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Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+ intestinal intraepithelial lymphocytes, which
are also impaired in IRF-1-/- mice, are not rescued by
expressing human Bcl-2. Our results indicate that IRF-1 differentially
regulates the development of these lymphocyte subsets and that survival
signals involving Bcl-2 are critical for the development of
thymus-dependent CD8+ T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bcl-2, an antiapoptotic protein, protects developing and mature T cells
against a variety of apoptotic signals such as glucocorticoids and
anti-CD3 cross-linking (9, 10, 11, 12). Within the thymus,
Bcl-2 is expressed in double-negative (DN) cells, single-positive (SP)
cells, and cells undergoing positive selection, but only at a low level
in the majority of double-positive (DP) cells that have failed positive
selection and committed to death by neglect (13, 14, 15). Mice
deficient in Bcl-2 showed a gradual disappearance of T (and B) cells
after the second week of life (16, 17). These results
suggest that Bcl-2 may protect thymocytes undergoing positive selection
from apoptotic stimuli (18, 19). In
IL-7R
-/- and common
-chain-/- mice, endogenous Bcl-2 expression
in thymocytes is greatly reduced, and forced expression of human Bcl-2
as a transgene rescues T lymphopoiesis, indicating the importance of
Bcl-2-mediated survival signals for T cell development (20, 21).
To examine whether survival signals involving Bcl-2 are critical for the IRF-1-mediated lymphocyte development, we introduced a human Bcl-2 transgene into IRF-1-/- mice. Forced expression of Bcl-2 successfully restored functional CD8+ T cells in the thymus and spleen. Unexpectedly, however, development of NK cells, NK T cells, and iIEL was not restored in Bcl-2+ IRF-1-/- mice, indicating that IRF-1 differentially regulates development of these lymphoid cells through Bcl-2-dependent and -independent pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human Bcl-2 transgenic (tg) mice, C57BL/6 Eµ-Bcl-2-25 (12) and C3H-lck-Bcl-2 (10), used for our studies were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 IRF-1-/- (5) mice were provided by T. W. Mak (Ontario Cancer Institute, Toronto, Canada). IRF-1-/- mice expressing the human Bcl-2 (hereafter Bcl-2+IRF-1-/-) were made as follows. IRF-1-/- mice and human Bcl-2 tg mice were crossed to generate Bcl-2+IRF-1+/- mice. F1 mice heterozygous for IRF-1 and Bcl-2 were backcrossed to IRF-1-/- mice to generate Bcl-2+IRF-1-/- mice. All mice were maintained in our specific pathogen-free animal facility, and experiments were conducted between 6 and 12 wk of age in accordance with our Institutional Guidelines.
PCR for genotype determination
IRF-1 knockout and Bcl-2 tg alleles were screened by PCR with tail DNA. PCR was performed in a total volume of 50 µl containing 1 µl DNA, 20 pmol of each primer, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 200 µM concentrations of each dNTP, and 1.5 U Taq polymerase (Promega, Madison, WI). PCR cycles used for IRF-1 were 94°C for 2 min, 60°C for 1 min, and 72°C for 2 min; those for Bcl-2 were 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min. A step for an initial denaturation for 2 min at 94°C before the first cycle and a final extension step at 72°C for 10 min were included. Primers for IRF-1 and Bcl-2 were purchased from Sci-Media (Tokyo, Japan). Their sequences were: IRF-1 sense, 5'-TTCCAGATTCCATGGAGGCACGC-3'; antisense, 5'-ATGGCACAACGGAAG TTTGCC-3' (for wild-type allele; product size, 900 bp), 5'-ATTCGCCAATGACAAGACGCTGG-3' (for knockout allele; product size, 700 bp); Bcl-2 sense, 5'-GTGTGTGGAGAGCGTCAACC-3'; antisense, 5'-TCACTTGTGGCTCAGATAGG-3' (product size, 250 bp). Ten microliters of final PCR products were analyzed by electrophoresis on a 1.5% agarose gel.
Abs and flow cytometric analysis
The following mAbs were purchased from PharMingen (San Diego,
CA): 145-2C11-FITC, -PE (anti-CD3
); H57-597-FITC, -PE, -biotin
(anti-TCR-
); GK1.5-PE (anti-CD4); PK136-PE, -biotin
(anti-NK1.1); 53-6.7-PE, -biotin (anti-CD8). Biotinylated
mAbs were detected with streptavidin red (Life Technologies,
Gaithersburg, MD). From 1 to 2 million cells were stained in PBS-2%
FCS, washed, and analyzed on a FACScan using the CellQuest program
(Becton Dickinson, San Jose, CA).
Detection of human and mouse Bcl-2 was effected by intracellular staining. After staining for surface Ags, cells were fixed with PBS containing 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.03% saponin for 10 min at room temperature, and stained with anti-human Bcl-2-FITC (Dako, Glostrup, Denmark), or purified anti-mouse Bcl-2 (PharMingen) plus anti-hamster IgG-FITC (PharMingen). Stained cells were washed with PBS containing 0.03% saponin and PBS-2% FCS, and cells were analyzed on a FACScan (Becton Dickinson).
Allogeneic MLR
CD8+ T cells were purified from
splenocytes using an AutoMacs magnet separation system (Miltenyi
Biotec, Bergisch Gladbach, Germany). Cells (1 x
105) were cultured with irradiated C57BL/6 or
BALB/c splenocytes (1 x 105) in
96-well flat-bottom plates. In some experiments, purified cells were
cultured in the presence of 5 µg/ml Con A or 1 µg/ml anti-CD3
plus 1 µg/ml anti-CD28. After 3 days of culture, 3.7 kBq
[3H]thymidine (Amersham Pharmacia, Piscataway,
NJ) per well were added. After 16 h, cells were harvested onto
filter papers and 3H incorporation was measured
by a liquid scintillation counter. Amounts of IFN- and IL-2 in
culture supernatants were determined with a Quantikine M ELISA kit (R&D
Systems, Minneapolis, MN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that IRF-1 is required for positive selection
of CD8+ T cells in the thymus (5, 6). Consistent with previous studies (5, 6),
selective reduction of CD8+ T cells was observed
in all lymphoid organs as well as blood of
IRF-1-/- mice (Fig. 1
A and data not shown).
Because IRF-1 is expressed mainly in thymocytes after the DP stage
(6), it is possible that IRF-1 controls positive selection
of CD8+ T cells by regulating survival signals.
Several experiments suggest that thymocyte survival signals for
positive selection involve Bcl-2, which is up-regulated during positive
selection and enhances thymocyte survival (12, 13, 14, 15, 16, 17). We
thus examined the expression of endogenous mouse Bcl-2 in wild-type and
IRF-1-/- thymocyte subsets (Fig. 1B,
left panels). As reported previously, endogenous Bcl-2 is
expressed predominantly in CD8SP, CD4SP as well as immature DN
thymocytes, whereas DP thymocytes express Bcl-2 at low levels in
wild-type mice. In IRF-1-/- thymocytes, levels
of endogenous Bcl-2 expression in CD4SP, DN, and DP thymocytes were
indistinguishable from those of wild-type mice. In contrast, the
majority of IRF-1-/- CD8SP cells expressed
endogenous Bcl-2 at significantly lower levels than those of wild-type
CD8SP cells (Fig. 1B, left panels). Similar results were
obtained when IRF-1-/-CD8SP cells expressing
high levels of TCR were gated and analyzed for Bcl-2 expression (data
not shown).
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To examine whether IRF-1-mediated survival signals critical for
CD8+ T cell development involve Bcl-2, we
introduced a human Bcl-2 transgene driven by a
lck-proximal promoter into IRF-1-/- mice. Fig. 1
B (right panels) shows expression of the human
Bcl-2 protein in thymocyte subsets and splenic T
cells of lck-Bcl-2+
IRF-1-/- mice. Virtually all thymocytes and
splenic T cells expressed the Bcl-2 protein, and no difference was
observed in the levels of the transgene expression between each
thymocyte subpopulation. As reported previously (10),
levels of the human Bcl-2 transgene expression in splenic T
cells was significantly lower than those in thymocytes, which is likely
due to the use of lck-proximal promoter to drive the transgene. As
shown in Fig. 1C, introduction of human Bcl-2 resulted in
increasing numbers of CD8+ T cells as well as
CD8:CD4 ratio to a level similar to those of
IRF-1+/- mice (Fig. 1C). Similarly,
introduction of an Eµ-Bcl-2 transgene also
restored the development of CD8+ T cells in
IRF-1-/- mice (Figs. 3 and 4A and
data not shown).
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We next examined by MLR whether the CD8+ T
cells restored by the Bcl-2 transgene expression are
functionally mature. Because
lck-Bcl-2+IRF-1-/- mice
bear a mixed background of H-2K and
H-2b, we used C57BL/6
Eµ-Bcl-2+IRF-1-/-
(H-2b) mice. CD8+ T cells
were purified from the spleens of IRF-1-/-,
Bcl-2+IRF-1-/-, and
IRF-1+/- mice by magnetic separation, and
allogeneic MLR was set up where isolated CD8+ T
cells were cocultured with either syngeneic C57BL/6
(H-2b) or fully allogeneic BALB/c
(H-2d) spleen cells. As shown in Fig. 2, Bcl-2+IRF-1-/-CD8+
T cells were capable of proliferating and producing IFN- and IL-2 in
response to BALB/c splenocytes, which was comparable with those of
IRF-1+/-CD8+ T cells (Fig. 2). As expected, CD8+ T cells isolated from mice
of any genotype did not proliferate or produce the cytokines, if any,
in response to syngeneic C57BL/6 spleen cells.
Bcl-2+IRF-1-/-CD8+
T cells were also able to proliferate and secrete IL-2 in response to
anti-CD3 plus anti-CD28 mAb as well as Con A stimulation at
levels comparable with those of
IRF-1+/-CD8+ T cells (data
not shown). We demonstrated for the first time that
CD8+ T cells isolated from
IRF-1-/- mice were functionally mature (Fig. 2). These results indicate that the expression of Bcl-2 restores the
number of CD8+ T cells in
IRF-1-/- mice and that the restored
CD8+ T cells are functional as wild-type
CD8+ T cells.
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+ iIEL are not rescued by
Bcl-2 transgene expression in IRF-1-/-
mice
We and others have previously reported that the numbers of NK, NK
T, and TCR-+ iIEL are substantially reduced
in IRF-1-/- mice that are defective in IL-15
production (7, 8) (Fig. 3A). It was later shown that
the development of these cells is impaired in
IL-15R-/- as well as
IL-15-/- mice (22, 23). One of the
important roles of IL-15 is to deliver survival signals involving Bcl-2
family members such as Bcl-2 and Bcl-xL in these
lymphocyte subsets (24, 25, 26). Consistent with these
studies, we found that endogenous Bcl-2 was hardly detectable in
TCR-+ iIEL prepared from
IRF-1-/- mice (Fig. 3B). However,
expression levels of endogenous Bcl-2 in NK cells and NK T cells of
IRF-1-/- mice were comparable with those of
wild-type mice (data not shown). We then examined whether the
expression of Bcl-2 restores the development of
TCR-+ iIEL as well as NK and NK T cells
using Eµ-Bcl-2+ IRF-1-/-
mice. Although Eµ-derived human Bcl-2 is expressed in these
lymphocyte subsets (Fig. 3C and data not shown), none of
these lymphocyte subsets was restored in
Eµ-Bcl-2+IRF-1-/-
mice (Fig. 3A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ iIEL, which are also reduced
in IRF-1-deficient mice, remained low in Bcl-2+
IRF-1-/- mice. Targeted disruption of the IRF-1 gene in mice revealed that whereas CD4+ T cells are present normally, number of CD8+ T cells is reduced in the thymus and in the periphery, demonstrating IRF-1 as a transcription factor essential for the development of CD8+ T cells (5). Because IRF-1 regulates TAP1 and latent infection membrane protein 2, critical components for the expression of MHC class I-peptide complex, down-regulation of these molecules may result in the reduction of MHC class I expression and the lack of positive selection of CD8 lineage (4). However, results from bone marrow chimeras and thymocyte reaggregation cultures performed between wild-type mice and IRF-1-/- mice have indicated that IRF-1-/- stromal cells can support the development of CD8+ lineage and that the reduction of CD8+ T cells was not simply due to the defect of thymic stromal cells (6). Using IRF-1-/- mice expressing TCR specific for p33, a lymphocytic choriomeningitis virus glycoprotein, and those expressing TCR specific for H-Y, it has been shown that positive selection of CD8+ T cells is impaired in the absence of IRF-1. Importantly, IRF-1 expression is developmentally regulated in the thymus and detected mainly in CD4SP and CD8SP cells after the DP stage (6). Because Bcl-2 is also expressed in SP thymocytes (12, 13, 14, 15, 16, 17), it is possible that engagement of MHC class I-peptide with TCR induces IRF-1 which subsequently up-regulates cell survival signals involving Bcl-2 in SP thymocytes. Although Bcl-2 may augment positive selection (18, 19), up-regulation of this molecule alone does not promote thymocyte differentiation (18). It is possible that the prolonged survival induced by Bcl-2 may give these cells "an appropriate duration" to obtain sufficient signals necessary for positive selection.
Because no appropriate IRF-1-binding motif was identified in promoter
region of the mouse Bcl-2 gene by computer analysis (T.
Ohteki and S. Koyasu, unpublished data), it is unlikely that IRF-1
controls the expression of Bcl-2 gene directly
but induces target gene(s) critical for the Bcl-2 up-regulation in
cells of CD8 lineage. In this context, certain cytokine(s) are potent
in up-regulating Bcl-2 levels in thymocytes and lymphocytes (20, 21, 24, 25, 26). Among various cytokines, previous studies have shed
light on IL-15 as an important cytokine controlling the
CD8+ T cell development. An IRF-binding element
exists within the 5'-upstream region of mouse IL-15 gene where IRF-1
binds and regulates the induction of IL-15 gene expression
(7). Indeed, the IL-15 gene is not induced in
IRF-1-deficient bone marrow cells (7, 8), demonstrating
that IRF-1 is a positive regulator for the IL-15 induction. Consistent
with these observations, CD8+ T cells are
substantially reduced in the thymus and/or periphery of
IL-15R-/- as well as
IL-15-/- mice (22, 23).
Furthermore, recent studies indicated that IL-15 provides survival
signals involving Bcl-2 family members such as Bcl-2, Bax, and
Bcl-xL in T as well as NK cells
(24, 25, 26). IL-15 secreted by bone marrow-derived dendritic
cells and macrophages may contribute to the induction of survival
signals involving Bcl-2 during the development of
CD8+ T cells in the thymus, although we cannot
exclude possible roles of other cytokines or cytokine-independent
mechanisms.
Alternatively, the IL-15/IL-15R interaction may be critical for the
maintenance of peripheral CD8+ T cell pool. It
was reported that CD8+ T cells bearing the
activated/memory phenotype that comprise 10–20% of
CD8+ T cells in the spleen and lymph nodes are
absent in IL-15-/- and
IL-15R-/- mice (22, 23).
Previous reports have shown that Bcl-2 is up-regulated in memory-type
CD8+ T cells and that IL-15 is critical in the
long term maintenance of memory CD8+ T cells in
vivo (27, 28), supporting the above notion. In mice
deficient in Bcl-2, the number of lymphocytes decreased within a few
weeks after birth (16, 17). Among T cells,
CD8+ T cells disappeared first, followed by
CD4+ T cells, in both thymus and periphery of
Bcl-2-/- mice. These results and our data
collectively suggest that IL-15-induced Bcl-2 up-regulation is also
important for survival of peripheral CD8+ T
cells.
Studies using mice deficient in IL-15R as well as IL-15 have proved
that IL-15 is also essential for the development of NK cells, NK T
cells, and TCR-+ iIEL (22, 23). Because Bcl-2 expression was minimal in
TCR-+ iIEL of
IRF-1-/- mice (Fig. 3B), IL-15
induced by IRF-1 may provide TCR-+ iIEL
with survival signals. Indeed, we have observed that addition of IL-15
to TCR-+ iIEL of
IRF-1-/- mice induced the expression of Bcl-2
and proliferation of these cells (Ref. 8 and T. Ohteki and
S. Koyasu, unpublished observations). However, forced Bcl-2 expression
does not restore these lymphocytes in vivo (Fig. 3), suggesting that
IL-15 does not simply induce Bcl-2 to provide these cells with survival
signals. Rather, IRF-1/IL-15 likely controls the development and/or
expansion of mature TCR-+ iIEL as well as
NK and NK T cells by multiple pathways (8, 25).
In conclusion, survival signals involving Bcl-2 induced by IRF-1 appear
to act as a critical factor for the development and survival of
thymus-derived CD8+ T cells. In contrast, IRF-1
controls the development of NK, NK T, and
TCR-+ iIEL through distinct mechanisms
in vivo.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shigeo Koyasu, Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: koyasu{at}microb.med.keio.ac.jp
3 Abbreviations used in this paper: IRF, IFN-regulatory factor; iIEL, intestinal intraepithelial lymphocytes; DN, double-negative; SP, single-positive; DP, double-positive; tg, transgenic.
Received for publication October 13, 2000. Accepted for publication March 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene regulatory elements. Cell 54:903.[Medline]
+ (NK1+T) cells, natural killer cells, and intestinal intraepithelial T cells. J. Exp. Med. 187:967. in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia. Proc. Natl. Acad. Sci. USA 91:3700. chain-deficient mice. Immunity 7:155.[Medline]
T cell receptor in mice. Eur. J. Immunol. 27:2885.[Medline]
+ intestinal intraepithelial lymphocytes. J. Immunol. 162:1896.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or BALB/c (
) splenocytes. After 3 days of culture,
proliferation measured by [3H]thymidine incorporation for
16 h was examined. B, Amounts of IL-2 and IFN-