By
From the Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Lineage commitment is a developmental process by which individual CD4+CD8+ (double positive, DP) thymocytes make a decision to differentiate into either CD4+ or CD8+ T cells. However, the molecular event(s) that defines lineage commitment is controversial. We have previously proposed that lineage commitment in DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi). In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule. In addition, the present results identify three distinct subpopulations of DP thymocytes that define the developmental progression of the lineage commitment process and demonstrate that lineage commitment is coincident with upregulation of TCR and bcl-2. Thus, this study supports a molecular definition of lineage commitment and uniquely identifies TCRhibcl-2hi DP thymocytes as cells that are already committed to either the CD4 or CD8 T cell lineage.
Thymocytes develop through a series of stages which
can be distinguished by variations in surface expression
of the coreceptor molecules CD4 and CD8 (reviewed in
reference 1). Double-negative (DN)1 cells expressing neither
CD4 nor CD8 (CD4 The cellular and molecular signals that induce lineage
commitment in DP thymocytes remain controversial (reviewed in reference 3). Studies to identify such signals have
not focused directly on DP thymocytes in which lineage
commitment signals are generated but rather have assessed
lineage commitment either by the successful generation of
SP T cells or by the appearance of populations that appear
to be in transition to a SP phenotype (4). Indeed, lineage commitment for T cells is best defined developmentally as an irreversible commitment to differentiate into
either a CD4+ or CD8+ T cell. Unfortunately, such a developmental definition does not permit lineage commitment to be recognized in signaled DP thymocytes when it
occurs, but only after it has resulted in its successful differentiation into a SP T cell. Consequently, we have formulated a molecular definition of lineage commitment that
can be applied directly to DP thymocytes. We have proposed that lineage commitment be molecularly defined as
the selective termination of synthesis of one coreceptor
protein in a cell that expresses both coreceptor proteins on
its surface (12). That a DP thymocyte expresses both CD4
and CD8 coreceptor proteins on its surface is definitive evidence that it was recently synthesizing both coreceptor
molecules, and whether or not it continues to actively synthesize both coreceptor molecules we think reflects its lineage commitment status.
We have recently described an assay, the coreceptor reexpression assay, which assesses individual DP thymocytes
for coreceptor synthesis (12). By our molecular definition
of lineage commitment, DP thymocytes synthesizing both
CD4 and CD8 coreceptors are uncommitted cells, whereas
DP thymocytes synthesizing only CD4 or CD8 coreceptor molecules are lineage committed cells. However, it has
been argued that selective termination of synthesis of one
coreceptor protein may occur sporadically in DP thymocytes, at random developmental points, and not reflect a
developmentally regulated lineage commitment event.
We now report that selective termination of CD4 or
CD8 coreceptor synthesis does not occur capriciously in
DP thymocytes, but is developmentally restricted to the
most differentiated subpopulation of DP thymocytes which
is characterized as CD5hiCD69hiTCRhibcl-2hi. In fact, all
CD5hiCD69hiTCRhibcl-2hi DP thymocytes have terminated
synthesis of one coreceptor molecule despite their surface
expression of both CD4 and CD8 proteins. As a result, it is
the immediate precursors of these cells, i.e., CD5hiCD69hi
TCRlobcl-2lo DP thymocytes, that appear to be coordinately signaled in the thymus to terminate synthesis of one
coreceptor molecule and to upregulate both TCR and bcl-2
expression. Thus, the present study identifies the phenotype of cells within the DP population that have undergone
lineage commitment and shows that lineage commitment is
coincident with high levels of TCR and bcl-2 expression.
Mice and Thymocyte Preparations.
C57BL/6 (B6) and TCR Staining and Sorting.
Thymocytes were stained and sorted as
previously described (12). In brief, 5 × 105 cells were distributed
in wells of a 96-well plate (Nunclon, Roskike, Denmark), pelleted, and stained for 30 min at 4°C with saturating concentrations of PE-conjugated anti-CD4 mAb (GK1.5; Becton Dickinson & Co., Mountain View, CA), FITC-conjugated anti-CD8 mAb (53-6-7; Becton Dickinson), and, where indicated, biotinylated anti-CD5 mAb (53.7.3; Becton Dickinson & Co.), biotinylated anti-CD69 mAb (H1.2F3; PharMingen, San Diego, CA) or
biotinylated anti-TCR Pronase Treatment.
The coreceptor reexpression assay was performed as previously described (12). In brief, sorted cells were
washed two times with PBS and resuspended at 1-5 × 106/ml
with or without 0.04% Pronase (Calbiochem Novabiochem, La Jolla, CA) in PBS. They were incubated for 15 min at 37°C and then pelleted and incubated with fresh pronase for another 10 min at 37°C. Cells were subsequently washed three times with
complete medium then distributed in 0.5-ml vol into 24-well
plates at a final concentration of 0.5-2.0 ×106 per ml. Cells were
incubated overnight (12-16 h) at 4°C or 37°C, stained, and analyzed.
Because
CD4 and CD8 surface proteins can survive for hours on
the surface of DP thymocytes, DP thymocytes can selectively terminate synthesis of one or the other coreceptor
molecule without an obvious change in surface phenotype.
To detect the coreceptor molecules that DP thymocytes
are actively synthesizing, we have utilized the coreceptor
reexpression assay (Fig. 1). In brief, thymocytes are stripped
of surface CD4 and CD8 proteins by extracellular treatment with low concentrations of the protease pronase,
washed, and then cultured overnight at 37°C in medium
alone to allow surface reexpression. Surface reexpression of
coreceptor molecules in this assay has previously been
shown to require new transcription and active protein synthesis and so provides a convenient demonstration of the
coreceptor molecules that individual DP thymocytes are actively synthesizing (12). In the present study we utilized the coreceptor reexpression assay to determine if any relationship existed between the selective termination of coreceptor synthesis and developmental maturity. As well-characterized markers of developmental maturity we utilized
surface expression of CD5 and CD69 (9, 16).
Surface expression of CD5 and CD69 is markedly heterogeneous among DP thymocytes, with DP thymocytes
contained within cell populations expressing low, intermediate, and high levels of each marker (Fig. 2, a and b). More
importantly, surface CD5 and CD69 expression is directly
correlated with developmental maturity such that the least
differentiated DP thymocytes express low amounts of CD5
and CD69 and the most differentiated DP thymocytes express high levels of CD5 and CD69 (17, 19, 20). Consequently, to determine if termination of coreceptor synthesis
occurred in a developmentally restricted subpopulation of
DP thymocytes, we performed the coreceptor reexpression
assay on thymocytes that were electronically purified by
3-color cell sorting into DP thymocytes expressing low/intermediate versus high levels of surface CD5 and CD69
(Fig. 3, a and b). All DP thymocytes expressing low/intermediate levels of CD5 or CD69 either synthesized both
coreceptor molecules or synthesized no coreceptor molecule (Fig. 3, a and b, upper panels); essentially none of these
cells selectively synthesized only one coreceptor molecule
(Fig. 3, a and b). In contrast, DP thymocytes expressing
high levels of surface CD5 or CD69 did contain cells that
were selectively synthesizing only one coreceptor molecule (Fig. 3, a and b, lower panels). DP thymocytes selectively
synthesizing CD8 usually outnumbered thymocytes selectively synthesizing CD4. We suspect this is because thymocytes that have selectively terminated coreceptor synthesis lose surface CD8 expression more quickly than
surface CD4 expression, a possibility consistent with observations by other investigators (22, 23). We also observed a
significant number of cells that have terminated or diminished synthesis of both CD4 and CD8. The fate of these
cells is not yet known but they may represent precursors of
mature DN T cells. Most importantly, these data indicate
that cells selectively synthesizing only one coreceptor molecule (either CD4 or CD8) are not present among the vast
majority of DP thymocytes, but are only present among DP thymocytes expressing high surface levels of CD5 and
CD69.
Even though all DP thymocytes selectively synthesizing
only one coreceptor molecule were CD5hiCD69hi, most of
the cells that were CD5hiCD69hi actively synthesized both
coreceptor molecules (Fig. 3 a). Consequently, we wished
to determine if markers existed that could uniquely identify
CD5hiCD69hi DP thymocytes that had selectively terminated synthesis of one coreceptor molecule and distinguish
them from the majority of CD5hiCD69hi DP thymocytes
that continued to synthesize both coreceptors. As a first attempt we wished to quantitatively compare CD5 and CD69 surface expression on these different DP subpopulations. DP thymocytes that had undergone the coreceptor
reexpression assay could be subsequently assessed for surface CD5 but not CD69 expression because CD5 is resistant to pronase treatment whereas CD69 is not. We found
that surface CD5 expression was essentially equivalent on
all CD5hi DP thymocytes, regardless of whether they were
actively synthesizing one or both coreceptor molecules
(Fig. 3 c) and therefore could not be used to distinguish the
subpopulations.
However, we observed a clear difference when we examined the small subset of CD5hiCD69hi DP thymocytes
that had selectively terminated synthesis of one or the other
coreceptor molecule for expression of two other differentiative markers, surface TCR and internal bcl-2 proteins
(24) (Fig. 4). CD5hi DP thymocytes that were selectively synthesizing only one coreceptor molecule (either
CD4 or CD8) expressed significantly higher levels of both
surface TCR
These results demonstrate that selective termination of
synthesis of one coreceptor molecule is a stringently regulated event during development which is evident only
within the small, most differentiated subset of DP thymocytes defined as CD5hiCD69hiTCRhibcl-2hi. In addition, these results identify three subsets of DP thymocytes that differ in their state of coreceptor synthesis: (a) CD5lo
CD69lo DP thymocytes that actively synthesize both coreceptor molecules; (b) CD5hiCD69hiTCRlobcl2lo DP thymocytes that synthesize both coreceptor molecules; and (c)
CD5hiCD69hiTCRhibcl2hi DP thymocytes that actively
synthesize only one coreceptor molecule having terminated
expression of the other (Table 1). We propose there may
be a precursor/progeny relationship among these subpopulations such that they define the progression of DP thymocytes from uncommitted to precommitted to lineagecommitted thymocytes.
As an independent test of our conclusion
that selective termination of coreceptor synthesis is a developmentally regulated event that only occurs in the most
differentiated subset of CD5hi DP thymocytes, we examined DP thymocyte populations from TCR
Having confirmed that selective termination of coreceptor synthesis occurs only in CD5hi DP thymocytes, we
wished to examine the developmental relationship between
CD5hi DP thymocytes that continue to synthesize both
coreceptor molecules and those that have selectively terminated coreceptor synthesis. In particular, if CD5hi DP thymocytes that continue to synthesize both coreceptor molecules are the precursors of those that have selectively
terminated synthesis of one coreceptor molecule, it would
be expected that these populations would appear sequentially in development. To examine this possibility, we performed the coreceptor reexpression assay on CD5hi DP
thymocytes from newborn mice less than 12 h old (Fig. 6). Interestingly, newborn mice contained CD5hi DP thymocytes at a frequency comparable to that of adult mice (10-15% of all DP cells). However, essentially all neonatal
CD5hi DP thymocytes actively synthesized both coreceptor
molecules; very few, if any, had terminated synthesis of either or both coreceptor molecules (Fig. 6). Thus, CD5hi
DP thymocytes that synthesize both coreceptor molecules
appear earlier in development than CD5hi DP thymocytes
that synthesize only one coreceptor molecule, consistent with (but not proving) a precursor/progeny relationship.
Lineage commitment is a developmental process by
which positively selected DP thymocytes are induced to
differentiate into either CD4+ or CD8+ T cells. Identification of the intrathymic signals that induce lineage commitment would be significantly facilitated if the molecular consequences of lineage commitment could be recognized
as soon as they occurred in DP thymocytes. We have proposed that lineage commitment be molecularly defined as
the selective termination of synthesis of one or the other
coreceptor molecule in a DP thymocyte expressing both
coreceptors on its surface. Lineage commitment by any
definition must require that DP thymocytes shift from synthesizing both coreceptor molecules to synthesizing only
one. However, the validity of defining lineage commitment in DP thymocytes by coreceptor synthesis has been
questioned because of the possibility that selective termination of coreceptor synthesis may occur randomly in DP
thymocytes and, therefore, may be unrelated to their differentiation state.
The present study, however, demonstrates that selective
termination of CD4 or CD8 synthesis as determined by
the coreceptor reexpression assay does not occur randomly
in DP thymocytes, but, instead is a highly regulated event
which has occurred exclusively within a developmentally discrete subpopulation of DP thymocytes that is CD5hiCD69hi
TCRhibcl-2hi. DP thymocytes expressing high levels of
CD69, TCR and bcl-2 have each been shown by other investigators to have undergone positive selection (19,
24) and TCRhi subpopulations of DP thymocytes, specifically, have been shown to contain the immediate precursors of SP thymocytes (23). That termination of
coreceptor synthesis was only evident within that population of thymocytes which has undergone positive selection is compelling evidence that selective termination of coreceptor synthesis is not a capricious event in DP thymocytes
but is a molecular indicator of lineage commitment. Importantly, selective termination of coreceptor synthesis as
detected by the coreceptor reexpression assay, unlike other
markers identifying lineage committed DP thymocytes, reveals the T cell lineage to which individual DP thymocytes
are committed since it reveals the coreceptor molecule that
committed thymocytes continue to synthesize.
The present study has identified three distinct subpopulations of DP thymocytes that are distinguishable by surface
phenotype and lineage commitment status: (a) CD5lo CD69lo
DP thymocytes that have not selectively terminated synthesis of either CD4 or CD8 coreceptor molecules, (b)
CD5hiCD69hiTCRlobcl-2lo DP thymocytes that continue to
synthesize both coreceptor molecules, and (c) CD5hiCD69hiTCRhibcl-2hi DP thymocytes that actively synthesize only
one or the other coreceptor molecule, but not both. A
striking and unanticipated finding of the present study is
that all DP thymocytes that have selectively terminated either CD4 or CD8 synthesis are CD5hiCD69hiTCRhibcl-2hi
and, furthermore, that essentially all CD5hiCD69hiTCRhibcl-2hi DP thymocytes have selectively terminated either
CD4 or CD8. This precise overlap suggests that intrathymic
signals induce the immediate precursors of these lineage
committed DP thymocytes (presumed to be CD5hiCD69hiTCRlobcl-2lo DP thymocytes) to coordinately: (a) upregulate TCR expression, (b) increase bcl-2 content, and (c) selectively terminate synthesis of one coreceptor molecule.
Consequently, CD5hiCD69hiTCRintbcl-2lo DP thymocytes
synthesizing both coreceptor molecules appear to be the
targets of lineage commitment signals in the thymus. A summary of these findings is presented in Table 1.
We think that a precursor/progeny relationship exists
among all three DP thymocyte subpopulations which is
consistent with their sequential appearance during development, although it is difficult to prove such precursor/progeny relationships conclusively. From a lineage commitment
perspective, we consider CD5loCD69lo DP thymocytes to be
uncommitted; CD5hiCD69hiTCRlobcl-2lo DP thymocytes
to be potential targets of lineage commitment signals and
so are precommitted; and CD5hiCD69hiTCRhibcl-2hi DP
thymocytes to be committed. The differentiation of CD5lo
CD69lo DP thymocytes into CD5hiCD69hiTCRlobcl-2lo DP
thymocytes presumably requires intrathymic signals transduced by surface TCR/CD3 complexes as CD5 and CD69
upregulation is mediated by TCR/CD3 signaling (17, 20,
32). Notably, the present study indicates that such intrathymic TCR/CD3 signals are not sufficient to selectively terminate synthesis of either CD4 or CD8 coreceptor molecules, as CD5hiCD69hiTCRlobcl-2lo DP thymocytes continue
to synthesize both coreceptor molecules. Rather, we think
additional signals are required in CD5hi CD69hiTCRlobcl-2lo
DP thymocytes to increase TCR expression, to increase
bcl-2 expression, and to selectively terminate either CD4
or CD8 coreceptor synthesis. It is important to note, however, that the present data indicate that TCR/CD3 signals
are necessary for selective termination of CD4 or CD8 synthesis. Whether these signals are generated by TCR engagement by antigen or by ligand engagement of non-clonotypic receptors that signal via the TCR/CD3 signaling
complex, such as Thy-1 (33), is not known.
In the present study the coreceptor reexpression assay has
been performed on DP thymocytes, whereas we have previously utilized the coreceptor assay on transitional populations of DP thymocytes that have quantitatively downregulated surface expression of one or the other coreceptor
molecule (12). The results of our previous studies indicated
that termination of CD4 synthesis was more stringently regulated during development than termination of CD8
synthesis, and we advanced a model, the asymmetric commitment model, to explain our findings. Specifically, we
proposed that DP thymocytes progress to a stage in development (termed the commitment checkpoint) when they
are assessed for the presence or absence of a CD8-commitment signal. The presence of a CD8-commitment signal
results in selective termination of CD4 synthesis (CD8commitment), whereas the absence of a CD8-commitment
signal results by default in selective termination of CD8
synthesis (CD4-commitment). The present study provides a phenotype that can be assigned to each of the commitment stages in the asymmetric commitment model (Fig. 7):
CD5loCD69lo DP thymocytes are uncommitted cells that are
preselection; CD5hiCD69hiTCRlobcl-2lo DP thymocytes are
cells at the commitment checkpoint and so are precommitment; CD5hiCD69hiTCRhibcl-2hi DP thymocytes have
committed to either the CD4 or CD8 T cell lineages (Fig. 7).
However, the present finding that all lineage-committed DP
thymocytes, even those that have selectively terminated
CD8 synthesis, are CD5hiCD69hi indicates that commitment to either lineage occurs only in TCR/CD3 signaled
DP thymocytes. Hence, we would like to clarify the asymmetric commitment model to state that: (a) DP thymocytes
that fail to receive TCR/CD3 signals remain CD5loCD69lo
and die from neglect as uncommitted DP thymocytes; and
(b) CD4-commitment occurs in CD5hiCD69hi DP thymocytes that have not received CD8-commitment signals.
In conclusion, the present study demonstrates that selective termination of CD4 or CD8 coreceptor synthesis in
DP thymocytes is a highly regulated developmental process
that occurs in strict parallel with other events that define
positive selection. It is anticipated that the molecular definition of lineage commitment validated in this study will
enhance identification of the intrathymic signals that induce lineage commitment in developing thymocytes.
CD8
) mature into double-positive
(DP) cells expressing both CD4 and CD8 (CD4+CD8+)
which, in turn, develop into single-positive (SP) cells that selectively express CD4 (CD4+CD8
) or CD8 (CD4
CD8+).
Development of immature DP thymocytes into mature SP
thymocytes is a highly regulated process in which only
those DP thymocytes with TCR of appropriate specificities
are positively selected to further differentiate into SP T
cells. The process of positive selection (reviewed in reference
2) involves at least three major cellular events: (a) conversion of a DP thymocyte expressing both CD4 and CD8
coreceptor molecules into a SP T cell expressing only one
coreceptor molecule, referred to as lineage commitment; (b) conversion of a short-lived DP thymocyte into a longlived SP T cell; and (c) conversion of a functionally incompetent DP thymocyte into a functionally competent SP T
cell. The cellular and molecular bases for these processes remain largely a matter of speculation. In this study we have
addressed the first issue of lineage commitment.
/
mice (14) were obtained from Jackson Laboratories (Bar Harbor,
Maine) and were housed and bred in a specific pathogen-free facility. Thymuses were dissected from mice between 6 and 10 wk
of age and single cell suspensions were made by gently teasing
dissected lobes with forceps and filtering over nylon.
mAb (H57-597; PharMingen) in 30 µl
final volume of staining medium (0.5% BSA, 0.5% NaN3 in
HBSS). PE-conjugated, FITC-conjugated, and biotinylated antihuman CD3 mAb (Leu 4; Becton Dickinson & Co.) were used as
controls for nonspecific staining. All staining was also performed in
the presence of anti-FcReceptor (FcR) antibody (2.4G2; Becton
Dickinson & Co.) to block nonspecific antibody binding to
FcRs. Cells were washed twice with 150 µl of staining medium
and if a second step were required, were stained again in 30 µl final volume of saturating concentrations of either Texas-red streptavidin or RED670-streptavidin (GIBCO BRL, Gaithersburg, MD)
for 5 min at 4°C. Cells were washed twice again and analyzed by
flow cytometry. For sorting, cells were stained at a final concentration of 108/ml in 12 × 75 mm tubes (no. 2058; FALCON,
Becton Dickinson & Co.) using saturating amounts of PE-conjugated anti-CD4 mAb, FITC-conjugated anti-CD8 mAb and biotinylated anti-CD5 mAb. After washing two times with staining
medium, cells were resuspended at a concentration of 108/ml and
stained with saturating concentrations of Texas red (TR)-streptavidin (GIBCO BRL). After sorting, pronase treatment, and culture cells were typically restained with the same antibody combinations for analysis. In those cases when it was necessary to stain
for an additional marker, we restained with PE anti-CD4 and
FITC anti-CD8 as usual, but used distinct biotinylated antibodies
(biotinylated anti-TCR
mAb (H57-597, PharMingen) or antimurine bcl-2 mAb (3F11, PharMingen) followed by a biotinylated anti-hamster mAb (G94-56, PharMingen) in combination
with RED670-conjugated streptavidin to avoid confusion with
any residual anti-CD5/TR-streptavidin conjugates. Staining with
anti-bcl-2 required that the cells be permeabilized by treatment
with 0.03% saponin and we followed the method described by
Veis et al. (15). Flow cytometry was performed on a Becton
Dickinson FACStar® Plus and analyzed using software designed
by the Division of Computer Research and Technology at the
National Institutes of Health except in cases where RED-670 was
used as a second step, when cells were analyzed on a Becton
Dickinson FACScan® with Cell Quest software. Dead cells were
excluded electronically by gating on forward scatter and propidium iodide intensity when analyzed on the FACStar® Plus and by
gating on forward and side scatter when analyzed on the FACScan®.
Selective Termination of Either CD4 or CD8 Coreceptor Synthesis in Defined Subpopulations of DP Thymocytes.
Fig. 1.
Coreceptor reexpression assay. Thymocytes that express both
CD4 and CD8 on their surfaces may no longer be actively synthesizing both CD4 and CD8 coreceptor molecules. To reveal the coreceptor(s) that individual CD4+CD8+ thymocytes are actively synthesizing, thymocytes are treated with low concentrations of the protease pronase
which strips the surface of the cell of existing CD4 and CD8 protein molecules. Cells are then allowed to reexpress the coreceptor proteins they are actively synthesizing during overnight culture at 37°C. In the schematic, surface CD4 and CD8 proteins are indicated by 4 or 8 outside the
circle, and internal CD4 and CD8 mRNAs are indicated by 4 or 8 inside
the circle.
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
CD4+CD8+ thymocytes are heterogeneous for expression of
CD5 and CD69. Freshly isolated thymocytes from B6 mice were analyzed by three color flow cytometry for surface expression of CD4, CD8 and CD5 (a) or CD69 (b). Gates subdividing cells into low, intermediate and high expression of CD5 (a) or CD69 (b) are indicated. Contour plots
displaying CD4 and CD8 expression of cells in each gate are shown below each gate and the frequency of cells falling into CD4+CD8+,
CD4CD8+ and CD4+CD8
subpopulations are indicated. Solid line indicates CD5 or CD69 staining and dashed line indicates negative control
(Leu 4) staining.
[View Larger Versions of these Images (25 + 26K GIF file)]
Fig. 3.
Coreceptor synthesis in CD4+CD8+ thymocytes. Populations of thymocytes from B6 mice were electronically sorted by three color flow
cytometry into purified populations of CD5hi versus CD5lo/int CD4+CD8+ cells (a) or CD69hi versus CD69lo/int CD4+CD8+ cells (b). The sorting gates
are indicated and the phenotype of the sorted cells after overnight culture at either 4°C or 37°C is shown (left panels). Sorted cells were assessed for coreceptor synthesis using the coreceptor reexpression assay, and the phenotype of pronase stripped cells after overnight culture at either 4°C or 37°C is
shown (right panels). The frequency of cells falling into each boxed region is indicated. It is evident after pronase treatment and 37°C culture that
CD4+CD8+ thymocytes that express only one coreceptor molecule are present only among the CD5hi (a) and CD69hi (b) CD4+CD8+ thymocyte subpopulations. The CD5 expression levels of sorted CD5hi CD4+CD8+ cells from (a) which only expressed CD8 (***), CD4 (**) or which continued to
synthesize both coreceptors (*) were compared (c). Staining with a negative control antibody on unsorted populations is shown as a shaded histogram.
This staining shows that the populations continuing to synthesize both CD4 and CD8 could not be distinguished from cells that had selectively terminated CD4 or CD8 synthesis by CD5 expression levels.
[View Larger Versions of these Images (30 + 32 + 33K GIF file)]
and internal bcl-2 protein than CD5hi DP
thymocytes that continued to synthesize both coreceptor
molecules (Fig. 4). Thus, DP thymocytes that have selectively terminated synthesis of one coreceptor molecule
(either CD4 or CD8) are exclusively contained within a
small subpopulation of DP thymocytes that are not only
CD5hiCD69hi, but are also TCRhibcl-2hi.
Fig. 4.
TCR and bcl-2
expression identify CD4+CD8+
thymocytes that have selectively
terminated synthesis of one coreceptor molecule. Purified CD5hi
CD4+CD8+ thymocytes were
obtained by electronic sorting and
assessed by the coreceptor reexpression assay. The CD4 and
CD8 profile of sorted CD5hi
CD4+CD8+ cells treated with
pronase and cultured overnight
at 37°C is shown as a contour
plot. TCR
and bcl-2 expression of sorted cells which only
reexpressed CD8 (***), CD4
(**) or which continued to synthesize both CD4 and CD8 (*)
are displayed. The shaded profiles represent staining of unsorted whole thymocytes that
had been similarly pronase
treated and cultured at 37°C
overnight.
[View Larger Version of this Image (21K GIF file)]
/CD3 Complexes and Occurs Late in
Development.
/
mice that
are unable to assemble and express surface TCR
complexes and, consequently, lack CD5hi DP thymocytes (Fig.
5 a). In this experiment, we attempted to increase our ability to detect DP thymocytes synthesizing only one coreceptor molecule by performing the coreceptor reexpression assay on purified populations of CD4+8lo and CD4lo8+
transitional DP thymocytes that may be developmentally
more advanced than the bulk of DP thymocytes (Fig. 5 b).
Even so, we failed to detect any DP thymocytes in TCR
/
mice that were selectively synthesizing only one coreceptor
molecule (Fig. 5 b). Thus, surface expression of TCR
/
CD3 complexes and the signals they transduce are required
for generation of CD5hi DP thymocytes and for selective
termination of a coreceptor molecule.
Fig. 5.
Selective termination of coreceptor synthesis requires
TCR/CD3 expression. (A) CD5 expression of CD4+CD8+ thymocytes from wild-type and TCR
deficient mice was assessed by flow
cytometry. It is evident that the CD5hi CD4+CD8+ thymocyte subpopulation is absent in TCR
-deficient mice. (B) CD4+CD8lo and CD4lo
CD8+ subpopulations of thymocytes from a TCR
-deficient mouse were electronically sorted according to the gates indicated and were assessed for
coreceptor synthesis by the coreceptor reexpression assay. Frequency of
cells falling into each quadrant are indicated. No cells that had selectively
terminated CD4 or CD8 synthesis are evident in either population.
[View Larger Versions of these Images (13 + 25K GIF file)]
Fig. 6.
CD5hi CD4+CD8+ cells within a newborn thymus have not
yet selectively terminated CD4 or CD8 synthesis. Neonatal thymi were
harvested within 12 h of birth and CD5hi CD4+CD8+ cells were electronically sorted according to the gate indicated in the left panel. The
coreceptor reexpression assay was performed on the sorted subpopulation
and the frequency of cells falling into each quadrant is indicated. It is evident that the vast majority of CD5hi CD4+CD8+ thymocytes continued
to synthesize both CD4 and CD8; no discrete populations of cells that
had selectively terminated CD8 or CD4 synthesis was evident. (For comparison, Fig. 4 displays an experiment performed with CD5hi CD4+CD8+
thymocytes from adult mice.)
[View Larger Version of this Image (28K GIF file)]
Fig. 7.
Subpopulations of CD4+CD8+ thymocytes, their commitment status, and their relationship to the Asymmetric Commitment
Model (12). The results of the present study provide a phenotype that can be assigned to each of the commitment stages proposed in the asymmetric commitment model (12). Preselection CD4+CD8+ thymocytes are too
immature to undergo lineage commitment and express low levels of
CD5, CD69, TCR and bcl-2. Signals transduced by the TCR/CD3
complex (as result of engagement of those surface molecules that signal
through the TCR/CD3 complex, e.g. TCR, Thy-1, etc.) increase CD5
and CD69 expression and promote the differentiation of preselection
CD4+CD8+ thymocytes to the point that they are able to undergo lineage commitment (commitment checkpoint). At the commitment checkpoint TCR-signaled CD4+CD8+ thymocytes whose TCR and CD8
molecules are coengaged become CD8-committed, whereas TCR-signaled CD4+CD8+ thymocytes whose TCR and CD8 molecules are not
coengaged become CD4-committed. Upregulation of TCR and bcl-2
occurs coincident with lineage commitment so that lineage committed
thymocytes express TCR and bcl-2 at high levels.
[View Larger Version of this Image (14K GIF file)]
Address correspondence to Alfred Singer, Experimental Immunology Branch, NCI/NIH, Building 10 Room 4B-17, Bethesda, MD 20892. Dr. Punt's current address is Haverford College, Biology Department, 370 Lancaster Avenue, Haverford, PA 19041.
Received for publication 4 September 1996
1 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive.1. | Robey, E., and B.J. Fowlkes. 1994. Selective events in T cell development. Annu. Rev. Immunol. 12: 675-705 [Medline] . |
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