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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3491-3498
 -Selection Is Associated With the Onset of CD8 Chain Expression
on CD4+CD8 + Pre-T Cells During Human
Intrathymic Development
By
Yolanda R. Carrasco,
César Trigueros,
Almudena R. Ramiro,
Virginia G. de Yébenes, and
María L. Toribio
From the Centro de Biología Molecular "Severo Ochoa,"
Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
 |
ABSTRACT |
T-cell precursors that undergo productive rearrangements at the
T-cell receptor (TCR)  locus are selected for proliferation and
further maturation, before TCR expression, by signaling through a
pre-TCR composed of the TCR chain paired with a pre-TCR (pT) chain. Such a critical developmental checkpoint, known as
-selection, results in progression from CD4
CD8 double negative (DN) to CD4+
CD8+ double positive (DP) TCR
thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4+ CD8
intermediate stage. Here we show that the CD4+
CD8 to CD4+ CD8+ transition
involves the sequential acquisition of the and chains of CD8 at
distinct maturation stages. Our results indicate that CD8, but not
CD8, is expressed in vivo in a minor subset of DP
TCR thymocytes, referred to as CD4+
CD8+ pre-T cells, mostly composed of resting cells
lacking cytoplasmic TCR chain (TCRic). In contrast,
expression of CD8 heterodimers was selectively found on DP
TCR thymocytes that express TCRic
and are enriched for cycling cells. Interestingly, CD4+
CD8+ pre-T cells are shown to be functional
intermediates between CD4+ CD8
TCRic and CD4+
CD8+ TCRic+
thymocytes. More importantly, evidence is provided that onset of CD8
and TCRic expression are coincident developmental events associated with acquisition of CD3 and pT chain on the cell surface. Therefore, we propose that the CD4+
CD8+ to CD4+
CD8+ transition marks the key control point of
pre-TCR-mediated -selection in human T-cell development.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
BONE MARROW-DERIVED lymphoid progenitors
seed the postnatal thymus in which they undergo the sequential
rearrangement of the and  T-cell receptor (TCR) genes to finally
generate mature T lymphocytes bearing an TCR.1-3
Early during the intrathymic developmental process, pre-T cells that
have succeeded in productive rearrangements at the TCR locus are
rescued from apoptotic cell death and selected for further maturation,
before TCR expression, by signaling through a pre-TCR composed of
the TCR chain paired with a pre-TCR (pT) chain and associated
with CD3.4-7 Expression of this pre-TCR complex promotes
at the same time a cell-cycle transition that results in the expansion
of those thymocytes expressing a functional TCR chain, a process
that has been termed -selection.7-9
In both mice and humans, such a critical developmental checkpoint is
associated with the acquisition of the CD4+
CD8+ double-positive (DP) phenotype, which is then followed
by a first round of TCR gene rearrangement and
expression.3,7-10 Differences exist, however, in the
developmental timing of appearance of CD4 and CD8 in both species.
Progression to the DP compartment occurs in mice through the
CD44 CD25+ stage of double-negative (DN;
CD4 CD8) thymocytes via
CD44 CD25 DN
thymocytes.8,9 The latter subset, although currently referred to as the last DN stage, includes cells that are already transcribing and expressing low (barely detectable) levels of both CD4
and CD8, so that they spontaneously differentiate in vitro to the DP
stage within 24 hours.11-13 Isolated
CD44 CD25+ DN cells, in contrast, are
unable to generate CD4+ CD8+ DP cells in
vitro13; however, they can acquire the CD8 chain in
response to certain combinations of cytokines,14 thus
expressing the CD8 homodimeric form. Interestingly, such
CD8+ cells remain negative for CD3 and CD4 in vitro,
but can differentiate into conventional CD4+
CD8+ DP thymocytes expressing the CD3-associated
TCR under the influence of the thymic
microenvironment.15 In contrast to mice, developing DN
thymocytes in humans acquire CD4 before CD8 and, therefore, the
DN-to-DP transition occurs in humans via CD3
CD4+ CD8 intermediates.16
These thymocytes, similarly to their mouse CD44
CD25+ DN counterparts, differentiate in vitro into cells
expressing exclusively the CD8 homodimer17 but
generate common CD4+ CD8+ DP thymocytes
with the mature CD3-TCR complex in vivo.16
Current data on the physiologic expression of CD8 either as an
homodimer or as an heterodimer on distinct CD8+ cell
types support the notion that CD8+ T cells, such as
intestinal intraepithelial lymphocytes (IEL), can be generated by an
extrathymic maturation pathway independent of CD8 expression,
whereas induction of CD8 and, hence, the generation of
CD8+ cells is thymus-dependent.18,19 This
is further supported by functional studies showing that the CD8
polypeptide is critically involved in the maturation of CD8-lineage
cells inside the thymus, so that both positive and negative selection
of major histocompatibility complex (MHC) class-I-restricted T cells
is impaired in CD8/ mice.19-22
All these data concur with the fact that the CD8+
phenotype is prominent among CD8+ T cells from athymic mice
and rats, whereas normal thymocytes and CD8+ T cells from
euthymic animals are virtually all
CD8+.15,19,23 Nonetheless,
CD8+ cells have been reported to exist
physiologically, although in a very low proportion, in the human
thymus,17 raising the possibility that they constitute an
important intermediate in the pathway of generation of thymus-derived T
cells in humans. Supporting this notion, results from a very recent
study by Blom et al24 have shown that such
CD8+ thymocytes, which coexpress surface CD4 but
still lack a mature TCR,7 have extensive TCR gene
rearrangements. However, functional studies on the developmental
potential and precursor-product relationships of isolated
CD4+ CD8+ thymocytes are still lacking,
precluding a better understanding of the physiological relevance of
this particular cell subset in the context of thymocyte development and
-selection in humans.
Here we show that CD4+ CD8+ human
thymocytes are actually functional intermediates between
CD4+ CD8 and CD4+
CD8+ thymocytes. More importantly, evidence is
provided that onset of CD8 expression and -selection are
coincident events associated with the acquisition of CD3 and pT
chain on the cell surface during intrathymic development in humans.
These results suggest that the pre-TCR-mediated check-point of
-selection is placed in humans at the CD4+
CD8+ to CD4+
CD8+ transition.
| |
MATERIALS AND METHODS |
Isolation of thymocyte subsets.
Postnatal thymus samples were removed during corrective cardiac surgery
of patients aged 1 month to 3 years. Thymocyte suspensions obtained by
Ficoll-Hypaque (Nycomed, Oslo, Norway) centrifugation were fractionated
on stepwise Percoll (Pharmacia, LKB, Uppsala, Sweden) density gradients
as previously described.25 Thymocytes recovered from the
1.068 and 1.08 density layers, referred to as large and small
thymocytes, respectively, were depleted (>99% purity) of mature T,
B, NK, and myeloid cells (Lin cells) by
immunomagnetic sorting as described elsewhere.10 Thymocytes
coexpressing CD4 and CD8 (DP) were then magnetically sorted from the
remaining large and small Lin cell pool with
anti-CD8-coated magnetic beads (Dynabeads, Dynal Corp, Oslo, Norway),
whereas CD3 CD4+ CD8
thymocytes were sorted from the CD8-depleted pool of large cells by
treatment with anti-CD4-coated magnetic beads (Dynal).10
Small DP thymocytes thus isolated were all negative for TCR and
CD3 expression, and will be hereafter referred to as small DP
CD3 thymocytes.10 In contrast, large DP
thymocytes included CD3 and CD3low
pT+ thymocytes. Both subsets were independently isolated
from the whole pool of large DP TCR thymocytes
as described previously.10 These cells will be referred to
as large DP CD3 and large DP CD3low
thymocytes, respectively. Large DP CD3 thymocytes
were fractionated into CD8+ and
CD8+ cells by cell sorting as described below.
Flow cytometry and cell sorting.
Directly labeled monoclonal antibodies (MoAb) against the following
antigens were used: CD3 (Leu4-PE), CD8 (Leu2a-fluorescein isothiocyanate [FITC]), and CD5 (Leu1-FITC) from Becton Dickinson & Co, San José, CA; CD4 (CD4-PE-Cy5), CD44 (CD44-FITC), CD69 (CD69-FITC), and CD3 (CD3-PE-Cy5) from Caltag Laboratories, South San
Francisco, CA; CD71 (T9-FITC) from Coulter Corp, Hialeah, FL; and CD28
(CD28-FITC) from Serotec Ltd, Oxford, UK. Unlabeled MoAb against CD8
(2ST8-5H7, kindly provided by Dr E.L. Reinherz, Dana-Farber Cancer
Institute, Boston, MA)26 as well as MoAb recognizing
monomorphic determinants of TCR (BMA031, generously provided by
Dr R. Kurrle, Behringwerke AG, Marburg, Germany),27 were
used in combination with FITC- or PE-coupled goat-antimouse F(ab)'2 immunoglobulin (Ig) (Caltag). Either
unlabeled or directly labeled isotype-matched irrelevant MoAb (Caltag)
were used as negative controls. For detection of cytoplasmic TCR,
cells were treated with 0.5% saponin (Sigma, St Louis, MO), incubated
with the anti-TCR chain F1 MoAb28 (generously
provided by Dr M. Brenner, Brigham and Women's Hospital, Boston, MA)
and labeled with PE- or PE-Cy5-coupled goat antimouse IgG1
(Caltag). To define background fluorescence, cells were sequentially
treated with a nonreactive mouse IgG1 MoAb plus PE- or
PE-Cy5-conjugated goat antimouse IgG1. Surface expression
of pT chain was determined by sequential staining with a rabbit
polyclonal antibody (ED-1) previously described,10 and
FITC-conjugated goat antirabbit F(ab)'2 Ig (Southern
Biotechnology Associates, Inc, Birmingham, AL). Preimmune rabbit serum
was used as negative control. Two- or three-color staining was
performed as described elsewhere.25 Stained cells were
analyzed in an EPICS XL flow cytometer (Coulter Electronics Inc,
Hialeah, FL). Cell cycle analyses were performed by flow cytometry
using a doublet discrimination function in cells treated with 0.05%
digitonin (Sigma), washed, and stained with 50 µg/mL of propidium
iodide (PI) (Sigma), as described elsewhere.10 Cell sorting
of CD8+ and CD8+ cells was
performed in an EPICS Elite Cell Sorter (Coulter Electronics, Inc) on
isolated large DP CD3 thymocytes after labeling with
anti-CD8 plus PE-labeled goat antimouse IgG2a. An irrelevant IgG2a
mouse MoAb was used as negative control. Sorted cells were greater than
98% pure as determined by post-sort analysis.
Western blot analysis.
Cells were lysed for 30 minutes at 4°C in lysis buffer containing
0.5% Deoxicholate, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 50 mmol/L NaF, 1 mmol/L Na3VO4 (Sigma Chemical
Co), 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0) (Merck, Darmstadt,
Germany), 1 mmol/L PMSF, and 1 µg/mL each of leupeptin, pepstatin A,
and aprotinin (Sigma). The protein concentration was determined by micro-bicinchoninic acid (BCA; Pierce, Rockford, IL) assay. Defined quantities (~7 µg/lane) were electrophoreses in 7%
SDS-polyacrylamide gel electrophoresis (PAGE) under reducing
conditions. Western blotting was performed as previously
described.25 Blots were incubated with either the
anticyclin B (Transduction Laboratories, Lexington, KY), the anti-RAG1,
the anti-RAG2 (Pharmingen, San Diego, CA), or the anti--tubulin
(Amersham International) mouse MoAb, or with a polyclonal rabbit
anticyclin A antibody, or a polyclonal goat anti-Retinoblastoma
(anti-Rb) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), as
primary reagents. Specific signals were shown with either
horseradish perioxidase (HRPO)-labeled polyclonal sheep
antimouse Igs, or HRPO-labeled polyclonal goat antirabbit Igs (Amersham
International) or HRPO-labeled polyclonal rabbit antigoat Igs (Jackson
Immunoresearch, West Grove, PA), respectively, and an enhanced
chemiluminescence (ECL)-detection kit (Amersham International).
Hybrid human/mouse fetal thymic organ cultures.
The in vitro generation of mature TCR+ human T cells
was analyzed using a modification of the previously described hybrid human/mouse fetal thymic organ culture (hu/mo FTOC).29
Briefly, thymuses removed from 15-day-old embryos of Swiss mice were
precultured for 5 to 6 days in the presence of 1.35 mmol/L dGuo
(Sigma). Afterwards, the thymic lobes were washed and cocultured in
hanging drops in Terasaki plates (Nunc, Inc, Roskilde, Denmark) with
either CD8+ or CD4+ (3 × 104 to 105 cells/lobe) human thymocytes. After
2 days, lobes were transferred to Millipore filters (Millipore Corp,
Bedford, MA), that were layered over gelfoam rafts and cultured in
Iscove's modified Dulbecco's medium (IMDM) supplemented with 2%
human AB serum and 5% fetal calf serum (FCS; Gibco BRL, Paisley,
Scotland). Surface staining of human cells was performed at the
indicated culture periods, and flow cytometric analyses (FCA) were then
performed on electronically gated CD45+ human cells.
| |
RESULTS |
Characterization of CD3 CD4+
CD8+ DP cells in the
human thymus.
We have recently reported that surface CD3 expression and cell size
define three subsets of human TCR thymocytes,
namely large CD3low, large CD3, and
small CD3, which represent distinct pre-T-cell
developmental stages.10 All three pre-T-cell types
expressed surface CD4 and were reactive with conventional anti-CD8
antibodies (directed against an epitope on the CD8 molecule), so
that they were characterized as DP thymocytes.10 However,
as shown in Fig 1A, analysis on the
correlated expression of CD8 and CD8, performed with the
anti-CD8 MoAb 2ST8-5H7,26 showed that large
CD3low and small CD3 pre-T cells were
homogeneously CD8++, whereas a
significant proportion (32 ± 4%) of large CD3
pre-T cells expressed CD8 but lacked CD8 chain. Such a
differential expression of the and chains of CD8 defines two
distinct subsets of large CD3 pre-T cells in which
CD8 may be expressed either as a CD8 homodimer or as
a CD8 heterodimer,26 so that they will
hereafter be referred to as either CD8+ or
CD8+, respectively. Both cell subsets could be
independently isolated by cell sorting, and were then compared for
their developmental status by analyzing the intracytoplasmic expression
of TCR chain (TCRic). As shown in Fig 1B, a clear
phenotypic pattern was obtained: TCRic expression was
not detectable in CD8+ thymocytes, whereas
essentially all CD8+ pre-T cells coexpressed
TCRic. These results confirm our previous data showing a
bimodal distribution of TCRic within the pool of large
CD3 pre-T cells as a whole10 but, in
addition, they suggest that expression of TCRic
parallels acquisition of the CD8+ phenotype.
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| Fig 1.
Differential expression of CD8 and CD8 chains on
distinct subsets of human pre-T cells: Intracytoplasmic TCR
expression and cell-cycle progression are associated with CD8 chain
expression. (A) Large CD3, large CD3low
pT+, and small CD3 pre-T cells,
isolated as described in Materials and Methods, were analyzed by
two-color flow cytometry for CD8 versus CD8
expression. (B) Large CD3 CD4+
CD8+ (DP) pre-T cells were fractionated by cell sorting
into CD8+(top panels) and
CD8++ (bottom panels) cells after
labeling with the 2ST8-5H7 anti-CD8 MoAb plus PE-coupled goat
antimouse IgG2a. Reanalysis of surface CD8 expression
postsorting is shown (shaded histograms). Sorted cells were then
analyzed by flow cytometry for intracytoplasmic TCR chain (TCRic)
expression (shaded histograms) and DNA content. Cytoplasmic background
fluorescence was determined on sorted cells stained with a nonreactive
mouse IgG1 MoAb plus PE-Cy5-coupled goat antimouse
IgG1 (unshaded histograms). Percentages of cycling cells
(in S and G2/M phases) are indicated. Results are
representative of four independent experiments.
|
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Because TCRic expression is currently envisaged as a
marker of -selection, we concluded that the CD8+
and the CD8+ subsets could represent two distinct
pre-T-cell developmental stages placed on either side of the
-selection process. This was confirmed by FCA showing that both
populations differed dramatically in their cell cycle status. As
expected of -selected thymocytes, CD8+ pre-T cells
featured a high proportion (up to 55%) of cycling cells, whereas
essentially all (>90%) CD8+ thymocytes were
"unselected" cells arrested in the G0/G1
phases of the cell cycle (Fig 1B).
The distinct developmental status of the CD8+ and
CD8+ cell subsets of large CD3
pre-T cells, prompted us to examine in more detail their phenotypic profiles, as compared with that of large cycling CD3low
pre-T cells, previously shown to represent the particular stage of
-selected thymocytes at which the CD3-associated pre-TCR is expressed in vivo.10 Regardless of CD3 expression, the two
subsets (CD3 and CD3low) of -selected
CD8+ TCRic+ thymocytes
were homogeneously positive for the expression of CD44, whereas
CD8+ pre-T cells were CD44 (Fig
2). In addition, all three pre-T-cell
types displayed surface CD4, but levels of expression were consistently
lower in the latter. Expression levels of activation markers such as
CD71 (transferrin receptor) and, to a lesser extent, CD28 were also
significantly lower in CD8+ pre-T cells, whereas CD5
and CD69 were similarly expressed in the three populations (Fig 2).
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| Fig 2.
Cell-surface phenotype of individual subsets of
CD8+and CD8+ human large pre-T
cells. Large CD3 DP pre-T cells shown in Fig 1 were
stained with directly labeled MoAb against the indicated cell-surface
molecules. FCA was performed on electronically gated
CD8+ (shaded areas) or
CD8++ (unshaded areas, bold line)
cells. Large CD3low pT+ pre-T cells, shown
to be homogeneously CD8++ (see Fig
1), were included in the study for comparison (unshaded areas, thin
line). Background values (vertical lines) were determined with
isotype-matched irrelevant MoAb. A representative analysis out of three
independent experiments is shown.
|
|
CD4+ CD8+ pre-T cells are the immediate
precursors of CD4+ CD8+ pre-T cells that
coexpress surface pT and CD3.
To provide direct evidence that CD4+ CD8+
thymocytes do in fact represent a minor, but physiologically relevant,
population of pre-T-cell intermediates, they were next examined for
their developmental potential in a hybrid hu/mo FTOC system. Highly purified CD4+ CD8+ thymocytes (>98%
pure after cell sorting) were consistently found to give rise to
conventional DP cells coexpressing CD4 and the CD8 heterodimer at
early periods of culture (20% by day 5 in this experiment; Fig
3A, upper panel). Of notice, essentially all CD4+ CD8+ progeny (>95%) generated
by this time in different experiments had acquired low surface CD3 with
minimal differentiation (<2%) into TCR+ cells (Fig
3A, lower panel). Interestingly, such CD3low
TCR CD4+ CD8+
pre-T cells (20% of total cells recovered by day 5) were all positive
for the expression of TCRic and, more importantly, they displayed low surface levels of the pT chain (Fig 3B), as assessed with a polyclonal anti-pT antibody.10
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| Fig 3.
Phenotypic analysis of the cellular progeny generated
after differentiation of CD4+ CD8+
human pre-T cells in FTOC. (A) CD4+
CD8+ pre-T cells, isolated by cell sorting as
described in Materials and Methods, were cultured in a hybrid hu/mo
FTOC and analyzed after 5 and 14 days for the expression of CD8,
CD8, TCR, and CD3. Analysis of TCR versus CD3 was
performed by three-color flow cytometry after electronic gating on the
CD8+ progeny. (B) Intracytoplasmic TCR (TCRic)
and surface pT expression (shaded areas) was analyzed after gating
on the CD3low progeny recovered by day 5. Background
fluorescence was determined with isotype-matched irrelevant MoAb and
with a rabbit preimmune serum. A representative experiment out of three
is shown.
|
|
We have previously shown that CD4+ CD8+
pre-T cells with this particular CD3low pT+
phenotype are functional progenitors of common DP thymocytes that
already express surface TCR.25 Accordingly,
CD4+ CD8+ cells generated in the thymic
lobes increased in numbers throughout culture (up to 9- to 10-fold in 6 to 7 days) to become the major cell subset (>95%) by day 14, and
acquired simultaneously intermediate to high levels of both CD3 and the
mature TCR (50% of total cells recovered by day 14 in the
experiment shown in Fig 3A). These results provide evidence that
CD4+ CD8+ thymocytes represent the
immediate precursors of the first intrathymic cells with a conventional
CD4+ CD8+ DP phenotype. Moreover, they
indicate that the developmental onset of CD8 chain expression is
closely associated with the -selection process and parallels the
acquisition of a surface pre-TCR.
CD4+ CD8+ pre-T cells are functional
intermediates between CD4+ CD8
progenitors and CD4+ CD8+ pre-T cells.
It is currently believed that CD4+ CD8
thymocytes that still lack the CD3-TCR are the immediate
precursors of DP thymocytes in the human thymus.16 However,
we show in this study that CD4+ CD8+
pre-T cells are efficient progenitors of CD4+
CD8+ DP thymocytes, suggesting that they represent
very transient intermediates between CD4+
CD8 and CD4+ CD8+
cells in vivo. To seek direct evidence that CD4+
CD8+ thymocytes represent the normal progeny of human
CD4+ CD8 thymocyte precursors in the
pathway of T-cell differentiation, highly purified
CD3 CD4+ CD8
thymocytes (>98% pure) were analyzed for their developmental fate in
the hu/mo FTOC system. The pattern of differentiation obtained in
different experiments was identical (Fig
4A): CD4+ cells acquired
rapidly the CD8 chain, while remaining CD8 negative (30%
CD8+, <2%
CD8+ by day 4) and, later on, CD8 chain was
coexpressed with CD8 in a significant cell fraction (40% by day 11;
Fig 4A) that became the major population (>90%
CD8+) by day 15 to 16 (data not shown). More
importantly, three-color flow cytometry of the cells harvested on days
4 and 11 of culture extended our findings ex vivo, and confirmed that
acquisition of the CD8+ phenotype was linked to the
expression of TCRic, so that all CD8+
progeny generated in the thymic lobes remained negative for
TCRic expression (Fig 4B). As observed when thymic lobes
were reconstituted with CD4+ CD8+ cells,
cellular proliferation lasted for 6 to 7 days in lobes injected with
CD3 CD4+ CD8
progenitors. Regardless of the starting population, cellular expansion
in the FTOC involved preferentially (if not exclusively) the
TCRic+ CD8+ population, so
that generation of CD8+ cells may ocurr essentially
in the absence of cell proliferation. Taken together, our results
provide formal proof that CD4+ CD8+
thymocytes are the direct progeny of CD3
CD4+ CD8 thymocytes and the immediate
precursors of the first DP thymocytes expressing the CD8
heterodimeric form. In addition, they are compatible with a model in
which the CD8 chain is acquired by developing cells in the human
thymus before the process of -selection, whereas expression of
CD8 is induced as a developmental consequence of the -selection
process after signaling through the pre-TCR.
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| Fig 4.
CD4+ CD8+ human
thymocytes are functional intermediates between CD3
CD4+ CD8 progenitors and
CD4+ CD8+ -selected pre-T cells.
(A) CD3 CD4+ CD8
thymocytes, isolated as described in Materials and Methods, were
cultured in a hybrid hu/mo FTOC and analyzed after 4 and 11 days for
the coexpression of CD4, CD8, and CD8. (B) Intracytoplasmic
TCR (TCRic) expression (shaded areas) was analyzed by three-color
flow cytometry in the cellular progeny recovered at day 4 of culture.
By day 11, analysis was performed after electronic gating on the
CD8+ and the
CD8++ progeny. Background
fluorescence was determined with isotype-matched irrelevant MoAb. A
representative experiment out of five is shown.
|
|
Developmental events associated with -selection parallel the onset
of CD8 chain expression.
The above data allowed us to propose that CD8 expression is a cell
marker of -selection. Further support for this notion came from
molecular studies aimed at analyzing individual CD8+
and CD8+ pre-T-cell stages for the expression of
several proteins known to be regulated as a consequence of the
-selection process. Particularly, phosphorylation of Retinoblastoma
(Rb), which is tightly associated with cell-cycle
activation,30 expression of cell-cycle-associated cyclin A
and cyclin B, and downregulation of RAG2 protein expression were
analyzed by Western blotting, as indicators of -selection (Fig
5 and Table
1).8 As shown in Fig 5, a
mixture of both the hyperphosphorylated and the hypohosphorylated Rb
forms was detected in CD3 CD4+
CD8 as well as in CD4+
CD8+ thymocytes. This pattern changed dramatically at
the next CD3low pT+ DP stage that showed an
exclusive expression of the slow hyperphosphorylated Rb form, which was
also the predominant form displayed by large CD3
CD4+ CD8+ pre-T cells. Expectedly,
noncycling, small CD3 DP pre-T
cells10 displayed exclusively the fast hypophosphorylated Rb form. Of note, expression of the 60-kD cyclin A, which was barely
detectable in CD3 CD4+
CD8 and CD4+ CD8+
thymocytes, was markedly upregulated in both the CD3low
pT+ and the CD3 large DP pre-T-cell
subsets displaying an exclusive hyperphosphorylated Rb form, whereas it
dropped to basal levels in the CD3 small DP cells.
An identical expression pattern was observed for the 62-kD cyclin B
(Fig 5).
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| Fig 5.
Analysis of Rb phosphorylation, and expression of Cyclins
A and B and RAG proteins in distinct subsets of human pre-T cells.
Cellular lysates from human thymocytes of the indicated phenotypes were
isolated as described in Materials and Methods and analyzed by Western
blotting for the expression of Rb, Cyclin A, Cyclin B, RAG1, and RAG2.
Expression of -tubulin was analyzed as an internal control.
Molecular sizes are indicated on the left (kD).
|
|
Analysis of RAG2 protein content in individual pre-T-cell subsets
indicated that, as reported in mice,8 RAG2 downregulation may be a functional developmental consequence of -selection also in
humans. As shown in Fig 5, we found that the relative expression levels
of RAG2 (as compared with those of -tubulin) was reduced in both
subsets (CD3low and CD3) of
CD8+ pre-T cells, and became almost undetectable in
downstream small pre-T cells. However, RAG2 protein was reexpressed at
high levels at the next stage of CD3-TCR-expressing DP
thymocytes (data not shown) indicating that, similarly to mice, RAG2
downregulation is transient in thymocyte development in humans.
Parallel analysis showed that RAG1 was not subjected to a similar
developmental regulation at the protein level, because relatively high
levels of RAG1 were expressed in all pre-T-cell subsets. Taken
together, these results provide additional support to our proposal that -selection occurs in humans during the transition from
CD4+ CD8+ to CD4+
CD8+ DP pre-T cells and allow us to conclude that
CD8 chain induction may be one of the direct consequences of the
-selection process.
| |
DISCUSSION |
The progression of thymocytes through distinct developmental stages is
marked by the ordered pattern of expression of a number of cell surface
molecules of which the coreceptors CD4 and CD8 are particularly
relevant. In humans, the developmental expression of these two
molecules marks progression of thymocytes from immature CD4 CD8 DN cells through
CD4+ CD8 intermediates and finally to
CD4+ CD8+ DP thymocytes, before expression of a
surface TCR and commitment to either CD4+
CD8 or CD4 CD8+
mature T cells.3,16 We have previously shown that
essentially all CD4+ CD8 intermediates
display a germline configuration at the TCR locus and lack
expression of TCR protein, whereas both TCR gene rearrangements and cytoplasmic TCR chain expression occur in the vast majority of
thymocytes at the downstream CD4+ CD8+
TCR stage. The finding that the latter subset
is highly enriched in large cycling cells25 allowed us to
propose that the CD4+ CD8 to
CD4+ CD8+ transition represents the critical
developmental point at which -selection promotes the clonal
expansion and further differentiation of human pre-T cells independent
of TCR.25 Molecular support to that notion came from
additional studies showing that expression of surface CD3 and pT
chain is restricted in vivo to a significant proportion of
cells within the population of CD4+ CD8+
TCR cycling thymocytes,10
indicating that the human pre-TCR is actually expressed on pre-T cells
in transit to the CD4+ CD8+ DP
stage.25 However, we show in this study that the DP
compartment is heterogeneous regarding the expression of CD8, so that
it is mostly composed of cells with the expected CD4+
CD8+ phenotype, but also includes a minor fraction of
cells that coexpress CD4 and CD8 without CD8. The
physiological existence of such CD4+
CD8+ thymocytes suggests that
expression of the CD8 glycoprotein either as an homodimer or as
an heterodimer26 |
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ABSTRACT
INTRODUCTION