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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. 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