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

1 Department of Immunology and Host Defenses, Ehime University School of Medicine, Shigenobu, Onsen-gun, Ehime 791-0295, Japan

Correspondence to: Y. Asano; E-mail: asanoy{at}m.ehime-u.ac.jp
Transmitting editor: A. Singer


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 first 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 influences 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 influences 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 influence functional T cell commitment before T cells encounter nominal antigen.

Keywords: antigen-presenting cell, GATA-3, pathogen infection, T cell commitment, T-bet


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pathogens stimulate the immune response of a host resulting in a clearance of microbes (14). T cells are divided into two types according to the set of lymphokines they produce, i.e. IFN-{gamma}-producing type 1 T cells and IL-4-producing 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 (712). 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-specific fashion under the influence of IL-12 (16,9,11,1418). 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 (111,14,15,18).

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-{gamma}-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 wild-type 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.

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 first time a two-step induction mechanism for T cell subsets during pathogenic infection. The first step is induced by the basal function of macrophage/dendritic cells, and is antigen independent and non-specific. This step affects the precursor frequency of type 1 and type 2 T cell subsets. Although this first step does not involve activation through the TCR, the step influences GATA-3 and T-box expressed in T cells (T-bet) gene expression which is thought to regulate type 1/type 2 T cell subset differentiation (2125). The second step requires antigenic stimulation of T cells together with IL-12 or IL-4, and is antigen-specific 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 specificities are influenced by infection by a single pathogenic species. This finding could lead to new insights into T cell responses during pathogenic infections.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 specific 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-specific 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 specific 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 x 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 x 106) were stimulated in vitro with 4 x 106 T-cell depleted splenic APC from uninfected syngeneic BALB/c mice in the presence of 1 µM specific OVA peptides. T cells of uninfected TCR-Tg mice were also cultured with APC from Lm-infected mice. In specific experiments where stated, rIL-12, rIL-4, anti-IL-12 mAb and anti-IL-4 mAb were added to the culture. The medium used was RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1 x non-essential amino acids, 50 µM 2-mercaptoethanol and 10% heat-inactivated FCS. After 5 days of cultivation at 37°C in a 5% CO2 humidified air atmosphere, cells were collected and washed, and re-stimulated with APC and homologous antigen for 2 days. Amounts of IL-4 and IFN-{gamma} 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 µM specific 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-{gamma} 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-{gamma} 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 C{alpha} 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 fluorescein-labeled anti-CD11b, anti-CD11c, anti-CD40, anti-CD80, anti-CD86, anti-CD25, anti-CD69 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
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
APC of pathogen-infected mice induce a type 1 T cell response
We used OVA-peptide specific 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 specific 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-{gamma}-producing type 1 T cell subset. When uninfected naive T cells were stimulated with infected APC, the T cells shifted to the IFN-{gamma}-producing T cell subset.



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Fig. 1. Influence 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 µM OVA peptide. The cultured cells were re-stimulated with uninfected BALB/c APC and homologous peptide, and cytokines were subsequently detected. (B) T cells of uninfected TCR-Tg 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 µM OVA peptide. The cultured cells were re-stimulated with uninfected BALB/c APC and homologous peptide, and cytokines were subsequently detected.

 
To determine whether Lm-infected APC cells influence the function of uninfected APC, TCR-Tg T cells were stimulated with a mixture of APC from uninfected and Lm-infected 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 influences 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 efficiency of the antigen presentation by infected APC.



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Fig. 2. Proliferative response of in vitro-shifted T cells to antigen presented by uninfected and Lm-infected APC. (A) T cells of uninfected TCR-Tg 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 µM OVA peptide, and cytokines were subsequently detected. (B) T cells of uninfected TCR-Tg mice (naive precursors for Th cells) were stimulated with uninfected ({circ}) or Lm-infected (•) 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 µM 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 ({circ}, {square}) or Lm-infected (•, {blacksquare}) 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-{gamma} production by type 2 T cells (D) was less than the detection level.

 
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-{gamma} 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 specific effect of Lm-infected APC to stimulate T cells to differentiate into a type 1 T cell subset.

No significant 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 significant difference in the pattern and level of the chemokine receptor gene expression on purified T cells of uninfected and Lm-infected mice (Fig. 3B).



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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, ß-actin; lanes 3 and 4, IP-10; lanes 5 and 6, Mig; lanes 7 and 8, MCP-1; lanes 9 and 10, MIP-1ß; 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, C{alpha}; 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 purified T cells of uninfected (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and 7-day Lm-infected 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 anti-B220 mAb. Negatively stained cells were further analyzed for the expression of the indicated cell surface molecules. (D) Uninfected and Lm-infected spleen cells of TCR-Tg mice were stained with the combination of the indicated antibodies.

 
The expression level of cell surface molecules on non-T, non-B spleen cells of uninfected and Lm-infected mice was compared by flow cytometry. As shown in Fig. 3(C), there was 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 Vß8+ 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 influenced 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 specific antigen increased the proportion of the IFN-{gamma}-producing type 1 T cell subset and reduced that of the IL-4-producing type 2 subset. This process is antigen specific, 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 subset-inducing 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-{gamma} 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 influenced by Lm infection.



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Fig. 4. Accessory functions of APC are differentially influenced during Lm infection. T cells of uninfected TCR-Tg mice were cultured for 5 days with uninfected or Lm-infected APC and 1 µM 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 µM OVA peptide, and cytokines were subsequently detected.

 
A commitment of T cell subsets is observed prior to exposure to specific antigen in Lm-infected mice
APC of uninfected mice stimulate uninfected TCR-Tg T cells which differentiate predominantly into the type 2 subset. This differentiation to type 2 T cells was disturbed in T cells of Lm-infected 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 first 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 anti-IL-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 Lm-infected mice did not produce either IFN-{gamma} or IL-4 by ex vivo stimulation with nominal antigen and APC. The IFN-{gamma}-producing T cells were induced during in vitro stimulation (Fig. 5D). These findings 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.



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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-1---/--- TCR-TG mice (B) were cultured for 5 days in the presence of uninfected APC from syngeneic BALB/c mice and 1 µM 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 µM 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 µM OVA peptide. The cultured cells were re-stimulated with uninfected BALB/c APC and homologous peptide, and cytokines were subsequently detected.

 
This possibility was directly tested by measuring the precursor frequencies of the IFN-{gamma}-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-{gamma} 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-{gamma} producers was 18.4% in Lm-infected TCR-Tg mice. The frequency of double (IFN-{gamma} 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 specific antigen.


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Table 1. The precursor frequencies observed in T cells of TCR-Tg mice uninfected or infected for 7 days with Lm
 
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 T-bet gene and GATA-3 gene have been shown to be selectively expressed by type 1 and type 2 T cells respectively (2124). 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 influenced the T cells of unrelated specificity.



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Fig. 6. Expression of T-bet and GATA-3 mRNA by splenic T cells of Lm-infected mice. (A) T-bet, GATA-3 and C{alpha} 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 RAG-1---/--- TCR-Tg mice.

 
Neither IL-12 nor NK cells are required for the first step of type 1 T cell precursor induction
We used IRF-1–/– TCR-Tg mice, which were deficient in IL-12 production and were deficient 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-{gamma} 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 first step of type 1 T cell precursor induction in vivo during pathogen infection.



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Fig. 7. Neither IL-12 nor NK cells are required for the first step of type 1 T cell precursor induction. IRF-1---/--- 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 µM OVA peptide. The cultured cells were re-stimulated with uninfected BALB/c APC and homologous peptide, and cytokines were subsequently detected.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pathogen infections induce a shift in functional T cell subset balance toward type 1 T cell dominance (16,9,11,1418). However, the mechanisms involved in this shift have yet to be clarified, i.e. when and how T cells are committed to the type 1 subset (111,14,15,18). 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 first step is mediated by the basal function of APC cells, and is antigen-independent and non-specific. 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 influenced by infection by a single pathogenic species.



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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 first 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-specific. 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 specific antigen presented by APC. Therefore, this step is antigen specific.

 
T cells of TCR-Tg mice infected with Lm exhibited a shift to type 1 T cell dominance. The proportion of double (IFN-{gamma} 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 antigen-independent fashion. In addition, the increase of type 1 T cell precursors during Lm infection was responsible for this deviation. This process influences 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 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 first 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 finding also supports the idea that the first 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 deficiency (16,37). It was also shown that a type 1 T cell response was induced in V{alpha}14 gene-disrupted mice which lack NK1.1+ T cells (Y. Asano, unpublished). Taking all these observations together, it can be concluded that the first step of functional T cell subset commitment is profoundly dependent on the function of APC.

In contrast to the first 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 efficiency 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 Lm-infected APC is not due to the change of APC function in the second step. Rather, changes in APC function of the first 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 finding that the addition of IL-4 plus anti-IL-12 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 influencing the expression of T-bet and GATA-3 genes. It is further suggested that the molecules involving in the first 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 first 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 first step is independent of both specific antigen and IL-12, since a single pathogenic species has an effect on TCR-Tg+ T cells with unrelated specificity and, in addition, IL-12-deficient mice produce high levels of IFN-{gamma}-producing 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-specific manner. T cells are committed to type 1 T cells in this step by influencing 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 specific antigen presented by APC accompanied by the changes in the expression of T-bet and GATA-3 genes. This process is strictly antigen specific. 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—antigen-presenting cell

IRF—IFN-regulatory factor

Lm—Listeria monocytogenes

OVA—ovalbumin

T-bet—T-box expressed in T cells

Tg—transgenic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 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.[ISI][Medline]
  2. Kaufman, S. H. E. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129.[ISI][Medline]
  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.[ISI][Medline]
  4. Medzhitov, R. and Janeway, C. A. 1997. Innate immunity: impact on the adaptive immune response. Curr. Opin. Immunol. 9:4.[ISI][Medline]
  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.[ISI][Medline]
  6. Mosmann, T. and Sad, S. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17:138.[ISI][Medline]
  7. LeGros, G. and Paul, W. E. 1990. Generation of Interleukin 4-producing 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.[Abstract]
  8. Swain, S. L. and Huston, G. 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145:3796.[Abstract/Free Full Text]
  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.[ISI][Medline]
  10. Scott, P. 1993. IL-12: initiation cytokine for cell-mediated immunity. Science 260:496.
  11. Magram, J. 1996. IL-12-deficient mice are defective in IFN-{gamma} production and type I cytokine responses. Immunity 4:471.[ISI][Medline]
  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.[ISI][Medline]
  13. Seder, R. A. and Paul, W. E. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12:635.[ISI][Medline]
  14. Abbas, A., Murphy, K. and Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[ISI][Medline]
  15. O’Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275.[ISI][Medline]
  16. Taki, S., Sato, T., Ogasawara, K., Fukuda, T., Sato, M., Hida, S., Suzuki, G., Mitsuyama, M., Shin, E.., Kojima, S., Taniguchi, T. and Asano, Y. 1997. Multistage regulation of Th1-type immune responses by the transcription factor IRF. Immunity 6:673.[ISI][Medline]
  17. 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.[Abstract/Free Full Text]
  18. Harty, J., Lenz, L. and Bevan, M. 1996. Primary and secondary immune responses to Listeria monocytogenes. Curr. Opin. Immunol. 8:526.[ISI][Medline]
  19. Karp, C. L. 1999. Measles: immunosuppression, interleukin-12, and complement receptors. Immunol. Rev. 168:91.[ISI][Medline]
  20. 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.[Abstract/Free Full Text]
  21. 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.[ISI][Medline]
  22. Zheng, W. and Flavell, R. A. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.
  23. 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-specific expression of the interleukin-5 gene. J. Biol. Chem. 272:21597.[Abstract/Free Full Text]
  24. 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.
  25. Rengarajan, J., Szabo, S. J. and Glimcher, L. H. 2000. Transcriptional regulation of Th1/Th2 polarization. Immunol. Today 21:479.[ISI][Medline]
  26. Karasuyama, H. and Melchers, F. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4, or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97.[ISI][Medline]
  27. 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.[ISI][Medline]
  28. Trinchieri, G. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13:251.
  29. Finkelman, F., Katona, I., Urban, J., Snapper, C., Ohara, J. and Paul, W. 1986. Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B-cell stimulatory factor 1. Proc. Natl Acad. Sci. USA 83:9675.[Abstract]
  30. Murphy, K., Heimberger, A. and Loh, D. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[ISI][Medline]
  31. 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.[Abstract/Free Full Text]
  32. 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.[ISI][Medline]
  33. 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.[Abstract/Free Full Text]
  34. 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.[ISI][Medline]
  35. 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.[ISI][Medline]
  36. 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.[Abstract/Free Full Text]
  37. Ogasawara, K., Hida, S., Azimi, N., Tagaya, Y., Sato, T., Yokochi-Fukuda, 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.[ISI][Medline]
  38. 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.[Abstract/Free Full Text]
  39. Wysocka, M., Kubin, M., Vieira, L., Ozmen, L., Garotta, G., Scott, P. and Trinchieri, G. 1995. Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[ISI][Medline]