really contradict those from the transfer and coreceptor reexpression studies (18, 19, 21, 22). The transfer experiments (18, 19, 58) demonstrated that the CD4+CD8int population from II0 mice contained CD8-committed thymocytes, but did not rule out that it also includes CD4-committed cells because, having mismatched receptors and coreceptors, these cells should not have survived in the host wild-type thymus any better than in the II0 donor. As for the coreceptor reexpression assays, the data actually did show that cells committed to both lineages were present within the CD4+CD8int population of II0 mice (Fig. 4 A in reference 21). The existence of CD4-committed cells was discounted by these authors in favor of a CD4 "default selection" pathway, hypothesized to occur independently of MHC engagement. However, no evidence for such a pathway has been observed in several studies (22, 41; Fig. 6 B), so that it appears reasonable to equate the CD4-committed CD4+CD8int cells described in this report with those that reexpressed only CD4 after pronase treatment.
The existence of CD4-committed, but class I-reactive, intermediates is consistent with results from the older experiments forcing expression of coreceptors via transgenesis (11, 12, 14). Our data (Figs. 7 and 8) extend the older data by fulfilling, in two TCR transgenic systems, the prediction that the numbers of intermediates with mismatched receptors and coreceptors should correlate with their ability to be rescued to full maturity by artificially expressing the appropriate coreceptor. Our results also explain the range in observations made in previous rescue experiments, relatively efficient rescue being obtained in some cases (11, 14, 15, 59) but not in others (49, 50). Inefficient rescue has been cited repeatedly as evidence for instructive or hemi-instructive models (11, 12, 21). It is clear now that rescue experiments are not inherently inefficient but one must choose an appropriate TCR tg system that provides enough intermediates to be rescued.
The Asymmetry of Commitment.The present data reaffirm the existence of CD4 lineage-committed transitional
intermediates that were nudged down the differentiation pathway by engagement of their TCRs by MHC class I
molecules in the absence of class II molecules. An analogous population of CD8 lineage-committed cells was
hypothesized when CD4intCD8+ cells were found in class
I-deficient mice (10, 16), but their existence has been
questioned (20, 42). Our findings confirm that transitional intermediates committed to the CD8 lineage in the
absence of MHC class I molecules do exist, as evidenced by
the absence of gal expression in CD4+CD8intCD3hi cells
in I0 mice (Fig. 6 A). This result is consistent with reports
that transgenic mice expressing class II-restricted TCRs
(17, 60) and MHC-deficient mice complemented with
MHC class II genes delivered by an adenovirus vector
injected intrathymically (Rooke, R., manuscript in preparation) can give rise to mature bona fide CD8+ cells. However, the numbers of CD8-committed transitional intermediates in class I-deficient mice are low: 2.3 ± 0.9% of the
numbers in normal mice (n = 5), compared with 20 ± 5%
the normal numbers for CD4-committed intermediates in
class II-negative animals. The rarity of the CD4intCD8+
cells may explain why they were not detected by other
groups
either because three-color bromodeoxyuridine labeling experiments are inherently maladapted for the detection of small populations (42), or because the analysis/
sorting gates that were used included DP cells, thereby masking the transitional intermediates (20, 21). Thus, CD4/
CD8 lineage commitment seems to exhibit qualitative symmetry in that receptor/coreceptor-mismatched commitment
occurs in both pathways, but there is quantitative asymmetry in that such "mistaken" commitment to the CD4 lineage
is far more frequent than that to the CD8 lineage
actually
somewhat in line with the earlier assertions of asymmetry in
the positive selection process (21, 61).
Asymmetry is also evident in the fact that no CD4-committed equivalent of CD8-committed CD4+CD8int thymocytes (waltzers) has been described. Here, we detected
no galhi cells in the CD4intCD8+ population, in good agreement with findings from several groups (18, 21, 22). Thus,
while commitment to the CD8 lineage can launch at least
two different modes of differentiation, either direct progression to the CD4
CD8+ phenotype as seen for cells in the
HY system, or transit through the CD4+CD8int stage as seen
for OVA T cells, commitment to the CD4 lineage seems to
provoke a quite direct progression to the mature SP stage.
What explains the diverse modes of differentiation exhibited by the CD8 lineage? A hint may come
from the results of recent analyses of cis-control elements of
the CD8 gene (62, 63) and past studies on CD4 gene
control elements (35, 36, 64). Distinct enhancer elements
promote expression of CD8, and perhaps CD4, in DP versus mature SP cells; regulation is further complicated by a
silencer located in the CD4 gene. It may be that, concomitant with positive selection, transcriptional activity driven
by the DP cell-specific enhancer is shut off, and then
whether the cells destined to become CD8 SP proceed directly or transit through the CD4+CD8int stage may simply
reflect the speed with which the transcription factors binding to the "mature" CD8 SP-specific enhancer and/or the
CD4 silencer become operational.
Differential mobilization or demobilization of transcription factors could result from different TCR affinities/avidities. It was suggested some time ago that the affinity/avidity of a DP thymocyte's TCR for the positively selecting ligand or stromal cells might play a role in determining its pathway of differentiation (14, 49), and recent data support this notion (60, 65). It seems likely that the divergent behaviors of OVA and HY TCR tg thymocytes reflect different strengths of interaction between the two TCRs and their positively selecting ligands (18). Pircher and colleagues have also attributed the differing numbers of CD4+CD8int cells found in the P14 TCR tg line on the H-2b and H-2bm13 backgrounds to different avidities for the selecting ligands (13). At present, it is not clear whether stronger signals promote differentiation along the CD4 or CD8 pathway, as arguments have been presented on both sides (14, 49, 60, 66). Thymocyte-extrinsic factors within the thymus milieu as well as intrinsic factors of a more generalized nature also play a role in determining cell fate. Concerning the former, it should be kept in mind that the thymic stroma is very heterogeneous, containing niches where the expression of MHC molecules can vary markedly (4, 67); concerning the latter, recent results on Notch are intriguing (68).
CD4 Lineage Cells in the Absence of CD4.Finally, we confirmed that the population of MHC class II-restricted
CD4CD8
T lymphocytes previously observed in CD4-deficient mice (26, 52) do indeed belong to the CD4
lineage. In CD4
mice (CD40/L), a subset of DN
/
T
cells in both the thymus and the peripheral lymphoid organs expresses high levels of
gal (Fig. 9). Thus, commitment to the CD4 lineage does not require the CD4 molecule; in fact, it is quite efficient in its absence. Interestingly, although the numbers of DN CD3+
gal+ thymocytes generated after selection were quite high (~30% the numbers
in normal mice), their relative abundance was lower in the
periphery (~12%). A possible explanation for this diminution is that signals from the CD4 molecule contribute (albeit in a dispensable fashion) both to the thymic selection
of CD4+ T cells and to their later export and persistence in
the peripheral lymphoid organs. This would be consistent
with the idea that maintenance of naive CD4+ T cells in
the periphery requires continued "tickling" by MHC molecules (69, 70).
The results presented here argue that CD4/CD8 lineage commitment is fundamentally symmetrical, in that engagement of either class of MHC molecule by a differentiating DP thymocyte can give rise to transitional intermediates committed to either lineage. There does not appear to be a CD4 lineage default pathway, nor any special requirements to provoke commitment to the CD8 lineage. Along both routes of differentiation, the initial lineage choice is validated at a later stage, when only those cells expressing appropriately matched T cell receptors and coreceptors are permitted to survive.
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Footnotes |
---|
Address correspondence to Christophe Benoist or Diane Mathis, Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 163, 67404 Illkirch Cedex, France. Phone: 33-3-88-65-32-00; Fax: 33-3-88-65-32-46; E-mail: cbdm{at}igbmc.u-strasbg.fr
Received for publication 24 August 1998 and in revised form 8 October 1998.
We thank S. Gilfillan for the D4 EcoRI genomic library; P. Soriano for the geo cassette; D. Littman for the
CD4-deficient mice; H. von Boehmer and P. Kisielow for the HY TCR tg mice; R. Dubridge and D. Capon for the
2m
mice; A. Baron for the CD8 transgenics; P. Kastner for critically reading the manuscript;
C. Waltzinger for performing the cytofluorimetric analyses and cell sorting; P. Bohn-Marchal, C. Ebel, P. Gerber, J. Hergueux, and Corinne Bronn for excellent assistance; and P. Michel, F. Fischer, and V. Louerat
for maintaining the mice.
This work was supported by institute funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, and Bristol-Myers Squibb, and by grants to D. Mathis and C. Benoist from the Human Frontier Science Program and the Ministère de la Recherche. S. Chan was supported by the Association Nationale pour la Recherche sur le SIDA, the American Cancer Society, and the Ligue National contre le Cancer. M. Correia-Neves received fellowships from the Programa Gulbenkian de Doutoramento em Biologia e Medicina and the Junta Nacional de Investigaçao Cientifica e Tecnologica.
Abbreviations used in this paper
2m,
2-microglobulin;
B6, C57Bl/6;
gal,
-galactosidase;
DN, double-negative;
DP, double-positive;
ES, embryonic stem;
FDG, fluorescein digalactopyranoside;
I0, MHC class
I-negative;
II0, MHC class II-negative;
I0II0, MHC double-deficient;
PGK, phosphoglycerate kinase;
RT, reverse transcriptase;
SP, single-positive;
tg, transgenic.
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