An alternate pathway for type 1 T cell differentiation

Chiguang Feng, Shohei Watanabe, Saho Maruyama, Gen Suzuki1, Mitsuharu Sato3, Takahisa Furuta2, Somei Kojima2, Shinsuke Taki3 and Yoshihiro Asano

Department of Microbiology and Immunology, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan
1 National Institute of Radiological Science, Chiba 263-8555, Japan
2 >Department of Parasitology, Institute of Medical Science, University of Tokyo, Tokyo 108-0071, Japan
3 >Department of Immunology, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan

Correspondence to: Y. Asano


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
IFN-regulatory factor-1 (IRF-1) gene-disrupted mice are defective in IL-12 and IL-18 gene expression at the transcriptional and post-translational level respectively. The mutant mouse mounts a type 2 T cell response upon bacterial infection because of the impaired induction of the IL-12 p40 gene and IFN-{gamma}-producing type 1 T cells are not induced. We showed here, however, that different pathogens activate a novel pathway for inducing IFN-{gamma}-producing type 1 T cells even in an IRF-1-deficient mouse. This pathway is independent of IL-12 and IL-18, and is mediated by a distinct function of macrophage lineage cells. Macrophages of the mutant mice fail to activate the IL-12-dependent pathway, but they function in the IL-12-independent pathway in Plasmodium-infected mice. This leads to the hypothesis that the IL-12-independent novel pathway for inducing IFN-{gamma}-producing T cells is distinct from the classical type 1/type 2 T cell subset differentiation pathway.

Keywords: IFN-regulatory factor-1, IL-12, Leishmania major, Listeria monocytogenes, Plasmodium berghei, T cell subsets


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Immune responses characterized by the production of distinct sets of lymphokines are optimally protective against different types of pathogens during bacterial, parasitic and viral infections. T cells are divided into two types based on the set of lymphokines they produce, i.e. IFN-{gamma}-producing type 1 T cells and IL-4-producing type 2 T cells (15). Responses dominated by the production of IFN-{gamma} provide a defense against microorganisms that establish intracellular infections (6). On the other hand, responses dominated by the production of IL-4 protect against infections by extracellular pathogens (6). The process of differentiating T cells into type 1 or type 2 is controlled by the cytokines produced during the innate immune response in its early phase (712). Cytokines present at the initiation of the immune response at the TCR ligation stage determine type 1 and type 2 T cell differentiation from the precursor (6,13).

The type 1 differentiation program is initiated by the production of IL-12 by macrophages. Bacterial stimuli activate macrophages, and subsequently NK cells in the innate immune response to produce IL-12 and IFN-{gamma} respectively (9,14). IL-12 subsequently induces NK cells to produce IFN-{gamma} (1517), which in turn activates macrophages to present antigens to antigen-specific T cells (18). This type of innate immune response and its accompanying antigen-specific T cell response are appropriate for the eradication of microbial pathogens (14,19,20). Conversely, production of IL-4 early in an immune response directs the development of type 2 T cells from naive precursors (20). The critical role of IL-12 and IL-4 in the induction of subsets of T cells was demonstrated by gene targeting studies in the mouse (11,12,21).

Macrophages and NK cells function to link the innate immune system with the acquired immune system during pathogenic infections. In previous studies, it was demonstrated that IFN-regulatory factor (IRF)-1 gene-disrupted mice fail to mount a type 1 response in vitro (22,23). These mutant mice are defective in the production of IL-12 and the activation of NK cells. This defect resulted in a failure to induce an IFN-{gamma}-producing type 1 T cell subset. In the present study, we further analyzed this defect found in IRF-1–/– mutant mice during in vivo infections with various pathogens. We show here that the defect in the IL-12 production is due to the transcriptional level of the gene by IRF-1 and that the IL-18 protein is also deficient in IRF-1–/– mutant mice at the post-translational level. We also found that IL-12 is required for the induction of type 1 T cells in bacterial infections as observed in the previous in vitro experiment with protein antigen (22). On the other hand, neither IL-12 nor IL-18 is essential in the induction of type 1 T cells in a pathogenic infection. We propose a novel IL-12-independent pathway for inducing type 1 T cells that requires a distinct function of innate immune cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Cytokines and antibodies
Recombinant murine IL-12 was provided by the Genetic Institute. The mAb used for treating splenic T cells were anti-Thy-1 (30-H12) (24), anti-CD4 (GK1.5) (25) and anti-CD8 (83-12-5) (26). Selected rabbit serum was used as a complement source.

Mice
The IRF-1-deficient mice which were used have been previously described (27) and they were maintained by backcrossing to C57BL/6 mice. Mice used for experimental infections were backcrossed 5–8 times. IFN-stimulated gene factor-3{gamma} (ISGF3{gamma}, p48)-deficient mice have been described previously (28). The littermates of each mutant strain were used as control mice. These mutant mice and their littermates were reared under specific pathogen-free conditions in the animal facility of either the University of Tokyo or Ehime University School of Medicine. BALB/c and C57BL/6 mice were purchased from Charles River Japan (Yokohama, Japan). All mice were used according to our institutional guides for animal experimentation.

Experimental infections and pathogens
Leishmania major (MHOM/SU/73/5ASKH) protozoa were provided by Dr Himeno (Tokushima University School of Medicine, Tokushima, Japan) and maintained in our animal facility by in vivo passages. Protozoa (5x106) were inoculated intradermally into the right hind footpads of mice. The footpad swelling of the mice was monitored by a micrometer. After 6 weeks of infection, cytokine production of spleen cells was assessed by stimulation in vitro with concanavalin A (Con A; 10 µg/ml). A pathological examination of the footpads and lymph nodes of these infected mice was carried out at 6 wk post-inoculation by hematoxylin & eosin staining and periodic acid Schiff staining. Plasmodium berghei (ANKA strain) protozoa were maintained in our animal facility by blood passages and mice were infected i.p. with 1x107 parasitized erythrocytes. Listeria monocytogenes (EGD strain) was provided by Dr Mitsuyama (Niigata University School of Medicine, Niigata, Japan), and bacteria in the exponential phase were harvested, resuspended in PBS, heat-killed by incubation for 90 min at 74°C and stored at –80°C until use (29). Treated bacteria cells were thus used as heat-killed L. monocytogenes (KLm). Inactivated Bordetella pertussis (Tohama strain) was obtained from Chiba Kessei Institute (Chiba, Japan). Either 2x103 of L. monocytogenes and 1x109 of KLm or 5x109 of B. pertussis were inoculated i.p. and spleen cells were prepared from the mice on day 7.

Culture conditions for splenocytes
Spleen cells were isolated from mice, and were cultured in a volume of 2 ml/16 mm diameter flat-bottomed well (3524; Costar, Data Packaging, Cambridge, MA) at a density of 5x106/ml and were incubated for 24 h at 37°C in 5% CO2 humidified air atmosphere. The medium used was RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1xnon-essential amino acid, 50 µM 2-mercaptoethanol and 10% heat-inactivated FCS. In specific experiments where stated, splenocytes were cytotoxically treated with anti-Thy-1 mAb, anti-CD4 mAb or anti-CD8 mAb in the presence of complement, and the residual cells were used as a responder. The efficiency of the treatment was monitored and confirmed the efficiency of the treatment by flow cytometry.

Cytokine ELISA
Spleen cells were stimulated with Con A (10 µg/ml) in vitro for 24 h. The amounts of IFN-{gamma} and IL-4 in the culture supernatant were determined by sandwich ELISA established with mAb that were purchased from PharMingen (San Diego, CA). Recombinant mouse proteins were purchased from Genzyme (Cambridge, MA) and were used for standards.

Induction of IL-18 protein and IL-18 mRNA
Mice were injected i.p. with Propionibacterium acnes (10 mg/head) and were challenged with lipopolysaccharide (LPS; 10 µg/head) on day 7. The serum was collected 2 h after the challenge and the IL-18 protein was detected by ELISA. Total RNA was prepared from spleen cells 2 h after the challenge with LPS, and subjected to Northern blot analysis for IL-18 mRNA and IL-1ß-converting enzyme (ICE) mRNA.

Isolation and stimulation of peritoneal exudate cells (PEC) for IL-12 induction
Peritoneal cells taken from mice treated with thioglycolate 3 days earlier were allowed to adhere on tissue culture plates for 1 h and non-adherent cells were removed. Adherent cells were cultured in the presence of medium alone, LPS (10 µg/ml) or LPS plus recombinant mouse IFN-{gamma} (100 U/ml; Genzyme). Total RNA was extracted 16 h later and subjected to Northern blot analysis with probes for IL-12 p40 (a gift from Dr H. Yamamoto, Osaka University) and tumor necrosis factor (TNF)-{alpha}.

For in vivo administration of PEC, PEC were cytotoxically treated with anti-Thy-1 mAb plus complement and the residual cells were used for inoculation. More than 95% of these cells were CD11b+ and <5% were B220+.

RNA isolation and RNA blot analysis
Total cellular RNA was isolated by the guanidinium thiocyanate method. The procedure for RNA blot analysis was described in Harada et al. (30). Fragments of IFN-{gamma}, TNF-{alpha}, IL-4, IL-12 p40, IL-18 and ICE cDNA were labeled by the random primer method (Amersham, Little Chalfont, UK) to prepare probe DNAs.

mRNA detection by RT-PCR
Equal amounts of RNA (1 µg) were reverse transcribed using 5 U of reverse transcriptase (RAV-2; Takara Shuzo, Otsu, Shiga, Japan), 2.5 mM of dNTP and 300 ng of random primers (Takara Shuzo) in a total volume of 20 µl. Reverse transcription was carried out at 42°C for 60 min and 30 µl of RNase-free TE buffer was added to each sample. PCR amplification was carried out in a GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT) using 1 µl of the reverse transcribed product and 0.5 µl of DNA polymerase (Takara Shuzo) in a final volume of 50 µl. The reaction condition was as follows: DNA denatured at 94°C for 5 min, 35 cycles at 94°C for 1 min, 53°C for 1 min and 72°C for 2 min, and DNA extension at 72°C for 10 min. The oligonucleotide primers used were: IL-12 p40, 5'-TGCTCGAGTTGTAGAGGTGGACTGG-3' and 5'-CGGGTACCTTCCACATGTCACTGCC-3'; ICE, 5'-GATTCT- AAAGGAGGACATCC-3' and 5'-GTACATAAGAATGAACTGGA-3'; IFN-{gamma}, 5'-GAAAGCCTAGAAAGTCTGAATAACT-3' and 5'-ATCAGCAGCGACTCCTTTTCCGCTT-3'; V{alpha}14J{alpha}281, 5'-CCCAAGTGGAGCAGAGTCCT-3' and 5'-AATCCCTCCGACGTAAAACC-3'; C{alpha}, 5'-CATCCAGAACCCAGAACC-3' and 5'-CGGAACTTGGAAGTCAGGC-3'; C{delta}, 5'-CAGGCTTCCAACTTCTCAG-3' and 5'-TCGCCTCAGGAGAGG-3'. The PCR products were visualized and recorded, and the intensities of the bands were determined by NIH Image software.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Impaired in vivo type 1 T cell differentiation upon infection with various pathogens in mice lacking the IRF-1 gene
Mice lacking the IRF-1 gene were assessed for their susceptibility to L. major parasitic infection. We compared the results obtained using L. major-resistant strain C57BL/6 and wild-type littermates of IRF-1–/– mutant mice, and used susceptible strain BALB/c mice as a control. Transient swelling of footpads was observed after inoculation with L. major in the resistant strain C57BL/6 and wild-type littermates of IRF-1–/– mutant mice. The footpad swelling in these mice peaked at 4 weeks of infection and gradually diminished by 6 weeks of infection, while the footpad swelling in the susceptible strain BALB/c mice showed the clearest difference in the peak magnitude of response. However, the footpad swelling observed in IRF-1–/– mice was severer than that seen in BALB/c mice (Fig. 1Go). The mutant mice had been backcrossed to C57BL/6 and were thought to have a L. major-resistant trait. Therefore, IRF-1 is the factor that determines the susceptibility to L. major infection.



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Fig. 1. Footpad swelling of mice injected with L. major. (A) BALB/c ({square}), C57BL/6 ({blacksquare}), IRF-1-deficient (IRF-1–/–) (•) and wild-type (IRF-1 WT) ({circ}) mice were injected with 5x106 L. major parasites into the right hind footpads. Footpad swelling was measured by subtracting the swelling of control left hind footpad that had been injected with saline from the swelling of infected right foot pad. Each group consisted of six mice and the average footpads swelling was plotted. The results are representative of two independent experiments. (B) Representative swellings of three groups is shown. The arrowheads indicate the infected footpads.

 
The characteristic feature of the infected footpad in wild-type mice was inflammatory granulation tissue associated with the accumulation of neutrophils, lymphocytes, plasma cells and macrophages. In contrast, a granuloma formation was seen in the footpads of IRF-1–/– mice. This granuloma is characteristic of a marked accumulation of ballooned macrophages filled with L. major protozoa in their cytoplasm and is not associated with other inflammatory cell infiltrates. A similar difference in the characteristics of histology was revealed in popliteal lymph nodes (data not shown). The pathway mediating destruction of parasites by murine macrophages involves production of nitric oxide by inducible nitric oxide synthase (iNOS) (3135). It was also shown that the iNOS gene is transcriptionally regulated by IRF-1 and the expression of the gene is impaired in IRF-1–/– mutant mice (36). Therefore, macrophages in IRF-1–/– mutant mice were not effectively activated to kill the intracytoplasmic parasites during infection.

In our previous report, we noted that IRF-1 gene disruption was accompanied by a failure of type 1 T cell differentiation in vitro (22). The present study was undertaken to investigate the role of IRF-1 in in vivo differentiation of T cell subsets during bacterial and protozoal infection. Wild-type and IRF-1–/– mice were inoculated with L. monocytogenes, B. pertussis and L. major, and their splenic T cells were stimulated with Con A to measure IL-4 and IFN-{gamma} production. Splenic T cells of uninfected wild-type and mutant mice exhibited a similar cytokine production pattern as shown in Fig. 2Go(A). The inoculation with L. monocytogenes and B. pertussis induced T cells producing IFN-{gamma} but not IL-4 in wild-type mice, suggesting that type 1 T cells were induced in wild-type mice by these pathogens. In contrast, the same inoculum induced IL-4-producing T cells in the mutant mice without inducing IFN-{gamma}-producing T cells. The results indicated a shift to a type 2 T cell subset instead of a shift to a type 1 T cell subset in the mutant mice (Fig. 2BGo). These observations are consistent with our previous findings that IRF-1–/– mutant mice are susceptible to L. monocytogenes infection (22). A type 1 T cell cytokine, IFN-{gamma}, functions to protect mice from the L. monocytogenes infection through the activation of macrophages (37,38). It is also noted in Fig. 2Go(B) that the skewing of T cell differentiation occurred during the priming of the mice by pathogens, since type 1 and type 2 T cell cytokines were produced equally in unprimed wild-type and mutant mice. Therefore, IRF-1 gene disruption had an effect on macrophage function and the differentiation of functional T cell subsets.



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Fig. 2. Cytokine production patterns of T cells from wild-type and IRF-1–/– mice. Uninfected (naive) (A) and pathogen-inoculated (B) mice (three mice per group) were examined for their production of IFN-{gamma} and IL-4 by splenocytes. Spleen cells of IRF-1–/– (filled bar) and wild-type (open bar) mice were stimulated with Con A (10 µg/ml) in vitro. The amounts of cytokines produced in the culture supernatants were measured by ELISA after 24 h cultivation. Representative results of two to 10 independent experiments are shown.

 
The results of footpad swelling and the pathological examination of L. major-infected mutant mice suggest that T cells might shift to the type 2 subset because of the failure to activate macrophages. As shown in Fig. 2Go(B), T cells of L. major-infected wild-type mice produced IFN-{gamma} but not IL-4 as seen with L. monocytogenes and B. pertussis, while T cells of L. major-infected mutant mice produced predominantly IL-4. The results confirmed that the infection of mutant mice with L. major induces type 2 T cells. This finding is consistent with the result shown in Fig. 1Go, where the infection of L. major in IRF-1–/– mice was the most pronounced among the four mouse strains tested. It should be also noted, however, that some IFN-{gamma} was detected in the spleen cell culture supernatant of L. major-infected mutant mice. The production of IFN-{gamma} by T cells of bacteria-infected mutant mice was marginal or not detectable as shown in Fig. 2Go(B). This result suggests the possibility that T cell deviation may be differentially regulated in protozoa-infected and bacteria-infected mutant mice. This point will be further discussed in a later section.

IRF-1 functions to regulate transcription of the IL-12 p40 gene and processing of pro-IL-18 protein
The involvement of IL-12 in type 1 T cell differentiation is well established in many systems (6,9,11,12). It has also be shown that the gene expression of IL-12 p40 is regulated by NF-{kappa}B in the presence of IFN-{gamma} (39). In the current study, we further examined the need for IFN-{gamma} in IL-12 p40 gene expression by two mutant mouse strains, IRF-1–/– mice and ISGF3{gamma}–/– (p48–/–) mice. Both IRF-1 and p48 molecules are activated by IFN-{gamma} and regulate the transcription of IFN-{gamma}-regulated genes (40). Therefore, IL-12 p40 gene induction with LPS plus IFN-{gamma} was evaluated by Northern blot analysis using thioglycolate-induced peritoneal adherent cells from IRF-1–/– mice, p48–/– mice and their wild-type mice littermates (Fig. 3AGo). Co-stimulation with LPS and IFN-{gamma} was required for efficient IL-12 p40 gene induction in IRF-1 and p48 wild-type mice, while the same stimulation failed to effectively induce the gene in IRF-1–/– mice. The gene induction in p48–/– mice was comparable to their wild-type littermates. The results suggest that IRF-1 but not p48 is involved in the regulation of the IL-12 p40 gene.



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Fig. 3. IRF-1 regulates the gene expression of IL-12 p40. (A) Peritoneal macrophages of IRF-1–/–, p48–/– mice and their wild-type mice were collected by i.p. injection of thioglycolate. The collected cells were incubated on plastic dishes for 1 h at 37°C, and adherent cells were stimulated with medium alone (C), LPS (10 µg/ml) alone (L) or LPS (10 µg/ml) and 100 U/ml of IFN-{gamma} (L + {gamma}) for 16 h at 37°C. Total RNA was prepared from these treated cells and was subjected to Northern blot analysis for IL-12 p40 mRNA and TNF-{alpha} mRNA expressions. (B) DNA fragment containing putative IRF-1 binding sequences in the 5' upstream region of the IL-12 p40 gene was cloned by PCR and inserted into the upstream of the basic vector (pLuc) of the luciferase assay (IL-12 p40-promoter-pLuc). (C) The constructed IL-12 p40-promoter-pLuc plasmid was transfected to Cos7 cells with or without the expression plasmid of IRF-1 (pAct-1). The resulting enzyme activity of luciferase was compared with that of the basic vector plasmid (pLuc). The results in (A) and (C) are representative of three independent experiments.

 
Since we found the putative IRF-1 binding site in the published DNA sequence (ACTTTGGGTTTCC) at –68 to –56 upstream of the IL-12 p40 gene (39,41,42), we directly tested the possibility that IRF-1 transcriptionally regulates the expression of IL-12 p40 by luciferase assay. The –133 to +51 DNA region of the IL-12 p40 gene was obtained by PCR and inserted 5' upstream of the luciferase gene (basic vector, pLuc) lacking the promoter region (Fig. 3BGo). The constructed plasmid (IL-12 p40-promoter-pLuc) was electrically transfected to Cos7 cells and luciferase activity was measured (Fig. 3CGo). Co-transfection of IRF-1-expression plasmid (pAct-1, containing actin-promoter and the full length of IRF-1 cDNA) with the constructed plasmid to Cos7 cells augmented the luciferase activity. Although the data is not shown, recombinant IRF-1 protein was shown to bind to synthetic oligonucleotides corresponding to sequences at –77 to –49 of the IL-12 p40 gene by electrophoretic mobility shift assay. Taking these results together, IRF-1 activates the transcription of the IL-12 p40 gene by binding to the promoter region of the gene. Therefore, IRF-1–/– mutant mice are defective in the induction of the IL-12 p40 gene, which in turn impairs the induction of type 1 T cell subset upon a bacterial infection.

The production of IFN-{gamma} by T cells is augmented by an IFN-{gamma}-inducing factor (IGIF or IL-18) in bacterial infections and IL-18 synergizes with IL-12 for IFN-{gamma} induction (4346). Therefore, we also tested the gene expression of IL-18 in IRF-1–/– mutant mice by stimulation with P. acnes and LPS as described by Matsui et al. (47). A large amount of IL-18 protein was found in the serum of the wild-type mice, while no IL-18 protein was detected in the mutant mice (Fig. 4AGo). On the other hand, Northern blot analysis showed a comparable level of transcription of the IL-18 gene in both IRF-1–/– and wild-type mice.



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Fig. 4. Mature IL-18 protein was not detected in IRF-1–/– mice. (A) Three mice of each group were injected i.p. with P. acnes (10 mg/head). On day 7, the mice were challenged with LPS (10 µg/head) 2 h before sampling. The amounts of IL-18 protein in the serum of wild-type (open bar) and IRF-1–/– (filled bar) mice were measured by ELISA. IL-18 and ICE mRNA in spleen were assessed by Northern blot analysis using the total RNA extracted from spleen cells of wild-type (open bar) and IRF-1–/– (filled bar) mice. ND, not detected. (B) Wild-type and IRF-1–/– mice were infected with P. berghei or P. acnes. On day 7 of infection, RT-PCR was carried out to detect ICE mRNA using total RNA extracted from spleen cells of uninfected (C) and infected (I) mice.

 
The results suggested a defect in the post-translational process of IL-18 gene expression. The processing of inactive pro-IL-18 protein by ICE is required to produce the active mature IL-18 protein (4850). In addition, mitogen induction of the ICE gene is IRF-1 dependent (51). Indeed, expression of ICE mRNA was low in IRF-1–/– mice as shown in Fig. 4Go(A). It should be also noted that mRNA of the ICE gene was not induced by pathogenic infection in the mutant mice (Fig. 4BGo). Therefore, the impaired induction of the ICE gene in IRF-1–/– mice might result in the failure to activate the IL-18 protein. It has been shown in the present study that IRF-1–/– mutant mice are deficient in both IL-12 and IL-18 (Figs 3 and 4GoGo). Thus, the deficiency of IRF-1 had a profound influence on the IFN-{gamma} response of the mutant mice and explains the results shown in Figs 1–4GoGoGoGo.

Wild-type PEC bypass the IL-12 and IL-18 defect in IRF-1–/– mutant mice and induce IFN-{gamma}-producing T cells
Since the patterns of cytokine production by wild-type littermates and IRF-1–/– mutant mice were similar in the naive unprimed state, IRF-1 molecules appeared to be functioning during exposure to pathogens. To restore the IL-12 and IL-18 defect in IRF-1–/– mice, mice were administered recombinant IL-12 or wild-type PEC when they were inoculated with L. monocytogenes and B. pertussis. IFN-{gamma} mRNA expression of splenic T cells was assessed by Northern blot analysis (Fig. 5AGo). Recombinant IL-12 did not restore the impaired IFN-{gamma} mRNA induction, though the administration of IL-12 in an in vitro system has been shown to partially restore the response (22). It was shown, however, that the wild-type peritoneal cells overcame the IFN-{gamma} mRNA induction defect in IRF-1–/– mutant mice (Fig. 5AGo).



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Fig. 5. Restoration of IFN-{gamma} production by the administration of wild-type peritoneal macrophages. (A) Wild-type (open bar) and IRF-1–/– (filled bar) mice were primed by the indicated pathogen in the presence of either recombinant IL-12 protein (1 µg/head) or wild-type peritoneal macrophages (2.5x107/head). Spleen cells of these mice were stimulated with Con A in vitro for 16 h and the total RNA was subjected to a Northern blot analysis to determine the mRNA of IFN-{gamma}. The relative amount of mRNA expressed was evaluated by BAS2000. ND, not done. (B) The wild-type and IRF-1–/– mice were inoculated with B. pertussis together with (filled bars) or without (open bars) wild-type peritoneal macrophages. Spleen cells were prepared from these mice 1 week after inoculation and the cells were stimulated with Con A in vitro. The cytokines produced in the culture supernatants were determined by ELISA. A representative result out of two independent experiments is shown.

 
Restoration of this defect was also seen in the cytokine production pattern. The administration of PEC at the time of inoculation of pathogens restored the IFN-{gamma} production and reduced IL-4 production in the mutant mice, while the same treatment did not change the cytokine production pattern in the wild-type mice (Fig. 5BGo). Therefore, cells, probably macrophages, functioning in the innate immune system are responsible for the failure to induce IFN-{gamma}-producing type 1 T cells and these cells are defective in IRF-1–/– mutant mice. The result shows that the IL-12 production deficiency is not the only reason for the defect seen in the mutant mice. In addition, the results suggest the possibility of the involvement of another factor that may be regulated by the IRF-1 gene. A candidate for this factor is IL-18, since IRF-1 regulates IL-18 expression as shown in the previous section (Fig. 4Go). We will show, however, in the following section that the IL-18 is not essential for the induction of IFN-{gamma}-producing T cells.

IFN-{gamma}-producing T cells induced in IRF-1–/– mutant mice upon infection with P. berghei
IFN-{gamma} was produced in the spleen cell culture supernatant of L. major-infected mutant mice, although the amount was much less than that found in wild-type mice. This finding is in striking contrast to the condition observed in bacteria-infected mutant mice in which IFN-{gamma} production was marginal or not detectable (Fig. 2BGo). The results suggest the possibility that T cell deviation may be differentially regulated in protozoa-infected and bacteria-infected mutant mice.

This possibility was further examined using P. berghei blood-stage protozoa as a pathogen. To learn whether P. berghei infection activates the IL-12 p40 gene in vivo, wild-type mice were infected with P. berghei blood-stage parasites and thioglycolate-induced PEC were collected on day 2. The PEC were stimulated in vitro with Con A or LPS plus IFN-{gamma} for 16 h, and were subjected to Northern blot analysis for IL-12 p40 and TNF-{alpha} mRNA expression. IL-12 p40 mRNA was not detected in the RNA of ex vivo PEC. The treatment of PEC with LPS plus IFN-{gamma} induced the expression of IL-12 p40 gene in PEC of uninfected and infected mice. The magnitude of the induction was, however, down-regulated in the infected mice, though the reason for this down-regulation was unknown. On the other hand, TNF-{alpha} mRNA induction was comparable in the two groups (Fig. 6A and BGo). We should also note that the IL-12 p40 gene was not expressed in IRF-1–/– mutant mice at any point during P. berghei infection even using the RT-PCR method (Fig. 6C and DGo) and that the ICE gene was not induced during infection (Fig. 4BGo).



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Fig. 6. Impaired induction of IL-12 p40 mRNA in P. berghei-infected wild-type and mutant mice. (A and B) Expression of IL-12 p40 and TNF-{alpha} mRNA in thioglycolate-induced peritoneal macrophages of uninfected and P. berghei-infected wild-type mice was determined by Northern blot analysis (A), and the relative amount of the expression was calibrated by BAS2000. The open and filled bars indicate uninfected and infected mice respectively (B). (C and D) Expression of IL-12 p40 mRNA in thioglycolate-induced peritoneal macrophages of uninfected and P. berghei-infected wild-type and mutant mice was determined by Northern blot analysis (C). In (D), IL-12 p40 mRNA expression in spleen cells was determined by RT-PCR using P. berghei-infected wild-type and IRF-1–/– mice at day 0 (lanes 3 and 6), day 1 (lanes 4 and 7) and day 3 (lanes 5 and 8). As a positive control, IL-12 p40 expression in unstimulated (lane 1) and LPS plus IFN-{gamma}-stimulated (lane 2) PEC of uninfected wild-type mice (D). A representative result out of three independent experiments is shown.

 
P. berghei blood-stage parasites grow in red blood cells and re-enter using specific receptors on red blood cells, and they do not penetrate macrophages (52). However, the above results show that infection with blood-stage parasites influences the gene activation in macrophages. In addition, these results demonstrate that the gene expression of IL-12 p40 and TNF-{alpha} is differentially regulated in PEC of P. berghei-infected mice. Alternatively, it is also possible that IL-12 p40 and TNF-{alpha} were produced by the different subset of cells, though there was no difference in number and profile of PEC from uninfected and infected mice (data not shown). In any case, IL-12 p40 gene expression is suppressed in these mice. This suppression may result in the impairment of IFN-{gamma} production in the infected mice. However, it is known that IFN-{gamma}-producing T cells are activated in Plasmodium parasite-infected mice. Therefore, we decided to test whether IFN-{gamma}-producing type 1 T cells might be induced without participation of IL-12 in P. berghei-infected mice.

Since IL-12 p40 gene expression was suppressed in Plasmodium-infected wild-type mice, we investigated whether IFN-{gamma}-producing T cells are induced in Plasmodium-infected IRF-1–/– mutant mice that are defective in IL-12 p40 gene expression. P. berghei-infected wild-type mice exhibited a pattern of cytokine response similar to bacteria-infected wild-type mice (Fig. 7AGo). A large amount of IFN-{gamma} was produced by Plasmodium-infected wild-type mice, although the IL-12 p40 gene was suppressed in these mice. IFN-{gamma}-producing T cells were induced in P. berghei-infected wild-type mice which had been injected with anti-IL-12 antibody (data not shown). To our surprise, the production of IFN-{gamma} was pronounced in P. berghei-infected mutant mice. Thus, IFN-{gamma} can be induced without IRF-1 in Plasmodium-infected mice. The production of IL-4 was also observed in the mutant mice. Therefore, the pattern of cytokine production by splenic cells of the mutant mice was different from that of bacteria-infected mutant mice.



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Fig. 7. IFN-{gamma}-producing T cells were induced in P. berghei-infected IRF-1–/– mice. (A) Three mice per group were uninfected (open bar) or infected (filled bar) with P. berghei blood-stage parasites. Four days after infection, spleen cells of IRF-1–/– and wild-type mice were stimulated with Con A (10 µg/ml) in vitro. After 24 h cultivation, the amounts of cytokines produced in the culture supernatants were measured by ELISA. (B–D) Spleen cells of P. berghei-infected wild-type and IRF-1–/– mice were stimulated with Con A in vitro. After 16 h, the cultured spleen cells were cytotoxically treated with anti-CD4, anti-CD8 and anti-Thy-1 mAb followed by extraction of total RNA from the residual cells. RT-PCR was carried out to detect IFN-{gamma} mRNA (B), V{alpha}14J{alpha}281 mRNA (C), and C{alpha} mRNA and C{delta} mRNA (D). Intensities of the bands were determined using a digital image analyzer and were expressed as percent response to the intensity of non-treated control (B) and as relative intensity (C and D). The results are representative of five (A) and two (B–D) independent experiments.

 
To investigate whether these IFN-{gamma}-producing cells were T cells, splenic cells of P. berghei-infected mice were first stimulated in vitro with Con A for 16 h, and then cytotoxically treated with anti-Thy-1 mAb and complement. Total RNA was extracted from the residual cells and subjected to determination of IFN-{gamma} mRNA by RT-PCR. It was shown that cells producing IFN-{gamma} in P. berghei-infected wild-type and mutant mice were in fact T cells. Cytotoxic treatment of the T cells showed that CD4+CD8 T cells in the wild-type and CD4CD8 T cells in IRF-1–/– mutant mice were responsible for IFN-{gamma} production (Fig. 7BGo). These results suggest that IFN-{gamma}-producing T cells may be induced via two different pathways in pathogen-infected mice: one pathway is activated in the wild-type mice infected with P. berghei and induces CD4+ type 1 T cells, and the other is activated in the mutant mice infected with P. berghei and induces CD4CD8 type 1 T cells.

The phenotype of IFN-{gamma}-producing T cells in Plasmodium-infected mutant mice resembles that of NKT cells (53,54). TCR-mediated activation of NKT cells is CD1-restricted and the activation of NKT cells results in the production of IFN-{gamma} and IL-4 (37,55,56). A formyl-peptide of bacterial origin is presented by CD1 molecules on macrophages during bacterial infection (5759). In addition, NKT cells are present in IRF-1–/– mutant mice, though IRF-1–/– mice lack NK cells (22). If NKT cells are responsible for the IFN-{gamma} production observed in Plasmodium-infected mutant mice, a similar pattern might be found in L. monocytogenes-infected mutant mice. However, the inoculation of L. monocytogenes did not induce IFN-{gamma} secretion in the mutant mice (Fig. 2BGo). In addition, there was no or marginal activation of IL-4-producing T cells in Plasmodium-infected wild-type mice (Fig. 7AGo), suggesting that NKT cells are not responsible for the observed IFN-{gamma}-production in these mice.

To directly test the possibility of whether NKT cells are responsible for IFN-{gamma} production observed in Plasmodium-infected mutant mice, RT-PCR was used to test for the presence of V{alpha}14J{alpha}281 mRNA, which is used in invariant TCR of NK1.1+ T cells (Fig. 7CGo) (6063). NKT cells were strongly induced in Plasmodium-infected wild-type mice and were of the CD4CD8 phenotype (Fig. 7CGo). It should be noted that anti-CD4 treatment reduced the level of IFN-{gamma} mRNA expression (Fig. 7BGo). On the other hand, NKT cells were not induced in Plasmodium-infected mutant mice (Fig. 7CGo). From these results taken together, it is clear that IFN-{gamma}-producing T cells in the wild-type and mutant mice do not belong to NKT cells. We also tested the possibility that IFN-{gamma}-producing T cells in P. berghei-infected mutant mice belong to {gamma}{delta} T cells (Fig. 7DGo). Abundant C{delta} mRNA was detected in the wild-type mice, while that in the mutant mice was relatively low. The C{alpha} mRNA was detected in both mice at comparable levels. This result suggests that the {gamma}{delta} T cells are not the major subset of IFN-{gamma}-producing T cells in P. berghei-infected mutant mice.

The results shown in Figs 3 and 4GoGo prove that IL-12 and IL-18 were deficient in IRF-1–/– mutant mice. Studies using IL-12-deficient mice, IL-18-deficient mice and their offspring showed that these cytokines had an important role for IFN-{gamma} production (11,46). The induction of IFN-{gamma}-producing T cells in P. berghei-infected IRF-1–/– mice is, however, neither due to the effect of IL-12 nor IL-18. Considering the fact that IFN-{gamma}-producing T cells were not induced in bacteria-infected IRF-1–/– (i.e. IL-12-deficient) mice, we suggest that two distinct pathways are present which induce IFN-{gamma}-producing T cells, an IRF-1-regulated, IL-12-dependent pathway and an IL-12-independent (IRF-1-independent) pathway. In addition, we should note that marked IL-4 production was observed in Plasmodium-infected mutant mice. Despite the fact that both type 1 and type 2 T cell induction are co-regulated in the IL-12-dependent pathway, type 1 or type 2 cytokine-producing T cells are both activated in Plasmodium-infected mutant mice. This finding leads to the hypothesis that the IL-12-independent novel pathway for inducing IFN-{gamma}-producing T cells is distinct from the classical type 1/type 2 T cell subset differentiation pathway.

The restoration experiment with PEC (Fig. 5Go) shows that the lack of IFN-{gamma}-producing T cells in bacteria-infected IRF-1–/– mutant mice is due to a functional defect in macrophage lineage cells related to the activation of the IL-12-dependent pathway. These cells are, however, functional in the IL-12-independent pathway activated in Plasmodium-infected mice. We should also note that deviation of T cells is evident after priming with pathogens. Thus, the function of macrophage lineage cells that are involved in innate immunity may determine the outcome of the T cell response.


    Acknowledgments
 
We would like to thank Dr Masao Mitsuyama for providing us with L. monocytogenes, Dr Kunihiro Himeno for L. major, Dr Hiroshi Yamamoto for the IL-12 p40 probe and the Genetic Institute for recombinant mouse IL-12. We also would like to thank Dr Masato Nose, Dr Kenji Nakanishi, Dr Tomohiro Yoshimoto and Ms Taeko Fukuda for their comments on histology, detection of IL-18 protein and animal care. We would also like to acknowledge helpful discussion with and critical comments by Dr Alfred Singer, Dr Richard J. Hodes, Dr Tadatsugu Taniguchi, Dr Makoto Kanoh and Dr Hiroto Shinomiya. This work was supported in part by a 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
 
Con Aconcanavalin A
Klmheat-killed Listeria monocytogenes
ICEIL-1ß-converting enzyme
IGIFIFN-{gamma}-inducing factor
iNOSinducible nitric oxide synthase
IRF-1IFN-regulatory factor-1
ISGF3{gamma}IFN-stimulated gene factor-3{gamma}
LPSlipopolysaccharide
PECperitoneal exudate cell
TNFtumor necrosis factor

    Notes
 
Transmitting editor: M. Taniguchi

Received 28 December 1998, accepted 5 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

  1. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. 1996. Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2346.
  2. Mosmann, T. R. and Coffman, R. L. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145.[ISI][Medline]
  3. 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]
  4. Kaufman, S. H. E. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129.[ISI][Medline]
  5. Mosmann, T. R. and Sad, S. 1996. The expanding universe of T-cell subsets: Th1 and Th2 and more. Immunol. Today 17:138.[ISI][Medline]
  6. Seder, R. A. and Paul, W. E. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12:635.[ISI][Medline]
  7. LeGros, G. G., Ben-Sasson, S. S., Seder, R. Finkelman, F. D. and Paul, W. E. 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J. Exp. Med. 172:921.[Abstract]
  8. Swain, S. L., Weinberg, A. D., English, M. 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.[ISI][Medline]
  11. Magram, J., Connaughton, S. E., Warrier, R. R., Carvajal, D. M., Wu, C. Y., Ferrante, J., Stewart, C., Sarmiento, U., Faherty, D. A. and Gately, M. K. 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. Abbas, A. K., Murphy, K. M. and Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[ISI][Medline]
  14. 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.[ISI][Medline]
  15. Tripp, C. S., Wolf, S. F. and Unanue, E. R. 1993. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon {gamma} production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl Acad. Sci. USA 90:3725.[Abstract]
  16. Afonso, L. C., Scharton, T. M., Vieria, L. Q., Wysocka, M., Trinchieri, G. and Scott, P. 1994. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 263:235.[ISI][Medline]
  17. Scharton-Kersten, T., Afonso, L. C., Wysocka, M., Trichieri, G. and Scott, P. 1995. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol. 154:5320.[Abstract/Free Full Text]
  18. Farrar, M. A. and Schreiber, R. D. 1993. The molecular cell biology of interferon-{gamma} and its receptor. Annu. Rev. Immunol. 11:571.[ISI][Medline]
  19. Sher, A. and Coffman, R. L. 1992. Regulation of immunity to parasites by T cells and T cell-derived cytokines. Annu. Rev. Immunol. 10:385.[ISI][Medline]
  20. O'Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275.[ISI][Medline]
  21. Kopf, M., Le Gros, G., Bachmann, M., Lamers, M. C., Bluethmann, H. and Kohler, G. 1993. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature 362:245.[ISI][Medline]
  22. Taki, S., Sato, T., Ogasawara, K., Fukuda, T., Sato, M., Hida, S., Suzuki, G., Mitsuyama, M., Shin, E.-H., Kojima, S., Taniguchi, T. and Asano, Y. 1997. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1. Immunity 6:673.[ISI][Medline]
  23. Lohoff, M., Ferrick, D., Mittrucker, H. W., Duncan, G. S., Bischof, S., Rollonghoff, M. and Mak, T. W. 1997. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6:681.[ISI][Medline]
  24. Ledbetter, J. A. and Herzenberg, L. A. 1979. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47:63.[ISI][Medline]
  25. Dialynas, D. P., Wide, D. B., Marrack, P., Pierres, A., Wall, K. A., Harvan, W., Otten, G., Loken, M. R., Pierres, M., Kappler, J. W. and Fitch, F. W. 1983. Characterization of the murine antigenic determinant, designated L3T4 recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen-reactivity. Immunol. Rev. 74:29.[ISI][Medline]
  26. Leo, O., Foo, M., Segal, D. M., Shevach, E. M. and Bluestone, J. A. 1987. Activation of murine T lymphocytes with monoclonal antibodies: deletion of Lyt-2+ cells on an antigen not associated with the T cell receptor complex but involved in T cell activation. J. Immunol. 139:1214.[Abstract/Free Full Text]
  27. Matsuyama, T., Kimura, T., Kitagawa, M., Preffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham, A., Potter, J., Furlonger, C. L., Narendan, 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]
  28. Kimura, T., Kadokawa, Y., Harada, H., Matsumoto, M., Sata, M., Kashiwazaki, Y., Tarutani, M., Tan, R. S., Takasugi, T., Matsuyama, T., Mak, T. W., Noguchi, S. and Taniguchi, T. 1996. Essential and non-redundant roles of p48 (ISGF3{gamma}) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies. Genes Cells 1:115.[Abstract/Free Full Text]
  29. Koga, T., Mitsuyama, M. Handa, T. Yayama, T. Muramori, K. and Nomoto, K. 1987. Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice. Immunology 62:241.[ISI][Medline]
  30. Harada, H., Fujita, T., Willson, 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]
  31. Green, S. J., Melter, M. S., Hibbs, J. B. and Nacy, C. A. 1990. Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism. J. Immunol. 144:278.[Abstract/Free Full Text]
  32. Assreuy, J., Cunha, F. Q., Epperlein, M., Noronha-Dutra, A., O'Donnel, C. A., Liew, F. Y. and Moncada, S. 1994. Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major. Eur. J. Immunol. 24:672.[ISI][Medline]
  33. Bookvar, K. S., Granger, D. L., Poston, R. M., Maybodi, M., Washington, M. K., Hibbs, J. B. and Kurlander, R. L. 1994. Nitric oxide produced during murine listeriosis is protective. Infect. Immun. 62:1089.[Abstract]
  34. Bogdan, C., Gessner, A., Solbach, W. and Rollinghoff, M. 1996. Invasion, control and persistence of Leishmania parasites. Curr. Opin. Immunol. 8:517.[ISI][Medline]
  35. Reiner, S. L. and Locksley, R. M. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.[ISI][Medline]
  36. Kamijo, R., Harada, H., Matsuyama, T., Sosland, M., Gerectiano, J., Shapiro, D ., Le, J., Koh, S. I., Kimura, T., Green, S. J., Mak, T. W., Taniguchi, T. and Vileck, J. 1994. Requirement for transcription factor IRF-1 in NO synthetase induction in macrophages. Science 263:1612.[ISI][Medline]
  37. Harty, J. T., Lenz, L. L. and Bevan, M. J. 1996. Primary and secondary immune responses to Listeria monocytogenes. Curr. Opin. Immunol. 8:526.[ISI][Medline]
  38. 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]
  39. Murphy, T. L., Cleveland, M. G., Kulesza, P., Magram, J. and Murphy, K. M. 1995. Regulation of interleukin 12p40 expression through an NF-{kappa}B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
  40. Reis, L. F., Harada, H., Wolchok, J. D., Taniguchi, T. and Vilcek, J. 1992. Critical role of a common transcription factor, IRF-1, in the regulation of IFN-ß and IFN-inducible genes. EMBO J. 11:185.[Abstract]
  41. Tanaka, N., Kawakami, T. and Taniguchi, T. 1993. Recognition DNA sequences of interferon regulatory factor I (IRF-1) and IRF-2, regulators of cell growth and the interferon system. Mol. Cell. Biol. 13:4531.[Abstract]
  42. Yoshimoto, T., Kojima, K., Funakoshi, T., Endo, Y., Fujita, T. and Nariuchi, H. 1996. Molecular cloning and characterization of murine IL-12 genes. J. Immunol. 156:1082.[Abstract]
  43. Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., Akita, K., Namba, M., Tanabe, F., Konishi, K., Fukuda, S. and Kurimoto, M. 1995. Cloning of a new cytokine that induces IFN-{gamma} production by T cells. Nature 378:88.[ISI][Medline]
  44. Robinson, D., Shibuya, K., Mui, A., Zonin, F., Murphy, E., Sana, T., Hartley, S. B., Menon, S., Kastelein, S., Bazan, F. and O'Garra, A. 1997. IGIF does not drive Th1 development but synergizes with IL-12 for Interferon-{gamma} production and activates IRAK and NF-{kappa}B. Immunity 7:571.[ISI][Medline]
  45. Okamura, H., Kashiwamura, S.-I., Tsutsui, H., Yoshimoto, T. and Nakanishi, K. 1998. Regulation of interferon-{gamma} production by IL-12 and IL-18. Curr. Opin. Immunol. 10:259.[ISI][Medline]
  46. Takeda, K., Tsutsui, H., Yoshimoto, T., Adachi, O., Yoshida, N., Kishimoto, T., Okamura, H., Nakanishi, K. and Akira, S. 1998. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8:383.[ISI][Medline]
  47. Matsui, K., Yoshimoto, T., Tsutsui, H., Hyodo, Y., Hayashi, N., Hiroishi, K., Kawada, N., Okamura, H., Nakanishi, K. and Higashino, K. 1997. Propionibacterium acnes treatment diminishes CD4+NK1.1+ T cells but induces type 1 T cells in the liver by induction of IL-12 and IL-18 production from Kuppfer cells. J. Immunol. 159:97.[Abstract]
  48. Ghayur, T., Banerjee, S., Hugunin, M., Butler, D., Herzog, L., Carter, A., Quintal, L., Sekut, L., Talanian, R., Paskind, M., Wong, W., Kamen, R., Tracey, D. and Allen, H. 1997. Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386:619.[ISI][Medline]
  49. Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K., Fleming, M. A., Hayashi, N., Higashino, K., Okamura, H., Nakanishi, K., Kurimoto, M., Tanimoto, T., Flavell, R. A., Sato, V., Harding, M. W., Livingston, D. J. and Su, M. S. 1997. Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme. Science 275:206.[Abstract/Free Full Text]
  50. Fantuzzi, G., Puren, A. J., Harding, M. W., Livingston, D. J. and Dinarello, C. A. 1998. Interleukin-18 regulation of interferon gamma production and cell proliferation as shown in interleukin-1 beta-converting enzyme (caspase-1)-deficient mice. Blood 91:2118.[Abstract/Free Full Text]
  51. Tamura, T., Ishihara, M., Lamphier, M. S., Tanaka, N., Oishi, I., Aizawa, S., Matsuyama, T., Mak, T. W., Taki, S. and Taniguchi, T. 1995. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature 376:596.[ISI][Medline]
  52. Hadley, T. J. 1996. Invasion of erythrocytes by malaria parasites: a cellular and molecular overview. Annu. Rev. Microbiol. 40:451.[ISI][Medline]
  53. Fowlkes, B. J., Kruisbeck, A. M., Ton-That, H., Weston, M. A., Coligan, J. E., Schwartz, R. H. and Pardoll, D. M. 1987. A novel population of T cell receptor {alpha}ß-bearing thymocytes which predominantly express a single Vß gene family. Nature 329:251.[ISI][Medline]
  54. Budd, R. C., Miescher, G. C., Howe, R. C., Lees, R. K., Bron, C. and MacDonald, H. R. 1987. Developmentally regulated expression of T cell receptor ß chain variable domains in immature thymocytes. J. Exp. Med. 166:577.[Abstract]
  55. Cui, J., Shin, T., Kawano, T., Sato, H., Kondo, E., Toura, I., Kaneko, Y., Koseki, H., Kanno, M. and Taniguchi, M. 1998. Requirement for V{alpha}14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623.[Abstract/Free Full Text]
  56. Kawano, T., Cui, J., Koezuka, Y., Toura, I., Kaneko, Y., Motoki, K., Ueno, H., Nakagawa, R., Sato, H., Kondo, E., Koseki, H. and Taniguchi, M. 1998. CD1d-restricted and TCR-mediated activation of V{alpha}14 NKT cells by glycosylceramides. Science 278:1626.[Abstract/Free Full Text]
  57. Kurlander, R. and Nataraj, C. 1997. Characterization of the murine H2-M3wt-restricted CD8 response against a hydrophobic, protease-resistant, phospholipid-associated antigen from Listeria monocytogenes. Immunol. Rev. 158:123.[ISI][Medline]
  58. Pamer, E. G., Sijt, A. L. A. M., Villanueve, M. S., Busch, D. H. and Vijh, S. 1997. MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses. Immunol. Rev. 158:129.[ISI][Medline]
  59. Princiotta, M. E., Lenz, L. L., Bevan, M. J. and Staerz, U. D. 1998. H2-M3 restricted presentation of a Listeria-derived leader peptide. J. Exp. Med. 187:1711.[Abstract/Free Full Text]
  60. Lantz, O. and Bendelac, A. 1994. An invariant T cell receptor {alpha} chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD48 T cells in mice and humans. J. Exp. Med. 180:1097.[Abstract]
  61. Makino, Y., Kannno, R., Ito, T., Higashino, K. and Taniguchi, M. 1995. Predominant expression of invariant V{alpha}14+ TCR{alpha} chain in NK1.1+ T cell populations. Int. Immunol. 7:1157.[Abstract]
  62. Moretta, L., Ciccone, E., Mingari, M. C., Biassoni, R. and Moretta, A. 1994. Human natural killer cells: origin, clonality, specificity, and receptors. Adv. Immunol. 55:341.[ISI][Medline]
  63. Yokoyama, W. M, 1995. Natural killer cell receptors specific for major histocompatibility complex class I molecules. Proc. Natl Acad. Sci. USA 92:3081.[Free Full Text]