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Department of Molecular Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, Japan
Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo, Japan;
Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan;
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Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan;
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Department of Immunology, Tokai University School of Medicine, Isehara, Japan; and
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Institute for Genome Research and PRESTO Research Project, University of Tokushima, Tokushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human and murine AML1, also known as Pebpa2b, Cbfa2, or Runx1, encode the DNA-binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) (2, 3). The DNA binding domain of AML1, called the Runt domain, shows homology to the products of Drosophila developmental genes, runt and lozenge. The AML1 gene is known to be involved in human leukemogenesis (reviewed in Ref. 4) as well as in the physiological development of definitive hemopoiesis in the murine fetal liver (5, 6, 7).
There have been some observations suggesting that AML1 plays
a significant role in the T lymphocytes. First, expression of
AML1 is detected abundantly in embryonic and adult thymuses.
Based on the in situ hybridization and morphological observation, the
cells positive for AML1 expression represent T lymphocytes
rather than stromal cells (8). Second, PEBP2/CBF has been
implicated in the T cell-specific expression of several genes including
TCRs. The enhancer elements of the TCR
,
TCRß , TCR
, and TCR
genes
harbor PEBP2/CBF binding sites (reviewed in Ref. 9).
PEBP2/CBF and Ets-1 or c-Myb bind to their adjacent sites in the
enhancer elements of TCRs and activate transcription
synergistically (10, 11, 12, 13, 14). PEBP2/CBF was also identified as
a binding factor for the enhancer core of a T cell tropic strain of
murine leukemia virus (15, 16). Finally, we reported
recently that overexpression of AML1 protein can render a T cell
hybridoma resistant to TCR-mediated apoptosis (17). This
is achieved by down-regulating the expression of the apoptotic
Fas-ligand gene and simultaneously up-regulating
IL-2R expression. We proposed that AML1 might play a
pivotal role in TCR-mediated growth and/or the death of T
lymphocytes.
Based on the circumstances and the rationales described above, we were motivated to examine the role of AML1 in the later stages of T lymphocyte development in vivo. Unfortunately, AML1-/- embryos die on embryonic day 12.5 due to the concomitant and massive hemorrhage of primitive erythrocytes in the CNS (5, 6, 7) and, thus, cannot provide any information on the role of AML1 in T lymphocyte development. Therefore, we used transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. The results obtained reveal a new and unexpected aspect of the gene regulation involved in the fate of SP T cells. The AML1 gene plays a key role in the maturation of SP thymocytes, and furthermore functions differentially to maintain the pool of CD4 SP (CD4+CD8-) vs CD8 SP (CD4-CD8+) cells in peripheral lymphatic tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression plasmid pCX2neoBS harbors the Runt domain of
murine AML1b/PEBP2B1 protein and was described previously
(18). The hemagglutinin (HA) tag that represents the
epitope of flu virus HA and the nuclear localization signal (NLS) of
SV40 were fused to the amino terminus and carboxyl terminus,
respectively, of the Runt domain by the PCR method, as follows. PCR was
performed on pCX2neoBS as template DNA. The sense primer was
5'-ccggaattcggccaccatgtatccatatgatgttccagattatgctatggtggaggtactagctgaccac-3',
in which the singly and doubly underlined sequences represent the HA
epitope and the codon for the initiating methionine, respectively. The
antisense primer was
5'-ccggaattctacaccttacgcttcttctttggcaccttacgcttcttctttggatcatctagtttctgccgatg-3',
in which the singly and doubly underlined sequences represent the NLS
in tandem and the termination codon, respectively. The PCR product was
digested by EcoRI and subcloned into the EcoRI
site of human pCD2 minigene (19). The resulting plasmid
was designated pCD2-HA/Runt/NLS. The authenticity of the modified
sequences in the plasmid was confirmed by sequencing.
Mice
Transgenic mouse lines expressing the Runt protein were generated as follows. The DNA of pCD2-HA/Runt/NLS was digested by SalI and XbaI, and the purified fragment containing the insert was microinjected into fertilized eggs of C3H/HeN mice. Litters possessing the transgene were backcrossed with C57BL/6J mice. The presence or absence of the transgene was examined by PCR using genomic DNA as a template. The sense and antisense primers were 5'-atggtggaggtactagctg-3' and 5'-ggatcatctagtttctgcc3', respectively, and a 410-bp fragment was amplified from the transgene. Three independent transgenic lines, namely 48, 65, and 81, were established. Targeting of the AML1 gene was described previously (7). In case, mice were given bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) in the drinking water (0.8 mg/ml) for various periods. The anti-CD3 Ab was purified from the tissue culture supernatant of 145-2C11 hybridoma, as described previously (17). The Ab solution containing 100 µg protein was injected i.p. into individual mice. Eight-week-old mice were used for analysis, unless otherwise specified.
Flow cytometrical analysis
Cells were liberated from the thymus, spleen, or lymph node and suspended in PBS containing 0.2% (w/v) BSA. The single cell suspensions were incubated with appropriately diluted mAbs on ice for 30–60 min. Fluorescein-conjugated Abs used were as follows: anti-CD4 PE (H129.19; Sigma), anti-CD4 FITC (RM4-5; PharMingen, San Diego, CA), anti-CD4 Cychrom (RM4-5; PharMingen), anti-CD8a PE (53-6.7; PharMingen), anti-CD8a FITC (53-6.7; PharMingen), anti-CD8a RED613 (53-6.7; Life Technologies, Rockville, MD), anti-TCRß PE (H57-597; PharMingen and Caltag, South San Francisco, CA), and anti-heat-stable Ag (HSA) FITC (M1/69; PharMingen).
The procedure to detect BrdU-incorporated cells was as follows. Thymocytes or splenocytes were first incubated with anti-CD4 Cychrom and anti-CD8a PE. After washing, cells were fixed and permeabilized with 1% (w/v) paraformaldehyde and 0.1% (v/v) Tween 20 in PBS at 4°C overnight. Cells were then digested by 0.7 U/µl of DNase I (Takara, Ohtsu, Japan) in 150 mM NaCl, 100 mM sodium acetate, and 5 mM MgCl2 at 37°C for 15 min. After washing, cells were labeled with anti-BrdU FITC (Becton Dickinson, San Jose, CA). To detect apoptotic cells, cells were stained with annexin V according to the procedure supplied by the manufacturer (Medical and Biological Laboratories, Nagoya, Japan).
The analytical flow cytometer used was EPICS-XL (Coulter, Palo Alto, CA), and the data were analyzed by the software, MacLAS. In most cases, dead cells were excluded by propidium iodide gating.
Cell culture
Cells were liberated from the thymus, suspended in RPMI 1640 medium containing 10% (v/v) FCS, 10 mM HEPES-KOH, pH 7.4, 2 mM L-glutamine, and 50 µM 2-ME, and subjected to culture at a density of 5 x 105 cells/well in a 96-well plate. The plate was coated beforehand with the anti-CD3 Ab and anti-CD28 Ab (PharMingen), according to the procedure, as described previously (17). In case of splenocytes, cells were liberated from the spleen, and erythrocytes were lysed by treatment with 0.15 M NH4Cl, pH 7.2. The cells were suspended in media and subjected to culture described as above. For TCR stimulation, splenocytes were treated by 10 µg/ml of Con A (Sigma).
Immunoblot analysis
The procedure to prepare the CD4 SP fraction from thymocytes was as follows. The thymocytes’ suspension was incubated with the anti-CD8a Ab (AD4 (15); Cedarlane Laboratories, Hornby, Ontario, Canada) and Low-Tox-M guinea pig complement (Cedarlane) at 37°C for 60 min. The surviving CD4 SP cells were adsorbed to anti-CD4 Ab (GK1.5)-coated magnetic microbeads and purified by passing the suspension through a Mini MACS separation column (Miltenyi Biotec, Auburn, CA). To prepare the CD8 SP fraction from thymocytes, the CD4 SP cells were killed by using the anti-CD4 Ab (RL172) and the remaining CD8 SP cells were adsorbed to anti-CD8a Ab (53-6.7)-coated magnetic microbeads (Miltenyi Biotec). The CD4 SP and CD8 SP fractions from spleen were prepared by using the respective Ab-coated microbeads and a separation column. The purity of the recovered SP fractions was over 90%, as judged by flow cytometrical analysis.
All the procedures necessary for immunoblot analysis, including protein
extraction, electrophoresis, transfer to the filters, and
immunoreactions, were as described previously (20, 21).
The anti-HA mAb, 12CA5, was obtained from Boehringer Mannheim
(Indianapolis, IN). The anti-AML1 peptide Ab was raised in rabbits
using as the Ag peptide the carboxyl-terminal 15-aa sequence of murine
AML1b/PEBP2B1 (22). The anti-AML1 peptide antiserum
was used at 500-fold dilution and, when necessary, was preabsorbed with
the 20 µg/ml of Ag peptide.
EMSA
The procedures for preparing whole cell extract and for EMSA were as described previously (23). The PEBP2/CBF-binding sequence from Polyomavirus enhancer was used as a probe to detect PEBP2/CBF and Runt DNA binding activities. In competition analysis, a 100-fold molar excess of unlabeled oligonucleotide was included in the binding mixture for EMSA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the study was mainly designed to follow the fate of SP T
lymphocytes, we examined the expression of AML1 polypeptides in such
cells by immunoblot analysis (Fig. 1
a). The CD4 SP and CD8 SP
cells were purified from thymocytes and splenocytes, and their protein
extracts were probed by the anti-AML1 peptide Ab. One band was
detected at a position corresponding to a protein of 56 kDa
(lanes 1–4). The bands represent specific
immunocomplexes, because they were abolished by including an excessive
amount of the corresponding peptide in the incubation mixture
(lanes 5–8). The 56-kDa species comigrated with the
AML1 polypeptide purified from a bacterial lysate and was missing from
a protein extract of the AML1-/- embryo
(22). Thus, we were able to confirm the expression of AML1
polypeptides in thymic and splenic CD4 SP and CD8 SP subsets.
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To explore the role of AML1 in the development of T lymphocytes,
we generated transgenic mouse lines expressing a dominant interfering
form of AML1. Because the DNA binding domain of AML1, the Runt domain,
is known to dominantly interfere with PEBP2/CBF-dependent
transactivation (18), it was placed downstream of the CD2
promoter and upstream of the CD2-poly(A) addition sequence in the
microinjected plasmid (19). Three transgenic mouse lines,
namely lines 48, 65, and 81, were established. The expression of the
Runt domain in thymuses and spleens of transgenic mice was examined by
immunoblot analysis (Fig. 1
b). The anti-HA epitope Ab
detected the HA-tagged Runt protein at the expected 17.5-kDa position.
The expression of the HA-Runt protein was confirmed in both of the
tissues of all transgenic mouse lines. Nontransgenic mice did not
express it.
EMSA was performed using the PEBP2/CBF-binding sequence as a probe
(Fig. 1c). Endogenous PEBP2/CBF DNA-binding activity was
detected in the thymus and spleen of nontransgenic mouse
(lanes 1 and 3), whereas Runt
domain-derived DNA-binding activity was detected in the tissues of
transgenic line 81 (lanes 2 and 4).
DNA-binding specificity was confirmed for both complexes by competition
analysis (data not shown). Absence of slower migrating complex
indicates that the Runt domain indeed functioned to interfere with the
endogenous PEBP2/CBF DNA-binding activity in the tissues of transgenic
mice. In the following sections, only the data obtained for the
transgenic line 81 will be presented, but essentially similar results
were obtained for the other two lines as well.
Generation of SP thymocytes is partially impaired in Runt-transgenic mice
The profiles of T lymphocytes in the thymus, spleen, and lymph
nodes were examined using 8-wk-old transgenic mice (Fig. 2a). When thymocytes were
analyzed by two-color flow cytometry, the percentages of CD4 SP and CD8
SP cells in the transgenic mouse (3.6 and 0.8%, respectively) were
reduced compared with those of the nontransgenic mouse (9 and 3.4%,
respectively). The total number of cells present in the thymus did not
vary significantly between the nontransgenic and transgenic mouse
lines, when a number of individuals from each group were examined
(Table I). Therefore, the
mean numbers of CD4 SP and CD8 SP thymocytes in the transgenic line
were 40 and 28%, respectively, of those seen in the nontransgenic
mice. The mean percentage and cell number of
CD4+CD8+ (double-positive,
DP) thymocytes in the transgenic line were slightly larger than those
seen in the nontransgenic mice (Fig. 2a and data not shown).
It is likely that transition of thymocytes from the DP to the SP stage
is partially impaired by the expression of a dominant interfering form
of AML1 and that this results in the observed decrease in the numbers
of SP thymocytes.
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Analysis of T lymphocytes in peripheral lymphoid tissues revealed
additional alterations in the transgenic mice. As described below,
these were a further decrease in the proportions of CD4 SP, but
not of CD8 SP cells, resulting in the overall reduction of the CD4
SP/CD8 SP ratio. The percentages of CD4 SP cells in the spleen or lymph
node of transgenic mouse were significantly smaller than those seen in
the nontransgenic mouse (Fig. 2a). Actually, the mean
numbers of CD4 SP cells in spleens and lymph nodes of the transgenic
line were not 40%, as simply extrapolated from the situation in the
thymus, but were 17 and 16%, respectively, of the mean numbers in the
nontransgenic mice (Table I). Therefore, in the transgenic line, the
decrease in the proportion of peripheral CD4 SP cells was more severe
than that of CD4 SP thymocytes.
In contrast, we encountered the different situation in the case of CD8
SP cells. The percentages of CD8 SP cells in the spleen and lymph node
of transgenic mouse were similar to and rather larger, respectively,
than those seen in the nontransgenic mouse (Fig. 2a). The
mean numbers of CD8 SP cells in spleens and lymph nodes of the
transgenic mice were 37 and 33%, respectively, of the mean numbers in
the nontransgenic mice (Table I). As mentioned above, the numbers of
CD8 SP thymocytes in the transgenic line were 28% of those in the
nontransgenic line. Thus, unlike the case of the CD4 SP cells, the
proportion of CD8 SP cells remained unchanged or became slightly larger
in the peripheral lymphoid tissues of transgenic mice.
The differential profiles of each SP subset were more apparent, when the ratios of CD4 SP to CD8 SP cells were calculated. The CD4 SP/CD8 SP ratios in the spleens and lymph nodes of transgenic mice were on average equal to 1, but were 2 in the case of nontransgenic mice. Thus, the second phenotypic change in transgenic mice is a further decrease in the proportions of CD4 SP, but not of CD8 SP cells in the peripheral lymphoid organs. This phenotype is considered to be distinct from the above-described alteration in thymocytes, in which both SP subsets were decreased in the transgenic mice. The CD4 SP/CD8 SP ratios of thymuses were rather higher in transgenic mice compared with nontransgenic mice.
Heterozygous disruption of AML1 causes similar phenotypes to those seen in Runt-transgenic mice
In addition to AML1, two other mammalian runt genes are known to exist, namely AML3/Pebpa2a/Cbfa1 and AML2/Pebpa2c/Cbfa3. The Runt domain of AML1 should interfere with the DNA-binding and transcriptional activation potential of all three of these mammalian proteins. A PEBP2/CBF DNA-binding activity detected in the protein extract of thymus is reported to be mainly due to the product of AML1 (24). Therefore, we examined whether the similar as above phenotypes could be observed in mice targeted for the AML1 gene. The AML1+/- mice apparently suffer from no developmental abnormalities and survive to adulthood (7).
When thymocytes were analyzed by flow cytometry, the
percentages of CD4 SP and CD8 SP cells were slightly lower in the
AML1+/- mouse compared with those in the
wild-type mouse (Fig. 2b). Reflecting this, the mean numbers
of each SP thymocyte in the heterozygotes were 
70% of those of the
wild-type mice (Table I). Therefore, the decrease in the number of both
SP subsets can be seen in the AML1 heterozygous thymocytes
as well, although this decrease was less severe compared with the
decrease seen in transgenic mice.
As for the SP cells in the peripheral lymphatic tissues of the AML1 heterozygous mice, changes similar to those of the Runt-transgenic mice were observed. This can be readily seen by comparing the ratios of CD4 SP to CD8 SP cells. The mean ratios in the AML1+/- mice (0.6) were half of those of the wild-type mice (1.3), reflecting the additional decrease in the proportion of CD4 SP cells and the slight increase in that of CD8 SP cells in peripheral tissues of heterozygous mice.
It must be noted that the surface expression of TCR was not altered
significantly in the AML1-diminished, CD4 SP splenocytes
(Fig. 3). TCR expression in the CD8 SP
splenocytes was not altered by AML1 dysfunction, either
(data not shown). Thus, the reduction in the peripheral CD4 SP/CD8 SP
ratios was observed for their TCRhigh
subpopulations. In addition, almost all of peripheral CD8 SP cells in
the AML1+/- mice were of the CD8ß
phenotype, indicating their thymic origin (data not shown).
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The peripheral CD4 SP/CD8 SP ratio is perturbed at the level of recent thymic emigrants in AML1-diminished mice
In all the experiments above, mice with an age of 8 wk were used.
At, or soon after birth, positively selected SP thymocytes start to
emigrate from the thymus and to settle in the peripheral lymphoid
tissues. We next examined whether a similar phenotypic change would be
observed in the peripheral T lymphocytes of neonatal mice, which
resemble recent thymic emigrants. As can be seen in Table II, the CD4 SP and CD8 SP cells in the
spleens of wild-type neonates represent only a small proportion of
total splenocytes. Interestingly, the mean percentages of splenic CD4
SP cells in the AML1-diminished neonates were significantly
lower than those of the corresponding SP subset in the wild-type
neonates. Thus, the CD4 SP/CD8 SP ratios in spleen of
AML1-diminished mice were roughly half of those in spleen of
wild-type mice. This suggests that the event leading to the reduction
of the CD4 SP/CD8 SP ratio by AML1 diminution occurs
probably at the time, or soon after the SP cells are released from the
thymus.
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a). The
BrdUlow subpopulation in spleen corresponded to
11% in the CD4 SP and 9% in the CD8 SP subsets, respectively. Because
this BrdUlow fraction disappears from spleens of
thymectomized mice, the cells in the splenic
BrdUlow subpopulation are considered to have
incorporated BrdU when they are staying in thymus as SP cells
(25). Therefore, the splenic BrdUlow
subpopulation represents recent thymic emigrants (see the figure legend
for the BrdUhigh subpopulation).
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b). In the wild-type mice, the
numbers of BrdUlow cells in the CD4 SP subset
were consistently larger than those in the CD8 SP subset, whereas, in
the transgenic mice, a converse situation was observed in most of the
mice examined. Essentially similar as above results were obtained when
comparing the AML1+/+ and
AML1+/- mice (Fig. 4c). Thus,
the larger numbers of recent thymic emigrants in the CD4 SP subset than
those in the CD8 SP subset most likely underlie the basis of CD4 SP/CD8
SP ratio seen in peripheral lymphatic tissues of wild-type mice.
Likewise, the smaller ratio of peripheral CD4 SP/CD8 SP observed in the
AML1-diminished mice appears to correlate with the reduced
number of emigrants in the CD4 SP subset compared with that in the CD8
SP subset (see the figure legend for the mean numbers of
BrdUlow cells).
The results presented in Table II and Fig. 4 suggest that the reduction
of the peripheral CD4 SP/CD8 SP ratio seen in the
AML1-diminished mice is at least partially due to the
perturbation of recent thymic emigration.
Maturation of SP thymocytes is affected in AML1-diminished mice
After SP cells are positively selected and before they are released into the circulation, the cells pass through a step called maturation in the thymic medulla. The more mature SP cells are, the more efficiently they proliferate and emigrate (reviewed in Ref. 26). To correlate maturation with the recent thymocyte emigration described as above, we examined the degree of maturation of SP thymocytes by measuring the expression of the HSA (27).
Thymocytes from the wild-type and AML1-diminished mice were
processed for four-color flow-cytometrical analysis (CD4, CD8, TCR, and
HSA). Fig. 5 depicts the profiles of HSA
expression in the
CD4+CD8-TCRhigh
and
CD4-CD8+TCRhigh
subfractions. Characteristically, the HSAlow
population in the CD4 SP subset was consistently less prominent in the
Runt-transgenic thymus (15%) compared with the nontransgenic thymus
(27%). Conversely, the HSAlow population in the
CD8 SP subset was more prominent in the transgenic line (58 vs 32% in
the wild type). Therefore, it appears that maturation is impeded in CD4
SP thymocytes and accelerated in CD8 SP thymocytes in the
Runt-transgenic mice. The effect of AML1 heterozygosity on
the HSA expression of SP thymocytes was relatively mild compared with
that of the Runt transgene (data not shown).
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). After 48 h of injection, the
percentages of DP cells became extremely low due to their death. In
contrast, the percentages of remaining SP cells became relatively high.
Interestingly, the percentages of CD4 SP cells in the
AML1-diminished thymus were about half of that in the
wild-type thymus. Thus, the CD4 SP/CD8 SP ratios in the
AML1-diminished thymus reduced to 2.9 and 1.1 compared with
those (3.9 and 1.6) in the wild-type thymus. This suggests that the
responsiveness to TCR stimulation was altered in the
AML1-diminished, SP thymocytes.
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). Thymocytes were
incubated with the varying concentration of anti-CD3 Ab and
processed for three-color flow cytometry (CD4, CD8, and annexin V).
Fig. 6a depicts the percentages of annexin V-positive cells
in the CD4 SP fraction. The Runt-transgenic CD4 SP subset contained
more apoptotic cells after the Ab treatment than the wild-type subset.
In the case of CD8 SP cells, they were first purified from thymocytes,
incubated in the presence of anti-CD3 Ab, and processed for
three-color flow cytometry (CD8, HSA, and annexin V, Fig. 6b). The Runt-transgenic CD8+
HSAlowmedium subset (this gating excluded the
immature CD8low SP cells) contained less
apoptotic cells than the wild-type subset. Thus, the CD4 SP and CD8 SP
thymocytes from the Runt-transgenic mice were more sensitive and less
sensitive, respectively, to TCR-mediated apoptosis than the
corresponding wild-type cells. A differential effect of anti-CD3
treatment in vitro was marginal when comparing the
AML1+/+ and
AML1+/- thymocytes (data not shown).
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) and TCR sensitivity
(Table III and Fig. 6), are considered to be reflected in the
perturbation of recent thymic emigration described in the previous
section. Altered proliferative response of AML1-diminished, peripheral T cells to TCR stimulation
We subsequently examined whether the peripheral T lymphocytes in
the AML1-diminished mice would suffer from any functional
abnormality, in addition to the pertubation of cell numbers.
Splenocytes from the wild-type and Runt-transgenic mice were incubated
in the presence of Con A. On following days after incubation, an
aliquot of cells was removed and processed for two-color flow cytometry
and the relative numbers of CD4 SP and CD8 SP cells were plotted,
taking the cell numbers at the beginning of culture to be equal to 1
(Fig. 7). The increase in the number of
Runt-transgenic CD4 SP cells (closed circles) was significantly lower
than that of nontransgenic cells (open circles). Conversely, the number
of Runt-transgenic CD8 SP cells increased more rapidly than the number
of nontransgenic cells. Overall, the difference in the CD8 SP/CD4 SP
ratio between the Runt-transgenic and wild-type splenocytes became
larger. Similar results were obtained when splenocytes were taken from
AML1+/+ and
AML1+/- mice or when splenocytes were
treated with the anti-CD3 Ab (data not shown). The results indicate
that the peripheral CD4 SP and CD8 SP cells respond to proliferate upon
TCR stimulation with weak and enhanced sensitivity, respectively, when
the function of AML1 is diminished. The altered TCR
responsiveness of peripheral T lymphocytes is, thus, an additional
phenotype seen in the AML1-diminished mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A role for AML1 in the generation of SP thymocytes
The numbers of both CD4 SP and CD8 SP thymocytes were reduced in the AML1-diminished mice. This reflects the partial impairment of transition of thymocytes from the DP to the SP stage, irrespective of the choice between the CD4 SP and the CD8 SP lineages. We confirmed this by introducing the class I- and class II-restricted, TCR transgenes, respectively, into the AML1-diminished mice (K. H. and M. S., unpublished observation). The higher CD4 SP/CD8 SP ratios observed in the Runt-transgenic thymuses may reflect an additional possibility that the AML1 dysfunction causes the commitment of DP cells to the CD8 SP lineage to be more severely impaired than that to the CD4 SP lineage.
A role for AML1 in the maturation of SP thymocytes
SP thymocytes generated by positive selection pass through a maturation step in the thymic medulla. The down-regulation of HSA expression that accompanies this maturation does not merely signify a change of the differentiation surface marker. The HSAhigh and HSAlow SP thymocytes, which correspond to less advanced and more mature stages of differentiation, respectively, differ from each other in their secreting cytokines (30, 31), their proliferating capacity, and their responsiveness to TCR stimulation (28, 29). We showed that the HSAlow population decreased in the CD4 SP and increased in the CD8 SP thymocytes in the Runt-transgenic mice compared with the wild-type mice. In addition, we observed that the CD4 SP thymocytes from the AML1-diminished mice contained TCR-sensitive and thus going to die cells more abundantly. Thus, the maturation was impeded in the CD4 SP and accelerated in the CD8 SP thymocytes as a result of AML1 diminution. AML1 is the first example of a transcription factor that regulates the maturation of SP thymocytes.
A role for AML1 to maintain the homeostasis of the peripheral T cell pool
The reduction of the peripheral CD4 SP/CD8 SP ratio in the AML1-diminished mice cannot be explained by simply extrapolating the fluctuations in the numbers of CD4 SP and CD8 SP thymocytes. Based on the observations with AML1-diminished neonates, we suggested that the event leading to the reduction of the peripheral CD4 SP/CD8 SP ratio most likely occurs at the time, or soon after the cells emigrate from thymus.
The thymic medulla is known to be the site in which the postselection expansion takes place. Although this expansion is more prominent in embryonic thymuses (32, 33), it is detected at a significant level in adult thymuses as well (34, 35). The proliferation tends to occur at a later and more mature stage of SP thymocyte differentiation and contributes to the homeostasis of peripheral T cell pool, because approximately half of the thymic emigrants are the progenies of proliferating, mature SP thymocytes (reviewed in Ref. 26). In this respect, our observation on the numbers of recent thymic emigrants appears particularly relevant. In the AML1+/- mice, the mean numbers of BrdUlow cells in the splenic CD4 SP subset decreased to 60% of those in the wild-type mice, whereas the BrdUlow numbers in the AML1+/-, CD8 SP subset recovered to almost a similar level as those in the wild-type mice. Therefore, the reduction of the peripheral CD4 SP/CD8 SP ratio seen in the AML1-diminished mice appears at least partially due to the perturbation in the recent thymic emigration.
However, it should be pointed out that continual contact between the TCR and correctly restricted MHC molecules within the context of peripheral tissues contributes to the maintenance (survival and expansion) of peripheral T cell homeostasis as well, even in the absence of a specific Ag (36, 37, 38, 39, 40). We observed that diminution of AML1 weakened and enhanced, respectively, the proliferative response of splenic CD4 SP and CD8 SP cells upon TCR stimulation. The reduction of peripheral CD4 SP/CD8 SP ratio seen in the AML1-diminished mice might be also attributable to yet unidentified alteration in the in vivo proliferation of peripheral T lymphocytes.
AML1 and TCR signaling in the CD4 SP vs CD8 SP cells
Diminution of AML1 appears in general to have caused converse effects on the fates of CD4 SP vs CD8 SP cells. This was particularly evident when peripheral T cells were stimulated via their TCRs. The molecular mechanism how AML1 causes differential effects on respective SP subset is not known at present. In our recent report using the DO11.10 cell line as a model of Ag-induced cell death, we observed that overexpression of AML1 changed the outcome of TCR signaling from the Fas-ligand to IL-2R induction (17). It would be a challenge to elucidate how differentially AML1 coordinates with TCR signaling in the CD4 SP vs CD8 SP cells. In any case, the AML1 transcription factor most likely stands as one of the key molecules regulating the fates of SP T cells at the levels of thymus and peripheral lymphatic tissues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masanobu Satake, Department of Molecular Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
3 Abbreviations used in this paper: SP, single-positive; BrdU, bromodeoxyuridine; DP, double-positive; HA, hemagglutinin; HSA, heat-stable Ag; NLS, nuclear localization signal; PEBP2/CBF, polyomavirus enhancer binding protein 2/core binding factor.
Received for publication November 29, 1999. Accepted for publication September 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes in T cells during thymic development. Mol. Cell. Biol. 15:1662.[Abstract]
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) and Runt-transgenic (line 81) mice (•) and
cultured in the presence of Con A. After 48- and 72-h incubation, the
cells were processed for two-color flow cytometry (CD4 and CD8). The
numbers of CD4 SP and CD8 SP cells were plotted, taking the number at
the initiation of culture to be 1. Each solid or dashed line represents
splenocytes from individual mice. Splenocytes from one mouse were
cultured in triplicate dishes, and mean medians (circle) and SDs (bar)
of cell number are presented.