Download A two-step model of T cell subset commitment: antigen

International Immunology, Vol. 14, No. 6, pp. 567±575
ã 2002 The Japanese Society for Immunology
A two-step model of T cell subset
commitment: antigen-independent
commitment of T cells before encountering
nominal antigen during pathogenic
infections
Makoto Kanoh1, Teruyoshi Uetani1, Hirokazu Sakan1, Saho Maruyama1,
Fengzhi Liu1, Kohsuke Sumita1 and Yoshihiro Asano1
1Department of Immunology and Host Defenses, Ehime University School of Medicine, Shigenobu,
Onsen-gun, Ehime 791-0295, Japan
Keywords: antigen-presenting cell, GATA-3, pathogen infection, T cell commitment, T-bet
Abstract
Pathogenic infections lead to activation of innate immunity followed by induction of a type 1 T cell
subset and, therefore, provide a good model to evaluate when T cells commit to type 1 T cells.
Here we show a two-step mechanism of T cell subset commitment during pathogenic infection.
The ®rst step is mediated by the basal function of macrophage/dendritic cells and is antigen
independent. This step modulates the committed precursor frequency of T cell subsets and
in¯uences the expression of T-box expressed in T cells (T-bet) and GATA-3 genes. IL-12 and NK
cells are not required for this step. The second step requires antigenic stimulation of T cells
together with IL-12 or IL-4, and in¯uences on the expression of T-bet and GATA-3. We propose a
two-step T cell subset commitment pathway based on these observations. Therefore, pathogenic
infections in¯uence functional T cell commitment before T cells encounter nominal antigen.
Introduction
Pathogens stimulate the immune response of a host
resulting in a clearance of microbes (1±4). T cells are
divided into two types according to the set of lymphokines
they produce, i.e. IFN-g-producing type 1 T cells and IL-4producing type 2 T cells (1,2,5,6). The differentiation
process of T cells into type 1 or type 2 is controlled by
cytokines produced during the innate immune response in
its early phase (7±12). Cytokines present at the initiation of
the immune response at the stage of ligation of the TCR
determine type 1 and type 2 T cell differentiation from the
precursor (13,14). Viral and bacterial infections lead to the
activation of innate immunity followed by the induction of a
type 1 T cell subset which is thought to be induced in an
antigen-speci®c fashion under the in¯uence of IL-12 (1±
6,9,11,14±18). However, IL-12 gene expression is suppressed at the transcriptional level during some infections
such as by Plasmodium or measles (17,19,20). Although
the T cell subset differentiation pathway has been
characterized, the effects of a pathogenic infection on T
cell subset commitment during infection have yet to be
elucidated (1±11,14,15,18).
Macrophages and NK cells function to connect the innate
immune system and the acquired immune system during
infections by pathogens. In previous studies, we demonstrated that IFN-regulatory factor (IRF)-1 gene disrupted mice
fail to mount a type 1 response in vitro (16). These mutant mice
were defective in the production of IL-12 and activation of NK
cells, resulting in a failure to induce the IFN-g-producing type 1
T cell subset. The defect found in IRF-1±/± mutant mice of
inducing type 1 T cells was restored by the addition of wildtype macrophages, suggesting the precursor of type 1 T cells
is normally differentiated in the mutant mice (17). The results
suggest that the induction of type 1/type 2 T cell subsets
occurs based on a two-step mechanism. Therefore, pathogenic infections provide a good model to evaluate when T
cells commit to type 1 and type 2 T cells.
Correspondence to: Y. Asano; E-mail: [email protected]
Transmitting editor: A. Singer
Received 5 April 2001, accepted 28 January 2002
568 A two-step model of T cell subset commitment
Fig. 1. In¯uence of APC of Lm-infected mice on T cell subset differentiation. (A) T cells of uninfected TCR-Tg mice were cultured for 5 days in
the presence of uninfected (open bars) or Lm-infected (shaded bars) BALB/c APC and 1 mM OVA peptide. The cultured cells were restimulated with uninfected BALB/c APC and homologous peptide, and cytokines were subsequently detected. (B) T cells of uninfected TCRTg mice were cultured for 5 days in the presence of uninfected BALB/c APC (open bars) or the mixture of uninfected and Lm-infected BALB/c
APC (shaded bars) and 1 mM OVA peptide. The cultured cells were re-stimulated with uninfected BALB/c APC and homologous peptide, and
cytokines were subsequently detected.
In the present study, we analyzed the effect of pathogenic
infection on T cell subset commitment using TCR-transgenic
(Tg) mice. Here we show for the ®rst time a two-step induction
mechanism for T cell subsets during pathogenic infection. The
®rst step is induced by the basal function of macrophage/
dendritic cells, and is antigen independent and non-speci®c.
This step affects the precursor frequency of type 1 and type 2
T cell subsets. Although this ®rst step does not involve
activation through the TCR, the step in¯uences GATA-3 and Tbox expressed in T cells (T-bet) gene expression which is
thought to regulate type 1/type 2 T cell subset differentiation
(21±25). The second step requires antigenic stimulation of T
cells together with IL-12 or IL-4, and is antigen-speci®c and
accompanied with T-bet and GATA-3 gene activation.
Therefore, T cells are committed to type 1 T cells before they
encounter nominal antigen involving T-bet and GATA-3 genes.
In addition, T cells with different speci®cities are in¯uenced by
infection by a single pathogenic species. This ®nding could
lead to new insights into T cell responses during pathogenic
infections.
Methods
Cytokines and antibodies
Recombinant murine IL-4 and IL-12 were obtained as a culture
supernatant of transfectants provided by Dr H. Karasuyama
(Tokyo Metropolitan Institute of Medical Science, Tokyo) and
Dr H. Yamamoto (Osaka University, Osaka) respectively
(26,27). mAb speci®c for IL-4 and IL-12 were provided by Dr
W. E. Paul (National Institutes of Health, MD) and by Dr G.
Trinchieri (Wister Institute, PA) (28,29).
Mice
Ovalbumin (OVA) peptide-speci®c TCR Tg mice were originally developed by Dr D. Loh and RAG-1 gene-disrupted mice
were originally developed by Dr F. L. Alt (30,31). These mice
were provided by Dr T. Nakayama (Chiba University). IRF-1
gene-disrupted mice were provided by Dr T. Taniguchi
(University of Tokyo) (32). Mice and their littermates were
reared under speci®c pathogen-free conditions in the animal
facility of Ehime University School of Medicine. BALB/c mice
were purchased from Charles River Japan (Yokohama,
Japan). All mice were used in accordance with the institutional
guides for animal experimentation.
Experimental infections and pathogens
L. monocytogenes (Lm) (EGD strain) was provided by Dr M.
Mitsuyama (Kyoto University, Kyoto, Japan) and 2 3 103
bacteria were inoculated i.p.
In vitro stimulation of T cells
T cells of Lm-infected TCR-Tg mice were prepared as surface
Ig± nylon non-adherent cells as described in the literature (33).
Antigen-presenting cells (APC) were prepared from spleen
cells by depleting T cells with anti-T cell antibody and
complement followed by 10 Gy X-irradiation (33). T cells (1
3 106) were stimulated in vitro with 4 3 106 T-cell depleted
splenic APC from uninfected syngeneic BALB/c mice in the
presence of 1 mM speci®c OVA peptides. T cells of uninfected
TCR-Tg mice were also cultured with APC from Lm-infected
mice. In speci®c experiments where stated, rIL-12, rIL-4, antiIL-12 mAb and anti-IL-4 mAb were added to the culture. The
medium used was RPMI 1640 supplemented with 2 mM Lglutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM
sodium pyruvate, 1 3 non-essential amino acids, 50 mM 2-
A two-step model of T cell subset commitment
mercaptoethanol and 10% heat-inactivated FCS. After 5 days
of cultivation at 37°C in a 5% CO2 humidi®ed air atmosphere,
cells were collected and washed, and re-stimulated with APC
and homologous antigen for 2 days. Amounts of IL-4 and IFN-g
in the culture supernatant were determined by ELISA assay
(16,17).
Detection of precursor frequencies for type 1 and type 2 T
cells
T cells of uninfected and Lm-infected TCR-Tg mice were
plated in 96-well round-bottomed microtiter plates at 1 cell/
well, and stimulated with APC from uninfected BALB/c mice in
the presence of 1 mM speci®c OVA peptide and 10 U/ml rIL-2.
The cultures were stimulated weekly with T-depleted splenic
APC, homologous antigen peptides and IL-2. After 4 weeks of
cultivation, IL-4 and IFN-g in the culture supernatant were
detected by ELISA assay. A well was considered positive
when the amount of cytokine exceeded the mean + 5 SD of the
background.
Cytokine ELISA
The IFN-g and IL-4 in the culture supernatant were detected by
sandwich ELISA established with mAb that were purchased
from PharMingen (San Diego, CA). Recombinant mouse
cytokines were purchased from Genzyme (Cambridge, MA)
and were used as standards.
RNA isolation and RNA blot analysis
Total cellular RNA was isolated by the guanidine thiocyanate
method. The procedure for RNA blot analysis was described
in Harada et al. (34). Fragments of T-bet, GATA-3 and Ca were
labeled by the random primer method (Amersham, Tokyo,
Japan) to prepare probe DNAs.
Flow cytometry
mAb used for staining were biotin-conjugated anti-CD90 and
anti-CD45R/B220, and ¯uorescein-labeled anti-CD11b, antiCD11c, anti-CD40, anti-CD80, anti-CD86, anti-CD25, antiCD69 and anti-I-Ad. These mAb were purchased from
PharMingen. The biotin-conjugated antibody was developed
with phycoerythrin-labeled streptavidin. Stained cells were
analyzed on a FACSCalibur with CellQuest software (Becton
Dickinson, Mountain View, CA).
Results
APC of pathogen-infected mice induce a type 1 T cell
response
We used OVA-peptide speci®c TCR-Tg mice (30) and the
intracellular infectious pathogen Lm to evaluate the effect of
pathogenic infections. When T cells of uninfected TCR-Tg
mice were stimulated with a speci®c antigen and APC in vitro,
the cells differentiated predominantly into an IL-4-producing
type 2 T cell subset (Fig. 1A). In contrast, Lm infection
appeared to stimulate T cells to differentiate into an IFN-gproducing type 1 T cell subset. When uninfected naive T cells
were stimulated with infected APC, the T cells shifted to the
IFN-g-producing T cell subset.
569
To determine whether Lm-infected APC cells in¯uence
the function of uninfected APC, TCR-Tg T cells were
stimulated with a mixture of APC from uninfected and Lminfected mice. As shown in Fig. 1(B), addition of a small
fraction of Lm-infected APC to uninfected APC rendered T
cells to shift to type 1 T cells. The results suggest that the
Lm-infected APC render the function of uninfected APC to
induce type 1 T cells. Therefore, it is suggested that Lm
infection in¯uences T cell differentiation through the action
of APC.
Antigen-presenting ability of APC of pathogen-infected mice
is comparable to that of APC of uninfected mice
The process of in vitro induction of T cell subset differentiation is determined by the antigenic concentration present
during the induction culture. In the present system, type 1
T cells are predominantly induced at a lower concentration
of antigen, while type 2 T cells are induced at a higher
concentration of the antigen (Fig. 2A). Therefore, the result
observed in Fig. 1, where Lm infection appeared to
stimulate T cells to differentiate into a type 1 T cell
subset, may be due to the low ef®ciency of the antigen
presentation by infected APC.
This possibility was tested by stimulating T cells of
uninfected TCR-Tg mice with the APC of uninfected and
Lm-infected mice (Fig. 2B±D). The APC functions of
presenting antigenic peptide to T cells and of inducing T
cell proliferation were not severely disturbed by Lm
infection. Naive T cells of uninfected TCR-Tg mice
responded at a comparable magnitude to peptide antigen
presented by uninfected and infected APC (Fig. 2B). In
addition, uninfected and Lm-infected APC induced T cell
proliferation and produced IFN-g at the same level in the
Th1 subset. Th2 subset T cells also responded equally to
uninfected and Lm-infected APC (Fig. 2C and D).
Therefore, no preferential stimulation of T cell subsets by
antigen presentation was observed during Lm infection.
Rather, these suggested that the results observed in Fig.
1(A and B) are due to the speci®c effect of Lm-infected
APC to stimulate T cells to differentiate into a type 1 T cell
subset.
No signi®cant difference in chemokine gene expression and
cell surface markers between infected and uninfected
splenic APC except CD11b expression
It has been suggested that chemokines and their receptors
are essential elements that regulate the T cells and their
partners for priming type 1 and type 2 T cell-mediated
responses (35). Therefore, we examined the expression level
of chemokine genes in non-T, non-B spleen cells of uninfected
and Lm-infected mice (Fig. 3A). Chemokines were expressed
at comparable levels in non-T, non-B spleen cells of both
groups. The chemokine receptor gene expression on T cells
was also determined. There was no signi®cant difference in
the pattern and level of the chemokine receptor gene expression on puri®ed T cells of uninfected and Lm-infected mice
(Fig. 3B).
The expression level of cell surface molecules on non-T,
non-B spleen cells of uninfected and Lm-infected mice was
compared by ¯ow cytometry. As shown in Fig. 3(C), there was
570 A two-step model of T cell subset commitment
Fig. 2. Proliferative response of in vitro-shifted T cells to antigen presented by uninfected and Lm-infected APC. (A) T cells of uninfected TCRTg mice were cultured for 5 days in the presence of uninfected BALB/c APC and the indicated amount of OVA peptide. The cultured cells
were re-stimulated with uninfected BALB/c APC and 1 mM OVA peptide, and cytokines were subsequently detected. (B) T cells of uninfected
TCR-Tg mice (naive precursors for Th cells) were stimulated with uninfected (s) or Lm-infected (d) APC in the titrated amount of OVA peptide
for 3 days. Proliferative responses were measured by counting the [3H]thymidine uptake by the cultures. (C and D) T cells of TCR-Tg mice
were stimulated every week for 4 weeks with 1 mM OVA peptide and uninfected APC in the presence of either rIL-4 plus anti-IL-12 mAb for
type 2 T cells or rIL-12 plus anti-IL-4 mAb for type 1 T cells. Thus shifted type 1 (C) and type 2 (D) T cells were stimulated with OVA peptide
and uninfected (s, h) or Lm-infected (d, j) APC. Proliferative responses were measured by counting the [3H]thymidine uptake by the
cultures and cytokines were subsequently detected. IL-4 production by type 1 T cells (C) and IFN-g production by type 2 T cells (D) was less
than the detection level.
no apparent difference in the expression level of CD11c, CD40
and CD86 between uninfected and Lm-infected non-T, non-B
spleen cells. The differences observed in the expression level
of cell surface molecules were for CD11b and CD80.
The proportions of CD69+ T cells and CD25+ T cells were
slightly increased in Lm-infected Vb8+ T cells (Fig. 3D). Since
in vitro stimulation of TCR-Tg T cells with heat-inactivated Lm
did not increase the expression of CD69 and CD25, the result
is not due to the cross-reactivity of the Tg TCR (data not
shown). Rather, the result suggests that naive T cells are
activated during Lm infection in the absence of nominal
antigen.
APC induction of the two T cell subsets is affected
differently during Lm infection
In addition to the APC, IL-12 and IL-4 are required to induce
the differentiation of naive T cells into mature type 1 and type 2
T cell subsets (5,6,9,11,14,15,18). The above results suggest
that APC function is in¯uenced by infection. The effects of IL-4
and IL-12 together with the effects of mAb on these IL were
therefore evaluated (Fig. 4). The addition of IL-12 plus anti-IL-4
mAb to cultures with uninfected APC and speci®c antigen
increased the proportion of the IFN-g-producing type 1 T cell
subset and reduced that of the IL-4-producing type 2 subset.
This process is antigen speci®c, since there was almost no
induction of either T cell subset without the addition of antigen
(data not shown). Although APC reduced the type 2 subsetinducing activity during a 2-day infection, the addition of IL-4
plus anti-IL-12 mAb during an in vitro culture restored it.
However, the ability of APC obtained from 3-day infected mice
to induce differentiation into the type 2 subset could not be
restored by the addition of IL-4 plus anti-IL-12 mAb during an
in vitro culture (Fig. 4). In contrast, the ability of APC to induce
the type 1 subset was not impaired even after a 3-day
infection. Rather, the infected APC induced IFN-g production
at a level equal to that of IL-12 plus anti-IL-4 mAb. This result
shows that APC induction of the two T cell subsets is affected
differently during Lm infection. In addition, the APC induction
of the type 2 subset in infected mice is not restored by IL-4,
which underscores that the accessory function of APC is
profoundly in¯uenced by Lm infection.
A commitment of T cell subsets is observed prior to
exposure to speci®c antigen in Lm-infected mice
APC of uninfected mice stimulate uninfected TCR-Tg T cells
which differentiate predominantly into the type 2 subset. This
A two-step model of T cell subset commitment
571
Fig. 3. Chemokine and chemokine receptor mRNA expression by APC and T cells, and cell surface marker expression of APC. (A) Chemokine
gene expression (lanes 1 and 2, b-actin; lanes 3 and 4, IP-10; lanes 5 and 6, Mig; lanes 7 and 8, MCP-1; lanes 9 and 10, MIP-1b; lanes 11
and 12, RANTES) was analyzed by RT-PCR using RNA extracted from T cell-depleted splenocytes of uninfected (lanes 1, 3, 5, 7, 9 and 11)
and 3-day Lm-infected (lanes 2, 4, 6, 8, 10, 12 and 14) mice. (B) Chemokine receptor gene expression (lanes 1 and 2, Ca; lanes 3 and 4,
CCR1; lanes 5 and 6, CCR2; lanes 7 and 8, CCR3; lanes 9 and 10, CCR4; lanes 11 and 12, CCR5; lanes 13 and 14, CCR7; lanes 15 and 16,
CXCR4) was analyzed by RT-PCR using RNA extracted from puri®ed T cells of uninfected (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and 7-day Lminfected TCR-TG mice (lanes 2, 4, 6, 8, 10, 12, 14 and 16). (C) Uninfected and Lm-infected spleen cells were stained with anti-Thy-1 and antiB220 mAb. Negatively stained cells were further analyzed for the expression of the indicated cell surface molecules. (D) Uninfected and Lminfected spleen cells of TCR-Tg mice were stained with the combination of the indicated antibodies.
Fig. 4. Accessory functions of APC are differentially in¯uenced
during Lm infection. T cells of uninfected TCR-Tg mice were
cultured for 5 days with uninfected or Lm-infected APC and 1 mM
OVA peptide in the absence of (open bars) or presence of either
rIL-4 plus anti-IL-12 mAb (light bars) or rIL-12 plus anti-IL-4 mAb
(dark bars). The cultured cells were restimulated with uninfected
APC and 1 mM OVA peptide, and cytokines were subsequently
detected.
differentiation to type 2 T cells was disturbed in T cells of Lminfected TCR-Tg mice. When T cells of Lm-infected mice were
used to induce T cell subset differentiation in vitro using APC
of uninfected mice, type 1 T cell differentiation became
predominant and type 2 T cell differentiation was reduced.
The effect of infection ®rst became apparent in the type 2 T cell
subset and then in the type 1 subset (Fig. 5A). Since APC used
in the experiment were of uninfected mice origin and
predominantly induced the type 2 T cell subset under
experimental conditions, the observed effect of infection
found in TCR-Tg T cells was thought to be created prior to
the exposure to nominal antigen. Similar results were obtained
in experiments utilizing T cells from RAG-1±/± TCR-Tg+ mice
(Fig. 5B). It was also shown that the addition of IL-4 plus antiIL-12 mAb did not induce a shift to type 2 T cell subset in 7-day
infected T cells (Fig. 5C). In addition, the shift to type 1 T cells
requires the in vitro stimulation with antigen. T cells of Lminfected mice did not produce either IFN-g or IL-4 by ex vivo
stimulation with nominal antigen and APC. The IFN-g-producing T cells were induced during in vitro stimulation (Fig. 5D).
These ®ndings suggest the possibility that the observed
change in the proportion of T cell subsets after Lm infection
may be due to a change in the precursor frequency of pre-Th
cells in each subset.
This possibility was directly tested by measuring the
precursor frequencies of the IFN-g-producing type 1 T cell
subset and IL-4-producing type 2 T cell subset (Table 1). The
precursor frequency of IL-4 producers in splenic T cells of
uninfected TCR-Tg mice was 18.9%, while that of IFN-g
producers was 5.8%. This pattern was reversed in the T
cells of Lm-infected TCR-Tg mice. The precursor frequency of
IL-4 producers was 4.3% and that of IFN-g producers was
18.4% in Lm-infected TCR-Tg mice. The frequency of double
(IFN-g and IL-4)-producing wells was 1.6% in uninfected mice
and 1.7% in Lm-infected mice. In addition, no difference was
found in T cell subset proliferation in the presence of
uninfected or Lm-infected APC as shown in Fig. 2(C and D).
The results show that Lm infection generates a shift in type 1
and type 2 T cell subset precursors before exposure to a
speci®c antigen.
572 A two-step model of T cell subset commitment
This point was further evaluated by measuring the pattern of
T-bet gene and GATA-3 gene expression on ex vivo splenic T
cells during Lm infection (Fig. 6). The transcription factors Tbet gene and GATA-3 gene have been shown to be selectively
expressed by type 1 and type 2 T cells respectively (21±24).
Splenic T cells of uninfected mice expressed relatively high
amounts of GATA-3 ex vivo. The expression of GATA-3
gradually decreased during Lm infection. In contrast, the
expression of T-bet gradually increased during Lm infection
(Fig. 6A and B). Similar results were obtained with RAG-1±/±
TCR-Tg T cells (Fig. 6C and D). The result is consistent with the
observation obtained by the precursor frequency analysis.
What is most important is that changes in precursor frequency
and T-bet and GATA-3 genes expression occurred in an
antigen-independent manner, i.e. T cells committed to type 1
subset prior to encounter nominal antigen. Thus, Lm infection
in¯uenced the T cells of unrelated speci®city.
Neither IL-12 nor NK cells are required for the ®rst step of
type 1 T cell precursor induction
We used IRF-1±/± TCR-Tg mice, which were de®cient in IL-12
production and were de®cient in functional NK cells (16), to
investigate whether IL-12 is required for the in vivo shift to type
1 T cell precursors during pathogen infection. As shown in Fig.
7, comparable IFN-g production was observed in T cells of
IRF-1±/± TCR-Tg mice and IRF-1+/± TCR-Tg mice. The result
shows that neither IL-12 nor NK cells are required for the ®rst
step of type 1 T cell precursor induction in vivo during
pathogen infection.
Discussion
Pathogen infections induce a shift in functional T cell subset
balance toward type 1 T cell dominance (1±6,9,11,14±18).
However, the mechanisms involved in this shift have yet to be
clari®ed, i.e. when and how T cells are committed to the type 1
subset (1±11,14,15,18). In the present study, we showed the
shift is mediated by a two-step induction of T cell subsets
during pathogenic infection (Fig. 8). The ®rst step is mediated
by the basal function of APC cells, and is antigen-independent
and non-speci®c. This step modulates the precursor frequency of type 1 and type 2 T cell subsets. The second step
requires antigenic stimulation of T cells together with IL-12 or
IL-4. Therefore, T cells are committed to type 1 and type 2 T
cells before they encounter nominal antigen. The entire
immune system is in¯uenced by infection by a single pathogenic species.
T cells of TCR-Tg mice infected with Lm exhibited a shift to
type 1 T cell dominance. The proportion of double (IFN-g and
IL-4)-producing wells was low, and comparable in uninfected
and Lm-infected groups. This result is consistent with the idea
that the T cell subset commitment occurs either at the stage
before T cells encounter nominal antigen or at the very early
stage of antigenic stimulation. Since the shift occurred in the
absence of nominal antigen for TCR-Tg+ T cells, the observed
deviation of T cell subsets was induced in an antigenindependent fashion. In addition, the increase of type 1 T
Fig. 5. Effect of Lm infection on TCR-Tg T cells and splenic APC. (A
and B) T cells of uninfected (open bars) and Lm-infected (shaded
bars) TCR-Tg mice (A) and RAG-1Ð/Ð TCR-TG mice (B) were
cultured for 5 days in the presence of uninfected APC from
syngeneic BALB/c mice and 1 mM OVA peptide. (C) T cells of
uninfected and Lm-infected TCR-Tg mice were cultured for 5 days in
the presence of uninfected APC from syngeneic BALB/c mice and
1 mM OVA peptide in the presence of the indicated cytokine and
antibody. (D) T cells of uninfected and Lm-infected TCR-TG mice
were cultured for 0, 2 and 5 days in the presence of uninfected APC
from syngeneic BALB/c mice and 1 mM OVA peptide. The cultured
cells were re-stimulated with uninfected BALB/c APC and
homologous peptide, and cytokines were subsequently detected.
Table 1. The precursor frequencies observed in T cells of
TCR-Tg mice uninfected or infected for 7 days with Lm
T cells from
IFN-g
producer (%)
IL-4
producer (%)
IFN-g + IL-4
producer (%)
Uninfected
Infected (7 days)
5.8
18.4
18.9
4.3
1.6
1.7
Puri®ed T cells were placed in microtiter plates at a density of 1
cell/well. They were stimulated once a week for 4 weeks with 1 mM
speci®c OVA peptides, 10 U/ml rIL-2 and T cell-depleted splenic
APC prepared from syngeneic BALB/c mice.
A two-step model of T cell subset commitment
cell precursors during Lm infection was responsible for this
deviation. This process in¯uences the expression of T-bet and
GATA-3 genes, although T cells do not see nominal antigen.
The molecules involved in this step may regulate the expression of T-bet and GATA-3 genes, but the nature of the
molecules is currently unknown. However, unprimed naive T
cells were induced to commit and to differentiate into type 1 T
Fig. 6. Expression of T-bet and GATA-3 mRNA by splenic T cells of
Lm-infected mice. (A) T-bet, GATA-3 and Ca gene expression was
analysed by Northern blot using RNA extracted from ex vivo splenic
T cells prepared from Lm-infected mice at the indicated days after
infection. As a control, RNA prepared from type 1 and type 2 T cell
lines was included. (B) The intensity of the bands was measured by
BAS2000 (Fuji Film, Tokyo, Japan) and the ratio of GATA-3 to T-bet
is shown. (C and D) A similar analysis was carried out using RAG1Ð/Ð TCR-Tg mice.
573
cells by APC of Lm-infected mice origin, and APC stimulation
of naive T cells appeared to be involved.
The question is whether IL-12 is required for the ®rst step.
The present study (Fig. 7) and our previous studies utilizing
IRF-1 gene-disrupted mice suggest that the step is independent of IL-12 (16,17). First, the IRF-1 gene-disrupted mice are
defective in induce IL-12 p40 gene activation and active IL-18
protein production (16,17), and, therefore, cannot mount a
type 1 T cell response upon Lm infection. However, the defect
was restored by the addition of wild-type normal functional
APC, but not by IL-12 protein. Second, it was also demonstrated that the IRF-1 gene-disrupted mice were able to
induce type 1 T cell response even in the absence of IL-12
production by the mice when infected with Plasmodium
parasites (17). In addition, it was reported that type 1 T cells
were induced in IL-12 p40 gene-disrupted mice during viral
infection (36). This ®nding also supports the idea that the ®rst
Fig. 7. Neither IL-12 nor NK cells are required for the ®rst step of
type 1 T cell precursor induction. IRF-1Ð/Ð TCR-Tg mice and their
littermates were infected with Lm. Five days after infection, T cells of
these treated mice were cultured in vitro with uninfected BALB/c
APC and 1 mM OVA peptide. The cultured cells were re-stimulated
with uninfected BALB/c APC and homologous peptide, and
cytokines were subsequently detected.
Fig. 8. A two-step model of T cell subset commitment during pathogenic infection: a hypothesis. The Lm-infected innate immune system shifts
the precursors for T cell subsets in a two-step manner. In the ®rst step, pathogenic infection leads to the activation of the innate immune
system and stimulates naive T cells (precursors of Th cells). The Lm-infected innate immune system shifts the precursors for T cell subsets
predominantly to precursors for the type 1 T cell subset. This step is antigen independent and therefore antigen non-speci®c. Thus, the entire
immune system shifts to a type 1-dominant status in Lm-infected animals. T cell commitment to the type 1 subset occurs during this step. In
the second step, IL-12 and IL-4 only induce maturation of type 1 and type 2 T cell subsets respectively in the presence of speci®c antigen
presented by APC. Therefore, this step is antigen speci®c.
574 A two-step model of T cell subset commitment
step of T cell subset differentiation is IL-12 independent.
Moreover, the step appeared to be NK1.1+ cell independent,
since IRF-1 gene-disrupted mice lack functional mature NK
cells because of an IL-15 de®ciency (16,37). It was also shown
that a type 1 T cell response was induced in Va14 genedisrupted mice which lack NK1.1+ T cells (Y. Asano, unpublished). Taking all these observations together, it can be
concluded that the ®rst step of functional T cell subset
commitment is profoundly dependent on the function of APC.
In contrast to the ®rst step, the second step is antigen
dependent. APC of Lm-infected mice induce the deviation of
type 1 dominance in uninfected naive TCR-Tg+ T cells in the
presence of nominal antigen. Although the antigen is absolutely essential for this step, the observed deviation during Lm
infection is not simply due to the change in antigen-presenting
ef®ciency of infected APC nor the preferential stimulation of
type 1 T cells over type 2 T cells by infected APC (Fig. 2).
Therefore, the preferential induction of type 1 T cells by Lminfected APC is not due to the change of APC function in the
second step. Rather, changes in APC function of the ®rst step
might be responsible for driving naive TCR-Tg+ T cells to type
1 T cell precursors during Lm infection. This conclusion is also
supported by the ®nding that the addition of IL-4 plus anti-IL12 mAb failed to induce the type 2 T cell subset in 7-day
infected TCR-Tg+ T cells. In addition, it is suggested that the
APC have separate and distinct functions which induce type 1
and type 2 precursors. The induction of the type 2 T cell subset
was disturbed in the early phase of Lm infection, while the
induction of the type 1 T cell subset increases. The failure to
induce type 2 T cells by 7-day infected APC even in the
presence of IL-4 plus anti-IL-12 mAb is not due to the failure of
the antigen-presentation ability of the APC. Rather, this result
indicates the possibility that the ability to support type 2 T cell
differentiation is abrogated during Lm infection. The Lm
infection modulates the expression of T-bet and GATA-3
genes of T cells without involving antigenic stimulation, while
the precursor frequency changed during Lm infection. These
results suggest that T cells are committed to type 1 T cells
before they encounter nominal antigen during Lm infection by
in¯uencing the expression of T-bet and GATA-3 genes. It is
further suggested that the molecules involving in the ®rst step
may regulate the expression of T-bet and GATA-3 genes.
We thus propose a two-step model of T cell subsets
differentiation pathway as described below based on the
observations presented in this report (Fig. 8). In the ®rst step,
pathogenic infection leads to the activation of the innate
immune system and stimulates naive T cells (precursors of Th
cells). We think that this ®rst step is independent of both
speci®c antigen and IL-12, since a single pathogenic species
has an effect on TCR-Tg+ T cells with unrelated speci®city and,
in addition, IL-12-de®cient mice produce high levels of IFN-gproducing T cells (16,17,38,39). The Lm-infected innate
immune system shifts the precursors for T cell subsets
predominantly to precursors for the type 1 T cell subset in
an antigen-non-speci®c manner. T cells are committed to type
1 T cells in this step by in¯uencing the expression of T-bet and
GATA-3 genes. In the second step, IL-12 and IL-4 play an
important role for maturation of the type 1 and type 2 T cell
subsets respectively in the presence of speci®c antigen
presented by APC accompanied by the changes in the
expression of T-bet and GATA-3 genes. This process is strictly
antigen speci®c. Thus, the entire immune system shifts to type
1 dominant status even in single pathogenic species-infected
animals.
Acknowledgements
We would like to acknowledge helpful discussion with and critical
comments by Dr Alfred Singer, Dr Richard J. Hodes, Dr Pascale
Cossart, Dr Gen Suzuki and Dr Hiroto Shinomiya. This work was
supported in part by a grant of Special Coordination Fund for
Promoting Science and Technology from the Science and
Technology Agency of Japan, a grant-in-aid from the Ministry of
Education, Science and Culture of Japan, and a grant from the Uehara
Memorial Foundation.
Abbreviations
APC
IRF
Lm
OVA
T-bet
Tg
antigen-presenting cell
IFN-regulatory factor
Listeria monocytogenes
ovalbumin
T-box expressed in T cells
transgenic
References
1 Scott, P. and Kaufman, S. H. E. 1991. The role of T-cell subsets
and cytokines in the regulation of infection. Immunol. Today
12:346.
2 Kaufman, S. H. E. 1993. Immunity to intracellular bacteria. Annu.
Rev. Immunol. 11:129.
3 Unanue, E. R. 1997. Studies in listeriosis show the strong
symbiosis between the innate cellular system and the T-cell
response. Immunol. Rev. 158:11.
4 Medzhitov, R. and Janeway, C. A. 1997. Innate immunity: impact
on the adaptive immune response. Curr. Opin. Immunol. 9:4.
5 Mosmann, T. R. and Coffman. R. L. 1989. Th1 and Th2 cells:
different patterns of lymphokines secretion lead to different
functional properties. Annu. Rev. Immunol. 7:145.
6 Mosmann, T. and Sad, S. 1996. The expanding universe of T-cell
subsets: Th1, Th2 and more. Immunol. Today 17:138.
7 LeGros, G. and Paul, W. E. 1990. Generation of Interleukin 4producing cells in vivo and in vitro: IL-2 and IL-4 are required for
in vitro generation of IL4-producing cells. J. Exp. Med. 172:921.
8 Swain, S. L. and Huston, G. 1990. IL-4 directs the development of
Th2-like helper effectors. J. Immunol. 145:3796.
9 Hsieh, C. S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., O'Garra, A.
and Murphy, K. M. 1993. Development of TH1 CD4+ T cells
through IL-12 produced by Listeria-induced macrophages.
Science 260:547.
10 Scott, P. 1993. IL-12: initiation cytokine for cell-mediated
immunity. Science 260:496.
11 Magram, J. 1996. IL-12-de®cient mice are defective in IFN-g
production and type I cytokine responses. Immunity 4:471.
12 Matter, F., Mattner, F., Magram, J., Ferrante, J., Launois, P.,
Padova, K. D., Behin, R., Gately, M. K., Louis, J. A. and Alber, G.
1996. Genetically resistant mice lacking interleukin-12 are
susceptible to infection with Leishmania major and mount a
polarized Th2 cell response. Eur. J. Immunol. 26:1553.
13 Seder, R. A. and Paul, W. E. 1994. Acquisition of lymphokineproducing phenotype by CD4+ T cells. Annu. Rev. Immunol.
12:635.
14 Abbas, A., Murphy, K. and Sher, A. 1996. Functional diversity of
helper T lymphocytes. Nature 383:787.
15 O'Garra, A. 1998. Cytokines induce the development of
functionally heterogeneous T helper cell subsets. Immunity 8:275.
16 Taki, S., Sato, T., Ogasawara, K., Fukuda, T., Sato, M., Hida, S.,
Suzuki, G., Mitsuyama, M., Shin, E.., Kojima, S., Taniguchi, T. and
A two-step model of T cell subset commitment
17
18
19
20
21
22
23
24
25
26
27
28
29
Asano, Y. 1997. Multistage regulation of Th1-type immune
responses by the transcription factor IRF. Immunity 6:673.
Feng, C., Watanabe, S., Maruyama, S., Suzuki, G., Sato, M.,
Furuta, T., Kojima, S., Taki, S. and Asano, Y. 1999. An alternative
pathway for type 1 T cell differentiation. Int. Immunol. 11:1185.
Harty, J., Lenz, L. and Bevan, M. 1996. Primary and secondary
immune responses to Listeria monocytogenes. Curr. Opin.
Immunol. 8:526.
Karp, C. L. 1999. Measles: immunosuppression, interleukin-12,
and complement receptors. Immunol. Rev. 168:91.
Sutterwala, F., Noel, G., Clynes, R. and Mosser, D. 1997. Selective
suppression of interleukin-12 induction after macrophage
receptor ligation. J. Exp. Med. 185:1977.
Ouyang, W., Lohning, M., Gao, Z., Assenmacher, M., Ranganath,
S., Radbruch, A. and Murphy, K. M. 2000. Stat6-independent
GATA-3 autoactivation directs IL-4-independent Th2 development
and commitment. Immunity 12:27.
Zheng, W. and Flavell, R. A. 1997. The transcription factor GATA-3
is necessary and suf®cient for Th2 cytokine gene expression in
CD4 T cells. Cell 89:587.
Zhang, D. H., Cohn, L., Ray, P., Bottomly, K. and Ray, A. 1997.
Transcription factor GATA-3 is differentially expressed in murine
Th1 and Th2 cells and controls Th2-speci®c expression of the
interleukin-5 gene. J. Biol. Chem. 272:21597.
Szabo, S. J., Kim, S. T., Costa, G. L., Zhang, X., Fathman, C. G.
and Glimcher, L. H. 2000. A novel transcription factor, T-bet,
directs Th1 lineage commitment. Cell 100:655.
Rengarajan, J., Szabo, S. J. and Glimcher, L. H. 2000.
Transcriptional regulation of Th1/Th2 polarization. Immunol.
Today 21:479.
Karasuyama, H. and Melchers, F. 1988. Establishment of mouse
cell lines which constitutively secrete large quantities of interleukin
2, 3, 4, or 5, using modi®ed cDNA expression vectors. Eur. J.
Immunol. 18:97.
Yoshida, Y., Tasaki, K., Kimurai, M., Takenaga, K., Yamamoto, H.,
Yamaguchi, T., Saisho, H., Sakiyama, S. and Tagawa, M. 1998.
Antitumor effect of human pancreatic cancer cells transduced
with cytokine genes which activate Th1 helper T cells. Anticancer
Res. 18:333.
Trinchieri, G. 1995. Interleukin-12: a proin¯ammatory cytokine with
immunoregulatory functions that bridge innate resistance and
antigen-speci®c adaptive immunity. Annu. Rev. Immunol. 13:251.
Finkelman, F., Katona, I., Urban, J., Snapper, C., Ohara, J. and
Paul, W. 1986. Suppression of in vivo polyclonal IgE responses by
30
31
32
33
34
35
36
37
38
39
575
monoclonal antibody to the lymphokine B-cell stimulatory factor 1.
Proc. Natl Acad. Sci. USA 83:9675.
Murphy, K., Heimberger, A. and Loh, D. 1990. Induction by
antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes
in vivo. Science 250:1720.
Swat, W., Shinkai, Y., Cheng, H. L., Davidson, L. and Alt, F. W.
1996. Activated Ras signals differentiation and expansion of
CD4+8+ thymocytes. Proc. Natl Acad. Sci. USA 93:4683.
Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami,
T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K.,
Wakeham, A., Potter, J., Furlonger, C. L., Narendran, A., Suzuki,
H., Ohashi, P. S., Paige, C. J., Taniguchi, T. and Mak, T. W. 1993.
Targeted disruption of IRF-1 or IRF-2 results in abnormal type I
IFN gene induction and aberrant lymphocyte development. Cell
75:83.
Asano, Y., Singer, A. and Hodes. R. J. 1981. Role of the major
histocompatibility complex in T cell activation of B cell
subpopulations. Major histocompatibility complex-restricted and
-unrestricted B cell responses are mediated by distinct B cell
subpopulations. J. Exp. Med. 154:1100.
Harada, H., Fujita, T., Willso, K., Sakakibara, J., Miyamoto, M.,
Fujita, T. and Taniguchi, T. 1990. Absence of the type I IFN system
in EC cells: transcriptional activator (IRF-1) and repressor (IRF-2)
genes are developmentally regulated. Cell 63:303.
Sllusto, F. A., Lanzavecchia, A. and Mackay, C. R. 1998.
Chemokines and chemokine receptors in T-cell priming and Th1/
Th2-mediated responses. Immunol. Today 19:568.
Oxenius, A., Karrer, U., Zinkernagel, R. M. and Hengartner, H.
1999. IL-12 is not required for induction of type 1 cytokine
responses in viral infections. J. Immunol. 162:965.
Ogasawara, K., Hida, S., Azimi, N., Tagaya, Y., Sato, T., YokochiFukuda, T., Waldmann, T. A., Taniguchi, T. and Taki, S. 1998.
Requirement for IRF-1 in the microenvironment supporting
development of natural killer cells. Nature 391:700.
Fehr, T., Schoedon, G., Odermatt, B., Holtschke, T., Schneemann,
M., Bachmann, M., Mak, T., Horak, I. and Zinkernagel, R. 1997.
Crucial role of interferon consensus sequence binding protein, but
neither of interferon regulatory factor 1 nor of nitric oxide synthesis
for protection against murine listeriosis. J. Exp. Med. 185:921.
Wysocka, M., Kubin, M., Vieira, L., Ozmen, L., Garotta, G., Scott,
P. and Trinchieri, G. 1995. Interleukin-12 is required for interferongamma production and lethality in lipopolysaccharide-induced
shock in mice. Eur. J. Immunol. 25:672.