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The Journal of Immunology, 2008, 180, 4109 -4115
Copyright © 2008 by The American Association of Immunologists, Inc.
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Deviation from a Strong Th1-Dominated to a Modest Th17-Dominated CD4 T Cell Response in the Absence of IL-12p40 and Type I IFNs Sustains Protective CD8 T Cells1

Nural N. Orgun*, Meredith A. Mathis{dagger}, Christopher B. Wilson*,{dagger} and Sing Sing Way2,*

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The differentiation of naive CD4 T cells into specific effector subsets is controlled in large part by the milieu of cytokines present during their initial encounter with Ag. Cytokines that drive differentiation of the newly described Th17 lineage have been characterized in vitro, but the cytokines that prime commitment to this lineage in response to infection in vivo are less clear. Listeria monocytogenes (Lm) induces a strong Th1 response in wild-type mice. By contrast, we demonstrate that in the absence of IL-12p40 (or IFN-{gamma}) and type I IFN receptor signaling, the Th1 Ag-specific CD4 T cell response is virtually abolished and replaced by a relatively low magnitude Th17-dominated response. This Th17 response was dependent on TGF-β and IL-6. Despite this change in CD4 T cell response, neither the kinetics of the CD4 and CD8 T cell responses, the quality of the CD8 T cell response, nor the ability of CD8 T cells to mediate protection were affected. Thus, generation of protective CD8 T cell immunity was resilient to perturbations that replace a strong Th1-dominated to a reduced magnitude Th17-dominated Ag-specific CD4 T cell response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naive CD4 T cells have the ability to differentiate into distinct effector lineages, each characterized by the production of a unique profile of effector cytokines. Until recently, these CD4 T cell effector lineages have included Th1 cells that produce IFN-{gamma} important for immunity to intracellular pathogens and Th2 cells that produce IL-4, IL-5, and IL-13 important for immunity to parasitic infections. The newly elucidated Th17 lineage produces IL-17A/F, IL-21, IL-22, and GM-CSF (1, 2) and fulfills a niche that provides immunity against extracellular bacterial or fungal pathogens, like Citrobacter, Klebsiella, or Candida (3, 4, 5, 6). However, uncontrolled activation of Ag-specific Th17 cells has been implicated in the pathogenesis of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis in humans and experimental autoimmune encephalomyelitis and adjuvant arthritis in mice (1, 7). Determining the biological parameters that govern the differentiation program of CD4 T cells into each specific effector lineage is critical for the design of vaccines and other immune-modulatory strategies to ensure that only the desired lineage of effector CD4 T cell is generated, while simultaneously avoiding potentially detrimental immune responses.

At least three interrelated signals are required for the activation and differentiation of naive CD4 into specific effector cell lineages. The first (TCR binding to a specific peptide-MHC complex) and second (physical interaction between costimulatory receptors on the T cell and APC) signals lead to T cell activation, whereas the third signal (the presence or absence of specific inflammatory cytokines) plays a more critical role in the differentiation of activated T cells into specific effector cell lineages (8). Moreover, the effector cytokines produced by each specific lineage of effector CD4 T cells further reinforces the differentiation of other naive T cells into that lineage, while simultaneously inhibiting differentiation into other cell lineages. For example, IFN-{gamma} produced by Th1 cells promotes T cell differentiation to Th1 lineage by inducing IL-12 receptor β2 and T-bet expression and inhibits Th2 differentiation by repressing GATA-3 and IL-4 (9). Conversely, IL-4 produced by Th2 cells induces GATA-3 expression, thereby inducing IL-4 production by other T cells and inhibiting IL-12 receptor expression (10). Characterized primarily in vitro with T cells activated in a non-Ag-specific manner, Th17 differentiation requires the presence of TGF-β and IL-6 and the absence of IFN-{gamma} (3, 11, 12). However, the general applicability of these results to the actual signals that are produced and are important for differentiation of Ag-specific Th17 CD4 T cells during infection in vivo is unclear.

Experimental Listeria monocytogenes (Lm)3 infection is a well-characterized model by which to examine priming of Ag-specific T cells in vivo (13). Following infection with either wild-type (WT) or live, attenuated Lm strains that retain the ability to gain access to the cell cytoplasm, a protective T cell response is readily detected and characterized by the expansion of Ag-specific, IFN-{gamma}-producing Th1 CD4 and CD8 effector T cells. For Lm infection, Ag-specific CD8 T cells confer the majority of the protective effects, whereas CD4 T cells have an important role in the generation of long-lived memory CD8 T cells (14, 15, 16). Using this infection model, we have recently demonstrated that priming Ag-specific CD4 T cells for IFN-{gamma} production requires either IL-12 or type I IFNs, while priming Ag-specific CD8 T cells requires neither IL-12 nor type I IFNs (17). Furthermore, for CD4 T cells activated in the absence of IL-12 and type I IFNs, the lack of IFN-{gamma} production is not associated with a reciprocal production of Th2 cytokines such as IL-4 or IL-13 (17). Accordingly, in the present study, we examined the possibility that Lm infection in the absence of both IL-12p40 and IFN-IR signaling could prime a Th17-dominated response. After comparing the relative expression of IFN-{gamma} and IL-17 by Ag-specific CD4 T cells in wild-type (WT), IL-12p40- deficient, IFN-IR-deficient, and mice deficient in both IL-12p40 and IFN-IR, our studies indicate that the presence of either IL-12p40 or IFN-I is required for Th1 differentiation of naive CD4 T cells. In the absence of both IL-12 and IFN-IR signaling, the normally robust Ag-specific Th1 CD4 T cell response is replaced by a Th17-dominated response that is of significantly lower magnitude. Using this model for priming of Ag-specific Th17 cells, we further characterized the specific cytokine milieu required for in vivo Th17 CD4 T cell differentiation, the dynamics of Ag-specific Th17 T cell expansion and contraction after infection, and the impact a drastically skewed CD4 Th response plays on CD8 T cell immunity.


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

IL-12p40-deficient (P40–/–) mice obtained from The Jackson Laboratory had been backcrossed 11 times to B6 before use. Type I IFN receptor-deficient (IFN-IR–/–) mice backcrossed to B6 mice for 12 generations were obtained from Dr. K. Murali-Krishna (University of Washington, Seattle, WA). Mice deficient in both IL-12p40 and IFN-IR (P40–/– IFN-IR–/– mice) were generated by intercrossing P40–/– and IFN-IR–/– mice (17). Mice were housed in a specific pathogen-free facility at the University of Washington. All experiments were performed under Institutional Animal Care and Use Committee-approved protocols.

Listeria monocytogenes

The recombinant Lm strain, Lm-OVA, and Lm-OVA {Delta}actA derived from this strain through targeted deletion in the actA gene have been described previously (17, 18). For infections, Lm were grown to early log phase (OD600, 0.1) in brain-heart infusion medium (BD Biosciences) at 37°C, washed, and diluted with saline to a 200-µl final volume and injected i.v. into mice.

Reagents, in vitro cultures, and cell staining

For in vivo depletion, 1.0 mg of purified rat anti-mouse IFN-{gamma} (XMG1.2), anti-mouse IL-6 receptor (15A7), anti-mouse TGF-β (1D11.16.8), or 0.5 mg of purified rat anti-mouse CD4 (GK1.5), anti-mouse CD8 (2.43), or the corresponding rat IgG isotype control Abs were injected i.p. 1 day before Lm infection. For in vitro culture, splenocytes were plated into 96-well round-bottom plates (5 x 106 cells/ml) and stimulated with the indicated peptides (10–6 M) for 5 h (intracellular cytokine staining) or 72 h (culture supernatants) as described elsewhere (17). For intracellular cytokine staining, brefeldin A (BD GolgiPlug reagent) was added to cell cultures before peptide stimulation. For some experiments, the CD8 T cell response to OVA257–264 was examined with H-2Kb dimerX loaded with OVA257–264 peptide according to the manufacturer’s instructions (BD Biosciences). The concentration of IFN-{gamma} and IL-17 in splenocyte culture supernatants was quantified by ELISA using reagents from R&D Systems.

Statistics

The differences in percentages and numbers of cytokine-producing cells, cytokine concentrations in culture supernatants, and geometric mean numbers of recoverable CFUs between groups of mice were evaluated by using the Student t test with p < 0.05 taken as statistically significant (GraphPad Prism software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lm primes a low magnitude Th17-dominated response in the absence of both IL-12 and IFN-IR

After primary Lm infection, IL-12p40 and type I IFN signaling each play nonessential roles in priming naive CD4 T cells for IFN-{gamma} production. However, combined defects in both IL-12p40 and type I IFN signaling completely abrogate IFN-{gamma} production and Th1 differentiation by Ag-specific CD4 T cells (17). For the optimal study of the adaptive T cell response using mice with targeted defects in immune response pathways known to play either protective or detrimental roles in innate susceptibility to WT Lm infection, we used infection with attenuated Lm strains with targeted deficiency in actA to prime adaptive T cell immunity and normalize Ag load after infection. This defect eliminates the actin-recruitment protein in Lm, thereby preventing the bacterium from establishing a productive infection by preventing spread from an infected cell into neighboring cells. We previously demonstrated that infection of control mice or mice with targeted defects in IL-12, IFN-IR, or both IL-12 and IFN-IR with the same inoculum of Lm {Delta}actA results in comparable numbers of recoverable bacteria (representative of Ag load) in the first 24 h after infection with similar kinetics of bacterial clearance within the first week after infection, whereas this is not the case with WT Lm (17, 19, 20, 21, 22, 23). Accordingly, Lm {Delta}actA was used to further investigate the individual and combined roles of IL-12p40 and IFN-IR in T cell differentiation after in vivo infection.

Splenocytes from B6, IFN-IR–/–, P40–/–, and P40–/–IFN-IR–/– mice were harvested and stimulated with the Lm-specific MHC class II-restricted peptide, LLO189–201, and the Ag-specific response was quantified beginning 8 days after infection (17). At this time point after infection with Lm {Delta}actA, no recoverable Lm CFUs could be detected in the organs of any mice. Although only background levels of IL-17-producing cells were detected in B6, P40–/–, and IFN-IR–/– mice, Lm-specific IL-17-producing CD4 T cells were readily detectable in P40–/–IFN-IR–/– mice (Fig. 1, A and B). Consistent with our previous studies, there was a drastic reduction in the numbers of IFN-{gamma}-producing CD4 T cells in P40–/–IFN-IR–/– mice compared with B6, P40–/–, or IFN-IR–/– mice (17). Despite the relatively low frequency and numbers of IL-17- producing CD4 cells observed in response to LLO189–201 peptide stimulation in splenocytes from P40–/–IFN-IR–/– mice compared with the frequency of IFN-{gamma}-producing cells from control mice, there was a highly significant increased frequency of IL-17-producing CD4 T cells in P40–/–IFN-IR–/– compared with B6 (10-fold increase, p < 0.001), P40–/–, or IFN-IR–/– mice (4-fold increases, p < 0.005). The concentrations of IL-17 and IFN-{gamma} in splenocyte culture supernatants after in vitro peptide stimulation corroborated this apparent shift from a Th1-dominated CD4 T cell response in B6 mice to a Th17-dominated CD4 T cell response in P40–/–IFN-IR–/– mice (Fig. 1C). Taken together, these data indicate that in the absence of both IL-12p40 and IFN-IR, the normally robust Th1 Ag-specific CD4 T cell response is virtually abolished and replaced by a Th17-dominated response of relatively low magnitude.


Figure 1
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  		FIGURE 1. Th17 response in the absence of both IL-12 and IFN-IR. A, FACS plot indicating IL-17 and IFN-{gamma} production in a CD4+ cell gate among splenocytes from mice day 8 after infection with 106 Lm-OVA {Delta}actA and in vitro stimulation with the Lm-specific MHC class II peptide LLO189–201 or no peptide control. B, Total numbers of IL-17- or IFN-{gamma}-producing, LLO189–201-specific CD4 T cells per mouse spleen as quantified by intracellular cytokine staining. C, Concentration of IL-17 and IFN-{gamma} in splenocyte culture supernatants 72 h after stimulation with the LLO189–201 peptide or no peptide. These data represent 6–10 mice for each experimental group combined from three independent experiments. Bar, SE. *, p < 0.05.

 
IFN-{gamma} and IFN-I inhibit Th17 differentiation in response to infection in vivo

IFN-{gamma} strongly inhibits Th17 differentiation in vitro (24, 25). Since infection with either Lm or Lm {Delta}actA triggers the production of IFN-{gamma} by NK1.1+ cells in the first 24 h after infection, the IFN-{gamma} these cells produce might be a key factor that inhibits CD4 Th17 differentiation while facilitating Th1 differentiation in WT mice (26, 27). Moreover, because both IL-12 and IFN-I can induce IFN-{gamma} production by NK cells (28, 29), we reasoned that a loss of early IFN-{gamma} production might contribute to the observed deviation from a Th1 to a Th17 response in mice with defects in these cytokine pathways. To address this question, we first assessed production of IFN-{gamma} by NK1.1+ cells 24 h after infection with Lm. Similar to control B6 mice, NK1.1+ cells were the predominant cell type that produced IFN-{gamma} after Lm infection in P40–/– or IFN-IR–/– mice (data not shown). Compared with B6 controls, P40–/– mice had a drastic reduction in the percentage of NK1.1+ cells that produced IFN-{gamma} (Fig. 2A). Whereas no defect was apparent in IFN-IR–/– mice, IFN-{gamma}-producing NK1.1+ cells were essentially absent in P40–/– IFN-IR–/– mice. These findings demonstrate a previously uncharacterized synergistic role for IFN-I and IL-12P40 in priming NK1.1+ cells for IFN-{gamma} production, which in turn suppresses the development of IL-17-producing cells and enhances the generation of IFN-{gamma}-producing Th1 cells.


Figure 2
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  		FIGURE 2. IFN-{gamma} or IFN-I inhibits Th17 differentiation. A, Histogram plot indicating the percentage of NK1.1+ cells that produce IFN-{gamma} 24 h after infection with 106 Lm-OVA {Delta}actA (open histograms) or in the absence of infection (filled histograms) for the indicated groups of mice. B, Total number of IL-17-producing CD4 T cells after stimulation with LLO189–201 peptide on day 8 after infection with 106 Lm-OVA {Delta}actA in B6 or IFN-IR–/– mice pretreated with anti-IFN-{gamma} or isotype control Ab. C, Total numbers of IL-17-producing CD4 T cells after stimulation with LLO189–201 peptide on day 8 after infection with 106 Lm-OVA {Delta}actA as quantified by intracellular cytokine staining in P40–/–IFN-IR–/– (Figure 2) or B6 ({square}) mice pretreated with either rat IgG control (isotype), anti-TGF-β, anti-IL-6R, or both anti-TGF-β and anti-IL-6R Abs. These data represent six to eight mice for each experimental group combined from two independent experiments. Bar, SE. *, p < 0.05.

 
Because CD4 T cells from IFN-{gamma} receptor-deficient mice have no obvious defects in Ag-specific IFN-{gamma}-production and Th1 differentiation (30), the absence of IFN-{gamma} alone is not likely to account for the shift from a Th1 to a Th17 response in P40–/–IFN-IR–/– mice. To test the possibility that a combined deficiency in both IFN-{gamma} and IFN-IR accounts for this shift, we examined CD4 T cell differentiation in IFN-IR–/– and control B6 mice treated with anti-IFN-{gamma} or isotype control Ab 1 day before infection with Lm-OVA {Delta}actA. Treatment of IFN-IR–/– mice but not B6 mice with anti-IFN-{gamma} led to the generation of IL-17-producing cells, whereas few to no such cells were detected in IFN-IR–/– treated with the isotype control Ab (Fig. 2B). Taken together, these results demonstrate that Th17 CD4 T cell differentiation during Lm infection requires the abrogation of both IFN-{gamma} and IFN-IR.

TGF-β and IL-6 are required for Th17 differentiation in P40–/–IFN-IR–/– mice

Whereas IFN-{gamma} inhibits Th17 differentiation, both TGF-β and IL-6 are required for Th17 differentiation in vitro (3, 31). To determine whether these two cytokines are also required for Th17 differentiation in response to infection in vivo, we treated P40–/–IFN-IR–/– and control B6 mice with Abs to TGF-β, the IL-6 receptor, or both or with isotype control Abs before infection with Lm-OVA {Delta}actA. In P40–/–IFN-IR–/– mice, the IL-6R-blocking Ab alone diminished the IL-17 response by ~50% (p < 0.05 compared with isotype control) and depletion of TGF-β led to an even more dramatic reduction (~70%, p < 0.05 compared with isotype control) while depletion of both abolished this response (Fig. 2C). These results indicate that during Lm infection TGF-β and IL-6 synergize for optimal Th17 CD4 T cell differentiation when inhibitory signals from IL-12p40 and IFN-IR are eliminated.

Dynamics of Ag-specific Th17 compared with Th1 CD4 T cells

To determine how the shift from a relatively high magnitude Th1 response to a relatively low magnitude Th17 response alters the dynamics of Ag-specific CD4 T cells, we compared the percentage and numbers of CD4 T cells that produce either IL-17 or IFN-{gamma} in response to in vitro peptide restimulation 8, 14, and 32 days after primary infection with Lm-OVA {Delta}actA and 3 days after secondary infection with Lm-OVA (Fig. 3). Remarkably, despite an apparent ~90% reduction in the overall magnitude of the total CD4 T cell response, the kinetics of expansion, contraction, and re-expansion after secondary infection by IL-17-producing CD4 T cells in P40–/– IFN-IR–/– mice was virtually identical to that observed for IFN-{gamma}-producing CD4 T cells in B6 mice. Moreover, the shift from a Th1-dominated response in B6 mice to a Th17-dominated response in P40–/–IFN-IR–/– mice observed at day 8 was maintained at all time points examined after primary infection and after secondary Ag challenge.


Figure 3
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  		FIGURE 3. Total number (A and B) and percentage (C and D) of IL-17 (A and C) and IFN-{gamma} (B and D)-producing CD4 T cells per spleen after stimulation with the MHC class II peptide LLO189–201 on days 8, 14, or 32 after primary infection with 106 Lm-OVA {Delta}actA (left panels), or on day 3 after challenge with 105 Lm-OVA in mice inoculated with Lm-OVA {Delta}actA 32 days before challenge (right panels). These represent 8–12 mice per time point combined from three independent experiments. Bar, SE.

 
Ag-specific CD8 T cells primed in the absence of IL-12p40 and IFN-IR

Our previous studies indicate that IL-12p40 and IFN-IR together are not required for priming Ag-specific CD8 T cells since mice with combined defects in both IL-12P40 and IFN-IR compared with control mice have similar numbers of Ag-specific CD8 T cells at the peak of the immune response after primary Lm infection (17). However, since CD4 T cells are required to sustain CD8 T cells into the memory phenotype, we sought to examine how replacing a normally robust Ag-specific Th1 CD4 response with a relatively low magnitude Th17 response in P40–/–IFN-IR–/– mice would influence the dynamics of Ag-specific CD8 T cells. Measuring IFN-{gamma} production after in vitro restimulation with the MHC class I OVA257–264 peptide by CD8 T cells from both P40–/– IFN-IR–/– and B6 control mice revealed that both the total number and percentage of Ag-specific CD8 T cells decreased from peak levels by day 14 and were maintained to day 32 after primary infection with Lm-OVA {Delta}actA (Fig. 4, A and B). In response to rechallenge with Lm-OVA day 32 after primary infection, the numbers and percentage of IFN-{gamma}-producing CD8 T cells readily re-expanded to the same degree in both P40–/–IFN-IR–/– and B6 mice (Fig. 4, A and B). Importantly, although IFN-{gamma} was readily produced by CD8 T cells from P40–/–IFN-IR–/– and control mice, these cells in response to Ag restimulation did not produce levels of IL-17 above background during any of these time points (Fig. 4C and data not shown). Therefore, the number and percentage of Ag-specific CD8 T cells in P40–/–IFN-IR–/– and B6 mice was similar, but in contrast to the CD4 T cells, CD8 T cells from P40–/– IFN-IR–/– mice produced IFN-{gamma} but not IL-17.


Figure 4
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  		FIGURE 4. Total number (A) and percentage (B) of IFN-{gamma}-producing CD8 T cells per spleen after stimulation with the MHC class I peptide OVA257–264 on days 8, 14, or 32 after primary infection with 106 Lm-OVA {Delta}actA (left panels), or on day 3 after challenge with 105 Lm-OVA in mice inoculated with Lm-OVA {Delta}actA 32 days before challenge (right panels). C, FACS plot indicating IL-17 and IFN-{gamma} production in a CD8+ cell gate among splenocytes from mice day 8 after infection with 106 Lm-OVA {Delta}actA after in vitro stimulation with the MHC class I peptide OVA257–264 or no peptide control. These data represent 9–12 mice per time point combined from three independent experiments. Bar, SE.

 
Of note, the rate of contraction after day 8 by IFN-{gamma}- producing CD8 T cells was dampened in P40–/–IFN-IR–/– compared with B6 mice resulting in a modest (~50%) increase in both the numbers and percentage of Ag-specific CD8 T cells on days 14 and 32, despite an ~25% reduction in numbers and percentage on day 8. Accordingly, in additional experiments, we sought to further characterize the phenotype and activation status of the Ag-specific CD8 T cells in P40–/–IFN-IR–/– compared with control mice. For these experiments, Ag-specific CD8 T cells were identified among total splenocytes by staining with H-2Kb dimer loaded with OVA257–264 peptide. Consistent with results obtained by quantifying the CD8 T cell response after in vitro peptide restimulation, the Ag-specific CD8 T cell response quantified by OVA257–264 dimer staining was similar in P40–/–IFN-IR–/– mice compared with P40–/–, IFN-IR–/–, or B6 control mice and was found to peak on day 8, contract by day 14 after primary infection, and readily re-expand following Ag rechallenge (Fig. 5A). Phenotypic characterization using the surface markers CD44 and L-selectin (CD62L) revealed no significant difference among Ag-specific CD8 T cells primed in P40–/–IFN-IR–/– mice compared with control mice (Fig. 5, B and C). By day 8 and extending through day 32 after primary infection and day 3 after rechallenge, the majority of Ag-specific CD8 T cells in B6, IFN-IR–/–, P40–/–, or P40–/–IFN-IR–/– mice are effectors (CD44highCD62Llow) (32). Interestingly, beginning day 14 and through day 32 after primary infection, Ag-specific T cells in all groups of mice up-regulated CD127 (IL-7R {alpha}-chain) expression (Fig. 5D). However, at each time point, the level of CD127 expression was modestly, but consistently higher for Ag-specific T cells in P40–/–IFN-IR–/– mice compared with each control group, further indicating differentiation into functional long-lived memory cells (33).


Figure 5
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  		FIGURE 5. A, Percentage of Ag-specific CD8 T cells among splenocytes after primary infection with 106 Lm-OVA {Delta}actA (left panel) or on day 3 after challenge with 105 Lm-OVA in mice inoculated with Lm-OVA {Delta}actA 32 days before challenge (right panel) as determined by staining with OVA257–264 dimer. Bar, SE. These data are from six mice per time point per group from two independent experiments. Expression of CD44 (B), CD62L (C), and CD127 (D) among OVA257–264 dimer-positive Ag-specific (open histograms) compared with non-Ag specific (filled histograms) CD8 T cells at the indicated time points after primary infection with 106 Lm-OVA {Delta}actA (days 8, 14, and 32) and secondary challenge with 105 Lm-OVA (day 3). The mean fluorescent intensity for CD127 expression among dimer-positive Ag-specific T cells is indicated.

 
Protective immunity to Lm challenge during a Th17-dominated CD4 T cell response

To determine whether replacement of a robust Th1-dominated to a relatively low magnitude Th17-dominated CD4 T cell response compromised protective immunity to Lm infection, B6, P40–/–, IFN-IR–/–, and P40–/–IFN-IR–/– mice were primed with Lm-OVA {Delta}actA, challenged on day 32 with 105 CFUs (1 LD50) of Lm-OVA, and CFUs were enumerated 3 days later (Fig. 6A). Compared with naive B6 mice, all groups of mice that had been primed with Lm-OVA {Delta}actA were highly protected and, for the majority of mice in each group, CFUs were below the limits of detection in both the liver and spleen. Because CD8 T cells provide the predominant source of protective adaptive immunity to Lm infection in WT mice, we hypothesized that the relatively normal CD8 T cell response present in P40–/–IFN-IR–/– mice may also mediate protection. Alternatively, the CD8 T cells generated in such an environment might be less "fit" and perhaps under these circumstances Th17 CD4 T cells could play a larger role in protective immunity. To examine these possibilities, P40–/–IFN-IR–/– and B6 mice that had been previously primed with Lm-OVA {Delta}actA were depleted of CD4 or CD8 T cells or treated with an isotype control Ab before challenge with Lm-OVA. In both P40–/–IFN-IR–/– and B6 mice, depletion of CD8 T cells significantly reduced protective immunity, whereas depletion of CD4 T cells did not (Fig. 6B). These results indicate that CD8 T cells play an important role in protective adaptive immunity in P40–/–IFN-IR–/– mice and that replacement of a Th1-dominated response present in normal mice to a Th17-dominated response of relatively low magnitude does not impair the ability of CD8 T cells to confer protection to Lm infection.


Figure 6
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  		FIGURE 6. Protective immunity in the absence of both IL-12 and IFN-I. A, Numbers of recoverable Lm CFUs in the liver and spleen day 3 after challenge with 105 Lm-OVA in either naive B6 mice ({blacksquare}) or the indicated mice that had been primed with 106 Lm-OVA {Delta}actA 32 days before challenge. B, Numbers of recoverable Lm CFUs in the liver and spleen day 3 after challenge with 105 Lm-OVA in either B6 (black bar) or P40–/–IFN-IR–/– (Figure 6) mice not previously infected with Lm (naive) or infected with 106 Lm-OVA {Delta}actA 32 days before challenge and treated with anti-CD8, anti-CD4, or isotype control Ab 1 day before challenge. These data represent 8–10 mice for each experimental group combined from two independent experiments. Bar, SE. *, p < 0.05; n.s., not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Since the conditions that promote Th1 differentiation of CD4 T cells (IL-12 and type I IFNs) also prime CD8 T cells to differentiate into Ag-specific effectors, immunity to many intracellular pathogens that prime a Th1-dominated CD4 T cell response can be mediated by the concerted actions of these two effector cell types or be dominated by one or the other (17, 34, 35). In Lm infection, effector CD8 T cells play the dominant role in protection (13), and here we used Lm infection to examine the requirement for a Th1-dominated CD4 T cell response to generate protective Ag-specific CD8 T cells. Our results demonstrate that although the Ag-specific Th1 response in P40–/–IFN-IR–/– mice following Lm infection is virtually abolished, the protective capacity of the Ag-specific CD8 T cell response remains intact. Thus, activation of CD8 T cells capable of conferring protective immunity to an intracellular pathogen requires neither IL-12, type I IFN signaling, nor help from a Th1-dominated CD4 T cell response.

The newly described Th17 lineage of CD4 T cells is believed to fill a void in the functional repertoire of adaptive T cells by providing immunity to extracellular bacterial and fungal pathogens not covered by traditional Th1 or Th2 CD4 T cells. Although some important parameters that drive Th17 differentiation of CD4 T cells after activation have been established in vitro, little is known regarding the development of these cells in response to infection in vivo. Our results demonstrate that in the absence of both IL-12p40 and IFN-IR signaling, the Th1-dominated response normally primed by Lm infection is replaced with a Th17-dominated response of lower magnitude. The generation and expansion of IL-17-producing, Ag-specific CD4 T cells required both the absence of IFN-{gamma} (achieved either using neutralizing Abs to IFN-{gamma} or by the absence of IL-12p40) and IFN-IR signaling and the presence of IL-6 and TGF-β. Thus, the generation of Ag-specific Th17 cells in response to infection in vivo required the same cytokine milieu as others have described after T cell activation in vitro (3, 12, 24, 25). Our findings are also consistent with those demonstrating an in vivo role for TGF-β in the generation of Th17 cells in the gut and in experimental autoimmune encephalomyelitis (36). We extend those findings by showing that both TGF-β and IL-6 are required to generate Ag-specific Th17 cells after in vivo Lm infection and demonstrate that the kinetics with which Ag-specific Th17 and Th1 CD4 T cells expand and contract after primary infection and re-expand after secondary infection are similar. Lastly, our findings that Ag-specific CD8 T cells are primed and expand after primary Lm infection, re-expand, and confer protective immunity to Lm rechallenge in the absence of both IL-12p40 and IFN-IR indicate that other "signal 3" molecules for CD8 T cell are triggered during Lm infection. Of interest in this regard is IL-21. A recent study has demonstrated that IL-21 provided with microsphere beads coated with MHC+ peptide (signal 1) and B7-1 for costimulation (signal 2) can, along with IL-12 and type I-IFN, provide the needed third for activation of naive CD8 T cells (37). Moreover, for naive CD4 T cells, IL-21 can initiate alternative IL-6- and IL-23-independent, ROR-{gamma}t-dependent pathways to induce Th17 CD4 T cell differentiation (31, 38, 39). Determining whether IL-21 or an as yet undescribed cytokine(s) can support the differentiation and maintenance of protective CD8 T cells in response to infection in vivo is an important area for future investigation.


   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 This work was supported by National Institute of Child Health and Human Development/National Institutes of Health Grant K08HD51584, Infectious Disease Society of America Young Investigator Award, Puget Sound Partners for Global Health, and March of Dimes Basil O’Conner Research Award. Back

2 Address correspondence and reprint requests to Dr. Sing Sing Way, Departments of Pediatrics and Microbiology, University of Minnesota School of Medicine, Center for Infectious Diseases and Microbiology Translational Research, 2001 6th Street Southeast, Room 3-212, Minneapolis, MN 55455. E-mail address: singsing{at}umn.edu Back

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

Received for publication October 31, 2007. Accepted for publication January 4, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bettelli, E., M. Oukka, V. K. Kuchroo. 2007. TH-17 cells in the circle of immunity and autoimmunity. Nat. Immunol. 8: 345-350. [Medline]
  2. Weaver, C. T., R. D. Hatton, P. R. Mangan, L. E. Harrington. 2007. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu. Rev. Immunol. 25: 821-852. [Medline]
  3. Mangan, P. R., L. E. Harrington, D. B. O’Quinn, W. S. Helms, D. C. Bullard, C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, C. T. Weaver. 2006. Transforming growth factor-β induces development of the TH17 lineage. Nature 441: 231-234. [Medline]
  4. Ye, P., F. H. Rodriguez, S. Kanaly, K. L. Stocking, J. Schurr, P. Schwarzenberger, P. Oliver, W. Huang, P. Zhang, J. Zhang, et al 2001. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J. Exp. Med. 194: 519-527. [Abstract/Free Full Text]
  5. Happel, K. I., M. Zheng, E. Young, L. J. Quinton, E. Lockhart, A. J. Ramsay, J. E. Shellito, J. R. Schurr, G. J. Bagby, S. Nelson, J. K. Kolls. 2003. Cutting edge: roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection. J. Immunol. 170: 4432-4436. [Abstract/Free Full Text]
  6. Huang, W., L. Na, P. L. Fidel, P. Schwarzenberger. 2004. Requirement of interleukin-17A for systemic anti-Candida albicans host defense in mice. J. Infect. Dis. 190: 624-631. [Medline]
  7. Kikly, K., L. Liu, S. Na, J. D. Sedgwick. 2006. The IL-23/Th17 axis: therapeutic targets for autoimmune inflammation. Curr. Opin. Immunol. 18: 670-675. [Medline]
  8. Gutcher, I., B. Becher. 2007. APC-derived cytokines and T cell polarization in autoimmune inflammation. J. Clin. Invest. 117: 1119-1127. [Medline]
  9. Szabo, S. J., B. M. Sullivan, S. L. Peng, L. H. Glimcher. 2003. Molecular mechanisms regulating Th1 immune responses. Annu. Rev. Immunol. 21: 713-758. [Medline]
  10. Ansel, K. M., I. Djuretic, B. Tanasa, A. Rao. 2006. Regulation of Th2 differentiation and Il4 locus accessibility. Annu. Rev. Immunol. 24: 607-656. [Medline]
  11. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235-238. [Medline]
  12. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, B. Stockinger. 2006. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24: 179-189. [Medline]
  13. Pamer, E. G.. 2004. Immune responses to Listeria monocytogenes. Nat. Rev. Immunol. 4: 812-823. [Medline]
  14. Harty, J. T., M. J. Bevan. 1992. CD8+ T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo. J. Exp. Med. 175: 1531-1538. [Abstract/Free Full Text]
  15. Shedlock, D. J., H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337-339. [Abstract/Free Full Text]
  16. Sun, J. C., M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300: 339-342. [Abstract/Free Full Text]
  17. Way, S. S., C. Havenar-Daughton, G. A. Kolumam, N. N. Orgun, K. Murali-Krishna. 2007. 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. J. Immunol. 178: 4498-4505. [Abstract/Free Full Text]
  18. Foulds, K. E., L. A. Zenewicz, D. J. Shedlock, J. Jiang, A. E. Troy, H. Shen. 2002. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J. Immunol. 168: 1528-1532. [Abstract/Free Full Text]
  19. Tripp, C. S., M. K. Gately, J. Hakimi, P. Ling, E. R. Unanue. 1994. Neutralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice: reversal by IFN-{gamma}. J. Immunol. 152: 1883-1887. [Abstract]
  20. Brombacher, F., A. Dorfmuller, J. Magram, W. J. Dai, G. Kohler, A. Wunderlin, K. Palmer-Lehmann, M. K. Gately, G. Alber. 1999. IL-12 is dispensable for innate and adaptive immunity against low doses of Listeria monocytogenes. Int. Immunol. 11: 325-332. [Abstract/Free Full Text]
  21. Carrero, J. A., B. Calderon, E. R. Unanue. 2004. Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. J. Exp. Med. 200: 535-540. [Abstract/Free Full Text]
  22. Auerbuch, V., D. G. Brockstedt, N. Meyer-Morse, M. O’Riordan, D. A. Portnoy. 2004. Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes. J. Exp. Med. 200: 527-533. [Abstract/Free Full Text]
  23. O’Connell, R. M., S. K. Saha, S. A. Vaidya, K. W. Bruhn, G. A. Miranda, B. Zarnegar, A. K. Perry, B. O. Nguyen, T. F. Lane, T. Taniguchi, et al 2004. Type I interferon production enhances susceptibility to Listeria monocytogenes infection. J. Exp. Med. 200: 437-445. [Abstract/Free Full Text]
  24. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, C. Dong. 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6: 1133-1141. [Medline]
  25. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, K. M. Murphy, C. T. Weaver. 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6: 1123-1132. [Medline]
  26. Chang, S. R., K. J. Wang, Y. F. Lu, L. J. Yang, W. J. Chen, Y. H. Lin, H. H. Chang, S. L. Wang. 2007. Characterization of early {gamma} interferon (IFN-{gamma}) expression during murine listeriosis: identification of NK1.1+CD11c+ cells as the primary IFN-{gamma}-expressing cells. Infect. Immun. 75: 1167-1176. [Abstract/Free Full Text]
  27. Way, S. S., C. B. Wilson. 2004. Cutting edge: immunity and IFN-{gamma} production during Listeria monocytogenes infection in the absence of T-bet. J. Immunol. 173: 5918-5922. [Abstract/Free Full Text]
  28. Newman, K. C., E. M. Riley. 2007. Whatever turns you on: accessory-cell-dependent activation of NK cells by pathogens. Nat. Rev. Immunol. 7: 279-291. [Medline]
  29. Lieberman, L. A., C. A. Hunter. 2002. Regulatory pathways involved in the infection-induced production of IFN-{gamma} by NK cells. Microbes Infect. 4: 1531-1538. [Medline]
  30. Haring, J. S., J. T. Harty. 2006. Aberrant contraction of antigen-specific CD4 T cells after infection in the absence of {gamma} interferon or its receptor. Infect. Immun. 74: 6252-6263. [Abstract/Free Full Text]
  31. Zhou, L., I. I. Ivanov, R. Spolski, R. Min, K. Shenderov, T. Egawa, D. E. Levy, W. J. Leonard, D. R. Littman. 2007. IL-6 programs TH-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat. Immunol. 8: 967-974. [Medline]
  32. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225-234. [Medline]
  33. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  34. Shen, H., M. K. Slifka, M. Matloubian, E. R. Jensen, R. Ahmed, J. F. Miller. 1995. Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity. Proc. Natl. Acad. Sci. USA 92: 3987-3991. [Abstract/Free Full Text]
  35. Curtsinger, J. M., C. S. Schmidt, A. Mondino, D. C. Lins, R. M. Kedl, M. K. Jenkins, M. F. Mescher. 1999. Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells. J. Immunol. 162: 3256-3262. [Abstract/Free Full Text]
  36. Li, M. O., Y. Y. Wan, R. A. Flavell. 2007. T cell-produced transforming growth factor-β1 controls T cell tolerance and regulates Th1- and Th17-cell differentiation. Immunity 26: 579-591. [Medline]
  37. Casey, K. A., M. F. Mescher. 2007. IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype. J. Immunol. 178: 7640-7648. [Abstract/Free Full Text]
  38. Nurieva, R., X. O. Yang, G. Martinez, Y. Zhang, A. D. Panopoulos, L. Ma, K. Schluns, Q. Tian, S. S. Watowich, A. M. Jetten, C. Dong. 2007. Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature 448: 480-483. [Medline]
  39. Korn, T., E. Bettelli, W. Gao, A. Awasthi, A. Jager, T. B. Strom, M. Oukka, V. K. Kuchroo. 2007. IL-21 initiates an alternative pathway to induce proinflammatory TH17 cells. Nature 448: 484-487. [Medline]



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