By
From the Max-Planck-Institut für Immunbiologie, 79108 Freiburg, Germany
After productive rearrangement of a TCR chain gene, CD4
8
double negative (DN) thymocytes express TCR
polypeptide chains on the cell surface together with pre-T
and the
CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select
TCR
+ DN thymocytes for further maturation to the CD4+8+ double positive stage, whereas
DN cells that fail to generate a productive TCR
gene rearrangement do not continue in development. This process is termed TCR
chain selection. Although it is likely that differences
between proliferation dynamics of TCR
+ and TCR
cells may play a role, the exact mechanisms of TCR
chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCR
+ and TCR
thymocytes during fetal development, i.e., when
TCR
chain selection takes place for the first time. We analyzed in situ accumulation of
TCR
+ thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCR
+ and TCR
populations by flow cytometry. About 600 TCR
+ cells/thymic
lobe are generated by independent induction events between days of gestation (dg) 13.5. and
15.5. As of dg 14.5, most TCR
+ cells have entered S/G2 phase of cell cycle, followed by
seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding
burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCR
+ cells has
slowed down to less than 1/d. About three quarters of TCR
cells divide at a slow rate of 1/d
on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d.
From dg 16.5 onwards, TCR
cells, but not TCR
+ cells, contain a significant proportion
of apoptotic cells. The results suggest that failure to become selected results in shutdown of
proliferation and eventual programmed cell death of fetal TCR
cells. Positive selection of
fetal TCR
+ cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.
During development in the thymus, Pre-TCR-induced maturation of DN to DP thymocytes
is associated with a complex cluster of differentiation events
(reviewed in reference 8), including allelic exclusion of the
TCR Cell division parameters of immature thymocytes have
previously been studied in adult mice. Shortman et al. (35)
studied proliferation and turnover of DN thymocytes by
thymidine labeling in vivo and propidium iodide (PI) staining, and suggested the following sequence: the dividing,
most immature DN CD44+ (PgP-1+) CD25 The thymic anlage of mice starts to become repopulated
with thymocyte precursors on day of gestation (dg) 11-12.
TCR Mice.
BALB/c mice were obtained from our own specific
pathogen-free animal facility. For timed pregnancies, mice were
allowed to mate overnight (5 PM to 7 AM) and screened for vaginal plug. Noon of the following day was defined as dg 0.5. For in
vivo labeling, pregnant mice were injected 18 h before analyses of
fetal thymocytes with 250 µg of BrdU (Boehringer Mannheim
GmbH, Mannheim, Germany), and drinking water was supplemented with 1 mg/ml of BrdU.
Fetal Thymic Organ Culture.
Fetal thymic organ cultures were
performed with dg 13.5 or 14.5 thymic lobes as previously described (23, 24). Cultures were carried out for 1-4 d, referred to
as day +1, +2, +3, and +4, respectively. BrdU labeling was
done by incubation for 18 h with 10 µM concentration before
analysis.
mAb.
The following mAb, labeled with FITC or PE, were
purchased from PharMingen (San Diego, CA): anti-CD44, clone
IMF; anti-TCR FCM.
For propidium iodide/intracellular (IC) TCR lineage thymocytes proceed through two main developmental
checkpoints. First, cells with a CD4/CD8 double negative
(DN)1 phenotype differentiate to CD4/CD8 double positive (DP) cells. Second, DP thymocytes develop into CD4
or CD8 single positive (SP) cells (reviewed in reference 1).
Progression through either of these checkpoints is the result
of a process of selection (reviewed in reference 2). Differentiation of DN to DP cells depends on the expression of
the pre-TCR, consisting of the TCR
chain, pre-T
(3, 4),
and components of the CD3 complex (reviewed in 5).
Differentiation of DP to SP cells depends on the expression of a mature TCR, consisting of the TCR
heterodimer
and the CD3 complex (2, 10). The TCR
genes rearrange
during the DN stage (11), and expression of the preTCR selects for the approximately 56% of rearranging cells
that statistically generate a productive TCR
gene (14).
The TCR
genes rearrange during the DP stage (15).
Thus, selection by the pre-TCR, also termed TCR
chain selection, generates the repertoire of TCR
chains to be
combined with TCR
chains in the DP stage. A putative
ligand for the selection by the pre-TCR is not known (16).
DP cells expressing the mature
TCR are selected, positively or negatively, by the ability of the
TCR to interact with MHC/peptide ligands, thus generating the self tolerant, self-MHC restricted repertoire of CD4 or CD8 SP
thymocytes and peripheral T cells (17, 18)
locus (10, 19, 20), initiation of germline transcription of the TCR
locus (20, 21), transient downregulation
of the expression of the RAG genes (20, 22), shutdown of
CD25 (23), and transient expression of the activation
marker CD69 (26). Moreover, a large number of DP thymocytes is generated from a small number of DN cells,
presumably by induction of cell proliferation. This expansion phase is impaired in the absence of TCR
(27) or
of pre-T
(33). However, TCR
-deficient DN thymocytes can be induced to proliferate and to differentiate to
DP cells by cross-linking CD3
, expressed on DN thymocytes before and independent of TCR
(23, 34). These results suggested that cell proliferation associated
with the differentiation of DN to DP thymocytes may be
induced by pre-TCR-mediated signal transduction (26).
(IL-2R
)
thymocytes mature sequentially through a nondividing
CD25+CD44
stage into dividing CD25
CD44
DN
cells before they enter the DP stage and exit cell cycle. Most of TCR
rearrangement has been subsequently shown
to take place at the nondividing CD25+CD44
transitory
stage (12, 13). More recently, Penit et al. (36) reported results from 5-bromo-2
-deoxyuridine (BrdU) labeling of thymocytes in vivo suggesting that 70% of the CD25+CD44
DN cells were resting while 30% were proliferating. Hoffman
et al. (37) showed that a subset of CD25+CD44
DN thymocytes expressed markers of cell division, such as CDK2 and cdc2. Since proliferating CD25+CD44
cells were not
seen in RAG-deficient mice (36, 37), these authors speculated that the proliferating cells might be those that had
produced functional TCR
genes. However, none of these studies directly addressed the relationship between TCR
expression and thymocyte proliferation. Parkin et al. (38)
observed mitotic activity in newborn DN TCR
+ thymocytes, but proliferation kinetics of TCR
cells have not
been determined. Moreover, cell cycle and division parameters in fetal thymic development have not been studied.
gene VDJ rearrangements were first detected in fetal thymocytes on dg 15.5 (39, 40). Cells expressing TCR
chains were also observed on dg 15.5 by immunohistochemistry (41) and flow cytometry (FCM) among CD25+CD44
thymocytes (42). In the present report, we studied the accumulation and cell cycle dynamics of TCR
+ thymocytes
in ex vivo fetal thymuses and in dg 14.5 fetal thymic organ
cultures (FTOC) during the following four d in vitro, using confocal laser scanning microscopy (CLSM) and flow
cytometric analyses of PI- and BrdU-labeled cells. We detect TCR
+ cells from dg 13.5 onwards. Before dg 15.5, TCR
+ cells appear to accumulate mostly by independent
induction events to reach approximately 600 independent
TCR
+ cells/thymic lobe at dg 15.5. Concomitantly,
TCR
+ cells rapidly enter the S/G2 phase of cell cycle. In
FTOC, this is followed by 3 d of rapid proliferation, comprising at least seven to eight cell divisions, and an increase in
total TCR
+ cells of almost 200-fold. In vivo data suggest
a corresponding burst of up to nine cell divisions and an approximately 500-fold expansion between dg 14.5 and 18.5. Thereafter, TCR
+ cells return to slow cycle conditions.
A majority of TCR
cells appears to be slowly cycling on
dg 14.5 with a progressive increase in the proportion of
noncycling TCR
cells to 50%, resulting in a 8-(in vitro)
to 14-(in vivo) fold overall increase in TCR
cells during
the 4 d of observation. A significant proportion of apoptotic cells is seen from dg 16.5 onwards among TCR
,
but not among TCR
+ thymocytes.
, clone H57-597; anti-TCRV
6, clone RR4-7;
and anti-TCRV
3, clone KJ25. Anti-CD25, clone 5A2, was labeled with FITC in our own laboratory. FITC labeled anti-BrdU
clone BMC9318 was from Boehringer Mannheim, GmbH.
double staining cells were first incubated with culture supernatant of
mAb 2.4G2 to block FC
RII/III. Cells were then fixed with 1%
paraformaldehyde for 30 min on ice, followed by three washing
steps in PBS/2% FCS, the last containing, in addition, 0.1% saponin (Lot No. P4170; Sigma Chemical Co., Heidelberg, Germany). FITC labeled anti-TCR
mAb in PBS/0.125% saponin
was added for 20 min on ice, followed by two quick washes with
PBS and 2 × 20 min on a rocking platform in PBS/2% FCS/
0.1% saponin at 4°C. Cells were then fixed again in 1% paraformaldehyde, washed, incubated for 30 min at 37°C with 100 µg/ml
RNAse (Boehringer Mannheim, GmbH) and stained with 20 µg/ml propidium iodide (Sigma Chemical Co.). After washing,
cells were analyzed by FCM on a FACScan® (Becton Dickinson,
Mountain View, CA) using gates (signal area versus signal width)
that exclude doublet signals.
as
described above, and were sorted into TCR
+ and TCR
populations using a FACStar® plus (Becton Dickinson). Cells were
then fixed with 70% ethanol for 30 min on ice, resuspended in
0.1 M HCl, 0.5% Triton X-100 for 10 min on ice, centrifuged,
and resuspended in H2O. DNA was denatured by heating for 10 min at 95°C (43) followed by rapid cooling with ice-cold H2O.
This heating step obliterated all TCR
fluorescence. After one
wash in 0.5% Triton X-100 in PBS, cells were incubated with
FITC-labeled anti-BrdU mAb for 30 min at RT. FCM was done
using a FACScan®.
Confocal Laser Scanning Microscopy.
Whole thymic lobes were
quickly rinsed three times in PBS and then fixed with 2% paraformaldehyde for 3 h at 4°C on a rocking platform. They were
washed twice in PBS, twice in PBS/1% FCS/1% Tween-100,
and then incubated for 7 min in acetone at 20°C. This was followed by resuspension in H2O and 2 × 5 min rocking in PBS/ FCS/Tween. For mAb staining fixed lobes were first incubated
for 30 min with unlabeled mAb 2.4 G2 and then for 30 min with
each fluorochrome-labeled mAb alternating with 2 × 30 min
rocking in PBS/FCS/Tween, all at 37°C. Lobes were then embedded on microscope slides in Fluoromount-C (Southern Biotechnology Assoc., Birmingham, AL), covered by a cover slip,
turned upside down, and analyzed by an inverted CLSM instrument (Leica Instrs. GmbH, Heidelberg, Germany). Lobes were
minimally compressed by this technique. Magnification was 400fold if not otherwise stated.
PCR for TCR rearrangement.
DNA was prepared from thymocytes as described (39). DNA was amplified in 25-µl reaction
buffer, containing 0.1 µg DNA, 0.5 µM 5
and 3
primers, 200 µM
of each dNTP (Pharmacia, Freiburg, Germany) and 0.2 U SuperTaq (Staehelin, Basel, Switzerland) for 34 cycles (40 s at 94°C, 60 s at 63°C, 120 s at 72°C). Primers were 5
V
3 (44), 5
V
8,
5
D
2, and 3
J
2 (45).
Fetal thymuses were obtained from
pregnant BALB/c mice from dg 14.5 to 18.5 and FTOC,
were set up using dg 14.5 thymic lobes cultured for one to
four d. Thymocytes were stained on each day for IC TCR
chain, and Table 1 gives the absolute numbers of TCR
+
and TCR
cells determined in both sets of experiments.
About 100 TCR
+ cells/lobe were detected on dg 14.5. This is not a staining artefact, as TCR
+ cells identified by
FCM on dg 14.5 differ drastically in PI staining pattern
from TCR
cells (see Fig. 5). Moreover, we have no difficulties in demonstrating VDJ rearrangements by PCR in
dg 14.5 thymic lobes (see below, Fig. 7 C). In vivo, numbers of TCR
+ cells increased about 10-fold each day until dg
17.5, at which time the majority turned into DP thymocytes
(data not shown; 42). This was followed by a 3-4-fold increase of TCR
+ cells until dg 18.5. TCR
cells increased
merely 14-fold over the entire time interval. In vitro, numbers of TCR
+ cells increased fourfold during the first day
of culture, and then eight-ninefold on each of the next 2 d
and two-threefold on day four. TCR
thymocytes increased about eightfold until day +4 in FTOC. The end
result is a proportion of TCR
+ cells of ~80% by day 18.5 in vivo or of ~50% after 4 d of FTOC. The comparison
between the cell increments in vitro and in vivo reveals the
main difference between dg 15.5 and day +1 of culture, presumably reflecting a lag due to the recovery of the thymic lobes from the handling procedures. The differences
later in the time course are smaller and may to some extent
be due to lack of influx of new cells in vitro.
|
Subset Relationships of Early Fetal TCR
Expression of intracellular TCR chains before dg 15.5 has
not been previously described and it was therefore nessessary to determine the subset relationships of these early
TCR
+ thymocytes. To this end, we prepared fetal thymic
lobes from BALB/c mice at dg 13.5 and 14.5 and analyzed
them by CLSM for TCR
and CD44. Fig. 1 shows that
TCR
+ cells are detected as early as dg 13.5. However,
even at this early timepoint, all unequivocally identified
TCR
+ cells were negative for CD44. In a second experiment (Fig. 2), fetal thymic lobes were prepared on dg 14.5, 15.5, and 16.5, and analyzed by CLSM for TCR
and
CD25. Out of 25 individually studied TCR
+ cells on dg
14.5, 14 were were weakly positive (upper panel), and 11 were negative for CD25 (second panel). On dg 15.5 and
16.5, CD25 staining changed from a homogeneous to a
spotty pattern, and the proportion of CD25+TCR
+ cells
decreased, in agreement with previous results from FCM (42). Together, these results suggest that even at this early stage in development, TCR
expression does not preceed
the CD25+CD44
stage of DN thymocytes.
Morphodynamics of the Accumulation of TCR
As shown in Fig. 2, TCR+ cells
were readily detected on dg 14.5, and increased in density until dg 16.5, in parallel with a decrease in CD25+ cells. TCR
+
cells on dg 14.5 are dispersed as rare individual cells whereas on dg 15.5 some of them appear as doublets or as large dividing cells, indicating that cell division may have started.
Ex vivo thymic lobes later than dg 16.5 were not readily
analyzed by CLSM. Therefore, FTOC were set up with
dg 14.5 thymic lobes and cultured for an additional 4 d. Individual lobes were fixed on each day of culture and double stained with mAbs against CD25 and against TCR
(Fig. 3). The density of TCR
+ cells after 1 d of culture
was higher than on dg 14.5, but appeared lower than on dg
15.5 in ex vivo thymic lobes (see Fig. 2), and most TCR
+
cells were still dispersed individually with doublets rarely seen. On each of the following 2 d of culture, an increase
in the number of TCR
+ cells was seen, with local accumulations within the field of observation on day +3. A further increase in TCR
+ cells was seen on day +4, with the
cells distributed densely over the entire field of observation.
The intensity of CD25 staining and the number of CD25+
cells decreased in parallel with the increase in TCR
+
cells, slightly delayed compared to the ex vivo analyses.
The accumulation of TCR+ cells in FTOC was also
studied using mAb to individual TCR V
gene products.
Fig. 4 shows analyses using mAb to V
3 and V
6, which
gave the best signal to noise ratios. For both TCR V
gene
products, rare individual cells and a few doublets could be
detected on day +1 and cellular clusters were detected
from day +2 onwards. These clusters are interpreted as dividing TCR
clones. For unkown reasons, cluster morphology differed between clones expressing TCRV
3 and
TCRV
6, the former showing tightly packed and the latter showing more loosely associated accumulations of cells.
Attempts were made to estimate cluster sizes by a combination of counting cells in individual scans taken at 3 µ intervals, and by superimposing 3 µ scans from top to bottom through a cluster. The results are compiled in Table 2 and
suggest that clusters on day +2 range from 4 to 14 cells
with clusters of 6 to 8 cells predominating. On day +3 estimates were more difficult, suggesting an average of 30 to
40 cells/cluster. Estimates of cluster sizes on day +4 were
not possible, but a further increase in cluster size was apparent
(not shown). These data suggest that individual TCR
+
cells begin to proliferate in FTOC during day +1 and run
through at least five cell divisions within the following 2 d,
with additional cell divisions thereafter.
|
FTOC were
used to focus the cell cycle analyses on a population that
entered the thymus within a limited time window and was therefore expected to develop in a quasi-synchronous fashion. To this end, FTOC were set up on dg 14.5, and cells
were double stained with anti-TCR mAb and PI immediately and on each of 4 d of culture. As shown in Fig. 5,
TCR
+ cells contain high proportions of cells with hyperdiploid concentrations of DNA, characteristic of the S/G2
phases of the cell cycle. The proportion of TCR
+ cells in
S/G2 is highest on dg 14.5, and decreases only slightly until
day +4. This suggests that virtually all TCR
+ cells are in
cell cycle for the entire period of observation. The percentages of cells in S/G2 among TCR
cells are considerably
lower, and decrease from ~30% on dg 14.5 to <10% on
day +4 of culture. Nevertheless, these data show that on
dg 14.5 and during the initial days of culture, the TCR
subset contains a significant proportion of cycling cells as well. Proportions of blastoid cells were in good agreement
with cells in S/G2 in all cases. Note, from day +2 onwards,
small proportions of cells with hypodiploid DNA content
are seen among the TCR
, but not among the TCR
+
population. The detection of hypodiploid cells in only one
of the two subsets argues against a tissue culture artefact
(also see below) and suggests that TCR
cells may eventually die by apoptosis.
For labeling
with BrdU, we sorted TCR+ and TCR
cells from
FTOC preincubated with BrdU, and stained them with
anti-BrdU mAb after denaturation of the DNA by heating
to 90° (43). This method reliably permitted the differentiation of two distinct levels of BrdU incorporation, apparently corresponding to partial and saturating DNA labeling.
Since the results from CLSM suggested that TCR
+ fetal
thymocytes divide at least 2-3 times/day between days +1 and +3 of FTOC, we chose a labeling period of 18 h before analysis. During this time interval, nearly all asynchronously dividing cells with a cycle time of less than nine h
should have acquired a saturating BrdU label, whereas
longer cycle times should have been revealed by increasing
proportions of partially labeled and unlabeled cells. The results of representative experiments for each day are compiled in Fig. 6. TCR
+ cells on day +1 fell into three
categories: one third remained unlabeled, one third incorporated a partial, and one third incorporated a saturating
dose of BrdU. On day +2, the vast majority of TCR
+
cells had incorporated a saturating dose, whereas this proportion dropped to ~50% on day +3. On day +4, nearly
all of the TCR
+ cells showed partial BrdU labeling.
About 20-25% of the TCR
cells remained unlabeled
from day +1 to d +3, whereas the rest of the cells incorporated a partial BrdU label. By day +4, the proportion of
unlabeled TCR
cells increased to 50%. Together, these
results suggest that TCR
+ cells enter a phase of rapid proliferation on day +1 of FTOC with some of the cells going
through at least one division. Essentially all of the cells
progress through at least three divisions on day +2, and
most of the cells through another three divisions on day
+3. On day +4, most TCR
+ cells divide at least once
more while turning back to slow cycle conditions. Most of
the TCR
cells maintain a slow cycle throughout the first
3 d of culture with no more than one division per day and
a reduction in the proportion of cycling cells to 50% until
day +4.
The results of BrdU incorporation suggested that the
phase of rapid divisions of TCR+ cells only begins on day
+1 in FTOC (Fig. 6). In contrast, PI staining showed
>60% of TCR
+ cells to be in S/G2 already in dg 14.5 ex
vivo thymic lobes (Fig. 5). The incomplete BrdU incorporation into TCR
+ cells on day +1 of dg 14.5 FTOC
could indicate an intermittant slowdown of proliferation
due to the transfer of the thymic lobes to in vitro conditions. Alternatively, it was possible that TCR
+ cells remain in a prolonged S/G2 phase through dg 14.5 and most of dg 15.5 before actually undergoing their first division. In the latter case, only unlabeled and partially BrdU labeled
TCR
+ cells should be seen in dg 13.5 FTOC incubated
with BrdU for 18 h before analysis on day 13.5+1. As
shown in Fig. 7 A, the BrdU incorporation patterns after 1 d
of culture of dg 13.5 FTOC were similar to those of dg
14.5 FTOC, with an even higher proportion of TCR
+
cells labeled to saturation. These data suggest that dg 13.5 TCR
+ thymocytes have at least a similar capacity for proliferation in vitro as have dg 14.5 TCR
+ cells. Moreover,
we reasoned that if the onset proliferation of TCR
+ cells
in vivo was delayed until late dg 15.5, only the cells in
S/G2, and not the cells in G1 phase of cell cycle on that
day, should have incorporated BrdU injected into the
pregnant mothers 18 h earlier. As shown in Fig. 7 B, labeling in vivo was not as efficient as in vitro, and did not permit a quantitative determination of unlabeled, as compared
to partially and completely labeled, cells. Nevertheless, the
data show that both G1 and S/G2 phase TCR
+ cells on
dg 15.5 incorporated BrdU under these conditions in vivo.
These results support our conclusions from CLSM analyses and suggest that TCR
+ thymocytes start cell divisions as
early as dg 14.5 in vivo, and that transfer to in vitro conditions causes a lag in the onset of proliferation. The data in
Fig. 7 B further reveal that dg 15.5 and 17.5 TCR
cells
in both G1 and S/G2 phases of cell cycle incorporated
BrdU injected 18 h earlier into the pregnant mothers.
These data support the in vitro results in that they are consistent with a division rate of once per day for the major
proportion of TCR
cells. Finally, and also in support of
the in vitro results, dg 17.5 TCR
, but not TCR
+, cells
contain a proportion of hypodiploid cells suggesting ongoing cell death by apoptosis in this population.
The experiment in Fig. 7 C demonstrates that PCR
analysis yielded fragments corresponding to TCR VDJ
rearrangements in dg 14.5 thymocytes, together with stronger signals for DJ rearrangements and a strong germ line
fragment. In contrast to adult thymus, VDJ rearrangements
at dg 14.5 display only some of the six J
2 segments, presumably reflecting the paucity of rearranged TCR V
genes at that time. These data support our serological detection of TCR
+ cells in dg 14.5 thymocytes.
During thymic development, a large number of DP thymocytes arise from a small number of DN cells. While this process most likely involves cell proliferation, the extent of cell division cannot be judged from the pool sizes of DN and DP cells, as these are strongly influenced by parameters unrelated to cell division, such as entry rates, exit rates, and mean survival times (46). The present paper describes an attempt to obtain numerical information on cell divisions that occur during the generation of DP cells, using a combination of morphodynamic studies in situ with cell cycle analyses. To focus on cells that have entered the thymus during a defined temporal window in fetal life, most analyses have been done in thymic organ cultures. While it is clear that the increment in thymic cellularity during fetal life in vivo exceeds that in vitro, a proportion of the additional increment in vivo is obviously due to continuous influx of new cells into the thymus. Another proportion of the difference may be due to suboptimal conditions in vitro, so that numbers and rates of cell divisions determined in these studies obviously represent lower estimates. However, our comparisons of cell accumulation in vitro and in vivo localize the major deficit of FTOC to the first day of culture so that our data provide a reasonable basis for estimates of fetal thymocyte proliferation in vivo.
Differentiation of DN to DP cells is dependent on the
expression of the TCR chain, most likely as part of the
pre-TCR. Fetal thymocytes expressing TCR
polypeptides have first been reported on dg 15.5 (41, 42). We were
surprised to see TCR
+ cells as early as dg 13.5 by CLSM
and attribute the increased sensitivity to the capacity to
search for individual fluorescent cells by scanning through
the entire thymic organ. Since thymic repopulation begins
around dg 11 to 12, it appears that TCR
rearrangement may either be complete within 2 d of thymic arrival, or
that some cells may enter the thymus with previous TCR
VDJ rearrangements. The latter possibility is not excluded
by our data; although we never saw CD44+TCR
+ thymocytes on dg 13.5 or 14.5, biosynthesis of the TCR
chain may be delayed after rearrangement. However, the
developmental stage of TCR
gene rearrangement previously determined in adult mice (11) seems to hold for
fetal development as well (39, 40).
What are the proportions of TCR+ cells arising by de
novo induction and by subsequent proliferation? During dg
13.5 and 14.5, most TCR
+ thymocytes appear as individually dispersed cells in CLSM. On dg 15.5, a greater number of TCR
+ cells with many doublets are seen, followed
by a further increase in density on dg 16.5. Thus, according
to CLSM, little cell division occurs for at least one day after
thymocytes first express a TCR
polypeptide. We therefore think that the ~100 TCR
+ cells in dg 14.5 fetal thymic lobes almost exclusively arise by independent induction events. In situ analyses of the TCR
+ population in
FTOC shows maintenance of the individually dispersed appearance of TCR
+ cells until day +1, and BrdU incorporation suggests that about one third of these cells have
arisen by cell division. Together, these results suggest that a
minimum of 300 of the 400 TCR
+ cells/thymic lobe after one day of FTOC arose by independent induction
events. Our data from CLSM and from BrdU incorporation in vivo suggest that at least half of the 1,200 TCR
+
cells/thymic lobe on dg 15.5 in vivo may be the products
of cell division, suggesting a maximum of 900 independently induced TCR
+ cells until that time. Together, our
data suggest about 600, certainly less than the 1,000 independently induced TCR
+ cells/thymic lobe until dg 15.5. Thereafter, as shown by BrdU incorporation, TCR
+ cells
accumulate predominantly by cell division. Productive
TCR
locus rearrangement thus seems to take place in
<10% of the cells that have entered the thymus until dg
14.5, i.e., in a rather small proportion of thymocytes. Even
after subtraction of ~10%
thymocytes present at that
time, this is significantly less than statistically expected if all
remaining thymocytes were induced to rearrange their
TCR
genes. Induction of TCR
locus rearrangement may thus be a relatively inefficient process in early fetal
thymocytes.
From day +1 of FTOC onwards, TCR+ cells undergo
a series of rapid cell divisions resulting in a nearly 200-fold
expansion of this population within the following 3 d. This
expansion phase is indeed burst-like; it acquires maximum
speed (
3 divisions/d) on days +2 and +3, and returns to
a slow rate of about one-two divisions during the fourth
day. Estimates of cell divisions are derived from three mutually supportive sources of information: cell counts by
FCM, BrdU incorporation, and morphodynamic studies on the expansion of TCRV
clones in situ. The rates of
cell division estimated from the latter source are slightly
lower, possibly due to some degree of underestimation of
clone sizes in CLSM. Alternatively, clones expressing different TCRV
products may expand with somewhat different kinetics. The total accumulation of TCR
+ thymocytes in 4 d of FTOC is ~1/6-1/7 of that determined
until the corresponding dg 18.5 in vivo. Half of this difference is due to a slowdown in proliferation during the first
day of FTOC, leading to threefold less TCR
+ cells on
day +1 of culture than on dg 15.5 in vivo. Most of the other half can perhaps be accounted for by lack of influx of
new precursor cells into the thymus. Thus, we estimate an
about 500-fold expansion of TCR
clones during the burst
in vivo, corresponding to about nine cell divisions within a
time frame of ~4 d.
The biological purpose of pre-TCR dependent proliferation appears to be the selection for further maturation of
thymocytes with a functional rearrangement of the TCR
locus, thus avoiding the further differentiation of useless
cells that have failed to productively rearrange a TCR
gene. Our analyses of TCR
cells in FTOC reveals an
initial proportion of ~30% in S/G2 which declines to
<10% over the 4 d of culture. Moreover, an initial proportion of 3/4, declining to 1/2, becomes partially labeled with BrdU during an 18 h pulse. Both sets of data are
roughly in agreement and suggest that the majority of
TCR
cells are indeed dividing between dg 14.5 and
18.5, but they do so at a slower rate than that of the
TCR
+ cells. With the exception of dg 14.5 to 15.5, the
increment of TCR
cells/day is < twofold in vitro and
in vivo. This results in an ~8-14-fold increment in
TCR
cells until day +4 in FTOC or dg 18.5 in vivo,
consistant with one division/day of most, but not all cells.
We observed small but significant proportions of apoptotic
TCR
cells in vivo and in vitro, indicating that cells that
have failed to generate a productive TCR
rearrangement
eventually die by programmed cell death. The increase in
the proportion of nondividing TCR
cells over the 4 d is
consistent with the notion that shutdown of proliferation
preceeds cell death. Taken together, it appears that the major mechanism of pre-TCR-dependent positive selection of fetal thymocytes is a burst of up to nine rapid cell divisions that begins within less than two day after TCR
expression and lasts for about 4 d. In contrast, lack of selection of TCR
cells is reflected in a limited maintenance
of slow proliferation, then shutdown of proliferation, and
eventual cell death by apoptosis.
What is the cell cycle status of TCR+ cells on dg 13.5 and 14.5, i.e., before we can see doublet formation in
CLSM? As judged by PI staining >60% of TCR
+ cells
have entered S/G2 phase on dg 14.5, and TCR
+ cells in
G1 of cell cycle on dg 15.5 have incorporated BrdU during an 18 h pulse. These in vivo results suggest that TCR
+
cells may begin to divide as early as dg 14.5. This conclusion is further supported by our finding that many TCR
+
cells incorporate saturating amounts of BrdU on day +1 of
dg 13.5 FTOC. Nevertheless, our CLSM results suggest a
delay period between TCR
chain expression and the start
of cell proliferation. We could not exactly define this interval but feel that for most thymocytes the burst of rapid divisions does not start until about one day following expression of the TCR
chain, and that most of this delay may
be spent in the G2 phase of cell cycle. An interesting set of
questions arising from these results concerns the potential biological role of a delay before proliferation sets in: Does proliferation perhaps require a second signal, for example
through cytokine-cytokine receptor interaction (47). Is
the delay phase important for DNA repair subsequent to
TCR
rearrangement, and how is this delay phase regulated? Recent experiments suggest that p53 may perhaps
have an important role in regulating pre-TCR dependent
differentiation events (50).
How do these results on fetal thymocytes compare to
previous studies on proliferation of adult thymocyte subsets? According to Shortman et al. (35) the maturation of
DN to DP thymocytes is accompanied by two phases of
proliferation, separated by the CD25+CD44 DN stage in
which the cells are resting. The latter subset, at which
TCR
rearrangement takes place, was found by Penit et al. (36) and by Hoffman et al. (37) to be split into dividing and
nondividing cells. Although not directly shown, the dividing and nondividing subsets were interpreted as TCR
+
and TCR
cells, respectively, and it was speculated that
the latter cells would die due to lack of positive selection. A
200-300 fold expansion takes place during the subsequent
proliferation phase which begins at the CD25
CD44
DN
stage, ends with small resting DP cells, and lasts 6 d (35, 36).
As far as they can be compared, our results are roughly in
agreement with these data. For example, the numbers of
cell divisions and the expansion factors do not appear to
drastically differ between fetal and adult thymocytes. Moreover, the dividing TCR
subset in fetal thymus could
correspond to the first proliferative phase involving the immature CD44+CD25
population whereas the resting
TCR
subset in fetal thymocytes could potentially correspond to the nondividing subset among the CD25+CD44
adult DN cells. The results described in this paper have
augmented previous knowledge primarily by firmly establishing the causal connection between the second phase of
thymocyte proliferation and the expression of the TCR
chain, in addition to providing numerical information on
fetal thymocyte proliferation.
Address correspondence to Klaus Eichmann, Max-Planck-Institut für Immunobiologie, Stübewe, 51, D-79108 Freiburg, Germany.
Received for publication 8 August 1996
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