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Blood, 15 January 2002, Vol. 99, No. 2, pp. 513-519
HEMATOPOIESIS
Enforced expression of the Ikaros isoform IK5 decreases the
numbers of extrathymic intraepithelial lymphocytes and natural
killer 1.1+ T cells
Sean N. Tucker,
Heidi K. Jessup,
Hodaka Fujii, and
Christopher B. Wilson
From the Departments of Immunology and Pediatrics,
University of Washington, Seattle; and the Basel Institute for
Immunology, Switzerland.
 |
Abstract |
The zinc-finger protein Ikaros plays an important role in lymphoid
homeostasis, and loss of Ikaros expression through germline disruption
impairs lymphoid development. However, the role played by Ikaros after
commitment to the T-cell lineage is unclear. To address this question,
this study used the lck proximal promoter to drive the expression in
T-cell progenitors of a naturally occurring short Ikaros isoform (IK5),
which lacks the DNA-binding domain, reasoning that IK5 will form
heterodimers with long isoforms and perturb their function. The IK5
transgene led to a selective and dramatic decrease in extrathymic
intestinal intraepithelial lymphocytes (IELs) and natural
killer 1.1+ T (NK T) cells with little effect on
conventional   T cells, which resembles the T-cell phenotype of
interleukin-15 receptor chain (IL-15R) and IL-2/IL-15 receptor
chain (IL-2R) knockout mice. The expression of IL-2R on
double-negative T-cell progenitors of bi-5 was reduced, but enforced
expression of IL-2R did not rescue IELs or NK T cells in bi-5
transgenic mice, suggesting that Ikaros or Ikaros family members
regulate the expression of additional genes that are essential for the
development of IELs and NK T cells. The study concludes that modest
changes in the ratio of short to long Ikaros isoforms can substantially
perturb T-cell development, and the development of IELs and NK T cells is particularly sensitive to such changes.
(Blood. 2002;99:513-519)
© 2002 by The American Society of Hematology.
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Introduction |
Ikaros and its related family members, Aiolos and
Helios, are DNA-binding proteins that appear to regulate the
development and function of lymphoid cells at multiple stages (reviewed
in Cortes et al1). Ikaros is expressed throughout lymphoid
development, in some myeloid lineages, and even in the earliest
hematopoietic stem cells (HSCs).2-4 Alternative splicing
of Ikaros messenger RNA (mRNA) leads to formation of multiple different
isoforms, which differ in biologic activity. Long Ikaros isoforms bind
to a core DNA-binding motif, whereas short isoforms do
not.5 However, short Ikaros isoforms form heterodimers
with long isoforms and may thereby inhibit the ability of long isoforms
to engage DNA.5 The predominant isoforms expressed
throughout T-cell development are the long isoforms (IK1 and
IK2),4 whereas short isoforms appear to be relatively more
abundant in some myeloid lineages and short-term engrafting
HSCs.3
Ikaros is critically important for the normal development of all
lymphoid cells. Ikaros null mice (IK /) lack B, natural
killer (NK), and lymphoid dendritic cells and are markedly deficient in
  T cells. Conventional T-cell development is almost
completely absent in the youngest IK/ mice, with about
100-fold fewer thymocytes at birth.1,4 Thymocyte number
increases in older mice such that the total can approach wild-type
numbers after 6 weeks of age. The thymocytes that develop in
these mice are aberrant, as characterized by extreme expansion of
CD4+ cells and a hyperproliferative response to T-cell
receptor (TCR) stimulation.4 Mice in which only the
DNA-binding domain of Ikaros is disrupted (IK-DNA/
mice) have an even more severe phenotype, in that all lymphoid cells are missing.3 The more severe phenotype in
IK-DNA/ mice is thought to reflect dominant-negative
inhibition by short Ikaros isoforms of long Ikaros isoforms and other
members of the Ikaros family of proteins.
Germline disruption of Ikaros may preclude a clear assessment of its
role at later stages by altering the development potential of HSCs for
both myeloid and lymphoid development.6,7 Thus, to explore
the role of Ikaros in committed lymphoid progenitors independent of
effects in HSCs and to determine the importance of the predominance of
long isoforms in developing T cells, we generated mice in which the
short Ikaros isoform IK5 was expressed exclusively in T-cell
progenitors. IK5 lacks the ability to bind the Ikaros core DNA-binding
motif but still contains the C-terminal zinc fingers required to form
dimers. Although IK5 transgene expression was modest and less than the
aggregate expression of the endogenous Ikaros isoforms, the development
of NK T cells and extrathymically derived intraepithelial lymphocytes
(IELs) was markedly and selectively impaired. These results indicate
that even after commitment to the T-lymphocyte lineage, the
predominance of long relative to short Ikaros isoforms is essential for
normal T-cell development.
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Materials and methods |
Mice strains and transgenic mouse construction
A full-length Ikaros complementary DNA (cDNA) was provided by K. Georgopoulos (Massachusetts General Hospital, Charlestown, MA).
The IK5 isoform was made from full-length cDNA by polymerase chain
reaction (PCR) with splice overlap extension to remove exons 4 to 6 and
was sequenced to verify that there were no mutations. The IK5 cDNA was
inserted into the BamHI site of the p1026 promoter (lck
proximal promoter plus Eµ enhancer) obtained from R. Perlmutter.8 Transgenic mice were generated as described
previously.9 Mice were screened by PCR of tail DNA by
using primers that amplify across the junction between the lck proximal
promoter and the 5' coding region of Ikaros. Mice expressing the murine
interleukin-2 receptor (IL-2R) chain under the control of the
CD2 promoter/enhancer have been described previously.10
Mice expressing a bcl-xL transgene under the lck proximal
promoter11 were a gift from S. Korsmeyer (Dana-Farber
Cancer Center, Boston, MA). C57BL/6 and
2M/ mice were obtained from
Taconic. CD1/ mice, which had been backcrossed
4 times onto C57BL/6 before being used, were obtained from M. Grusby (Harvard School of Public Health, Boston,
MA).12
Northern blot analysis
Cells were isolated from thymus, spleen, and bone marrow. After
ammonium chloride lysis, total RNA was extracted from the cells using
Trizol (Life Technologies). A probe containing exon 7 of Ikaros was
used to identify transgene expression. The Northern blot was stripped
and reprobed using an elongation factor-1 cDNA probe, as previously
described.13
Western blot analysis
Cells were collected and lysed in TNT buffer (50 mM Tris-HCL,
150 mM NaCl,1% Triton X-100).14 Total protein was
quantitated using a Coomassie protein assay (Pierce). Each lane
received either 40 µg protein or the lysates from 100 000 cells for
the cell equivalent Western blots. The samples were separated by a 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After
transfer to nitrocellulose by using a semidry transfer apparatus
(Ellard Instrumentation), Ikaros was detected by using 1:1000
anti-C-terminal Ikaros antisera (provided by S. Smale, University of
California, Los Angeles) and goat antirabbit-horseradish peroxidase at
1:3000 (Zymed). The enhanced chemiluminence system (New England
Nuclear) was used to visualize bands.
IEL preparations
The small intestine was removed and Peyer patches were excised.
After removing fecal matter, the intestines were first cut longitudinally and then cut into 1-cm pieces. The pieces were digested
by using 1 mM EDTA in Hanks balanced salt solution (HBSS) at 37°C
with repeated vortexing to separate lymphocytes from the epithelial
sheathes. Aliquots were removed over time as the epithelial sheathes
settled to the bottom. The first 2 aliquots were replaced with
EDTA/HBSS, and subsequent aliquots were replaced with 5% fetal bovine
serum in HBSS. Removed aliquots were pooled and concentrated before
running over a nylon wool (Sigma) column. A 40%/75% Percoll (Sigma)
gradient was used to further enrich for IELs before analysis.
Cell staining and processing
Cells were stained using anti-CD3 , CD4, CD5, CD8, CD24,
CD43, IL-2R, IL-2R, V8-TCR, TCR, TCR (Pharmingen),
B220, NK1.1, CD8, CD25, and CD44 (CalTag, South San Francisco, CA).
Antibodies to IL-7R were obtained from Andy Farr (University of
Washington, Seattle). The antibodies were directly conjugated to
phycoerythrin (PE), fluorescein isothiocyanate (FITC), Tricolor (TC),
CyChromeC (CyC), PharRed, Allophycocyanin (APC), or biotin. Those
antibodies labeled with biotin were secondarily stained with
streptavidin (SA)-TC. Bead depletions were performed following the
manufacturer's instructions by using Dynal SA-magnetic beads and
biotinylated antibodies.
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Results |
Generation of transgenic mice that express the Ikaros isoform
IK5
Transgenic mice were generated in which IK5 was expressed under
the control of the immunoglobulin heavy chain enhancer (Eµ and the
lck proximal promoter (Figure
1A),8 which hereafter are
referred to as bi-5 mice. The transgene mRNA was expressed in
thymocytes, with reduced expression in splenocytes and total bone
marrow (Figure 1B). After normalizing transgene expression to an
elongation factor control, the 2 lines of bi-5 mice (lines 747 and
5516) appeared to express transgene mRNA in thymocytes in approximately
similar amounts. By reverse transcriptase (RT)-PCR, expression of the
transgene mRNA in NK cells and in mature T cells was approximately 5- to 10-fold less than in CD4+CD8+
double-positive (DP) thymocytes (data not shown). This is consistent with previous data, indicating that expression directed by this promoter/enhancer is highest in T-cell progenitors.8,15
Western blot analysis (Figure 1C) indicated that the abundance of IK5 protein in the thymus of bi-5 mice was similar to or somewhat less than
the abundance of the full-length isoform IK1 and the slightly shorter
isoform IK2. IK5 protein was expressed in
CD4CD8 (double- negative [DN]) and DP
thymocytes of the bi-5 mice but was not detected in splenic T cells of
bi-5 mice or in thymocytes or splenic T cells of controls (Figure 1D).
Ikaros protein was not detected in the livers of bi-5 or littermate
control mice (Figure 1C). IK5 mRNA was detected by sensitive
isoform-specific RT-PCR in thymocytes from control mice, indicating
that this isoform is expressed normally but in low abundance relative
to the long isoforms (data not shown).
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| Figure 1.
Generation of transgenic mice expressing the Ikaros
isoform IK5.
(A) Representation of the construct used to generate IK5 (bi-5)
transgenic mice. The construct contains the lck proximal promoter and
the Eµ immunoglobulin heavy-chain enhancer to direct expression of
IK5. The IK5 cDNA lacks exons 4 to 6, which eliminates 3 of 4 possible
DNA-binding zinc fingers, yet retains all of the C-terminal
Zn++ fingers. The HgX minigene has been shown to enhance
the expression of transgenes and was part of the original p1026
promoter construct.9 (B) Northern blot of the 2 different
bi-5 lines, 747 and 5516, using a probe containing exon 7 of Ikaros.
Both endogenous Ikaros and transgene mRNA are marked in the figure.
Cells used include thymocytes (T), splenocytes (Sp), and total bone
marrow (M). The blot is slightly overexposed to better visualize marrow
and spleen expression of the transgene. The HgX minigene,
although not capable of being translated, contains both introns and
exons; therefore, the transgene mRNA is expressed as multiple,
overlapping bands. The lower panel shows the blot after stripping and
reprobing for elongation factor-1 (EF) to account for loading
differences. (C) Western blot using antisera against the C-terminal
portion of Ikaros, which recognizes all Ikaros isoforms. Samples
include protein isolated from the thymus (Thy) and liver (Liv) of bi-5
transgenic line 747 and a littermate control (wt). (D) Cell equivalent
Western blot using 100 000 sorted cells per lane. Samples include DN
and DP thymocytes and splenic T cells (Sp T) from a representative bi-5
mouse (Bi-5 Tg) and littermate control (littermate).
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NK T cells are reduced in the bi-5 mice
On the basis of the pattern of expression, the IK5 transgene was
predicted to target developing T cells. Yet, contrary to the
observations in Ikaros/ and IK-DNA/
mice, the bi-5 transgene had little effect on the development of
conventional CD4+ and CD8+ T cells,
thymic T cells, NK cells, or B cells. The proportions of these
cell populations in the spleen, thymus, and bone marrow were similar in
bi-5 transgenic mice and littermate controls (Figure 2). There was a slight reduction in
thymocyte numbers in bi-5 mice, which averaged 75% of normal
(Figure 2D).
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| Figure 2.
Representative flow cytometry dot plots of cells from
bi-5 mice and littermate controls.
(A) Thymus, (B) spleen, and (C) bone marrow. Scattergram plot
(D) of the percentage of normal cell number for spleen (Spl)
and thymus (Thy). Each data point on the plot represents the percentage
of total thymocyte number that a bi-5 mouse had to a littermate control
harvested on the same day. (E) Splenic NK cell number versus mouse age.
NK cells were scored by NK1.1+/CD3
staining.
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A more striking difference was found in the percentage and numbers of
NK T cells. These cells have both NK and T-cell markers and are capable
of developing in thymectomized mice.16 Thymic NK T cells
are defined as HSAlo/, NK1.1+,
TCRint, CD8, and
CD44hi,12,17,18 with 60% CD4+ and
the remainder coreceptor negative. The TCR repertoire of NK T cells is
highly limited, comprising the invariant chain V14J281 paired
either with V8, which is greatly favored,19 or with
V7 or V2. Thymic NK T cells were markedly reduced in the bi-5
mice, as indicated by an approximately 5-fold decrease in the
percentage of V8intCD44hi and
NK1.1+CD44hi cells after depletion of
CD8+ and HSA+ cells (Figure
3A). To estimate the severity of the
phenotype, bi-5 mice were compared with mouse strains known to lack NK
T cells. Development of NK T cells requires positive selection on the
nonclassical major histocompatibility complex (MHC) class I-like
molecule CD1.12,20 After depletion of HSA+
cells, CD1/ and bi-5 mice had similar percentages of
the V8intCD44hi cells (Figure 3B), whereas
wild-type mice had a much greater amount. The
2M/ mice, which lack MHC class I
expression and CD1 expression, had a greater reduction in this
population than CD1/ and bi-5 mice, most likely because
of the lack of a TCRintNK1.1 population that
does not require CD1 for positive selection.18
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| Figure 3.
NK T cells are reduced in bi-5 mice.
(A) Flow cytometric analysis of thymocytes from a representative bi-5
mouse and littermate control that were depleted of HSA+ and
CD8+ cells and analyzed in parallel. Unless otherwise
stated, all data are from line 747. (B) Flow cytometric analysis of
thymocytes depleted of HSA+ cells. This figure compares the
bi-5 mice to littermate controls, CD1/, and
2M/ mice. The greater percentage of NK T
cells in mice shown in (B) than in (A) is due in part to age-dependent
differences (NK T-cell numbers increase with age) and to experimental
variability. The mean percentage of HSA/CD8
cells before bead depletion was 1.7% ± 0.8% (mean ± SD) for
bi-5 and 2.8% ± 1.6% for littermate controls. Results are
representative of those obtained in 4 or more independent
experiments.
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Extrathymically derived IELs are severely reduced
Like NK T cells, IELs are derived extrathymically, at least in
part.16,21 Normal mice have relatively similar numbers of TCR+ IELs (-IEL) and TCR+
IEL (-IEL). The overwhelming majority of -IELs express the CD8 coreceptor as a homodimer, whereas -IELs may express
CD8, CD8, CD4, or no coreceptor.22 There is
considerable evidence to suggest that CD8 expression only occurs
on extrathymically derived IELs.23,24 The bi-5 mice had a
drastic reduction in the percentage and number of -IELs and a
corresponding increase in the percentage of -IELs (Figure
4A). Among the -IELs, there was a
decrease in the percentage of cells using CD8 and an increase in
the percentage of cells using CD8 cells in bi-5 mice compared with littermate controls (Figure 4B). Thus, bi-5 mice had reductions both in NK T cells and in extrathymically derived IELs.
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| Figure 4.
IEL populations in the bi-5 mice.
Flow cytometric analysis of the IELs isolated from bi-5+
and littermate control mice for (A) TCR versus TCR and
(B) CD8 versus CD8 gated on
TCR+ cells. Bi-5 -IELs averaged
8.3% ± 3.3% (mean ± SD), whereas littermate -IELs
averaged 23.5% ± 8.6% (P = .001 by unpaired Student
t test). IELs recovered from bi-5 mice were approximately
68% (range, 20%-100%) of the numbers recovered in parallel from
littermate controls. The average number of IELs recovered from bi-5
mice was 4.2 × 105, which is 68% (range, 20%-100%) of
the numbers recovered in parallel from littermate controls with
considerable variation between experiments in cell recovery because of
differences in age and the multiple processing steps. Results are
representative of a minimum of 6 or more independent experiments with
mice between 3 and 15 weeks of age.
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Reduced expression of IL-2R on thymocytes of bi-5 mice
IL-15 signals through IL-2R and the common cytokine receptor
chain.25,26 For IL-15-specific binding, IL-15R is
also required.26 Like bi-5 mice, knockouts of IL-2R and
IL-15R or IRF-1 (a molecule required for IL-15 to be produced) have
severely reduced extrathymically derived IELs and NK T cells but very
mild defects in conventional T-cell
development.27,28 Because IL-2R expression is
restricted to hematopoietic cells, whereas IL-15R and IL-15 have
more ubiquitous expression patterns,25,29 we first
evaluated IL-2R expression on progenitors common to both and
NK T cells.17 IL-2R is expressed on DN thymocytes and
is down-regulated as cells mature to the DP stage.9 For this reason, thymocytes were gated on the DN population and analyzed for the expression of IL-2R on CD44+ (pro T1/pro T2) and
CD44 (pro T3/pro T4) cells.30 The expression
of IL-2R on CD44+ and CD44 DN thymocytes
from both lines of bi-5 mice was substantially reduced compared with
control mice (Figure 5A). Among
CD44 DN thymocytes, IL-2R expression was restricted to
the proT4 (CD44CD25) subset and was
substantially reduced on cells from bi-5 mice compared with controls
(data not shown).
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| Figure 5.
Expression of IL-2R is decreased on
CD4CD8 (DN) thymocytes of bi-5 mice.
(A) Expression of IL-2R on thymocytes from bi-5 (lines 747 and
5516), 2M/, wild-type (littermate for
747 line), CD1/, and CD1/+ mice.
Thymocytes were stained with CD8-biotin, CD4-biotin, IL-2R-PE, and
CD44-FITC and SA-APC and were gated on the
CD4CD8 population. Results are
representative of the decrease in IL-2R observed in the DN
population of bi-5 thymocytes in 6 or more separate experiments. (B)
Expression of IL-2R on
CD3/CD4/CD8/NK1.1/B220
thymocytes. Thymocytes were stained with CD3-APC, CD4-CyC, CD8-FITC,
NK1.1-biotin, B220-biotin, SA-PharRed, and IL-2R-PE. The results are
representative of 2 or more separate experiments. (C) DN thymocytes
from bi-5 mice and littermate controls were evaluated for the
expression of a variety of cytokine receptors.
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Because NK T cells are known to express IL-2R, and some are
coreceptor negative, reduced expression of IL-2R on DN thymocytes might reflect a reduction in NK T cells that express this protein rather than a reduction in expression of this protein on T-cell progenitors.17 To address this possibility, 2 different
approaches were taken. First, CD1/ and
2M/ mice were studied, because these
mice have substantially reduced NK T cell numbers (Figure 3B) but no
known defect in IL-2R expression. The expression of IL-2R on
CD44+ and CD44 DN thymocytes from both lines
of bi-5 mice was substantially reduced compared with wild type,
CD1/, or 2M/ mice
(Figure 5A). As a second approach, we excluded NK T cells, T
cells, B cells, and NK cells by gating on
CD3/CD4/CD8/B220/NK1.1
thymocytes. Again, the expression of IL-2R was reduced on this population of T-cell progenitors in bi-5 transgenic mice compared with
littermate controls (Figure 5B). Together, these findings suggest that
the reduction in IL-2R on DN thymocytes does not result solely from
the loss of NK T cells or a population of mature hematopoietic cells
for which this receptor is a marker. The decrease in IL-2R
expression for bi-5 transgenic mice appeared to be selective, because
the expression of IL-2R, c, c-kit, or IL-7R by DN
thymocytes of bi-5 mice and littermate controls was similar (Figure
5C), as was the abundance of IL-15R mRNA (data not shown).
Partial restoration of T-cell development in bi-5 mice by a
bcl-xL transgene but not by an IL-2R transgene
These findings suggested that the bi-5 phenotype might result,
at least in part, from reduced expression of IL-2R expression on T-cell progenitors. Two approaches were used to address
this possibility.
IL-2R and c transduce multiple signals in response to
IL-2 or IL-15, which lead to T-cell proliferation and differentiation and are mediated in part through STAT3, STAT5, JAK1, and
JAK3,10,31 and which counter apoptosis by inducing
bcl-xL or bcl-2.32-35 The enforced expression
of bcl-2 or bcl-xL in lymphoid progenitors substantially
restores development of conventional T cells in mice lacking
IL-7R (or c) but does not restore development of
T cells, B cells, or NK cells 11,36-38; NK T cells
were not addressed in these studies. Thus, if IL-2R acts in an
analogous manner for extrathymic and NK T-cell development as does
IL-7R for intrathymic T-cell development, enforced expression of
bcl-xL in the appropriate T-cell progenitors should restore
NK T cells and CD8+ -IELs but not -IELs
in bi-5 mice. To explore these possibilities, bi-5 mice were crossed to
mice that expressed bcl-xL under the control of the lck
proximal promoter (bcl+ mice).11 As predicted,
because bcl-xL is downstream of IL-2R, the
bi-5+/bcl+ mice still had reduced IL-2R
expression and normal c expression on DN thymocytes
(Figure 6A). Enforced expression of
bcl-xL did not restore NK T cells (Figure 6B) or
-IELs (Figure 6C) in bi-5 mice, but CD8+
-IELs were rescued, because the ratio of CD8- to
CD8-expressing cells within the -IELs of
bcl+/bi-5+ mice and bcl+/bi-5
littermates was similar (Figure 6D). These findings are similar to
those obtained in mice that are IL-15 deficient because of disruption
of the IRF-1 gene, in which enforced expression of bcl-2 restored
CD8+ T-cell numbers but did not restore NK cells, NK T
cells, or -IELs.39
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| Figure 6.
A bcl-xL (bcl+) transgene
rescued CD8, -TCR IELs in bi-5 mice, but not -IELs or
NK T cells.
(A) IL-2R and c expression in
bcl+/bi5+ mice. IL-2R was reduced in
bi-5+/bcl+ pro-T thymocytes, but
c was expressed at normal levels. (B) NK T cells and (C)
-IELs were not rescued by bcl-xl in bi-5+ mice.
(D) CD8 versus CD8 on TCR+
IELs. Bcl-xL appeared to restore the number of CD8
TCR+ IELs in the bi-5 mice. The ratio of CD8 to
CD8 IELs was similar for both bcl+/bi-5+
and bcl+/bi-5 mice. Two plots of
bcl+/bi-5+ are shown to demonstrate the degree
of variability. There was also a substantial increase in the numbers of
, TCR+CD8 T cells in bcl-xL
transgenic mice. These could be either CD4+ T cells or
coreceptor-negative T cells.
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The restoration of CD8+ -IELs in b-5 mice by
the bcl-xL transgene suggested that loss of a survival
signal provided through IL-2R might contribute to the bi-5
phenotype. However, enforced expression of IL-2R10
failed to rescue CD8+ -IELs, -IELs, or NK
T cells in bi-5 mice (data not shown). There was a substantial
reduction in the number of NK T cells in the IL-2R single transgenic
mice and a substantial reduction in total thymic cellularity and the
percentage of DP thymocytes in bi-5/IL-2R double transgenic mice,
which may have masked our ability to detect an effect of the IL-2R
transgene on T-cell development in bi-5 mice. Nonetheless, these
observations suggest that more than the loss of IL-2R during T-cell
development led to the phenotype observed in the bi-5 mice.
| |
Discussion |
Comparison between bi-5 mice and mice with Ikaros gene
disruptions
Our studies demonstrate that enforced expression of IK5 in
committed T-cell progenitors of bi-5 mice alters cell fate by
selectively impairing the development or survival of NK T cells and
extrathymically derived IELs. This phenotype was observed in 2 independent lines of bi-5 mice and differs greatly from those of
IK/ and the IK-DNA/ mice. T-cell number
is markedly reduced and CD4+ T cells are enriched in adult
IK/ mice,4 and T cells are absent in
IK-DNA/ mice.3 The much more profound
defects in IK/ and IK-DNA/ mice
compared with bi-5 and IK-DNA+/ mice likely reflect
differences both in the magnitude and in the timing of the defect in
Ikaros expression. In IK-DNA/ mice, long Ikaros isoform
expression is abolished and only short isoforms are expressed beginning
in HSCs and continuing thereafter in their cellular progeny, whereas in
bi-5 mice IK5 transgene expression commences in committed T-cell
progenitors and declines in mature T cells (Figure 1B,D), paralleling
the normal pattern of activity of the lck proximal
promoter.8
Mice that are heterozygous for the Ikaros DNA-binding domain disruption
(IK-DNA+/ mice) have extremely hyperproliferative T cells
and eventually develop clonal T-cell lymphomas and
leukemias.40 Like bi-5 mice, IK-DNA+/ mice
have increased expression of short relative to long Ikaros isoforms, so
the differences in phenotype between these mice likely result primarily
from differences in the timing of altered Ikaros expression. It is also
possible that differences in the nature of the short isoform
contribute, because the predominant short isoform expressed in
IK-DNA+/ mice is IK7, whereas bi-5 mice express the IK5
isoform. However, there is at present no evidence for functional
differences between these short isoforms. Together the phenotypes of
the bi-5 mice and the IK-DNA+/ mice support the notion
that modest changes in the relative abundance of Ikaros isoforms can
differentially affect cell fate and that the effects observed reflect
processes most sensitive to modest changes in Ikaros biologic
activity at that stage of development. Similarly, when the ratio
between short and long Ikaros isoforms was altered in human
CD34+ HSCs by retrovirally mediated overexpression of IK7,
the development of lymphoid dendritic cells but not myeloid dendritic
cells was selectively impaired.41
Reduced IL-2R expression on T-cell progenitors of bi-5
mice
The impaired development of IELs and NK T cells in bi-5 mice was
associated with impaired expression of IL-2R on DN T-cell progenitors. Like bi-5 mice, the numbers of NK T cells and -IELs are dramatically reduced in IL-2R and IL-15R knockout
mice,27,42 suggesting that reduced expression of IL-2R
might account for these effects of the bi-5 transgene. Similarly,
although the genes downstream of Ikaros that contribute to the striking
phenotypes observed in Ikaros knockout mice have not been clearly
defined, recent studies suggest that impaired expression of the flt-3
receptor and of c-kit ligand on HSCs and early T-cell progenitors may
contribute.1,7,41 Consistent with this notion, the
impaired development of lymphoid dendritic cells from human
CD34+ progenitors that overexpress IK7 was associated with
and may have resulted in part from reduced flt-3 receptor
expression.7,41 It is possible that the reduced expression
of IL-2R on T-cell progenitors in bi-5 mice, and of flt-3 receptor
and c-kit ligand on hematopoietic progenitors in the studies with
Ikaros knockout mice,1,7,41 may be due in part to
reductions in progenitor populations that express these receptors and
cytokines. However, our studies on IL-2R (Figure 5) and the effects
of retrovirally transduced IK7 on flt-3 receptor in vitro41
suggest that this is unlikely to be the sole explanation. Rather, these
findings collectively suggest that Ikaros may regulate the expression
of cytokine and cytokine receptor genes that play sequential and necessary roles in the proliferation, survival, and differentiation of
lymphoid progenitors.43 Consistent with this model, there are 2 potential Ikaros binding motifs (GGGAA) in close proximity within
the promoter region of human IL-2R, and deletions that eliminate
these sites greatly reduced reporter expression in CAT transcription
assays.44,45 There is also at least one GGGAA motif within
the promoter of murine IL-2R (S.N.T., unpublished observations,
May 2000).
Although enforced expression of IL-2R failed to rescue IELs or NK T
cells in bi-5 transgenic mice, this finding does not exclude a role for
reduced IL-2R expression in the phenotype of bi-5 mice. It does
suggest that Ikaros or Ikaros family members regulate the expression of
additional genes that are essential for the development of IELs and NK
T cells. This is consistent with current models, which suggest
that Ikaros plays a broad role in the regulation of gene
expression in lymphoid cells through multiple mechanisms.
How might the IK5 transgene perturb expression of IL-2R and
other genes that affect T-cell fate?
Ikaros has been proposed to play both positive and negative roles
in the regulation of gene expression in lymphoid cells. The dichotomous
effects of Ikaros may result in part from the complexity of Ikaros and
Ikaros family isoforms and their molecular interactions with each other
and with other regulatory proteins. The Ikaros family of proteins
interact with each other through C-terminal zinc fingers, whereas the
N-terminal zinc fingers are required for direct DNA
binding.46,47 The IK5 isoform expressed in bi-5 mice lacks
3 of 4 N-terminal Zn++ fingers required to bind the Ikaros
core binding motif but still contains the C-terminal Zn++
fingers.5 Similarly, the truncated Ikaros isoforms
expressed in IK-DNA/ and IK-DNA+/ mice
lack N-terminal Zn++ fingers. On the basis of studies
performed in vitro,46 these short isoforms can form dimers
with and function as dominant-negative inhibitors of long isoforms that
contain all 4 N-terminal Zn++ fingers.
Although originally proposed to regulate transcription directly, most
recent studies suggest that Ikaros regulates transcription primarily by
recruitment to target genes of macromolecular complexes, including the
Mi-2/NURD (nucleosome remodeling and histone deacetylation), mSIN3A
(histone deacetylase), and SWI/SNF chromatin remodeling complexes.2,48,49 SWI/SNF complexes may help to open
chromatin and facilitate transcription, whereas the Mi-2/NURD and
mSIN3A compact chromatin and impede transcription.50 Thus,
in bi-5 mice, IK5 could function as a dominant-negative inhibitor by
partially sequestering the DNA-binding isoforms away from the promoters of genes required for proper T-cell development, thereby impeding the
ability of endogenous longer Ikaros isoforms to facilitate transcription through the recruitment of SWI/SNF chromatin-remodeling complexes to these loci. An alternative explanation is that Ikaros represses the transcription of genes in developing T cells, either by
recruitment of the Mi-2/NURD and mSIN3A complexes1,50 or by
direct competition with transcription factors needed for gene expression, as it does at the  5 locus in developing B
lymphocytes51 and at the TdT locus in DP thymocytes in
vitro.52 In this model, the short Ikaros isoforms in bi-5,
IK-DNA/, and IK-DNA+/ mice would
facilitate repression mediated by long isoforms, perhaps through the
assembly into multimeric complexes with long isoforms bound to
pericentromeric foci.52 It is also possible that the actions of short Ikaros isoforms are context dependent, acting as
dominant-negative inhibitors when long Ikaros isoforms are limiting, as
in the IK-DNA/ mice, but forming multimers and acting
in concert with long isoforms when long isoform abundance is not
compromised.52 The complexity of the Ikaros system may
account in part for the difficulty in defining specific target genes
that account for the phenotypes observed in Ikaros mutant mice in
this and other studies.
| |
Acknowledgments |
We thank Stephen Smale for discussions and Ikaros antisera; Michael
Grusby for the CD1/ mice; Katia Georgopoulos for the
Ikaros cDNA; R. Perlmutter for the p1026 promoter; Tadatsugu Taniguchi,
Michael Bevan, Brad Nelson, Mark Groudine, and Michael Farrar for
review and discussions; Zandrea Ambrose for the IEL preparation
protocol; Ben Jacobson for the transgenic injections; and Kathryn Allen
for flow cytometry support.
| |
Footnotes |
Submitted August 28, 2001; accepted September 24, 2001.
Supported in part by grants HD18184 and AI37107 and by grant T32CA09537
(S.N.T.) from the National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Christopher B. Wilson, Dept of Immunology,
University of Washington, Campus Box 357650, 1959 NE Pacific St,
Seattle, WA 98195; e-mail: cbwilson{at}u.washington.edu.
| |
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Cell-autonomous defects |
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Abstract
Introduction