 |
Introduction |
In addition to the conventional lymphocyte subsets, other
lineages have been identified as NK1.1+TCR-
/
+
(NK1+T) cells, NK cells, and intestinal intraepithelial lymphocytes (IELs). NK1+T cells have been recently classified
as a lymphocyte subset that shares common features with both
NK cells and conventional T cells. This lineage expresses NK
markers including NKR-P1, Ly-49, and IL-2R
/15R
as
well as an invariant V
14J281TCR-
chain in combination with V
8, V
7, or V
2 (1, 2). Expression of these
TCRs is required for NK1+ T cell development (3, 4). They
are positively selected by MHC class I-related CD1 or thymic
leukemia (TL) molecules (5). The majority of TCR-
/
+
or TCR-
/
+ intestinal IEL expresses CD8-
/
homodimers.
Both NK1+T cells and CD8-
/
+ intestinal IELs can develop through either extrathymic or alternative thymic pathways (1, 2, 8). Notably, the IL-2R
/15R
chain is required
for the development of NK1+T cells, NK cells, and CD8-
/
+ intestinal IELs (9, 10), and IL-15 preferentially promotes the proliferation of these lymphocyte subsets (10).
IFN regulatory factor 1 (IRF-1), an IFN-inducible transcriptional activator, was initially identified as a protein that
binds cis-acting DNA elements in the IFN-
promoter
(13) and the IFN-stimulated response element of IFN-
/
-stimulated genes (16, 17). Recent studies with IRF-deficient (IRF-1
/
) mice demonstrated a reduction of
CD8+TCR-
/
+ cells and decreased MHC class I levels
as a consequence of reduced expression of transporter associated with antigen processing 1 (TAP-1) and low molecular weight protein 2 (LMP-2; 18, 19).
Since IRF-1 deficiency has been related to T cell maturation, we examined the development of NK1+T cells, NK
cells, and IELs in IRF-1
/
mice. Data indicated that these
lymphocyte subsets were selectively reduced and IL-15
messenger RNA (mRNA) was barely detectable in IRF-1
/
mice. Therefore, IRF-1 regulates the IL-15 gene that
is required for survival and/or expansion of these lymphocyte subsets in vivo.
 |
Materials and Methods |
Mice.
Mice deficient in IRF-1 (18) were backcrossed five
times with C57BL/6 mice. Homozygous IRF-1
/
mice were
bred and identified by staining blood with anti-CD8 and -CD4
mAb. Wild-type or heterozygous mice were used as controls. All
mice were maintained in our animal facility according to institutional guidelines, and experiments were done between 8 and 14 wk
of age.
Cell Preparation and Culture.
Liver mononuclear cells (MNCs)
and IELs were prepared as previously described (20). In some experiments, liver MNCs or IELs obtained from IRF-1
/
mice
were cultured with 100 ng/ml mouse IL-15 (provided by Immunex Co., Seattle, WA) for 7 d.
Antibodies and Flow Cytometric Analysis.
The following mAb
conjugates were purchased from PharMingen (San Diego, CA)
and used in this study: M1/69-FITC (anti-HSA), 53-5.8-FITC
(anti-CD8
), H57-597-FITC and -PE (anti-TCR-
), TM-
1-PE (anti-IL-2R
), GL-3-PE (anti-TCR-
), 53-6.7-PE (anti-CD8), PK136-PE and -biotin (anti-NK1.1), 1B1-PE (anti-CD1),
27D-biotin (anti-LFA-1), IM7-biotin (anti-CD44), and KJ16-biotin (anti-V
8.1,8.2). B22-purified mAb (anti-H-2Db) was prepared in our laboratory. Biotinylated mAbs were detected with
streptavidin red 670 (GIBCO BRL, Gaithersburg, MD) and purified mAbs were detected with goat anti-mouse IgG-FITC or
goat anti-rat IgM-FITC; 106 cells were stained in 2% FCS PBS,
washed, and analyzed by FACScan® using the Lysis II program
(Becton Dickinson, Mountain View, CA).
Analysis for IL-15 mRNA Expression.
Bone marrow (BM) cells
were isolated and stimulated by 30 µg/ml LPS and 100 U/ml IFN-
for 6 h. Total cellular RNA was isolated with TRIZOL (GIBCO
BRL) according to the manufacturer's protocol. 10 µg of total
RNA were subjected to electrophoresis in a denaturing 1.0% agarose gel containing 2% formaldehyde and transferred to Hybond
N+ nylon membrane (Amersham Corp., Arlington Heights, IL). The filter was hybridized with mouse IL-15 cDNA probe radioactively labeled with [32P]dCTP. The mouse IL-15 cDNA used
as a probe was obtained by polymerase chain reaction using specific
primers: sense primer 5'-GCC AGC TCA TCT TCA ACA-3' and
antisense primer 5'-TAA GTC TGA GAC GAG CTC TTT-3'.
Radioactivity was assessed using phosphorimager (Molecular Dynamics, Sunnyvale, CA). The filter was stripped and rehybridized with a
-actin cDNA probe.
 |
Results and Discussion |
Impaired NK1+T Cell and NK Cell Development in IRF-1
/
Mice.
Mouse NK1+T cells are generally either
CD4+8
or CD4
8
cells that are primarily found in the
thymus, liver, and BM (1, 2). We examined the NK1+T
cell subset in mice deficient for IRF-1
/
. Surprisingly, the
percentages of thymic and liver NK1+T cells were decreased by 4-5 fold and 8-10 fold, respectively, in IRF-1
/
mice. The total number of thymic NK1+T cells obtained
from IRF-1
/
mice was 10-fold lower than in wild-type
control mice. Interestingly, a partial reduction of NK1+T
cells was also seen in IRF-1+/
mice (Fig. 1, Table 1). The
IL-2R
/15R
+TCR-
/
+ cells were also decreased, suggesting that the pronounced reduction of NK1+T cells detected in IRF-1
/
mice was not simply due to the loss of
NK1.1 molecules from the cell surface (data not shown).
The small number of NK1+T cells detected in IRF-1
/
mice expressed the IL-2R
/15R
chain and preferentially
expressed V
8+ TCR as seen in control mice (data not
shown). In addition, analysis of the thymus, liver, and
spleen using IRF-1+/+, IRF-1+/
, and IRF-1
/
mice
clearly demonstrated a reduction of NK cells (TCR-
NK1.1+) in IRF-1
/
mice (Fig. 1). This is consistent
with the lack of NK cell function previously reported in
IRF-1
/
mice (21). Interestingly, IRF-1+/
mice consistently showed an intermediate phenotype, reflecting the
dose-dependent requirement for genes regulated by IRF-1.
These analysis showed that IRF-1 is important for NK cell
and NK1+T cell development.

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Fig. 1.
IRF-1 is important for NK1+T cell and NK cell maturation.
Thymocytes, liver, and spleen MNCs from indicated strains were stained with M1/69-FITC (anti-HSA), H57-597-PE (anti-TCR- ), and
PK136-biotin (anti-NK1.1) plus streptavidin 670. HSA cells are shown.
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|
Previous reports have shown that CD4
8+TCR
/
+
cells were selectively reduced in thymus and periphery of
IRF-1
/
mice (18). The data demonstrated a crucial role
for IRF-1 in T cell development for the first time. A recent
paper suggested that IRF-1 controls MHC class I expression through the regulation of transporter associated with
antigen 1 and low molecular weight protein (19). Since
mouse NK1+T cells require
2-microgloblin-associated
CD1 and TL molecules for development (5, 20, 22, 23),
we examined CD1 expression on thymocytes from IRF-1
/
mice. Consistent with a recent paper (19), Fig. 2
showed that the lack of the IRF-1 gene clearly resulted in
reduced H-2Db expression. However, the mean intensities
of CD1 on IRF-1
/
thymocytes was comparable to littermate controls, suggesting that the IRF-1 gene does not
control NK1+T cell development through CD1 expression. In addition, we can further exclude the role of the TL
antigen in NK1+T cell development, since both IRF-1
/
and control mice are of the C57B1/6 background and do
not express TL.

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Fig. 2.
Normal CD1 expression on IRF-1 / thymocytes. Thymocytes from the indicated strains were stained with 1B1-FITC (anti-CD1), 57.6.7-PE (anti-CD8), and L3T4-biotin (anti-CD4) plus streptavidin 670, and double-positive CD4+8+ thymocytes were analyzed for
CD1 expression. For H-2Db expression, total thymocytes were stained
with B22 (anti-H-2Db) plus goat anti-mouse Ig-FITC.
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Maturation of Intestinal IELs Is Reduced in IRF-1
/
Mice.
The majority of IELs express CD8 and can be divided into two subsets. One population bears CD8-
/
+
heterodimers and expresses TCR-
/
+, whereas the other
expresses CD8-
/
+ homodimers consisting of TCR-
/
+
and TCR-
/
+ cells. Using thymectomized recombinase
activating gene (RAG)-deficient mice reconstituted with
BM cells from athymic (nude) mice, thymus-independent development of CD8-
/
+ IELs has been clearly demonstrated to occur (8). Surprisingly, in IRF-1
/
mice, the
percentage of intestinal CD8-
/
+ IELs was approximately eight- to ninefold less than in wild-type control
mice. As seen with NK1+T cells, mice heterozygous for
IRF-1+/
showed altered CD8-
/
+ IEL development.
TCR-
/
+ IELs were profoundly reduced by IRF gene
disruption (Fig. 3 A, Table 2). In addition, CD8-
/
+T
cells were also reduced as seen in periphery. The total cell numbers of IELs from IRF-1
/
mice (0.4 ± 0.1 × 106)
were three- to fourfold lower than those from littermate
controls (1.5 ± 0.3 × 106). Therefore, IRF-1 controls the
expression of genes important for IEL T cell development.
It is likely that the reduced development of intestinal
/
+T cells is controlled by other mechanisms unrelated to
MHC class I expression in IRF-1
/
mice. Previous studies
using
2-microglobulin-deficient (MHC class I
/
) mice
showed a reduction in TCR-
/
+ IELs, but not TCR-
/
+ IELs (24), demonstrating that TCR-
/
+ and TCR-
/
+ IELs have differential requirements for
2-microglobulin dependent selection.

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Fig. 3.
IRF-1 controls intestinal IEL development. (A) Intestinal
IELs were obtained from either IRF-1+/+ mice, IRF-1+/ mice, or IRF-1 / mice and stained with H57-597 (anti-TCR- ) and GL-3-PE (anti- TCR- ), or 53.6.7-FITC (anti-CD8 ) and Lyt3-PE (anti-CD8 ). (B) Thymocytes were stained with L3T4-FITC (anti-CD4), GL-3-PE (anti- TCR- ), and 53.6.7-FITC (anti-CD8 ). Histograms are gated on double-negative CD4 8 thymocytes and TCR- expression is shown.
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Since the majority of thymus-independent intestinal
TCR-
/
+ cells were absent in IRF-1
/
mice, we also
examined whether thymic TCR-
/
+ cells were present in
these mice. Although the number of intestinal TCR-
/
+
cells were decreased by 10-fold in IRF-1
/
mice, thymic
TCR-
/
+ cells were normal (Fig. 3 B, Table 2). Thus,
IRF-1 selectively affected the development of intestinal
TCR-
/
+ cells.
IL-15 mRNA Expression Is Impaired in IRF-1
/
BM
Cells.
As certain cytokines are crucial for lymphocyte development, it is possible that a reduction in the expression
of cytokine receptors or cytokines may result in poor selection, survival, or expansion of NK1+T cells, NK cells, and
intestinal IEL subsets in IRF-1
/
mice. IL-15 is one of the
most likely targets because NK1+T cells, NK cells and intestinal IEL subsets are severely reduced in IL-2R
/
15R
/
mice (9, 10), while present in normal numbers in
IL-2, IL-7R
, or IL-7-deficient mice (10, 25). Interestingly, IL-15 preferentially promotes the proliferation of
these T cell subsets (10). Thus, we examined IL-15
mRNA expression by Northern blot analysis (Fig. 4 A).
Wild-type BM cells cultured in the presence of LPS and
IFN-
for 6 h, clearly increased IL-15 mRNA levels. In
contrast, IL-15 mRNA remained undetectable in IRF-1
/
BM cells, even after induction with LPS and IFN-
. These
data demonstrate that IRF-1 regulates the expression of IL-15.

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Fig. 4.
Impaired lineage
development correlates with the
absence of IL-15. (A) Limited
IL-15 expression in the absence
of IRF-1. BM cells were isolated
from IRF-1 / mice or control
wild-type (WT) mice. Total
RNA was extracted from untreated BM cells or BM cells cultured for 6 h in the presence of
LPS (30 µg/ml) and IFN- (100 U/ml). Northern blot analysis was performed using IL-15
cDNA and -actin probes. (B)
IL-15 induces the expansion of
NK1+T cells, NK cells, and IEL
subsets. Liver MNCs and intestinal IELs were isolated from IRF-1 / mice and cultured with 100 ng/ml mouse IL-15 for 7 d.
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NK1+T Cells, NK Cells, and Intestinal IELs were Recovered
by IL-15 In Vitro.
To further examine the importance of
IL-15 for maturation of NK1+T cells, NK cells, and intestinal IEL subsets in IRF-1
/
mice, liver MNCs and intestinal IELs were isolated from these mice and cultured with
100 ng/ml mouse IL-15 for 7 d (Fig. 4 B). Recovery of
these lymphocyte subsets was observed. This suggested that
IL-15 is essential for the survival or expansion of NK1+T
cells, NK cells, and intestinal IELs, and not early development or commitment.
NK1+T cells, NK cells, and intestinal IELs share cell surface markers and other common features during development. In addition to the expression and developmental requirement of IL-2R
/15R
chain, they also express the
NK complex that encompasses NKR-P1 and Ly-49 (1, 2,
28, 29). In contrast, conventional T cells do not express
these products. Although the majority of T cells develops
in the thymus, NK cells develop normally in athymic nude
mice. The developmental origin of NK1+T cells can be either thymus dependent or independent (2, 30, 31). Thymus-independent development of intestinal CD8-
/
+ T cells has
been clearly demonstrated to occur (8). Thus, NK1+T cells
and intestinal CD8-
/
+ T cells are related to the NK lineage and can be distinguished from mainstream T cells. Our
results demonstrate that IRF-1 controls the expression of
IL-15, which is likely to be important for the maturation of the
related NK1+T cell, NK cell, and CD8-
/
+ IEL lineages.
Address correspondence to Toshiaki Ohteki, Ontario Cancer Institute, Departments of Medical Biophysics
and Immunology, 610 University Ave., Toronto, Ontario, Canada, M5G 2M9. Phone: 416-946-2000; Fax:
416-946-2086; E-mail: tohteki{at}oci.utoronto.ca
We wish to thank Dr. Hans-Willi Mittrucker (Amgen Institute, Toronto, Ontario, Canada) for providing the
C57BL/6 background IRF-1
/
mice, Dr. Yutaka Tagaya (National Cancer Institute, Bethesda, MD) for helpful discussion, and Arsen Zakarian (Ontario Cancer Institute, Toronto, Ontario, Canada) for technical assistance.
This work was supported by the Medical Research Council of Canada. P.S. Ohashi is a recipient of a Medical Research Council scholarship.
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