Differential Requirement for p56lck in Fetal and
Adult Thymopoiesis
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of June 18, 2017.
Thierry J. Molina, Jean-Yves Perrot, Josef Penninger, Amélia
Ramos, Josée Audouin, Pascale Briand, Tak W. Mak and Jacques
Diebold
J Immunol 1998; 160:3828-3834; ;
http://www.jimmunol.org/content/160/8/3828
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References
Differential Requirement for p56lck in Fetal and Adult
Thymopoiesis1
Thierry J. Molina,2*† Jean-Yves Perrot,* Josef Penninger,‡ Amélia Ramos,* Josée Audouin,*
Pascale Briand,† Tak W. Mak,‡ and Jacques Diebold*
T
hymocyte differentiation occurs in a series of stages defined by the expression of various surface markers (1).
Immature thymocytes progress from the double negative
(DN,3 CD42CD82) to an immature single positive (ISP,
CD81CD42CD32) stage and then to a double positive stage (DP,
CD41CD81). Cells at this latter stage constitute the major thymocyte population from the end of fetal life. DP thymocytes undergo positive and negative selection and differentiate into mature
CD41CD82 or CD42CD81 single positive (SP) thymocytes,
which are exported from the thymus. Based on surface expression
of CD44 and CD25, the DN population can be classified into four
subpopulations. The pathway of differentiation has been defined as
follows (2): CD441CD252, CD441CD251, CD442CD251, and
CD442CD252.
It has been shown that thymocyte maturation does not occur at
a steady linear pace state but in discrete waves (3). Thus, the first
phase of active growth in the thymus ends on fetal day 18 to 19,
which is followed by a plateau phase during the perinatal period.
Starting at about postnatal day 3, there is a second growth phase
until postnatal day 14 to 16, when the thymus has reached its
maximum size. At 8 to 10 wk of age, thymic involution
commences.
*Department of Pathology, Hôtel Dieu de Paris, AP-HR, Paris, France; †Unité 380,
Institut National de la Santé et de la Recherche Médicale, Génétique et Pathologie
expérimentale, Institut Cochin Génétique Moléculaire, Paris, France; and ‡Amgen
Institute, Ontario Cancer Institute, Toronto, Canada.
Received for publication March 26, 1997. Accepted for publication December
22, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grant No. 6933 from Association pour la Recherche sur
le Cancer, Villejuif, France.
2
Address correspondence and reprint requests to Dr Thierry J. Molina, Department of
Pathology, Hôtel Dieu de Paris, 1 place du Parvis Notre Dame, 75004 Paris, France.
Abbreviations used in this paper: DN, double negative CD42CD82 thymocytes;
ISP, immature single positive CD42CD81 thymocytes; DP, double positive
CD41CD81 thymocytes; PE, phycoerythrin.
3
Copyright © 1998 by The American Association of Immunologists
Analysis of this process in adult mutant mice generated by homologous recombination has identified a series of genetic checkpoints in thymocyte maturation. Mutation of the recombinase
genes (RAG1, RAG2) (4, 5), of CD3e (6), or a double mutation of
TCR-b 3 TCR-d (7), all lead to a block in the differentiation at the
immature DN CD251CD442 stage. On the other hand, mutations
of the TCR a-chain (7, 8), or of the accessory molecules CD4 (9)
and CD8 (10), did not modify the size of the DP population but
have profoundly affected the transition from the DP to mature SP
stage. Finally, mutation of CD3z (11–13) or lck (14) gave rise to
an intermediate phenotype characterized by the generation of 5 to
15% of the normal number of DP thymocytes when compared with
control littermates. The ability to generate normal numbers of DP
in RAG12/2 3 CD3z2/2 after anti-CD3e treatment (15), as well
as in mice transgenic for a signaling deficient CD3 z-chain (16),
suggests that CD3z is probably involved in this phase of maturation through TCR surface expression and not as a signaling molecule. On the other hand, only 10% of DP were generated in
RAG22/2 3 lck2/2 mouse after anti-CD3e treatment (15), showing that lck is important for the transduction of signals for DP
expansion. These observations imply that lck and CD3z have different roles during this stage of thymocyte maturation.
The prereceptor T a-chain (17) was identified as a TCR chain
expressed on late stages of the double negative population. This
chain may function as part of pre-TCR (pTab), which transduces
signals in CD251CD442 DN thymocytes required for the downregulation of CD25 and the expansion and differentiation of DN
cells into CD41CD81 thymocytes. A role for lck as a principal
signaling molecule involved in pre-TCR signaling was suggested
by the fact that the overexpression of lck in RAG12/2 mice promoted expansion of the DN population and the generation of DP
thymocytes (18). However, the presence of DP thymocytes in the
adult lck2/2 mutant mice indicates that lck is not essential for the
DN to DP transition (14).
Only few studies have adressed the effect of targeted mutations at
the different waves of thymocyte maturation during fetal and postnatal
life. To further assess the importance of lck signaling during the early
phases of thymocyte maturation, we have analyzed the kinetics of
0022-1767/98/$02.00
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The protein tyrosine kinase p56lck is critical for the generation of mature thymocytes in adult mice. However its requirement
during the maturation of thymocytes from the fetal to the adult stage has not been clearly defined. We analyzed prenatal and
postnatal thymocyte maturation in mice deficient for p56lck (lck2/2). Before birth, lck appears to play a crucial role in the
expansion and proliferation of CD41CD81 double positive thymocytes, whereas proliferation and absolute numbers of
CD42CD82 double negative thymocyte precursors remained within the normal range until the end of the second week postnatal.
Three weeks after birth, the total numbers of double negative and immature single positive thymocytes underwent a dramatic
reduction that correlated with a decrease in the double positive population. This ontogenic defect was associated with a significant
decrease in the proliferation rates of thymocyte precursors. Our data suggest that signaling via p56lck kinase is differentially
required within a given phenotypically defined thymocyte subpopulation, depending on its stage of thymocyte maturation. The
Journal of Immunology, 1998, 160: 3828 –3834.
The Journal of Immunology
3829
thymocyte differentiation during its various waves in lck2/2 mutant
mice. We report that lck has differential roles during fetal and postnatal maturation. During fetal development, lck is required for the
expansion of DP but has no apparent involvement in the differentiation and proliferation of DN precursors. However, after 3 wk of birth,
lck becomes an important regulator of DN proliferation. These results
demonstrate that lck kinase activity is differentially required during
distinct phases of thymic ontogeny.
Materials and Methods
Mice
Mice mutant for lck (lck2/2) have been previously described (14). Adult
mice between 6 and 10 wk of age were used in experiments. Timed matings
were performed to facilitate the analysis of fetal thymocyte development.
The day on which a vaginal plug was observed was taken as day 0 of
gestation. All mice were maintained in accordance with institutional
guidelines.
Monoclonal Abs and flow cytometry
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PE- or TRICOLOR-conjugated anti-CD4 mAb (CT-CD4), FITC- or TRICOLOR-conjugated anti-CD8a mAb (CT-CD8a), PE-conjugated antiTCR-ab (57–597), and anti-gd TCR (GL3) were purchased from Caltag
(South San Francisco, CA). Monoclonal Abs against CD25 (PC-61; FITCconjugated) and CD44 (JM7.8; PE-conjugated) were purchased from
PharMingen (San Diego, CA). Single cell suspensions of thymocytes were
prepared in HBSS and resuspended in staining buffer (PBS, 2% FCS, 0.1%
NaN3). Cells were stained with mAbs for 30 min at 4°C in staining buffer.
To analyze the expression of CD44 and CD25 on the DN thymocyte subset,
thymocytes were incubated with anti-CD4 TC, anti-CD8 TC, anti CD25
FITC, and anti-CD44 PE. Samples were analyzed on a FACScan by gating
on CD42CD82 viable cells and analyzing CD25 and CD44 staining. To
analyze the immature single positive subset, thymocytes were incubated
with anti ab-PE, anti CD8-FITC, and anti CD4-TC; samples were gated on
CD42CD81 cells and TCR levels were analyzed in lck1/2 mice to differentiate immature from mature single positive thymocytes. Data were acquired in list mode and analyzed using Lysis II software (Becton Dickinson, Cockeysville, MD).
In vivo BrdU labeling
Pregnant mice and adult mice were i.p. injected with 2 mg BrdU (Sigma
Chemical, St. Louis, MO) in PBS. For 2 week-old mice, only 1 mg was
injected in each mouse. Thymi were harvested 2 h after injection and processed as previously described (19). Briefly, cells were first stained either
with a mixture of PE-conjugated anti-CD4 and TRICOLOR-conjugated
anti-CD8a or PE-conjugated anti-gd and TRICOLOR-conjugated antiCD4 and anti-CD8 to gate out gd cells in the DN subset. Cells were then
washed and resuspended in cold 0.15 M NaCl, and fixed by dropwise
addition of cold 95% ethanol. The cells were incubated for 30 min on ice,
washed with PBS, then incubated with PBS containing 1% paraformaldehyde and 0.01% Tween-20 for 1 h. Cells were pelleted, and resuspended in
50 Kunitz units DNase I (in 0.15 M NaCl, 4.2 mM MgCl2, pH 5; Sigma)
for 10 min. After washing, cells were incubated with FITC-conjugated
anti-BrdU mAb (Becton Dickinson) and analyzed on a FACScan using
three-color analysis. At least 10,000 viable cells were acquired per sample.
The percentage of BrdU-positive cells in each subset (labeling index) and
the absolute numbers of BrdU-positive cells (calculated by multiplying the
total number of thymocytes of each subset by its labeling index) were
calculated.
Results
LCK regulates CD41CD81 thymocyte expansion during
ontogeny
Thymi from heterozygous (lck1/2) and control wild-type
(lck1/1) mice at day 16 of gestation to 6 wk postnatal were
similar in size and contained similar cell population as defined
by CD4 and CD8 staining (data not shown). Thymocyte populations in mutant lck2/2 mice within the same litter did not
show any significant differences of cell numbers compared with
heterozygous littermates until day 18 of gestation, implying that
lck has no major role in the early expansion of thymocytes (Fig.
1A). Moreover, after the perinatal plateau, the postnatal wave of
FIGURE 1. Total thymocyte numbers (A) and numbers of CD42CD82
(DN) (B) and CD41CD81 (DP) (C) thymocytes subpopulations in lck2/2
and lck1/2 mice from fetal day 16 to 6 wk postnatal. Mean values of three
to six experiments in which at least three mice were included in each age
group are shown. Cells were harvested and stained as described in Materials and Methods.
3830
lck IN FETAL AND NEONATAL THYMOCYTE MATURATION
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FIGURE 2. Perinatal thymocyte maturation in lck1/2 and lck2/2 mice. Thymocytes from the indicated mice were isolated and stained with PEconjugated anti-CD4 and FITC-conjugated anti-CD8a. Thymocyte phenotypes were determined on days 16, 17, 18 (D16, D17, D18), day of birth (D0),
1 day after birth (D1PN), and day 4 postnatal (D4PN). The percentage of cells in each quadrant is indicated.
The Journal of Immunology
3831
Table I. Total numbers (6SD) of immature CD42CD81 single positive
(ISP) thymocytes on gestation day 16 (GD 16), gestation day 17
(GD17), gestation day 18 (GD18), to 2 wk postnatal, and 6 wk
Age
lck1/2
lck2/2
GD16
GD17
GD18
2 wk
4–6 wk
3.4 6 1.2 3 104
2.8 6 1.4 3 105
3.5 6 1.3 3 105
11.9 6 3.5 3 105
8.6 6 2.7 3 105
2.3 6 1.2 3 104
2.6 6 0.7 3 105
4 6 1.6 3 105
9.6 6 3.5 3 105
1.2 6 0.3 3 104
Abnormality of the phenotype of the double negative population
throughout maturation
The total and relative numbers of DN thymocytes in lck2/2 mice
paralleled those of lck1/2 littermates until 3 wk after birth (Fig.
1B). Since the double negative DN population has showed a block
in the differentiation of CD442CD251 cells to CD442CD252
cells in adult lck2/2 mice (20), we analyzed whether this developmental defect could also be observed during fetal and early postnatal life. Surprisingly, although the population size and proliferation of DN mutant thymocytes were within normal values before
birth and at 14 days postnatal, there was a significant decrease in
CD442CD252 population and an increase in CD442CD251 cells,
indicating a block in early development at the CD442CD251 to
CD442CD252 transition (Fig. 3). These results suggest that during embryogenesis and early postnatal life, a developmental block
of CD442CD251 DN cells does not account for the observed decline in the DN population.
LCK affects S phase progression of CD41CD81 thymocytes
before birth, whereas it is mainly involved in double negative
proliferation at 3 wk postnatal
To assess the role of lck in the cell cycle progression of thymocytes, lck1/2 and lck2/2 mice were injected with BrdU (3, 19).
BrdU incorporation after administration in vivo was evaluated to
determine the S phase progression of thymocytes (21) at gestation
day 18, day 14 postnatal, and 5 to 6 wk postnatal (Fig. 4, Table II).
After exclusion of gd thymocytes, the numbers of proliferating
FIGURE 3. Comparison of DN thymocyte subsets from lck2/2 and lck1/2 mice. Thymocytes were isolated and labeled with anti-CD4 TC, CD8 TC,
CD44 PE, and CD25 FITC. Thymocytes were gated for viable CD42 CD82 cells. CD44 vs CD25 profiles on DN cells from three different ages (gestation
day 18, GD18; day 14 postnatal, D14; 6-wk-old, 6w) are shown. One experiment representative of at least three experiments is shown.
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thymocyte maturation still occurred in lck2/2 mice, showing
that these mice were still capable of a partial response to the
stimuli responsible for this growth. The mutant mice thymi
reached their largest size at 2 wk postnatal, then exhibited a
rapid decrease in thymocyte numbers that started at about 3 wk
of age and persisted throughout adult life (Fig. 1A).
Analysis of the different thymocyte subpopulations in lck2/2
mice (Fig. 1B) showed that the numbers of DN cells were close to
normal values until 3 wk after birth, at which point DN thymocytes
were decreased by a factor of two to three. The CD81 single positive immature population was within the normal range at day 17
and 18 of gestation and appeared at the same time as in the heterozygous mice (Fig. 2, Table I). However, at 4 to 6 wk after birth,
CD8 single positive cells significantly declined in numbers in the
absence of lck expression. No mature single positive CD41 or
CD81 were generated throughout this period, implying that the
CD81 population in lck2/2 mice is comprised of immature thymocytes (Fig. 2). The decreased size of the thymus in lck2/2 mice
was due to a defect in the expansion of the CD41CD81 DP thy-
mocytes starting at day 17 of gestation (Fig. 1C). In addition,
CD41CD81 thymocyte cellularity declined rapidly at 3 wk after
birth (Fig. 1C).
3832
lck IN FETAL AND NEONATAL THYMOCYTE MATURATION
double negative cells were almost similar among lck2/2 mice and
control lck1/2 mice at day 18 of gestation (Fig. 4B) and at 2 wk
after birth. However at 6 wk postnatal, a significant decrease in the
percentage and absolute numbers of BrdU-positive DN cells was
observed in lck2/2 mice.
Of the three major thymocyte populations of the fetal thymus,
the CD81CD42 immature single positive (ISP) thymocytes are the
cells that display the highest rates of proliferation. Although there
is a slight decrease in the proliferation index of this population in
mutant mice before birth (48.8% 6 5.8 in lck2/2 vs 58% 6 4.9 in
lck1/2) (Fig. 4A), lck does not appear to be crucial for proliferation of these cells at this stage of development. Whereas at two
weeks the ISP subset number was comparable in heterozygous and
mutant mice, this population was dramatically decreased at 4 to 6
wk in the mutant mice (Table I). Interestingly, the proliferation
index of the ISP was significantly decreased in lck2/2 mice between 2 wk (49% 6 7) and 6 wk postnatal (26.2% 6 1.5), predicting the defect in the proliferative capacity of ISP observed at 6
wk of age.
In contrast to DN and ISP thymocytes, the total and relative
numbers of DP thymocytes were already decreased before birth
(Fig. 4A and Table II). In parallel to the decrease in the DP population, the proliferation index and BrdU uptake of DP thymocytes
were significantly impaired in lck2/2 embryos (GD 18). However,
after birth, the labeling index of DP thymocytes was comparable
among lck2/2 and lck1/2 mice, suggesting that the lck mutation
affects proliferation of fetal DP thymocytes but not postnatal DP
thymocytes.
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FIGURE 4. DNA synthesis rate (BrdU incorporation) of thymocyte subsets in lck1/2 and lck2/2 mice. Heterozygous pregnant mice (gestation day 18)
received one i.p. injection of BrdU. Two hours after the pulse, fetal thymocyte suspensions were prepared and processed for CD4, CD8, ab, gd, and BrdU
staining. DP, DN, and ISP were defined on the basis of relative CD4/CD8 expression as indicated on the dot plots in Figure 4A, upper panels. A, Percentages
of BrdU1 cells were calculated in DP and ISP subpopulations as shown on histograms. B, Percentages of gd T cells among DN cells (upper panels) and
percentages of BrdU1 cells among total DN cells (middle panels) and DN cells excluding gd cells (lower panels). Genotypes of embryos were determined
by PCR.
The Journal of Immunology
3833
Table II. BrdU incorporation
Absolute Numbers BrdU1c
Labeling Indexb
Age
lck1/2
lck2/2
lck1/2
lck2/2
Double negative thymocytesa
GD18 (gd excl.)
2 wk
6 wk
22.7 6 5
27.6 6 6
17 6 5.6
19.8 6 4
21 6 3.3
8.44 6 0.8
1.49 6 0.6 3 105
1.79 6 0.8 3 106
8.1 6 3.2 3 105
1.98 6 0.5 3 105
1.82 6 0.5 3 106
1.7 6 0.6 3 105
Double positive thymocytesa
GD18
2 wk
6 wk
20.3 6 2.77
12.6 6 1.9
9.5 6 3
11.3 6 2.1
11.6 6 2.9
7.9 6 2
9.5 6 2.3 3 105
21 6 0.3 3 106
15.6 6 7 3 106
1.3 6 1.5 3 105
2.9 6 0.1 3 106
0.2 6 0.1 3 106
a
Thymocytes were harvested from lck1/2 and lck2/2 mice 2 h after the BrdU pulse and CD42 CD82 (DN), CD41 CD81 (DP) were determined by staining with anti-CD4
and anti-CD8 mAbs. Cycling studies were performed at gestation day 18 (GD18), 14 days postnatal, and 6 wk postnatal. Data shown are representative of at least three
experiments in which each group contained at least three individual mice.
b
Percentage of BrdU1 cells in each subset.
c
Absolute numbers of BrdU1 cells were obtained by multiplying the total number of thymocytes in each subset by the labeling index.
Our study indicates that in the absence of lck, the absolute numbers
and proliferation of DN and ISP thymocytes are close to normal
values before birth and at 14 days postnatal, whereas proliferation
and absolute numbers of DN and ISP cells are significantly decreased by 3 wk after birth. In contrast, proliferation of DP thymocytes was affected during embryonic development but appeared
normal after birth. These data imply that lck has differential roles
in the proliferation and maintenance of DN, ISP, and DP thymocyte populations, depending on whether embryonic or postnatal
lymphopoiesis is occurring.
In RAG22/2 thymocytes, which lack a functional pre-TCR and
hence arrest at the CD442/CD251 stage of differentiation, proliferation studies showed a strong decrease in BrdU incorporation
among the DN population (5% vs 24% of labeling index (21)).
This result contrasts with our results in lck2/2 mutant mice, in
which DN proliferation is impaired only beyond 3 weeks of age.
Since it has been shown that lck has an important role in pre-TCR
signaling in adult mice (18), these results imply that lck function
can be replaced by another kinase during the embryonic and early
postnatal stages but not in adult life. Nevertheless, analysis of
CD44/CD25 expression patterns in embryonic thymocytes revealed a significant decrease of the CD442CD252 population, indicating a partial block at the CD442CD251 stage of differentiation. This phenotypic, but not proliferative, defect in the DN
population during prenatal development may be due to abnormal
cell surface expression of these molecules in the absence of lck.
Indeed, lck is known to play an important role in regulating the
surface expression of molecules like CD4 (22), TCR-b (18), and
CD3e (14). Therefore the decrease or lack of CD442CD252 thymocytes does not preclude the next maturation step, a phenomenon
that has been observed in CD3z2/2 mice (23) and during fetal DN
maturation (24). Furthermore, although expression of CD25 defines different populations of DN thymocytes, CD25-deficient
mice display normal thymocyte maturation, implying that this phenotypic marker does not have a crucial role in DN thymocyte
maturation (25).
As was the case for DN cells, absolute numbers and the proliferation of immature single positive (ISP) thymocytes before birth
were close to normal values in lck2/2 embryos, suggesting that, at
this stage, lck is not essential for the proliferation of ISP cells and
the maturation of DN to ISP thymocytes. Moreover, the absolute
numbers of ISP thymocytes were within normal values at day 14
postnatal, and the kinetics of the decrease in the ISP population
paralleled those seen in DN thymocytes.
The principal defect in lck2/2 embryonic thymocytes is a pronounced block in the proliferation of the DP thymocyte population,
indicating that lck is required for the expansion of the prenatal DP
population. The increase of DP following the overexpression of an
lck transgene (18) underscores the mitogenic role of lck for proliferation and expansion of the DP population.
Overall, our experiments show that lck is mainly involved in the
expansion of DP thymocytes during fetal and early postnatal maturation. However, 3 wk after birth, lck also has an important role
in the proliferation and maintenance of DN and ISP thymocytes.
Our results imply either that the same kinase may target a different
cell subpopulation at a different stage of development, or that some
other kinase(s) may compensate for an absence of lck at some
stage of maturation. The fact that some tyrosine-phosphorylated
proteins are present in adult thymocytes (26) and in purified DP
thymocytes (27) of lck2/2 mice shows that lck is not the only
tyrosine kinase involved in thymocyte maturation. For example,
the src-kinase Fyn may partially compensate for the lck mutation
during this stage of development, since fyn2/2 lck2/2 double mutant mice completely lack DP thymocytes (27, 28) due to a block
in the CD251CD442 stage. Moreover the dominant negative lck
transgenic mice have a more severe phenotype (29) compared with
an lck2/2, mice which may be due to an inhibition of both fyn and
lck kinase activities in the transgenic mice through competition for
downstream targets.
However, a key finding of this study is that lck activity can be
compensated for DN thymocytes during fetal and early postnatal
maturation, whereas it is essential after 3 wk of age. Although the
amount of fyn protein is lower than that of lck in DP thymocytes
of 4-wk-old mice (30), the relative amounts of fyn and lck in fetal
and postnatal thymocytes subpopulations have yet to be
determined.
Hypothetically, it is also possible that the proliferation of fetal
and early postnatal DN cells is less tightly regulated than proliferation in adult thymocytes, allowing certain domains of fyn to
bind to some substrates of lck in neonatal thymocytes but not in
adult thymocytes. In addition, DN thymocyte proliferation may
involve substrates specific to both fyn and lck in fetal and early
postnatal life whereas the kinase substrates for lck and fyn might
be different after 3 wk of age. Our data provide the first evidence
that signal transduction molecules can be differentially required in
a stage-dependent manner for the proliferation and development of
a given defined cell subpopulation depending on the age of
the mice.
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Discussion
3834
Acknowledgments
We thank David Tough, Claude Penit, Florence Vasseur, Catherine Cavard, Virginie Joulin, and Nicolas Rouquet for technical help and helpful
comments and Mary Saunders for scientific editing.
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lck IN FETAL AND NEONATAL THYMOCYTE MATURATION