By
From the * Immunology Program, Memorial Sloan-Kettering Cancer Center, and the Graduate
School of Medical Sciences, Cornell University, New York 10021
T cell lymphopoiesis involves extensive cell division and differentiation; these must be balanced
by export and programmed cell death to maintain thymic homeostasis. Details regarding the nature of these processes, as well as their relationships to each other and to the definitive process of T cell receptor (TCR) gene recombination, are presently emerging. Two widely held
concepts are that cell cycle status is inherently and inversely linked to gene recombination and
that the outcomes of gene recombination regulate developmental progression. In this study, we
analyze TCR- recombination and cell cycle status with respect to differentiation during early
T cell ontogeny. We find that although differentiation, cell cycle fluctuations, and gene recombination are coincident during normal T cell development, differentiation and cell cycle status
are not inherently linked to the recombination process or its products. Rather, recombination
appears to occur in parallel with these events as part of a genetically patterned program of development. We propose that the outcome of gene recombination (i.e., TCR expression) may
not influence developmental progression per se, but instead serves to perpetuate those developing cells that have been successful in recombination. The potential consequences of this model
for the regulation of thymic lymphopoiesis and programmed cell death are discussed.
T cell production occurs in the thymus, a non-self-renewing lymphopoietic tissue that must be continually seeded
by bone marrow-derived precursors. In the young adult
mouse, an estimated 50-100 precursor cells enter the thymus each day (1). These cells undergo a complex process of
cell division and differentiation, requiring approximately 3 wk and resulting in about 1 × 106 progeny from each precursor (1). The resulting pool of cells is then screened for
TCR specificity and, if appropriate, selected for final maturation into T lymphocytes.
Historically, immature thymocytes have been organized
into two major groups based on surface expression of CD4
and CD8; less mature cells express neither of these markers
(CD4
Productive TCR- Mice.
C57BL/6 mice were either purchased from The Jackson Laboratory (Bar Harbor, ME) or were bred under specific
pathogen-free conditions at Memorial Sloan-Kettering Cancer
Center. RAG-1 Isolation of Immature CD4 Cell Sorting.
These procedures were performed essentially as
described (7). In brief, CD3/4/8-depleted thymocytes, or whole
thymic suspensions from recombination-deficient mice, were stained
with commercial antibodies against CD24 (PE conjugated; PharMingen, San Diego, CA) and CD44 (FITC conjugated; Caltag
Laboratories, South San Francisco, CA). Biotinylated antibodies
against CD25 (clone PC-61) were conjugated by the authors, and
were detected using allophycocyanin (APC)-streptavidin (Biomeda
Corp., Foster City, CA). Lymph node B and T cells were prepared by cell sorting using PE-conjugated antibody against CD24
(PharMingen) and FITC-conjugated antibody against CD90 (Caltag), respectively. Bone marrow macrophages were identified using anti-CD11b (biotin conjugate; Caltag) and PE-streptavidin. Nonviable cells were excluded during sorting through the addition of propidium iodide (PI; Molecular Probes, Eugene, OR) to
the sample buffer at 0.5 µg/ml. Cell sorting was performed using
a FACStarPlus® cell sorter (Becton Dickinson and Co., Mountainview, CA) equipped with argon ion and rhodamine dye lasers.
Southern Blot Analysis.
This procedure was performed as previously described (11). In brief, ~4 × 105 sorted cells were embedded in low melting point agarose plugs. Plugs were treated
with three successive cycles consisting of 12 h at 50°C in 50 mM
Tris, pH 8.0, 20 mM EDTA, pH 8.0, 1% sodium lauryl sarcosine
containing 1 mg proteinase K per ml, followed by 12 h at 4°C in
10 mM Tris, 1 mM EDTA. After the third cycle, proteinase K
was inactivated using 1 mM Pefabloc (Boehringer-Mannheim, Indianapolis, IN). RNAse and EcoRI endonuclease digestions were performed, and digested DNA was electrophoresed in 20 × 25-cm 0.6% agarose gels. Gels were depurinated, denatured, and transferred to nylon membranes. Hybridization was performed using
32P-labeled probes specific for noncoding regions between D Flow Cytometric Analysis of DNA Content by PI Staining.
All steps
were performed at 4°C unless noted. For DNA content analysis
of normal mice, freshly sorted cell suspensions were resuspended
in 50 µl of normal saline, and added to 1 ml of ice-cold 70% ethanol/H2O while vortexing. Fixation was allowed to proceed overnight. Fixed cells were pelleted by centrifugation and the supernatant was removed. DNA was nicked by incubation of fixed cells in 2 N HCl, 0.5% Triton X-100 for 30 min at room temperature. Acid was neutralized by washing the cells in 0.1 M
Na2B4O7, pH 8.5. Cells were washed twice in Dulbecco's PBS
(DPBS) containing 5% FBS and 0.5% Tween-20. Cells were resuspended in DPBS containing PI at 5 µg/ml and analyzed using
a FACScan® flow cytometer (Becton Dickinson). Relative cell
cycle distribution was calculated using Multicycle software (Phoenix
Flow Systems, San Diego, CA).
Sorting of Cells by DNA Content.
Purified populations of DN
III (generally 7-8 × 106 starting cells) were fixed, treated with
RNAse (1 µg/ml at room temperature for 30 min), then resuspended at 2 × 106 cells/ml in HBSS containing PI at 5 µg/ml.
Cells with 2n or >2n DNA content were identified and sorted
using PI-fluorescence area and width parameters and pulse processing software (Becton Dickinson). Sorted cells were used for
Southern blot analysis as described above.
To assess the relevance
of current paradigms regarding gene recombination, cell
cycle regulation, and developmental arrest during T cell
ontogeny (3, 17, 18), we first assessed the extent of specific
D-J and V-DJ recombination events at the TCR-
Some aspects of the cell cycle characteristics of
DN subsets have been previously described (for examples
see references 2, 14). However, a comprehensive evaluation of cell cycle distribution using standard DNA content analysis has not been published. To evaluate the relationship of cell division to gene recombination during T cell
developmental progression, we next assessed DNA content
and relative cell size in purified cells from each of the DN
divisions (Fig. 4). We find that cell cycle status fluctuates greatly during T cell development. The earliest DN stage
contains the smallest proportion of cells with >2n DNA
content, but nonetheless contains a fair proportion of cells
with >2n DNA. The next stage of development (DN II)
apparently contains a large proportion of cycling cells, with
~40% of all cells in S, G2, or M phases of cycle; not surprisingly, these cells are slightly larger than their more
slowly cycling precursors. Cells at the DN III stage of development are the first cells with detectable TCR-
To assess whether the
fluctuations in cell cycle distribution seen during normal
T cell development (Fig. 4) were related to recombination,
we performed a comparative assessment of cell cycle distribution in mice that cannot rearrange antigen receptor loci
due to targeted disruption of RAG-1 or RAG-2 genes (22, 23). Because these mice have an apparent block in development at the point where a TCR-
Cells undergoing V-DJ
Rearrangement of antigen receptor genes requires both
RAG-1 and RAG-2 gene products (26). The observation
that stable RAG-2 activity is restricted to cells with 2n DNA
content, together with a variety of other evidence (17), has
led to the general speculation that recombination occurs
during or induces a state of cell cycle arrest in G1 (i.e., G0).
This concept has great appeal for a number of reasons. First,
limiting recombination to G0/G1 would prevent the asymmetric distribution of chromosomes that could occur if recombination-induced DNA strand breaks persisted during
mitosis (17). Second, the preponderance of cells with 2n
DNA content (2, 11, 13; Fig. 4) at the time of V-DJ The presumption of cell cycle arrest during recombination, together with appearance of some cycling cells (i.e.,
cells with >2n DNA) at DN stage III in normal mice, has
led others to propose that the cycling (in this case, blastic)
contingent represents cells with productive TCR- Our data regarding the timing of D-J In the light of current findings, it may be worthwhile to
reconsider the nature of the T cell differentiation process as
a more generic one in which antigen receptor gene rearrangement, although definitive, is only one of a multitude
of coincidental developmental processes. Our data show that
in normal mice, D-J rearrangement precedes the stage at
which development is arrested in thymocytes from recombination deficient mice (i.e., DN III; see Fig. 3). This suggests that both phenotypic progression and proliferative
status can be regulated independently of individual developmental deficiencies, such as those TCR-8
, double negative, DN)1, while more mature cells
express both (CD4+8+, double positive, DP). These two
groups of immature cells account for ~3% and 80% of thymic cellularity, respectively; the remaining 15% of thymocytes are cells with a mature phenotype. Within each of
these major categories, additional distinctions can be made. In the case of DN cells, four subsets can be detected based
upon surface immunophenotype (2, 3). Cells at the DN
stage are characterized by early high level expression of
CD44, and by transient expression of CD25; all the lymphoid components are also CD24+. Thus, the developmental progression is CD25
44+
CD25+44+
CD25+
44lo
CD25
44lo; for the sake of brevity, these will subsequently be referred to as DN subsets I-IV, respectively.
DN subsets I and II each represent ~5% of all DN cells (see
Fig. 1), although the exact proportion of DN II is arbitrary,
because this subset is not completely distinct from DN III.
DN III represents ~60% of all DN cells (Fig. 1). However,
this proportion is also technically misleading, because the
final subset (DN IV, representing an apparent 35% of DN)
must be considered to represent incipient DP cells for several reasons. First, such cells already express trace but distinguishable levels of surface TCR-
and CD3 (4), CD4, and
CD8 (5, 6). Second, they rapidly acquire the phenotype of
cortical DP thymocytes in vitro without additional stimulation (5, 7). Finally, these cells have been selected for possessing productive TCR-
genes (8), a process that is
thought to mediate the DN-DP transition (9). Thus, DN
IV cells actually represent very early DP cells that segregate
together with DN during purification, owing to minimal
surface expression of the proteins used for depletion (i.e.,
CD4 and CD8). This phenomenon is fortuitous, as it permits the purification of cells very early after the DP transition, as is described here and previously (4, 7, 10, 11).
Fig. 1.
Surface phenotypic
analysis of CD48
thymocytes.
(Top) CD24 histogram for thymocytes depleted of cells expressing CD3, CD4, CD8, myeloid, and erythroid surface
antigens. (Bottom) CD25 and CD44 expression on cells gated
as being CD24+. The relative
proportions of each population
(dashed lines) and the approximate locations of gates for cell
sorting (rectangles) are shown (bottom).
[View Larger Version of this Image (22K GIF file)]
gene recombination is known to
correlate with the transition from DN to DP (9). TCR-
recombination is thought to proceed in several steps, with
D-J
rearrangements preceding those of V-DJ
(12, 13),
although the developmental stages corresponding to these
steps can only be inferred from such studies. It has also
been shown that cells undergoing TCR-
recombination
(3, 11) are relatively slowly dividing (2, 14). These observations, and a multitude of other studies (17), have led
to a general speculation that recombination requires a state
of cell cycle arrest, that recombination and cell cycle progression are mutually exclusive, and that cell cycle induction therefore may mediate allelic exclusion. Here, we test
this hypothesis by comparing relative cell cycle distributions
in developing thymocytes from normal and recombinationdeficient mice. We find that cell cycle fluctuations during
development are regulated normally in mutant mice, despite the failure to initiate gene recombination. Further, we
find that positive regulation of cell cycle progression among
cells undergoing recombination is not restricted to cells possessing productive gene rearrangements, as has been proposed (18). Together, our findings suggest that while fluctuations in cell cycle distribution occur simultaneously with
differentiation, neither of these are regulated intrinsically by
the gene recombination process.
/
and SCID mice were purchased from The Jackson Laboratory. RAG-2
/
mice were purchased from Taconic
Farms, Inc. (Germantown, NY). All mice used for experiments
described in this manuscript were 5-8 wk of age.
8
Thymocytes.
For the experiments
described herein, our previously described method for the isolation of early T cell precursors (7) was revised; all isolation steps,
including cell sorting, were performed at 4°C to minimize biochemical changes within the cell and maximize similarity to the
in vivo state. The initial steps involving the preparation of cell
suspensions and staining with antibodies to CD4, CD8, myeloid,
and erythroid surface markers were as described (7). However,
rather than incubation at 37°C with rabbit serum complement for
depletion of antibody-positive cells, treated cells were incubated
on ice for 25 min with anti-immunoglobulin-coated paramagnetic beads (PerSeptive Biosystems, Framingham, MA) at a ratio
of 5 beads/cell. Positive cells were then depleted with a strong
magnet. An additional selection against small cortical thymocytes
and nonviable cells was performed by centrifugation of depleted
cells for 10 min at 1,100 × g in mouse iso-osmolar density gradient medium (Nycodenz; GIBCO BRL, Gaithersburg, MD) at
1.087 g/cc. Finally, a third round of depletion using different antibodies against CD3, CD4, and CD8, followed by a second type of anti-immunoglobulin-coated paramagnetic beads (Dynal, Oslo, Norway), was performed as described (7). Cells recovered in this
manner represented ~2-3% of the initial cell number; all cells recovered were large lymphoblasts. The proportions of CD4
8
subsets, after staining with a definitive panel of monoclonal antibodies (Fig. 1), were identical to those described previously (7).
1
and J
1.1, D
2 and J
2.1, or V
7. The latter is the secondmost
D
-proximal V
gene region, and is deleted in the majority of
V-D
rearrangements (8). Thus, deletion of the genomic targets
for these probes can be used quantitatively to assess gene recombination. Lane-to-lane loading was corrected by including a
probe hybridizing to a noncoding region located 3
of the fourth
(untranslated) exon of the C
cluster, as described (11). Probe hybridization was quantitated by phosphor-screen imaging using a
GS-323 Molecular Imager and Molecular Analyst software (BioRad Laboratories, Hercules, CA). Scanning densitometry of x-ray
films was also performed, using the same instrument.
Quantitative Assessment of D-J and V-DJ
Gene Recombination during T Cell Development.
locus,
using multigene genomic Southern blotting as previously described (11). This method allows the quantitative measurement of multiple gene loci simultaneously, without the
potential for bias that may be induced by amplificationbased technologies (i.e., PCR). Probes hybridizing to the
intervening DNA sequence between D-J
1 and D-J
2
(see Fig. 2), and a probe hybridizing near the 3
end of the
V
cluster (V
7; reference 8) were used to analyze deletions of the corresponding regions during gene recombination. A probe for a nonrearranging DNA sequence (in this
case, noncoding DNA located 3
of the C
locus) was used
as a lane-to-lane loading control. A variety of somatic cells
were used as genomic controls, including lymph node B cells,
bone marrow macrophages, and SCID mouse kidney cells; all
genomic tissue controls gave comparable quantitation, and in
general lymph node B cells were used due to ease of preparation. The results of quantitative genomic Southern blotting are presented in Fig. 3. DN subsets I and II express germline allotments of all recombining regions, demonstrating that no rearrangements have occurred. As previously shown (11), DN subset III exhibits extensive D-J
recombination, representing an almost complete deletion
(~90% of alleles rearranged) of the intervening sequence
between D
1 and J
1, and a less extreme but nonetheless
substantial deletion (65-70%) of the corresponding region
between D
2 and J
2. By contrast, only limited V-DJ
recombination is seen at the DN III stage, representing
<10% of all alleles on average; the significance of this finding is discussed later in the manuscript. These levels of recombination (i.e., extensive D-J
, limited V-DJ
) are
substantiated by the differential in immature (1.0 kb) versus
mature (1.3 kb) TCR-
mRNA seen in DN III extracts
from either normal (2) or CD3
/
(19) mice. Productive
TCR-
gene rearrangement is a prerequisite for further
development into DN IV and DP cells (8, 9). The extent
of deletion of the 3
V
region in DN stage IV (~60% of
alleles rearranged) correlates roughly with the levels predicted after such selection (~70%; reference 20); exact correlation is seen in mature T cells. This pattern suggests that
some excised recombination products (~10% of the total,
by our data) may still persist immediately after the transition to DN stage IV, but are ultimately degraded before the
time of terminal differentiation. In this respect, it is important to note that the hybridization signals measured in DN
III cells are unlikely to be affected substantially by excised
DNA products for several reasons. First, as mentioned above,
all recombining regions (i.e., D-J or V-DJ) showed a precipitous drop in quantitation at some stage, followed by
relatively stable expression, regardless of the rate of subsequent cell division. This suggests that most DNA recombination products are fairly rapidly degraded, and that cellular
proliferation preferentially replicates chromosomal DNA.
Second, we are unable to detect any significant levels of hybridization of V
7 probe to sub-genomic bands in undigested DNA from DN I (unrearranged) or DN III (actively rearranging) cells, where a 130-kb control marker (courtesy
of G. Bannish, Memorial Sloan-Kettering Cancer Center,
New York) is clearly distinguishable (data not shown).
Taken together, our findings suggest that D-J
recombination occurs before the acquisition of a DN III phenotype,
while V-DJ
recombination occurs substantially later, during the 2-3-d period of residence at the DN III stage, but
before transition to DN IV. This finding contradicts a popular model, which speculates that D-J and V-DJ recombination occur together in a rapid burst (3), but is supported
by a number of other findings, as discussed further below.
Fig. 2.
Schematic representation of the murine TCR- locus. The approximate locations of coding sequences are filled in black. The approximate
sizes and locations of EcoRI fragments of genomic DNA that hybridize to probes detecting D-J
or V-DJ
rearrangements are indicated by large shaded
rectangles. Actual probe sites are indicated by arrows. Drawing is not representative of actual scale.
[View Larger Version of this Image (6K GIF file)]
Fig. 3.
Southern blot analysis of TCR- gene recombination. A typical autoradiographic
film image of a Southern blot is
shown in A. Probes for D-J
1,
D-J
2, and V
7 hybridize with
genomic DNA fragments of
~10, 2.4, and 2 kb, respectively.
A probe for a nonrearranging, noncoding DNA sequence was
used as a control for lane-tolane loading, as described (11). B
shows phosphor-screen densitometric analysis of this and other
Southern blots, including bone
marrow macrophages (unrearranged) and lymph node T cells
(fully rearranged) as controls. The
relative intensity of the genomic
loading signal is standardized to
allow direct visual comparison of
the other signals. C graphically depicts these quantitative changes in genomic DNA after TCR-
recombination, as assessed by phosphor-screen imaging. D-J
recombination is essentially complete upon the transition from DN II to DN III. However, relatively little V-DJ
recombination (~10%
of all alleles) is noted at this stage. V-DJ
recombination is finally completed upon transition to DN IV, as predicted from other studies (8, 17). Data
points represent the mean ± SE for three (D-J
) or four (V-DJ
) individual experiments for DN subsets I and II, and six individual experiments for
all loci at all later stages of development.
[View Larger Version of this Image (33K GIF file)]
gene rearrangements (see Fig. 3); this stage also has fewer cells
with >2n DNA content than its precursors or progeny,
and has the smallest average cell size of the DN subsets.
Cells at DN stage IV, which are post-TCR-
selection,
and which have ostensibly received a signal through the
pre-T cell receptor (21), are rapidly dividing large blast
cells, with the majority of cells (>50%) possessing >2n
DNA. This number is substantially higher than that measured by others (14, 18); we attribute these differences
to methodological discrepancies (e.g., multiple versus single
enrichment steps, different animal strains), and/or in the
types of analysis performed (e.g., PI staining versus BrdU
incorporation). In any case, because all other subsets of thymocytes are more slowly cycling, inflation of this measurement by a contaminating population is not possible, suggesting that this is an accurate assessment of cell cycle status in the very earliest cells after the DP transition.
Fig. 4.
Cell cycle status of CD48
thymocytes. (Left) Relative cell
size (forward light scatter) analysis among DN cells gated on CD24, 25, and 44, as appropriate. (Center) PI analysis of DNA content in purified
DN subsets I-IV. The relative locations of the diploid (2n) and tetraploid
(4n) peaks are indicated on the thymus control. (Right) Pooled cell cycle
distributions (mean ± SD) from n experiments, as indicated.
[View Larger Version of this Image (34K GIF file)]
chain is required, we
concentrated on DN subsets II and III, which are germline
or fully D-J
/partially V-DJ
rearranged in normal mice,
respectively. DN subset II cells from both types of mutant
mice showed proportions of cells with >2n DNA content
that were indistinguishable from controls (Figs. 5 and 4, respectively). Despite the inability to initiate recombination, an event that is characteristic of the transition from DN II
to DN III in normal mice (see Fig. 3), developing thymocytes from mutant mice acquire the DN III phenotype
and coordinately decrease cell cycle status. The relative
number of DN III cells with >2n DNA content in mutant
mice is slightly lower than in normal mice (~10% versus
20%, respectively), as has been observed previously with
this subset (15, 16, 24, 25). Nonetheless, substantial levels
of cells in cycle are seen among DN III cells from mutant mice. Analysis of SCID and TCR-
/
mice, which have
defects in other aspects of recombination, gave virtually
identical results (data not shown). Taken together, the data
presented in Figs. 3, 4, and 5 show that the induction of a
slowly dividing state at DN stage III is not dependent upon recombination, that DN III cells are not arrested in cell cycle, and that induction of cell cycle among cells with a DN
III phenotype is not dependent upon gene recombination
or the presence of a TCR-
chain.
Fig. 5.
Cell cycle status in early thymocytes from recombinationdeficient mice. A representative histogram for DNA content of purified DN II or DN III cells (i.e., before and during TCR- recombination in
normal mice) is shown in each panel, together with analysis of relative cell
cycle distribution (mean ± SD) for n experiments, as indicated. Cell cycle
fluctuates normally with respect to development in these mice, despite
the lack of recombination-induced DNA strand breakage.
[View Larger Version of this Image (18K GIF file)]
Recombination in Cells Undergoing Cell Division.
recombination (i.e., DN III; see
Fig. 3) are apparently more slowly dividing that their precursors (DN II) or progeny (DN IV; see Fig. 4); consistent
with this cell cycle status, cells at this stage are generally
smaller than other DN counterparts (see Fig. 4). Recently,
it has been proposed that the larger (i.e., blast) component
within this subset represent cells that have been selected for
productive TCR-
gene rearrangements, which are being
clonally expanded in conjunction with the transition to DP
cells (18). This proposal implies that such blast cells would
possess TCR-
gene rearrangements that quantitatively resemble post-TCR-
selection cells (i.e., DN IV, DP, and
mature T cells) more than they do the remaining majority
of small DN III cells. To test this, we performed quantitative genomic Southern blotting for TCR-
rearrangements in cells of the DN III phenotype, which have been
segregated based on DNA content. We have chosen DNA content as a discriminatory marker in place of cell size; because gating based on cell size alone is subjective (reference
18; Fig. 6), while gating on both size and DNA content is
fairly precise (Fig. 6 a). Further, we find that a blastic morphology and >2n DNA content are coincident events at
this stage (Fig. 6 a). The results of our experiments show
that TCR-
gene recombination among DN III blasts is
not significantly different than that found in smaller (2n
DNA) DN III cells, or among the DN III subset in general.
This is consistent with the finding of cycling cells at DN
stage III in recombination-deficient animals (see Fig. 5), as
well as previous descriptions of limited self-replicating cell division within the DN III subset (1, 14, 16), and suggests that blastogenesis per se is not unequivocal in defining
post-TCR-
selection cells.
Fig. 6.
TCR- gene recombination in DN III cells segregated by DNA content/cell size. A shows relative cell size (forward light scatter) versus
DNA content among sorted cells of the DN III phenotype; blastogenesis is clearly coincident with increased DNA content, as expected. B shows a DNA
content histogram of such cells before and after sorting by DNA content. C shows Southern blot analysis of these cell types for TCR-
gene recombination, as described in Fig. 3; quantitation of this blot, which is representative of two such experiments, is shown in D. TCR-
gene recombination is
not quantitatively different between the smaller (2n DNA) and larger (>2n DNA) cells of this phenotype, suggesting that blastogenesis is not prognostic
of productive TCR-
rearrangement.
[View Larger Version of this Image (22K GIF file)]
gene
recombination (Fig. 3) stands in stark contrast with the preceding and subsequent stages of development, and is reminiscent of a state of arrest. Finally, such a mechanism could
be used to explain how the TCR-
locus is allelically excluded, by proposing that the induction of cell cycle in arrested cells (ostensibly through pre-TCR signaling) would
effectively prevent further recombination. However, we
show here that recombination is not required for either
positive or negative regulation of the cell cycle, because
DN III cells from recombination-deficient mice decrease
their proliferative rate much like their normal counterparts,
but nonetheless divide at a substantial rate (Fig. 5). DN III
cells with >2n DNA content cannot represent the residual
proliferation of DN II cells recently transiting to DN III if
recombination is restricted to the 2n DNA stages of cycle
(17) and D-J recombination has already occurred (Fig. 3).
Therefore, we must conclude that DN III cells continue to
divide while undergoing recombination, albeit at a slower
rate. This is consistent with previous studies demonstrating
limited self-renewing cell division at the DN III stage (1,
14, 16). In reality, the need to invoke a state of arrest (i.e.,
G0) during antigen receptor gene recombination is superfluous, because DNA strand breakage and coding joint religation appear to occur very rapidly (27), and therefore are
easily accommodated by the length of G1. It should also be
pointed out that unlike other processes resulting in DNA
strand breakage (e.g., radiation), which require a period of
time (i.e., arrest) to initiate the transcription of repair enzymes (28), the rate of antigen receptor gene coding joint
religation (27) suggests that the necessary enzymes already exist in developing lymphocytes. Moreover, we have previously shown that approximately half of the RAG-1 mRNA
in CD4+8+ thymocytes is contained within a small proportion (10-15%) of rapidly dividing CD4+8+ blasts (10). Because these cells divide approximately once every 7-8 h despite active TCR-
gene recombination (11), it is difficult
to imagine that they undergo a state of arrest. These arguments are not meant to imply that antigen receptor gene
recombination is not restricted to the 2n DNA stage of cell
cycle. However, our findings suggest that a state of arrest is
unnecessary for this process and, further, that fluctuations
(upward or downward) in cell cycle status during T cell development occur independently of both the recombination
process and its products.
rearrangements that are undergoing the DN to DP transition
(18). The biological rationale is that recombination requires
a state of cell cycle arrest, and that induction of cell cycle
through the pre-TCR (21) thus would enforce allelic exclusion. This conclusion is inconsistent with data showing
that neither targeted mutation of the pre-TCR (29) nor the
inhibition of RAG-2 degradation upon S phase entry (30) influences allelic exclusion. Nonetheless, were this the case, then TCR-
rearrangements in such cells should resemble
that of more mature cells (i.e., extensively rearranged at
both D-J and V-DJ
loci). In contrast, we find that DN III
cells with >2n DNA content are quantitatively very similar
to their 2n DNA counterparts in terms of TCR-
gene rearrangement (i.e., few complete rearrangements; see Fig.
6), suggesting that they have not been selected to divide on
the basis of TCR-
expression. It is relatively easy to reconcile these apparently contradictory findings through several lines of reasoning. Most important is the demonstration
that self-replicating cell division (i.e., proliferation without
differentiation) clearly occurs among DN III cells (1, 14,
16). Because we have shown that very few TCR-
alleles
are fully rearranged at this stage (Fig. 3), it is therefore implicit that DN III blasts will include many cells that have
not made V to DJ
rearrangements. The PCR-based approach used by Hoffman et al. (18) amplifies only fully rearranged genes (8); thus, the measured frequency for inframe or out-of-frame rearrangements may be quite correct for those blasts possessing complete rearrangements. However, this assay would not detect the majority of dividing
blasts, which have not fully rearranged V to DJ
. Further,
because hyperphosphorylation of pRb, increased cyclin A,
cyclin B, CDK2, and cdc2 levels, and decreased p27 levels,
are all hallmarks of cells in S, G2, and M stages of cycle, it is
not surprising that these markers were observed in DN III
blasts (18), whether or not they had undergone V-DJ
recombination. We believe that blastogenesis per se is not diagnostic of the TCR-
selection process at DN stage III,
and that blastic cells at this stage probably represent a combination of both autonomous replication and the induction
of the DN III
DN IV transition.
and V-DJ
recombination during T cell ontogeny reveal other interesting clues about this process. Based on the assessment of
gene rearrangement in hybridomas (12, 13), D-J recombination has been thought to precede that of V-DJ. The time
lag between these two processes during adult lymphopoiesis has not been measured, although studies of fetal thymocytes suggest a delay on the order of 1-2 d (13, 31).
Therefore, it is surprising to find that these events have been proposed to occur in a single burst at DN stage III (3). Our data support the existence of a substantial lag between
D-J and V-DJ
recombination (Fig. 3), which is reflected
by an entire developmental stage (i.e., DN III), the mean
lifespan of which has been measured at 2-3 d (1). This period of development may be required for the execution of
maturational programs unrelated to recombination; temporal regulation of development that is independent from recombination has already been shown, by demonstrating
that a rearranged TCR-
transgene does not substantially
accelerate the kinetics of T cell development (16, 32). One
explanation for this delay may be related to the intrathymic
divergence of TCR-
/
and TCR-
/
cells (33), allowing time for the competitive rearrangement of TCR-
,
TCR-
, and TCR-
genes (31, 33); another may simply be the expansion of a pool of cells that are about to rearrange V-DJ
. These possibilities and others are presently being evaluated in our laboratory.
expression. Like most other developmentally important genes, only when a
gene product becomes obligatory (in this case, at the DN
III
DN IV transition) does such a deficiency become apparent. The observation that most recombination-deficient
mouse models are leaky, allowing some cells to transit to
the DP stage despite the lack of required gene products
(i.e., TCR-
), further demonstrates the uncoupling of developmental progression from recombination. In this regard, it is tempting to suggest that the signal delivered
through the pre-TCR may not directly induce proliferation, but rather transduces a survival signal in those cells
with a productive TCR-
gene. In such a model, the induction of a rapidly cycling phenotype would be developmentally programmed to occur after DN III maturation is
complete (2-3 d), roughly corresponding to the time of
V-DJ
gene recombination. All cells would then be induced to proceed with further development, i.e., into DN
IV and DP cells. However, only those cells which receive
appropriate survival signals (such as through the pre-TCR)
would be perpetuated, with the remainder falling victim to
cell cycle, i.e., activation-associated, programmed cell death.
This model eliminates the need for a cellular decision based
on whether or not a specialized differentiation signal is received, and allows both the selection of functional cells and the elimination of nonfunctional ones by the same mechanism. Further testing of this hypothesis will require the evaluation of cell division status in leaky cells from recombination-deficient animals; such studies are currently underway.
Address correspondence to Dr. Howard T. Petrie, Memorial Sloan-Kettering Cancer Center, Box 341, 1275 York Avenue, New York 10021.
Received for publication 15 October 1996 and in revised form 10 February 1997.
The authors are deeply grateful to Dr. F. Livak for many helpful discussions and for preliminary review of the manuscript.This work was supported by research grant R29 AI 33940 from the National Institutes of Health (to H.T. Petrie), and by Cancer Center Support Grant NCI-P30-CA-08748 (to Memorial Sloan-Kettering Cancer Center).
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