By
From the * Centro de Biología Molecular "Severo Ochoa," and the Departamento de Inmunología y
Oncología, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Cantoblanco,
28049 Madrid, Spain
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Abstract |
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During thymocyte development, progression from T cell receptor (TCR) to TCR
rearrangement is mediated by a CD3-associated pre-TCR composed of the TCR
chain paired
with pre-TCR
(pT
). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here,
we show that developmental expression of pT
is time- and stage-specific, and is confined in
vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3.
This restricted expression pattern allowed the identification of a novel subset of small CD3
thymocytes lacking surface pT
, but expressing cytoplasmic TCR
, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation
of T early
transcription are coincident events associated with cell cycle arrest, and immediately preceding TCR
gene expression. Importantly, thymocytes at this late pre-T cell stage
are shown to be functional intermediates between large pT
+ pre-T cells and TCR
/
+ thymocytes. The results support a developmental model in which pre-TCR-expressing pre-T
cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small
resting pre-T cells, before the onset of TCR
gene expression.
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Introduction |
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Early in T cell development, thymocytes that have succeeded in productive V-D-J rearrangements at the
TCR locus are selected for cellular expansion and further
maturation before the TCR
gene is expressed (1). This
process, termed "
-selection," is regulated by the pre-TCR, which comprises the CD3 complex in association
with the TCR
chain and the invariant pre-TCR
(pT
)1
chain (4). A key question has been whether surface expression is essential for the pre-TCR complex to exert its
regulatory function. Recent studies support that this may
be the case, since pre-TCR-induced thymocyte maturation involves both the extracellular constant region and the
transmembrane region of TCR
(7), and requires exit of
the pre-TCR from the endoplasmic reticulum/cis-Golgi
compartment (8). The question remains as to whether pre-TCR signaling is triggered by binding to an extracellular
ligand or, alternatively, as proposed recently (9), whether
pre-TCR complexes become constitutively active as soon as
they reach the plasma membrane, where signaling molecules
are available. In this latter situation, pre-TCR activity might
be regulated by control of membrane expression. However,
extremely low levels of the pre-TCR complex (~100-fold lower than those of the TCR
/
on mature T cells) appear
to reach the plasma membrane of immature thymocytes (10),
a fact that has hindered the development of monospecific
anti-pre-TCR reagents and, hence, the study of pre-TCR
expression patterns on normal thymocytes.
Current data support the notion that one of the first consequences of pre-TCR expression is the induction of a cell
cycle progression that results in the greatest expansion in
cell numbers that occurs in the developing thymus (1, 11).
In mice, this process is associated with differentiation of
CD44loCD25+ into CD44loCD25 double negative (DN)
thymocytes, suggesting that the pre-TCR is first expressed
on the cell surface at this developmental transition (11).
Accordingly, CD44loCD25
thymocytes from normal
mice are large-sized cells expressing trace but distinguishable levels of TCR
and CD3 (12). Similarly, the fraction
of large thymocytes present in TCR
-deficient mice as
well as in TCR
transgenic recombination activating gene
(RAG)-1 mutant mice expresses low but stoichiometric
levels of TCR
and CD3 (13, 14). A highly analogous
checkpoint may occur in T cell development in humans
during the transition from CD4+CD8
TCR
/
precursors to CD4+CD8+ TCR
/
double positive (DP) thymocytes, as the latter cells are mostly large cycling cells, in
which TCR
is part of a complex that is distinct from the
mature TCR
/
, and could be the pre-TCR (15).
The pre-TCR-induced cell cycle transition is, in turn,
associated with the downregulation of RAG-1 and RAG-2
gene transcription (16) and RAG-2 protein expression
(11), which is likely to be an important component of the
process of allelic exclusion at the TCR locus (3, 11).
However, RAG genes have to be reexpressed at a later
stage to allow rearrangements at the TCR
locus (16).
Likewise, TCR
germline transcription and, hence, expression of sterile T early
(TEA) transcripts, is later induced as an obligatory early event in the opening of the
TCR
locus for subsequent VJ
rearrangement (17). This
terminal program is rapidly triggered in mice during, or
immediately after, the transition from CD44loCD25
cycling thymocytes to CD4+CD8+ DP resting thymocytes
(16). Accordingly, surface expression of the mature
CD3-TCR
/
complex is first detectable on small nonproliferating DP thymocytes (19, 20). However, progress towards a more precise definition of the stages involved in
the transition from TCR
to TCR
locus rearrangement
has been hampered thus far, both in mice and in humans,
because previous attempts to demonstrate surface expression of the pre-TCR complex throughout normal thymocyte development have been unsuccessful.
In this study, analysis performed with a polyclonal rabbit
Ab that recognizes an exposed epitope of the native human
pT protein revealed a restricted pattern of surface pT
expression during normal human pre-T cell development.
On the basis of surface CD3/pT
expression and cell size,
we have identified a novel subset of small pre-T cells which
lack surface CD3/pT
expression and are mostly in a noncycling state. Transition to this developmental stage is
shown to be associated with the induction of specific developmental events that precede expression of the TCR
gene. Interestingly, such small pT
noncycling pre-T
cells are shown to be functional intermediates between
large pT
-bearing pre-T cells and the first thymocytes expressing the mature
/
TCR.
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Materials and Methods |
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Isolation of Thymocyte Subsets.
Postnatal thymocytes isolated from thymus samples removed during corrective cardiac surgery of patients aged 1 mo to 3 yr were fractionated by centrifugation on stepwise Percoll density gradients (LKB, Uppsala, Sweden), as described elsewhere (21). Thymocytes from the 1.068 and 1.08 density layers were designated as large and small thymocytes, respectively. Large thymocytes were depleted (>99% purity) of mature T cells (Flow Cytometry Analysis.
Directly labeled mAbs against CD3 (Leu4-PE) and CD8 (Leu2a-FITC) were obtained from Becton Dickinson; anti-CD4 (CD4-PE-Cy5) mAbs were purchased from Caltag Laboratories, Inc. (San Francisco, CA). A PE-labeled mAb against the human TCR VGeneration of Polyclonal Anti-human pT Abs.
Cell Transfections and Immunofluorescence Assays.
C-myc tagging was performed by PCR amplification of a complete pTNorthern Blot Analysis.
Preparations of total RNA (10 µg) isolated as described previously (15) were run on 1% agarose/ formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes corresponding to the TCR CReverse Transcription PCR Analysis.
Total RNA (1 µg) was reverse-transcribed into cDNA according to the manufacturer's protocol (Boehringer Mannheim, Mannheim, Germany). Equivalent amounts of cDNA among different samples was estimated by reverse transcription (RT)-PCR carried out for 18, 21, and 25 cycles withHybrid Human/Mouse Fetal Thymic Organ Cultures.
The in vitro generation of mature TCR ![]() |
Results |
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We have previously identified a subset of large cycling CD4+CD8+ human thymocytes in which the TCR chain is expressed as
part of a complex distinct from the mature
/
TCR,
likely the pre-TCR (15). According to their size, such
TCR
/
DP thymocytes could be selectively isolated
from the fraction of large cells recovered from Percoll density gradients (approximately one third of total unfractionated thymocytes), whereas conventional TCR
/
+ DP
thymocytes were more common with the small-sized cell
fraction (around two thirds of total thymocytes [15]). Since
human thymocytes typically coexpress CD3 and the
/
TCR in stoichiometric amounts, CD3 expression studies
similarly defined a differential distribution of cell subsets
among Percoll-fractionated thymocytes (Table 1). However, analysis of the correlated expression of CD3 versus
TCR
/
revealed that, although most large TCR
/
thymocytes were CD3
, a distinct proportion of them expressed low but detectable levels of CD3 (Fig. 1). These
large CD3low TCR
/
thymocytes did not represent
/
T cells, as expression of the
/
TCR was exclusively detected on large TCR
/
thymocytes with a CD3bright
phenotype (Fig. 1, and data not shown). Unexpectedly,
CD3
and CD3low cells were also recovered from the small
cell fraction (Fig. 1). Forward scatter (FSC) analyses ruled
out the possibility that such cells represented large-sized
contaminants (Table 1). Moreover, in contrast to large
CD3low thymocytes, essentially all CD3low small cells (15-
20% of total small thymocytes) coexpressed the
/
TCR.
However, small CD3
thymocytes were phenotypically
similar to CD3
large cells in that they expressed neither
the
/
nor the
/
TCR (Fig. 1, and data not shown). As
both large and small CD3low thymocytes displayed a homogeneous CD4+CD8+ DP phenotype (see below), they
were phenotypically indistinguishable except for the expression of
/
TCR on small DP thymocytes, but not on
large DP thymocytes.
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The above results prompted us to investigate whether large thymocytes with the CD3low TCR/
phenotype do represent pre-T cells expressing the pre-TCR. However, this issue was difficult to approach because no appropriate reagents such as anti-pT
Abs or Abs
able to recognize the human TCR
chain on the cell surface were available. Consequently, Abs were raised in rabbits against a synthetic peptide contained in the extracellular Ig-like domain of the human pT
molecule (15). The
specificity of the affinity-purified antisera was then assayed
by immunofluorescence microscopy of COS cells transfected with a pT
cDNA, tagged with a c-myc epitope
that is recognized by the specific 9E10 mAb. Results in Fig.
2 A show that one of these anti-pT
antisera (ED-1) was reactive against all c-myc+ transfectants (top panels), and that
both anti-c-myc and anti-pT
reagents displayed an identical intracellular recognition pattern (bottom panels), thus confirming the anti-pT
specificity of the ED-1 antiserum.
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To determine whether the anti-pT antiserum was also
able to recognize the pT
chain when expressed on the
cell surface, we next derived pT
stable transfectants from
the human T cell line SUP-T1, which expresses TCR
(V
1.1) in the absence of a functional TCR
chain and,
hence, lacks surface TCR
/
heterodimers (34). As a
pT
-GFP chimeric protein was used in these studies, reactivity of the anti-pT
antiserum could be analyzed by flow cytometry on stable transfectants traced by their GFP expression. As shown in Fig. 2 B, such GFP+ transfectants expressed low but detectable levels of CD3, but were unreactive with the BMA031 mAb which recognizes a common epitope of the TCR
/
dimer (22). In contrast, both
TCR
/
and CD3 were detected on SUP-T1 clones stably transfected with a TCR
chain (V
12.1), whose expression could be followed with the anti-V
12.1 mAb 6D6 (25).
Interestingly, a reciprocal expression pattern was observed
when surface staining was performed with the anti-pT
Ab
plus anti-CD3. Thus, pT
(GFP+) transfectants, but not
TCR
(V
12.1+) transfectants, were reactive with the anti-pT
antiserum and coexpressed CD3 in stoichiometric
amounts (Fig. 2 B). It is worth noting that expression of the
endogenous TCR
could be specifically detected with an
anti-V
1 mAb (24) on both cell types. Strikingly, levels of
TCR
expressed on pT
+ transfectants were consistently
lower than those on TCR
-expressing clones, although in
both cases, either pT
or TCR
was coexpressed with
TCR
in stoichiometric amounts (Fig. 2 B). As a whole,
these data suggest that the ED-1 antiserum was able to specifically detect pT
-containing surface complexes which
likely comprise CD3-associated TCR
-pT
heterodimers,
the hallmark of the pre-TCR complex.
Having established that
the anti-pT antiserum recognized specifically pT
-containing surface complexes, we wished to examine whether
pT
was actually expressed on the surface of TCR
/
primary thymocytes. To this end, Percoll-fractionated large
and small DP thymocytes depleted of CD3int and CD3bright
cells (including both TCR
/
+ and TCR
/
+ cells) were
analyzed by flow cytometry for their reactivity with the
anti-pT
Ab. As expected, both isolated DP cell subsets
were exclusively composed of CD3
and CD3low cells (Fig.
3 A). CD3low thymocytes made up ~50 and 30% of the
large- and small-sized DP thymocytes, respectively. Of
these, only small CD3low thymocytes coexpressed the
/
TCR (see above), albeit at low levels, suggesting that they
were representative of the developmental onset of TCR
/
expression. As shown in Fig. 3 A, such cells were unreactive with the anti-pT
Ab. Expression of pT
was negative as well on CD3
DP thymocytes, regardless of their cellular
size. In contrast, essentially all large CD3low cells displayed a
low but detectable reactivity with the anti-pT
Ab, thus
providing direct evidence that pT
-containing complexes are expressed in vivo on the surface of normal pre-T cells.
That this low level staining is specific was demonstrated by
showing that it could be completely inhibited by the specific pT
peptide (Fig. 3 B). It is worth noting that pT
and CD3 were coexpressed on large CD3low thymocytes in
a stoichiometric-like fashion similar to that observed on
SUP-T1 pT
transfectants (Fig. 2 B), suggesting that the
pT
-containing complex expressed on the former cells did
correspond to the CD3-associated pre-TCR.
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The above results allowed a novel subdivision of
the TCR/
DP compartment into three individual subsets of thymocytes defined as large DP CD3
, large DP
CD3low, and small DP CD3
. Because pT
expression was
restricted to large DP CD3low thymocytes, we wanted to
investigate further the developmental status of the distinct
pT
+ and pT
populations in order to improve definition
of their precursor-product relationships. To this end, the
three cell subsets were independently isolated and examined for their respective patterns of TCR
, TCR
, and
pT
gene expression. Northern blot analysis shown in Fig. 4 A revealed that both the 1.3-kb mature and the 1.0-kb
immature TCR
transcripts were expressed in the three
subsets of TCR
/
DP thymocytes, regardless of their
cellular size and CD3 phenotype. In contrast, TCR
transcription was undetectable in all of them, but occurred at
high levels in mature SP thymocytes included as control.
As expected, CD4+CD8
CD3
cells, which represent upstream precursors of the TCR
/
DP thymocyte pool
as a whole (15), lacked both TCR
and TCR
mature
transcripts, but expressed 1.0-kb TCR
mRNA. Therefore, we concluded that the three TCR
/
DP subsets
identified in this study include cells that have already completed TCR
, but not TCR
, gene rearrangement and
transcription, indicating that they represent discrete pre-T
cell stages along the pathway of T cell development. As expected of pre-T cells, all three cell types expressed pT
mRNA, with higher levels in the large CD3
subset. Maximal pT
expression was found in the more immature CD4+CD8
CD3
thymocytes (Fig. 4 A). A more sensitive RT-PCR analysis of these very same populations confirmed the patterns of TCR
and pT
expression obtained
by Northern blotting (not shown). However, it revealed that TCR
transcription had occurred, albeit at low levels,
in small CD3
thymocytes, whereas it was completely absent from both the CD3
and the CD3low subsets of large
pre-T cells (Fig. 4 B). Based on these results, we concluded
that small DP CD3
thymocytes represented the particular
stage at which TCR
gene rearrangement and transcription are initiated during human T cell development; therefore, this subset was placed at the latest pre-T cell stage,
downstream of both subsets of large pre-T cells.
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Additional support for the proposed model came from
studies aimed at investigating the TEA and RAG gene
transcription patterns displayed by the three pre-T cell subsets. TEA is a TCR germline transcript whose expression
seems to be an obligatory early event in the opening of the
TCR
locus for subsequent rearrangement (17). As V
to
J
recombination necessarily involves deletion of the TEA
region (17), we reasoned that the onset of TCR
gene expression should necessarily be accompanied by a reciprocal
shutdown of TEA transcription. Northern blot analysis
performed with a specific TEA probe generated in this
study revealed that TEA transcription had not been induced in early CD4+CD8
CD3
thymocytes, but occurred at high levels in large CD3
DP thymocytes, and
was maximal in pT
+ pre-T cells (Fig. 4 A). However, it
was sharply downregulated in small CD3
thymocytes,
thus supporting the notion that the onset of TCR
gene
expression concurs with a decrease in TEA transcription, these being coincident events in the transition from large to small pre-T cells. A reciprocal pattern of RAG gene expression was observed at this developmental point after hybridization with RAG-1- and RAG-2-specific probes
(Fig. 4 A). Thus, although RAG-1 and RAG-2 transcripts
were detected at similarly high levels in CD4+CD8
CD3
and large DP CD3
thymocytes, their expression was five-
to eightfold lower in pT
+ pre-T cells. Interestingly, maximal transcription levels of both genes corresponded to
small CD3
pre-T cells. These results suggest that RAG
gene expression is selectively turned down in pre-TCR-
expressing pre-T cells, and is later regained in small pre-T
cells, allowing rearrangements at the TCR
locus to occur.
Formation and expression of the pre-TCR
is claimed to immediately promote a cell cycle transition,
which results in expansion and selection (-selection) of the
pool of pre-T cells deemed useful by virtue of successful
TCR
chain expression (1, 11). Therefore, the prediction
would be that all cells downstream of the pre-TCR-expressing pre-T cell stage should show evidence of
-selection.
To address this issue, pre-T cell subsets were independently analyzed by flow cytometry for their DNA content as well
as expression of cytoplasmic TCR
protein. Results shown
in Fig. 5 A revealed that essentially all (>90%) large pT
+
as well as small CD3
pre-T cells expressed cytoplasmic
TCR
; therefore, both cell subsets comprise
-selected
pre-T cells. Unexpectedly, however, only 50-80% of large
CD3
DP thymocytes (50% in this particular experiment)
expressed cytoplasmic TCR
, whereas the remaining 30-
50% were TCR
. Such a differential expression of cytoplasmic TCR
defined two distinct cell subsets of large
CD3
pre-T cells which could thus be placed on either
side of the
-selection process.
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Formal support for this notion came from additional
flow cytometric studies that addressed directly the cell cycle
status of either the TCR+ or the TCR
subsets of large
CD3
pre-T cells. As shown in Fig. 5 B, double staining
with anti-TCR
and PI demonstrated that essentially all
(>90%) large CD3
pre-T cells lacking TCR
were arrested in the G0/G1 phase of the cell cycle, whereas, as expected of
-selected thymocytes, TCR
+ CD3
pre-T
cells featured a high proportion (up to 55%) of cells in S/
G2/M. This is consistent with 30% of bulk CD3
pre-T
cells being in S/G2/M (Fig. 5 A). Therefore, TCR
large
pre-T cells are strong candidates for cells immediately before
-selection, and most likely immediately downstream
of the CD4+CD8
CD3
precursor stage, which was essentially composed of TCR
thymocytes (>95%) displaying only a background level (<10%) of cells in S/G2/M
(Fig. 5 A). As expected of
-selected thymocytes, large
pT
+ pre-T cells were highly enriched in cycling cells
(~55% in S/G2/M). However, TCR
expression could
not be associated with an active cycling state in small CD3
pre-T cells. Rather, these cells typically displayed only
background levels of cells in S/G2/M (<15%), with a substantial fraction of them (>50%) in the G2/M phase (Fig. 5
A). As an additional indicator of their resting state, small
CD3
pre-T cells were shown to display exclusively the
fast hypophosphorylated form of retinoblastoma (not
shown). We thus concluded that most, if not all, small
CD3
DP thymocytes are noncycling pre-T cells that have
already passed through
-selection. This, in turn, suggests
that
-selected large pre-T cells may normally lose surface
pre-TCR expression and return to slow cycle conditions
before the onset of TCR
gene expression. As a whole,
these data provide strong evidence that small resting pre-T
cells represent the latest pre-T cell stage in human thymocyte development, immediately upstream of conventional DP TCR
/
+ resting thymocytes.
To seek direct evidence that small CD3 DP
thymocytes represent the normal progeny of large pre-TCR-expressing pre-T cells in the pathway of T cell differentiation, highly purified large CD3low pre-T cells
(>98% pure) were analyzed for their developmental fate in
a hybrid hu/mo FTOC system. The pattern of differentiation from several experiments was identical (Fig. 6 A): the
rapid appearance of CD3
DP cells (up to 60% by day 5 in
this experiment) with minimal differentiation into TCR
/
+
cells (>5%), followed by the generation of a major population of conventional DP thymocytes that coexpressed
CD3 and the
/
TCR at low to intermediate levels (85%
by day 17), and the later appearance of small numbers of
mature SP thymocytes (not shown). FSC analysis of the cells
harvested on day 5 in the experiment shown in Fig. 6 A
revealed that, by this stage, the cells that remained CD3low
had kept their original size, whereas the CD3
cells generated in the lobes were significantly smaller (mean FSC: 450 vs. 410, respectively). However, by day 17, essentially all
large cells had reverted to small cells, and thus, all TCR
/
+
progeny generated by this time (85%) were similar in size to the remaining (15%) CD3
DP cells (mean FSC: 330, 335, and 329, for CD3
, TCR
/
low, and TCR
/
int cells,
respectively). Interestingly, total yields of viable human cells
increased progressively during the initial phase of culture, resulting in a 15-20-fold increase of absolute cell numbers
by days 5-7, but cellular recoveries then stabilized or increased modestly (up to 2-3 times) through the next 10-12 d,
and declined steadily thereafter.
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The above data indicate that cell division in thymus
lobes reconstituted with large CD3low pre-T cells is extensive and skewed to the early stages of culture. Therefore,
the high yields of TCR/
+ DP progeny in FTOC are
mostly a reflection of cellular expansion of blast precursors,
presumably before transition to small CD3
pre-T cells.
This in turn suggests that differentiation into TCR
/
+
DP cells can occur in the absence of cell division from
small noncycling CD3
pre-T cells. To provide direct evidence of precursor activity, we tested the capacity of highly
purified (>98%) populations of small CD3
DP thymocytes to produce TCR
/
+ progeny in the FTOC system. As shown in Fig. 6 B, a high proportion of both
TCR
/
low (20%) and TCR
/
int (50%) progeny was already seen in the thymic lobes at day 1, the earliest sampling time. However, the number of TCR
/
+ progeny
did not increase in absolute terms thereafter, an expected finding considering that all cells recovered by day 1 were
small-sized cells (mean FSC: 300, 293, and 290, for CD3
,
TCR
/
low, and TCR
/
int cells, respectively). Thus, although kinetics of TCR
/
+ cell generation were more
pronounced with small CD3
than with large CD3low
pre-T cells, total cell yields were substantially lower with the small CD3
pre-T cell fraction. Based on the above
results, we concluded that small CD3
DP thymocytes
represent functional intermediates between large pre-T
cells and TCR
/
+ DP thymocytes.
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Discussion |
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Considerable progress has recently been made in defining the role that preantigen receptors, namely the pre-B
cell receptor and the pre-TCR, play in lymphocyte development. It is now established that both receptors direct in
an analogous way the survival, expansion, and clonality of
pre-B and pre-T lymphocytes by triggering cell cycle activation and the simultaneous downregulation of RAG genes (10, 11). However, less is known about the mechanisms
that control terminal differentiation of lymphocyte precursors thus selected, especially in the T cell lineage. This can
be partly attributed in both mice and humans to the lack of
experimental data concerning regulation of pre-TCR expression on the surface of primary thymocytes, a fact that
has hampered the definition of the developmental stages involved in the transition from TCR to TCR
rearrangement. In this study, analysis performed with a polyclonal
rabbit Ab that recognizes an exposed epitope of the native
human pT
protein has provided evidence for a restricted pattern of surface pT
expression during normal T cell development in humans. Surface pT
versus CD3 expression,
together with cell cycle analyses, enabled a novel subdivision of the whole compartment of TCR
-expressing pre-T
cells into three distinct subsets of increasing maturity, and
allowed the identification of a late stage of small noncycling
pre-T cells representing the immediate precursors of
TCR
/
-bearing thymocytes. The definition of the precursor-product relationships between such pre-T cell subsets, together with the characterization of the stage-specific
events associated with the developmental onset of TCR
gene expression, namely exit from cell cycle, reexpression
of RAG genes, and downregulation of TCR
germline
transcription, collectively support the developmental scheme
depicted in Fig. 7.
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The distinct pre-T cell stages defined in our model are
all included within a subset of CD4+CD8+ DP thymocytes
that lack the mature /
TCR and represent, as a whole,
the downstream progeny of CD4+CD8
CD3
thymocyte
precursors (15). About one third of such DP TCR
/
thymocytes are larger in size than the remaining two thirds
and, thus, the two cell types have been defined, respectively, as large and small pre-T cells. Although pT
transcription is common to all pre-T cell stages, surface expression of the pT
protein is shown to be restricted to a
limited fraction (50%) of large-sized pre-T cells that coexpress small but stoichiometric amounts of CD3. As neither
pT
nor CD3 is detectable on the rest of the large and
small pre-T cells, the coexpression of both molecules seems to define the particular subset of primary pre-T cells in
which the pT
chain is paired with TCR
and associates
with CD3 to form the pre-TCR. However, attempts to
demonstrate coexpression of surface TCR
in vivo were
unsuccessful, essentially because neither anti-TCR
mAbs
useful for flow cytometry nor anti-pT
reagents suitable
for biochemical studies are yet available. Despite this, the
possibility that TCR
-pT
heterodimers associated with
CD3 are indeed expressed on pT
+ pre-T cells is strongly
supported by several independent findings: (a) low but stoichiometric amounts of pT
and CD3 were specifically coexpressed with endogenous TCR
on pT
transfectants
derived from a TCR
-deficient cell line; (b) we have previously shown that heterodimeric complexes containing
TCR
without TCR
could be immunoprecipitated from
unfractionated large DP TCR
/
thymocytes (15); (c)
others have noticed that large thymocytes from TCR
-
deficient and TCR
transgenic RAG-1 mutant mice express low but stoichiometric amounts of surface TCR
and
CD3 (13), similar to what has been reported for mouse
thymocytes from which a CD3-associated pT
-TCR
heterodimeric complex has recently been characterized
(14); and (d) to date, no surface pT
expression has been
described without association with TCR
and CD3.
Therefore, expression of surface pre-TCR complexes is
proposed in our model to be confined to the minor subset
of large pT
+ CD3low pre-T cells, whereas large pre-T
cells lacking detectable amounts of surface pT
and CD3
(~50% of all large DP TCR
/
thymocytes) are proposed to be homogeneously negative for pre-TCR expression (Fig. 7). However, the latter cells represent a heterogeneous population in which a major fraction (50-80%)
have already passed
-selection, as indicated by their high
expression levels of intracellular TCR
(11), whereas the
remaining cells (20-50%) still lack cytoplasmic TCR
and
may thus represent intermediates between CD4+CD8
CD3
thymocytes and the first
-selected pre-T cells. It is highly likely that such intermediates include the pool of precursor
thymocytes undergoing rearrangements at the TCR
locus, although they may also include cells carrying nonproductive V
-D
-J
joints on both TCR
loci, which may
thus be destined to die. Both possibilities, illustrated in Fig.
7, are compatible with the hypothesis that the human pre-TCR does not participate, as does its murine counterpart, in the transition to the DP stage (2, 3, 18). Rather, expression of CD8 appears to precede pre-TCR expression during human T cell development.
An important aspect of our study was the observation
that virtually all large pre-T cells with cytoplasmic TCR,
whether or not they display surface pT
chain expression,
were actively engaged in cell cycle, a characteristic previously associated with the process of
-selection (11). Conversely, DP thymocytes lacking TCR
protein were nondividing cells arrested at G0/G1. The finding that up to 40%
of cycling,
-selected pre-T cells did not express the putative pre-TCR is apparently difficult to reconcile with the
current idea that cell cycle activation involves signaling mediated through the pre-TCR (2, 3, 11). However, the possibility that undetectable, but functional, amounts of the pre-TCR are expressed on the surface of such cycling
pT
pre-T cells cannot be formally excluded. Alternatively, it is likely that, as proposed for pre-B cells at the
equivalent developmental point (35),
-selected pre-T cells
rapidly downregulate expression of the pre-TCR from the
cell surface while they are still in cycle. In this latter situation, it could be expected that the maintained expression of
surface pre-TCR in a short developmental window is both
necessary and sufficient to provide a sustained proliferation signal that would allow pre-T cells to undergo a great cellular expansion before turning back to slow cycle conditions. Supporting this hypothesis, results from a recent
study have provided evidence that such a proliferation
phase corresponds in mice to nine rapid cell divisions that
last for ~4 d and end at the small resting DP thymocyte
stage (36). This concurs with our finding that a major fraction (about two thirds) of
-selected pre-TCR
pre-T
cells in humans are small-sized, nondividing cells. Interestingly, although such small pre-T cells do not yet express
the mature
/
TCR, they already transcribe low levels of
the TCR
gene. Thus, they are proposed to define the developmental point at which onset of TCR
gene rearrangement and transcription occurs, and are placed in our
model at the latest pre-T cell stage, immediately upstream
of the first TCR
/
-expressing DP thymocytes (Fig. 7).
Consistent with the above proposal, we found that indicators of V-J
recombinase activity, such as RAG gene
reexpression and downregulation of TEA transcription, are
coincident and stage-specific events induced after entry of
late pre-T cells into the pool of small, resting cells. Thus, it
was observed that expression of RAG genes, which is
turned down after pre-TCR signaling (11, 18), is regained
in small pre-T cells, allowing rearrangements at the TCR
locus to be initiated at this stage. Further, the demonstration that germline transcription of TCR
spans all cycling pre-T cell stages but drops significantly in resting pre-T
cells also supports the concept that these cells are actively
rearranging their V
genes. Similarly, Ig L chain gene rearrangement is restricted to small, resting pre-B cells that represent the equivalent precursor stage along the B cell pathway (35). In contrast to the proposal that small resting
TCR
DP thymocytes are functional intermediates in the
T cell differentiation pathway, it is currently assumed that
these cells represent the large pool of end-stage products of
failed rearrangement attempts. However, recently published data have shown that small noncycling TCR
DP
thymocytes in the mouse are actually the physiological targets of the multiple rearrangements that occur at the
TCR
locus (20), and are subject to positive selection (21,
37). Direct evidence of the physiological relevance of small
resting CD3
pre-T cells in humans was further provided
by the demonstration that these cells are functional intermediates between large pT
+ pre-T cells and TCR
/
+
DP thymocytes. Accordingly, as shown previously in mice
(19, 20), surface expression of the mature CD3-TCR
/
complex can be first detectable on small nonproliferating
DP thymocytes.
As a whole, our results suggest that, after pre-TCR-mediated cellular expansion, -selected large pre-T cells may
normally downregulate surface pre-TCR expression and
return to slow cycle conditions before the onset of TCR
gene expression. The proposed pattern of pre-TCR expression differs from previous hypothetical models in mice
postulating that mature TCR
/
and pre-TCR complexes are coexpressed on the cell surface of late pre-T cells (10). However, it is still possible that some of these cells are
cotranscribing pT
and TCR
genes. It is tempting to
speculate that, in that situation, both molecules compete
with each other for dimerization with TCR
, the affinity
of TCR
being higher than that of pT
. Alternatively, as
proposed in mice, another still unknown component of the
pre-TCR (i.e., the hypothetical VpreT) might be already shut off at the earliest TCR
+ stages, hence preventing
surface expression of the whole pre-TCR complex (3). Finally, it must be stressed that the restricted pattern of surface pT
expression shown in this study closely resembles
that of the surrogate light chain of the pre-B cell receptor
(35, 38). This emphasizes the similarities of early developmental events associated with the transient expression of
both preantigen receptors during T and B cell development.
![]() |
Footnotes |
---|
Address correspondence to María L. Toribio, Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Facultad de Biología, Universidad Autónoma de Madrid, Cantoblanco 28049, Madrid, Spain. Phone: 34-1-3978076; Fax: 34-1-3978087; E-mail: mtoribio{at}trasto.cbm.uam.es
Received for publication 17 November 1997 and in revised form 26 June 1998.
C. Trigueros and A.R. Ramiro contributed equally to this work.We wish to thank Drs. B. Alarcón, M.A. Alonso, M. Brenner, R. Kurrle, J.A. López de Castro, T.W. Mak, and L.A. Turka for the generous gift of Abs and cDNAs, Dr. S.G. Copín for invaluable advice on the FTOC, and the Pediatric Cardiosurgery Units from the Centro Especial Ramón y Cajal and Ciudad Sanitaria La Paz (Madrid) for the thymus samples.
This work was supported in part by Glaxo Wellcome S.A., and by grants SAF95-0006 and SAF97-0161 from Comisión Interministerial de Ciencia y Tecnología (CICYT) and CAM083/013/97 from Comunidad de Madrid. We would also like to thank the Fundación Ramón Areces for an Institutional Grant to the Centro de Biología Molecular "Severo Ochoa." C. Trigueros and A.R. Ramiro are fellows of Fondo de Investigaciones Sanitarias and the Ministerio de Educación y Ciencia, respectively.
Abbreviations used in this paper
DN, CD4CD8
double negative;
DP, CD4+CD8+ double positive;
GFP, green fluorescent protein;
PI, propidium iodide;
pT
, pre-TCR
;
RAG, recombination activating gene;
RT, reverse transcription;
SP, single positive;
TEA, T early
;
FSC, forward
side scatter;
FTOC, fetal thymic organ culture.
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