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*Listeria Infections
The Journal of Immunology, 2007, 178: 4498-4505.
Copyright © 2007 by The American Association of Immunologists, Inc.

IL-12 and Type-I IFN Synergize for IFN-{gamma} Production by CD4 T Cells, Whereas Neither Are Required for IFN-{gamma} Production by CD8 T Cells after Listeria monocytogenes Infection

Sing Sing Way1,2,*, Colin Havenar-Daughton2,{dagger},{ddagger}, Ganesh A. Kolumam2,{dagger},{ddagger}, Nural N. Orgun{dagger},{ddagger} and Kaja Murali-Krishna1,{dagger},{ddagger}

* Department of Pediatrics, {dagger} Department of Immunology, and {ddagger} Washington National Primate Center, University of Washington School of Medicine, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Differentiation of Ag-specific T cells into IFN-{gamma} producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-{gamma} production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-{gamma} in vitro. However, their role in priming Ag-specific T cells for IFN-{gamma} production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-{gamma} production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-{gamma} production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial (~50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75–95%) reductions in IFN-{gamma} production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-{gamma} production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR+/+ and IFN-IR–/– CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-{gamma} production by CD4 and CD8 T cells in response to bacterial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interferon-{gamma} plays an important protective role in both innate and adaptive immunity to infection with viruses, bacteria, and other intracellular pathogens. Ag-specific IFN-{gamma}-producing T cells are the hallmark of protective adaptive immunity to this group of pathogens. Inflammatory cytokines produced early during infection are believed to play an important role in the differentiation of naive T cells into IFN-{gamma}-producing effector cells. Two well-characterized inflammatory cytokines that can influence the differentiation of T cells are IL-12 and type I IFN (IFN-I) (1, 2, 3, 4, 5). Most of the studies examining the signals required for T cell activation and priming for IFN-{gamma} production were conducted in vitro by stimulating T cells with plastic beads coated with MHC ligands plus peptide in the presence of exogenously added cytokines (1, 2, 3). Although these experiments clearly demonstrate the direct effects of these cytokines on isolated T cells in vitro, they do not address the role of these cytokines in vivo, and especially in the context of infection when a myriad of other cytokines and immune stimulatory pathways is activated. Nevertheless, understanding the role and relative contributions of IL-12 and IFN-I in T cell activation during in vivo infection is important because of their known direct effects on T cell IFN-{gamma} production and augmentation or inhibition of the other cytokines during certain experimental infections (6, 7).

Previous studies using infection and vaccination models have limited their analysis to either IFN-I or IL-12. For example, IFN-IR-deficient (IFN-IR–/–) mice can generate IFN-{gamma}-producing CD8 T cells after lymphocytic choriomeningitis virus (LCMV)3 infection (7, 8); and because more IL-12 is produced after LCMV infection in IFN-IR–/– mice, these results suggest that IL-12 may substitute for the lack of IFN-I signaling in these mice (7). For IL-12-deficient mice, infection with various viral or other intracellular pathogens, unlike model Ags administered with adjuvants, shows that IL-12 is not required for IFN-{gamma} production by pathogen-specific T cells (7, 9, 10, 11, 12). Yet, in these infection models in which IFN-{gamma} is produced by Ag-specific T cells in the absence of IL-12, the role of IFN-I in bypassing the requirement for IL-12 has not been clearly demonstrated. Given that IFN-I may be compensating for the lack of IL-12, and vice versa, assessing the combined deficiency of IFN-I and IL-12 is critical to understanding the role of these cytokines in priming T cells for IFN-{gamma} production during infection, in vivo. To address this, we used a well-characterized mouse model of infection with the Gram-positive bacterium Listeria monocytogenes (Lm) in which both immune mediators of innate host resistance and generation of Ag-specific T cells in response to bacterial infection can be characterized (13). Following primary Lm infection, both IL-12 and IFN-I are produced (14, 15, 16, 17), and Ag-specific CD4 and CD8 T cells expand and produce IFN-{gamma}. In this study, we examined the individual and additive roles of IL-12 and IFN-IR signaling in priming CD8 and CD4 T cells for IFN-{gamma} production after Lm infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6) and Thy1.1 mice were obtained from The Jackson Laboratory. IL-12beta-deficient (IL-12P40–/–) (11) and IL-12{alpha}-deficient (IL-12P35–/–) mice (7), backcrossed to B6 mice for 11 generations, were purchased from The Jackson Laboratory. IFN-IR-deficient (IFN-IR–/–) mice (18) were backcrossed to B6 mice for 12 generations before use (8). IFN-IR–/–IL-12P40–/– double-deficient mice were obtained by intercrossing IL-12P40–/– with IFN-IR–/– mice. B6-transgenic (TcrLCMV)1Aox, LCMV-gp61–80-specific CD4 TCR transgenic mice (SMARTA) (19) were obtained from C. Surh (The Scripps Research Institute, La Jolla, CA) via M. Bevan (University of Washington, Seattle, WA) and intercrossed with Thy1.1 mice or IFN-IR–/– (Thy1.2+) mice to generate SMARTA transgenic IFN-IR+/+ (Thy1.1+) and SMARTA transgenic IFN-IR–/– (Thy1.1+Thy1.2+) mice, respectively. All mice were housed in a specific pathogen-free facility at the University of Washington, and experiments were performed under Institutional Animal Care and Use Committee-approved protocols.

Infections with Listeria and LCMV

The rLm strains Lm-OVA and Lm-gp61–80 were provided by H. Shen (University of Pennsylvania, Philadelphia, PA). Lm-OVA {Delta}actA was constructed from Lm-OVA using homologous recombination after cloning ~500-bp fragments of the Lm actA locus into the HindIII/KpnI sites of the temperature-sensitive plasmid pKSV7 with the following primers: upstream flanking region, forward primer 5'-aagcttgcagcgaccgatagcgaag-3', reverse primer 5'-gaattccgctgcgctatccgatgg-3'; downstream flanking region, forward primer 5'-gaattcgttaagtccaaaggtatcg-3', reverse primer 5'-ggtacctaaagagaacacgccaatag-3' (underlined sequences indicate introduced restriction sites). Lm were grown to early log phase (OD600 0.1) in brain heart infusion medium (BD Biosciences) at 37°C, washed in saline, and diluted in 200 µl final volume. For endogenous responses, 106 Lm-OVA {Delta}actA were injected i.v. into mice. For adoptive transfer experiments, 105 Lm-gp61–80 were injected i.p. into mice. LCMV strain Armstrong was plaque purified, grown in baby hamster kidney cells, and titered on Vero cells. A total of 2 x 105 PFU LCMV diluted in 200 µl final volume was injected i.v. into mice.

Reagents, Abs, in vitro cultures, adoptive transfer, and cell staining

For in vivo depletion, 1.0 mg of purified anti-mouse IL-12P40 (clone C17.8) or rat IgG2a isotype control (Bioexpress) was injected into mice 1 day before infection, as described previously (7). For in vitro culture, splenocytes were plated into 96-well round-bottom plates (5 x 106 cells/ml), and either stained directly with tetramer, or stimulated with the indicated peptides or heat-killed Lm for 5 h (intracellular cytokine staining) or 72 h (culture supernatants), as described (8, 20). Heat-killed Lm was prepared by washing and resuspending Lm in log-phase growth in PBS and incubating at 65°C for 30 min. Unless otherwise indicated, all peptide stimulations were performed at a concentration of 10–6 M. For intracellular cytokine staining, monensin (BD Biosciences; GolgiStop reagent) was added to cell culture before peptide stimulation. For adoptive transfer of SMARTA cells, CD4 T cells were purified by negative selection from SMARTA transgenic IFN-IR+/+ (Thy1.1+) or SMARTA transgenic IFN-IR–/– (Thy1.1+Thy1.2+) mice using CD4 T cell isolation kits (R&D Systems). These cells were transferred i.v. into recipient mice 1 day before Lm infection and contained 1 x 105 SMARTA transgenic IFN-IR+/+ (Thy1.1+) and 1 x 105 SMARTA transgenic IFN-IR–/– (Thy1.1+Thy1.2+) CD4 T cells per recipient mouse. The concentration of IL-12P40 and IL-12P70 in serum, and IFN-{gamma}, IL-4, and IL-13 in splenocyte culture supernatants was determined by ELISA using reagents from R&D Systems.

Statistics

The differences in percentages and numbers of stimulated splenocytes, percentage of cytokine-producing transgenic cells, and cytokine concentrations in culture supernatants between groups of mice were evaluated by using Student’s t test, with p < 0.05 taken as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of Ag-specific CD8 T cells in the absence of IFN-IR after Lm infection

IFN-I produced in response to viral infections confers protective effects through multiple mechanisms (21). These include IFN-I-mediated direct antiviral effects, activation of APCs, and enhancement of pathogen-specific CD8 T cell clonal expansion. As a consequence, mice deficient in IFN-IR become highly susceptible to most viral infections. By marked contrast, mice deficient in IFN-IR compared with control mice are more resistant to primary Lm infection (15, 16, 17). A possible mechanistic explanation for these results is that IFN-I produced during Lm infection sensitizes and enhances early lymphocyte apoptosis, which in turn leads to a cascade of events, including the production anti-inflammatory cytokines by the phagocytes, resulting in a more permissive niche for bacterial replication (22). Considering these early IFN-I effects on lymphocyte apoptosis, IFN-I-mediated effects on expansion of Ag-specific T cells, we compared the role of IFN-I in expansion and priming Ag-specific T cells following either Lm or LCMV infection. The expansion of LCMV-specific CD8 T cells was dramatically reduced in IFN-IR–/– mice compared with B6 control mice, confirming the important role of IFN-I for expansion of CD8 T cells in response to LCMV infection (Fig. 1). In contrast, following infection with Lm-OVA, expansion of OVA-specific CD8 T cells occurred with the same magnitude in both IFN-IR–/– and B6 mice (Fig. 1). These results suggest that although the presence of IFN-I signaling in the host plays an important role for the optimal expansion of CD8 T cell response to LCMV infection, it is not required for expansion of Ag-specific CD8 T cells in response to Lm infection.


Figure 1
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  		FIGURE 1. Ag-specific CD8 T cell expansion after LCMV or Lm-OVA infection. Mice were infected with LCMV Armstrong (2 x 105 PFUs) or Lm-OVA (104 CFUs), and splenocytes were analyzed by LCMV-gp33–41 and OVA257–264 tetramer and cell surface staining day 8 after infection. The number in each quadrant indicates the percentage ± SE of gated cells in each quadrant from three to four mice per group, and is representative of two independent experiments.

 
Increased production of IL-12 in IFN-IR-deficient mice

For CD8 T cells cultured in vitro, either IL-12 or IFN-I can provide, in addition to TCR and costimulation, a third signal allowing for proliferation and IFN-{gamma} production (2). Because Lm infection in vivo triggers the production of both IL-12 and IFN-I (16, 17, 23), we hypothesized that IL-12 produced early after Lm infection may overcome the requirement for IFN-I signaling and allow for the apparent normal expansion of Ag-specific CD8 T cells in IFN-IR–/– compared with B6 mice after Lm infection. Consistent with this hypothesis, beginning at 8 h after Lm-OVA infection, increased concentrations of both IL-12P40 and IL-12P70 were present in B6 and IFN-IR–/– mice; and by 24 h after infection, maximal levels of IL-12P40 and IL-12P70 are observed (Fig. 2, A and B). Of note, the serum concentration of both IL-12P40 and IL-12P70 was ~5-fold higher in IFN-IR–/– compared with B6 mice. In contrast, following LCMV infection, only minimal amounts of IL-12P40 and no IL-12P70 could be detected in either B6 or IFN-IR–/– mice (Fig. 2, A and B).


Figure 2
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  		FIGURE 2. Mean serum concentration of IL-12P40 (A and C) or IL-12P70 (B and D) within the first 96 h after infection with Lm-OVA (104 CFUs), LCMV (2 x 105 PFUs) (A and B), or Lm-OVA {Delta}actA (106 CFUs) (C and D) in B6 or IFN-IR–/– mice. Each data point represents three to four mice per group, and is representative of at least two independent experiments. Bar, one SE.

 
Relative roles of IL-12 and IFN-I signaling in the host for inducing IFN-{gamma} production by Ag-specific T cells during Listeria infection

To determine the relative contribution of IL-12 produced in response to Lm infection on the Ag-specific adaptive T cell response in normal and IFN-IR–/– mice, we examined the effects of IL-12 depletion on the expansion and priming of CD8 and CD4 T cells for IFN-{gamma} production in IFN-IR–/– compared with B6 mice. Because IL-12 plays a protective role, and IFN-I plays a detrimental role early in the course of wild-type (WT) Lm infection (10, 15, 16, 17, 24), a comparison of the adaptive T cell responses in mice that lack each of these cytokines (or cytokine receptors) is complicated by potential differences in Lm Ag load. To bypass this potential limitation, we examined the immune response to the attenuated Lm mutant containing a targeted deficiency in actA instead of WT Lm. Within an infected cell, unipolar expression of ActA by Lm facilitates and is required for bacterium to recruit host cell actin into organized tails, allowing for intracellular spread leading to a productive infection (25). We and others have demonstrated that infection with the {Delta}actA Lm mutant is able to prime Lm-specific CD8 and CD4 T cells, and yet is nonlethal even at relatively high inocula (up to 106 CFUs) in mice deficient in components of innate immunity such as MyD88, IFN-{gamma}, or TNF-{alpha} receptor normally essential for protection from WT Lm infection (20, 26, 27). To verify that the absence of either IL-12 or IFN-IR signaling does not significantly alter the Lm load following infection with Lm-OVA {Delta}actA, we examined the number of bacteria in the spleen of IFN-IR–/–, IL-12P40–/–, and control mice following infection with 106 CFUs of Lm-OVA {Delta}actA (Fig. 3). For Lm infection, the amount of Ag 24 h after infection as reflected by the amount of live bacteria is an important determinant of the magnitude of the ensuing T cell response because administration of antimicrobials before this time point abrogates the response and administration of antimicrobials after this time point has no effect (28). For either IFN-IR–/– or IL-12P40–/– mice compared with B6 mice, no differences in Lm CFUs could be detected in the spleen 24 h after infection with 106 CFUs of Lm-OVA {Delta}actA; and by day 8 after infection, no Lm could be recovered (Fig. 3). Moreover, consistent with what we observe for Lm-OVA infection, infection with this inocula of Lm-OVA {Delta}actA triggered the production of IL-12P40 and IL-12P70 in both B6 and IFN-IR–/– mice with similar kinetics, and conferred a similar reciprocal increase in IL-12 serum concentration in IFN-IR–/– compared with B6 mice (Fig. 2, C and D). Therefore, to overcome potential limitations related to differences in Ag load following infection in mice with increased resistance (IFN-IR–/– mice) or increased susceptibility (IL-12-deficient mice) to WT Lm infection, all subsequent experiments analyzing the endogenous adaptive T cell response were performed with the {Delta}actA mutant in Lm.


Figure 3
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  		FIGURE 3. Number of recoverable bacteria per mouse spleen 24 h (Figure 3) and 8 days ({square}) after infection with 106 Lm-OVA {Delta}actA in B6, IFN-IR–/–, IL-12P40–/–, and IFN-I–/–IL-12P40–/– mice. Each bar represents four to six mice per group, and is representative of two independent experiments. Bar, one SE.

 
To examine the relative contribution of IL-12 on the Ag-specific T cell response after Lm infection, IFN-IR–/– and B6 were treated with the anti-mouse IL-12P40 Ab (hybridoma clone C17.8) or an isotype control Ab 1 day before infection with Lm-OVA {Delta}actA. Eight days after infection, similar expansion of OVA-specific CD8 T cells and priming of OVA-specific CD8 T cells to produce IFN-{gamma} could be detected in B6, B6 depleted of IL-12, IFN-IR–/– mice, or IFN-IR–/– depleted of IL-12 (Fig. 4, A–D). These data suggest that during Lm infection, neither IL-12 nor IFN-I is required for expansion or priming Ag-specific CD8 T cells for IFN-{gamma} production. In contrast, examination of IFN-{gamma} production by Ag-specific CD4 T cells in these same mice revealed important and additive roles for IL-12 and IFN-I. In either IFN-IR–/– mice or B6 mice depleted of IL-12 compared with B6 control mice, there was a modest (~50%), but significant (p < 0.05) reduction in both percentage and absolute number of IFN-{gamma}-producing CD4 T cells following stimulation with the listeriolysin O (LLO)189–201 peptide (Fig. 4, E and F). However, for IFN-IR–/– mice depleted of IL-12 compared with B6 mice treated with control Ab, there was a substantial reduction (~75–80% reduction; p < 0.05) in both the percentage and number of IFN-{gamma}-producing CD4 T cells. To determine whether these differences in percentage and absolute numbers of IFN-{gamma}-producing CD4 T cells (and the lack of differences for CD8 T cells) reflect differences in the total amount of IFN-{gamma} produced by these cells, we measured the concentration of cytokine in culture supernatants of cells from B6 mice, B6 mice depleted of IL-12, IFN-IR–/– mice, and IFN-IR–/– depleted of IL-12 after stimulation with the MHC class II (LLO189–201) or MHC class I (OVA257–264) peptides (Fig. 4G). Following stimulation with the MHC class II, LLO189–201 peptide, there was a ~70% reduction in concentration of IFN-{gamma} in splenocytes from IFN-IR–/– mice or B6 mice depleted of IL-12 compared with splenocytes from B6 control mice (p < 0.05 for each group compared with B6), and a 90% reduction (p < 0.05) in IFN-{gamma} production by splenocytes from IFN-IR–/– depleted of IL-12 compared with splenocytes from B6 control mice. For these same splenocytes, stimulation with the MHC class I, OVA257–264 peptide revealed no differences in IFN-{gamma} production by splenocytes from IFN-IR–/– mice, B6 mice depleted of IL-12, or IFN-IR–/– mice depleted of IL-12 compared with splenocytes from B6 control mice (Fig. 4G). Furthermore, the reduction in IFN-{gamma} production by CD4 T cells in IFN-IR–/– mice, B6 mice depleted of IL-12, or IFN-IR–/– mice depleted of IL-12 was not associated with a reciprocal increase in Ag-specific IL-4 or IL-13 production (data not shown).


Figure 4
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  		FIGURE 4. Expansion and IFN-{gamma} production by CD8 and CD4 T cells day 8 after infection with 106 Lm-OVA {Delta}actA in B6 mice, B6 mice depleted of IL-12, IFN-IR–/– mice, or IFN-IR–/– depleted of IL-12. Percentage (A) and total number (B) of OVA257–264 tetramer-positive CD8 T cells per mouse spleen, and FACS plots demonstrating percentage (C) and total number (D) of IFN-{gamma}-producing CD8 T cells per spleen after stimulation with OVA257–264 peptide or no peptide. FACS plots demonstrating percentage (E) and total number (F) of IFN-{gamma}-producing CD4 T cells per spleen after stimulation with LLO189–201 peptide or no peptide. G, Concentration of IFN-{gamma} in splenocyte culture supernatants 72 h after stimulation with OVA257–264 peptide (MHC class I), LLO189–201 peptide (MHC class II), or no peptide determined by ELISA. Each data point represents six to eight mice per experimental group from two independent experiments. Bar, one SE.

 
In additional experiments, we sought to more stringently test the role of IL-12 for priming CD4 and CD8 T cells for IFN-{gamma} production. For these studies, the Ag-specific immune response to Lm infection in IL-12-deficient mice (resulting from targeted deficiency in either the IL-12P40 or IL-12P35 subunits) and mice deficient in both IL-12P40 and IFN-IR (IFN-IR–/–IL-12P40–/–) generated by intercrossing IL-12P40–/– mice with IFN-IR–/– mice was examined. Combined deficiency in both IFN-IR and IL-12P40 did not affect Lm Ag load because there was no difference in the number of bacteria recovered in the spleen 24 h after infection with 106 CFUs of Lm-OVA {Delta}actA in IFN-IR–/–IL-12P40–/– compared with IFN-IR–/–, IL-12P40–/–, or B6 mice (Fig. 3). Consistent with results using IL-12-depleting Ab, examination of the Ag-specific CD8 T cell response day 8 after infection with Lm-OVA {Delta}actA revealed similar percentages of IFN-{gamma}-producing CD8 T cells, absolute numbers of IFN-{gamma}-producing CD8 T cells, or accumulation of IFN-{gamma} in splenocyte culture supernatants after stimulation with OVA257–264 peptide between B6, IFN-IR–/–, IL-12P40–/–, IL-12P35–/–, or IFN-IR–/–IL-12P40–/– mice (Fig. 5, A, B, and F). In these experiments, we also examined the CD8 T cell response by stimulation with a subdominant MHC class I peptide within LLO, LLO296–304 (29, 30). Although the percentage and total number of CD8 T cells that respond to the LLO296–304 peptide are much lower than response to OVA257–264, no differences were observed in either percentage or absolute number of CD8 T cells that produce IFN-{gamma} in B6, IFN-IR–/–, IL-12P40–/–, IL-12P35–/–, or IFN-IR–/–IL-12P40–/– mice to this subdominant Ag (Fig. 5, A and C). These data demonstrate that in response to Lm infection, neither IL-12 nor IFN-I signaling is required for priming Ag-specific CD8 T cells for IFN-{gamma} production.


Figure 5
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  		FIGURE 5. IFN-{gamma} production by CD8 and CD4 T cells day 8 after infection with 106 Lm-OVA {Delta}actA in B6 mice, IFN-IR–/– mice, IL-12P40–/–, IL-12P35–/–, or IFN-IR–/–IL-12P40–/– mice. FACS plots demonstrating percentage (A) and total number (B and C) of IFN-{gamma}-producing CD8 T cells per spleen after stimulation with the MHC class I peptides, as follows: OVA257–264, LLO296–304, or no peptide control. FACS plots demonstrating percentage (D) and total number (E) of IFN-{gamma}-producing CD4 T cells per spleen after stimulation with LLO189–201 peptide or no peptide. F, Concentration of IFN-{gamma} in splenocyte culture supernatants 72 h after stimulation with OVA257–264 peptide (MHC class I), LLO189–201 peptide (MHC class II), or no peptide, as determined by ELISA. G, Percentage of maximal IFN-{gamma} production by CD4 T cells determined by intracellular cytokine staining after stimulation with LLO189–201 peptide (10–9 to 10–4 M) or no peptide for the indicated groups of mice. Bar, one SE. Each data point represents four to six mice per experimental group from two independent experiments.

 
In these same mice infected with Lm-OVA {Delta}actA, we examined the Ag-specific CD4 T cell response. Consistent with results from these previous studies using IL-12-depleting Ab, IL-12P40–/–, IL-12P35–/–, and IFN-IR–/– mice compared with B6 mice each had partial (40–60%) reductions in percentage and absolute numbers of IFN-{gamma}-producing CD4 T cells after stimulation with the LLO189–201 peptide, whereas combined deficiency of both IL-12 and IFN-IR signaling in IFN-IR–/–IL-12P40–/– mice revealed a dramatic (95%) reduction in both percentage and absolute numbers of IFN-{gamma}-producing CD4 T cells (Fig. 5, D and E).

In additional experiments, we examined whether the defects in IFN-{gamma} production by CD4 T cells in splenocytes from IL-12-deficient, IFN-IR-deficient, or mice with combined IL-12 and IFN-IR deficiency could be overcome by increasing the peptide concentration used for in vitro restimulation (Fig. 5G). Although raising the peptide concentration in restimulation (from 10–9 to 10–4 M) increased the percentage of IFN-{gamma}-producing CD4 T cells in B6, IL-12P40–/–, IL-12P35–/–, and IFN-IR–/– mice in a concentration-dependent manner, the ~50% reduction in IFN-{gamma} production by Ag-specific CD4 T cells from IL-12P40–/–, IL-12P35–/–, and IFN-IR–/– compared with B6 mice was consistently observed. Furthermore, because only minimal levels of IFN-{gamma} were produced by CD4 T cells from mice with combined defects in both IL-12 and IFN-IR at all peptide concentrations examined, the difference in IFN-{gamma} production in CD4 T cells from these mice compared with cells from B6 mice or mice with individual defects in either IL-12 or IFN-IR is more exaggerated at higher peptide concentrations (Fig. 5G). To examine whether differences in IFN-{gamma} production by CD4 T cells in IL-12 and IFN-IR-deficient mice after Lm infection were applicable to other MHC class II Ags other than the LLO189–201 peptide, we measured IFN-{gamma} production in splenocytes after restimulation with heat-killed Lm. Compared with CD4 T cells from B6 mice, similar defects (~50% reduction) in IFN-{gamma} production were observed for CD4 T cells in IL-12P40–/–, IL-12P35–/–, and IFN-IR–/– mice, and more dramatic (~80% reduction) defects for CD4 T cells in mice with combined defects in both IL-12 and IFR-IR were observed (data not shown). To determine whether defects in priming Ag-specific CD4 T cells in mice with combined deficiency in both IL-12 and IFN-IR could be explained by reduced MHC class II expression by APCs, we examined the surface expression of MHC-II on dendritic cells (CD11c+ splenocytes). For mice with combined deficiency in both IL-12 and IFN-IR compared with B6 or mice with individual defects in IL-12 or IFN-IR alone, no defects in MHC-II surface expression could be detected before Lm infection, or at 24 h and 7 days after infection. Taken together, these data demonstrate that during Lm infection, IL-12 and IFN-I each have important and together have additive roles for priming IFN-{gamma} production by CD4 T cells, whereas neither IL-12 nor IFN-I is required for priming CD8 T cells for IFN-{gamma} production.

Additive roles for IL-12 and IFN-I in priming transgenic CD4 T cells for IFN-{gamma} production

IFN-I can be produced and exert biological responses in multiple cell types, and thereby influence the T cell response to infection directly by signaling on T cells or indirectly through stimulation of other cell types (8, 31, 32). Therefore, a comparison of CD4 T cell responses in Lm-infected B6 and IFN-IR–/– mice does not specifically address the role of IFN-I signaling in Ag-specific T cells. To examine whether the modest reductions in percentage and numbers of IFN-{gamma}-producing CD4 T cells in IFN-IR–/– mice, and the more dramatic reductions in IFN-{gamma} production by CD4 T cells in mice with combined defects in both IL-12 and IFN-IR are due to defects in IFN-IR signaling on CD4 T cells, we measured expansion and IFN-{gamma} production by IFN-IR+/+ and IFN-IR–/– TCR transgenic CD4 T cells after adoptive transfer into normal or IL-12-deficient mice. For these experiments, we used CD4 SMARTA cells specific for LCMV gp61–80, and transferred an equal number of IFN-IR+/+ SMARTA and IFN-IR–/– SMARTA cells into either B6 or IL-12P35–/– mice. These mice were then infected with a recombinant strain of Lm expressing the MHC class II LCMV Ag, Lm-gp61–80. As expected, for cells transferred into B6 or IL-12P35–/– mice that were uninfected, SMARTA donor cells were recovered at a relatively low frequency (~5–10% of transferred cells) (Fig. 6A). By day 5 postinfection, both IFN-IR+/+ and IFN-IR–/– SMARTA cells expanded ~10- to 20-fold in both B6 and IL-12P35–/– recipient mice, and at day 8 postinfection, the numbers of SMARTA cells had undergone significant contraction in both B6 and IL-12P35–/– mice. At the peak of expansion, the response of SMARTA donor T cell population to stimulation with the gp61–80 peptide was examined in splenocytes from B6 and IL-12P35–/– mice. In both B6 and IL-12P35–/– mice, there was only a modest reduction in the percentage of IFN-{gamma}-producing SMARTA IFN-IR+/+ compared with IFN-IR–/– cells (Fig. 6B); however, there was a more dramatic (37 ± 6 compared with 14 ± 4; p < 0.05) reduction in IFN-{gamma} production for SMARTA IFN-IR+/+ cells in B6 mice compared with IFN-IR–/– cells in IL-12P35–/– mice. Importantly, the reduction in IFN-{gamma} production by IFN-IR–/– SMARTA cells in either IL-12P35–/– or B6 mice compared with IFN-IR+/+ cells in B6 mice is not due to general defects in cell activation because under the same stimulation conditions, there were no differences in CD40L expression by these cells (Fig. 6B). Taken together, these results demonstrate that during Lm infection, IFN-I acting directly on CD4 T cells primes them for IFN-{gamma} production, and IFN-I and IL-12 synergize to prime CD4 T cells for maximal IFN-{gamma} production.


Figure 6
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  		FIGURE 6. A, Number of SMARTA CD4 T cells (IFN-IR+/+, Thy1.1+, {diamondsuit}; IFN-IR–/–, Thy1.1+Thy1.2+, {square}) in congenic (Thy1.2+) B6 or IL-12-deficient (IL-12P35–/–) recipient mice before infection (day 0), and days 5 and 8 after infection with rLm-gp61–80. B, IFN-{gamma} production and CD40L expression by IFN-IR+/+ (Thy1.1) or IFN-IR–/– (Thy1.1, Thy1.2) SMARTA transgenic cells day 5 after infection with rLm-gp61–80 in B6 or IL-12-deficient (IL-12P35–/–) mice after in vitro stimulation with gp61–80 peptide (line histogram) or no peptide (filled histogram). The numbers in each histogram represent the mean ± SE for three mice per experimental group, and are representative of three independent experiments with similar results. Bar, one SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we used the well-characterized Lm infection model in mice to examine the individual and combined roles of IFN-I and IL-12 in priming Ag-specific T cells for IFN-{gamma} production after in vivo infection. We first compared the number and percentage of Ag-specific IFN-{gamma}-producing CD8 and CD4 T cells in Lm-infected B6 mice, IFN-IR–/– mice, B6 mice depleted of IL-12, and IFN-IR–/– mice depleted of IL-12. These experiments showed that the percentage and number of IFN-{gamma}-producing Ag-specific CD8 T cells are not substantially affected by individual or combined defects in IL-12 or IFN-IR signaling. In contrast, both the percentage and number of IFN-{gamma}-producing CD4 T cells were partially reduced (~50%) in both IFN-IR–/– and B6 mice treated with anti-IL-12 Ab, and substantially reduced (~90%) in IFN-IR–/– mice treated with anti-IL-12 Ab. These results with IL-12-depleting Ab in normal and IFN-IR–/– mice were confirmed using mice with targeted deficiency in IL-12, and in mice deficient with targeted deficiency in both IL-12 and IFN-IR. We then performed additional experiments to examine the direct action of IFN-I on Ag-specific CD4 T cells by comparing the response of adoptively transferred IFN-IR+/+ vs IFN-IR–/– TCR transgenic CD4 T cells in Lm-infected B6 or IL-12-deficient mice. These experiments demonstrated that in the presence of IL-12, IFN-I stimulation of CD4 T cells contributes to IFN-{gamma} production only partially, whereas in the absence of IL-12, IFN-I stimulation of CD4 T cells plays a more significant role in IFN-{gamma} production. Together these results demonstrate that during Lm infection, IL-12 and IFN-I act synergistically for priming CD4 T cells for IFN-{gamma} production, whereas neither IFN-I nor IL-12 is required for priming CD8 T cells for IFN-{gamma} production.

The precise mechanisms that govern the differentiation of CD4 T cells into IFN-{gamma}-producing Th1 or IL-4/IL-13-producing Th2 cells after infection in vivo are incompletely understood. IL-12 is essential for IFN-{gamma} production and Th1 differentiation by CD4 T cells in the context of immunization with dead Ags (e.g., keyhole limpet hemocyanin or toxoplasma extract) and some infections (e.g., Lm, Mycobacterium avium, Leishmania major), whereas for other infections (e.g., LCMV) a deficiency of IL-12 appears to have little or no effect on IFN-{gamma} production by Ag-specific CD4 T cells (9, 10, 11, 12, 33). In this study, we demonstrate that this alternative IL-12-independent pathway for IFN-{gamma} production by Ag-specific CD4 T cells during Lm infection is mediated by IFN-I. To our knowledge, this is the first report demonstrating that IL-12 and IFN-I can synergize in priming IFN-{gamma} production and Th1 differentiation of CD4 T cells in response to any infection or immunization. Importantly, in the absence of IL-12 and IFN-I signaling, Lm-specific CD4 T cells do not have a reciprocal increase in production of the Th2 cytokines IL-4 or IL-13. These data suggest that for Lm infection, despite the absence of signals that prime CD4 T cells to undergo a Th1 differentiation pathway, additional Th2-promoting signals are necessary for priming Ag-specific CD4 T cells to produce cytokines such as IL-4 and IL-13.

The reciprocal regulation of IL-12 and IFN-I and the ability of these divergent pathways to prime CD8 T cell for IFN-{gamma} production have been described previously after infection with LCMV (7). In this study, depletion of IL-12 or IFN-I, or targeted deficiency in IL-12 or IFN-IR signaling reciprocally increased the concentration of the other cytokine. Therefore, depletion of either IL-12 or IFN-I alone had little or no effect, whereas combined ablation of both IL-12 and IFN-I resulted in dramatic reductions in IFN-{gamma} production by LCMV-specific CD8 T cells. Overall, the data we present in this study are consistent with their conclusions that IL-12 and IFN-I play functionally redundant roles for priming CD8 T cells during LCMV infection. However, our finding that IFN-IR–/– mice (backcrossed extensively to B6) compared with B6 mice have reduced CD8 T cell expansion after LCMV infection is inconsistent with their finding of a relatively normal CD8 T cell response in IFN-IR–/– mice on a 129s/v background compared with 129s/v mice. The reasons for this difference are unclear, but may relate to mouse strain-specific differences in IL-12 production following LCMV infection. Indeed, in our hands, even after infection with a ~10-fold increased inocula of virus used in our present study, we could not detect biologically active IL-12P70 production in response to LCMV infection in either B6 or IFN-IR–/– mice. Thus, the dramatic reduction in LCMV-specific CD8 T cells in IFN-IR–/– compared with B6 mice that we describe is most likely due to the lack of biologically active IL-12 produced in response to LCMV infection in these mice.

Stimulation of naive CD8 T cells in vitro with artificial APCs coated with TCR plus peptide and costimulation molecules does not trigger proliferation or IFN-{gamma} production, but under these same conditions, addition of either IL-12 or IFN-I results in both proliferation and IFN-{gamma} production (2, 34, 35). These findings reflect important roles for these cytokines in vivo because addition of exogenous IL-12 during initial in vivo priming of TCR transgenic CD8 T cells with peptide results in increased expansion and IFN-{gamma} production by these cells after secondary Ag priming. Moreover, CD8 T cells initially primed without exogenous IL-12 cannot lyse target cells coated with cognate peptide despite having only modest defects in expansion and IFN-{gamma} production (35). Accordingly, our ongoing studies are directed at further characterizing the function of Ag-specific CD8 T cells after Lm infection that appear to expand and produce IFN-{gamma} normally in the absence of IL-12 and/or IFN-IR by examining their ability to confer protective immunity to Lm rechallenge in memory time points. Furthermore, our finding that Ag-specific CD8 T cells have no defects in expansion or IFN-{gamma} production in the absence of both IL-12 and IFN-IR suggests that Lm infection triggers other cytokines or immune pathways that can activate alternative pathways for priming CD8 T cells. A likely candidate for this cytokine is IL-18 because it can trigger activation and IFN-{gamma} production by T cells in the absence of IL-12 in other contexts (36, 37, 38). However, when tested in vitro with artificial APCs coated with TCR and costimulation molecules, IL-18 could not restore activation of naive CD8 T cells (2). We are currently investigating whether this reflects inherent differences between priming during in vivo infection and culture in vitro, and whether priming T cells in the absence of IL-12 and/or IFN-IR will impart a functional defect in their ability to confer protective immunity in response to WT Lm rechallenge.


   Acknowledgments
 
We thank Drs. C. B. Wilson, M. Mathis, T. Kollmann, and A. Hajjar for helpful discussions and critical reading of the manuscript; Drs. M. J. Bevan and C. Surh for providing SMARTA mice; and Dr. H. Shen for providing rLm strains.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Sing Sing Way, 1959 Northeast Pacific Street, Box 357650, Seattle, WA 98195; E-mail address: singsing{at}u.washington.edu or Dr. Kaja Murali-Krishna, 1959 Northeast Pacific Street, Box 357650, Seattle, WA 98195; E-mail address: mkaja{at}u.washington.edu Back

2 S.S.W., C.H.-D., and G.A.K. contributed equally to this work. Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; LLO, listeriolysin O; Lm, Listeria monocytogenes; WT, wild type. Back

Received for publication August 8, 2006. Accepted for publication January 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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