IL-23 Promotes the Production of IL-17 by Antigen-Specific CD8 T Cells in the Absence of IL-12 and Type-I Interferons

Meredith M. Curtis, Sing Sing Way and Christopher B. Wilson
J Immunol July 1, 2009, 183 (1) 381-387; DOI: https://doi.org/10.4049/jimmunol.0900939

Abstract

In contrast to CD4 T cells, CD8 T cells inherently differentiate into IFN-γ-producing effectors. Accordingly, while generation of IFN-γ-producing Th1 CD4 T cells was profoundly impaired in mice deficient for both type-I IFN and IL-12 signaling in response to infection with Listeria monocytogenes, generation of Ag-specific, IFN-γ-producing CD8 T cells was unimpaired. However, a fraction of these CD8 T cells also produced IL-17 in an IL-23-dependent manner. Furthermore, the addition of IL-23 in vitro was sufficient for some naive CD8 T cells to differentiate into IFN-γ/IL-17 dual-producing cells and was associated with increased expression of ROR-γt and ROR-α. Addition of IL-6 and TGF-β to IL-23 further augmented ROR-γt and ROR-α expression and suppressed Eomes expression, thereby enhancing IL-17 production by CD8 T cells. A loss of cytotoxic function accompanied the production of IL-17, as the addition of IL-6 and TGF-β resulted in a marked reduction of granzyme B and perforin expression. Thus, CD8 T cells retain sufficient plasticity to respond to environmental cues and can acquire additional effector functions in response to their environmental context.

The adaptive immune system provides protection against a broad and diverse array of potential pathogens by activating an Ag-specific response tailored against the invading pathogen. In response to infection with each specific pathogen, the immune response is intricately regulated, allowing for fine-tuning of the pathogen-specific response that balances efficient pathogen eradication with minimal collateral damage to host tissues. Cytokines produced by APCs play important roles in fine-tuning the differentiation program of pathogen-specific T cells (1). For example, during Listeria monocytogenes (Lm)3 infection, the cytokines IL-12 and type-I IFNs each can prime naive CD4 T cells to differentiate into IFN-γ-producing Th1 cells, while the absence of both of these cytokines permits Th17 CD4 T cell differentiation provided that IL-6 and TGF-β are present (2, 3). Interestingly, while the presence and/or absence of specific cytokines readily alters the differentiation program for Ag-specific CD4 T cells induced in response to Listeria infection, Ag-specific CD8 T cells maintain IFN-γ production even under conditions where IFN-γ production is repressed in CD4 T cells.

Recently, however, additional plasticity in the differentiation program for pathogen-specific CD8 T cells has been demonstrated. The T-box transcription factors T-bet and Eomes were shown to limit CD8 T cell plasticity in a collaborative manner, because in their absence, infection with LCMV induced the development of virus-specific CD8 T cells that produced IL-17 and had impaired IFN-γ production and cytotoxicity (4). These studies illuminate CD8 T cell intrinsic pathways that actively suppress IL-17 production to promote an IFN-γ-producing cytotoxic effector program. Additionally, CD8 T cells that produce IL-17 have been observed in situ in humans with psoriasis (5) and multiple sclerosis (6), and in animal models of these and other disorders (7, 8), as well as in healthy adults (9), but the external signals that fostered the development of these IL-17-producing CD8 T cells are incompletely understood. In vitro, IL-21, a cytokine produced by Th17 and T follicular helper cells, can prime naive CD8 T cells to repress production of IFN-γ while maintaining cytotoxicity (10), and CD8 T cells cultured in cytokine conditions that allow naive CD4 T cells to differentiate into Th17 effectors, e.g., IL-6 plus TGF-β, can produce substantial amounts of IL-17 and repress IFN-γ production (11, 12). Additionally, IL-23 or IL-23 plus IL-1 promote IL-17 production by total CD8 T cells in vitro (7, 13). In this study, we describe the cell extrinsic factors regulating the differentiation of Ag-specific CD8 T cells into an IL-17/IFN-γ-dual-producing population and demonstrate how the balance of cell intrinsic factors, such as T-bet, Eomes, and RORγt, is altered to achieve this shift in function.

Materials and Methods

Mice

C57BL/6 mice, IL12Rβ2-deficient and IL-12/23p40-deficient mice backcrossed 10 or more times to B6 were obtained from The Jackson Laboratory and bred in house. P14 TCR-transgenic (Tg) mice specific for the LCMV epitope GP33–41 and congenic for Thy1.1 and type-I IFN receptor-deficient (IFNAR−/−) mice were each provided by Dr. Murali-Krishna Kaja (University of Washington, Seattle, WA). IFNAR−/− × IL12Rβ2−/− and IFNAR−/− × IL12/23p40−/− mice were generated by intercrossing. All 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

For infection, 106 colony forming units (CFU) of the previously described Lm-OVAΔactA strain (2, 14) was grown to early log phase (OD600 0.1) in brain heart infusion medium (BD Biosciences) at 37°C, washed, and diluted in 200 μl final volume and injected i.v.

Reagents, in vitro cultures, and cell staining

For in vivo depletion, 1.0 mg of purified anti-mouse IFN-γ (clone XMG1.2) or IgG1 isotype control (Bio-X-Cell) was injected i.p. into mice 1 day before infection. The peptides OVA257–264: SIINFEKL and GP33–41: KAVYNFATC were each purchased from United Biochemical Research. To assess endogenous CD8 T cell responses to Lm-OVAΔactA, splenocytes were harvested on day 7 and stimulated with SIINFEKL peptide (1 μM) for 4.5 h in the presence of Brefeldin A for intracellular cytokine staining or for 72 h for assessment of culture supernatants as described (2). For in vitro polarization cultures, IL12/23p40-deficient APCs (CD4−CD8−NK1.1−CD44low) and naive (CD62LhighCD44low) T cells from P14 Tg mice were purified on a FACS Aria. In brief, 2.5 × 105 T cells and 7.5 × 105 APCs were plated into 96-well round-bottom plates and stimulated with the P14 GP33–41 peptide (300 ng/ml) in Iscove’s modified Dulbecco’s medium supplemented with 10% FBS (v/v), penicillin, streptomycin, gentamicin, and 50 nM 2-ME. Culture conditions, as indicated, included 10 ng/ml rIL-23 (eBioscience), 20 ng/ml rIL-6 (PeproTech), 10 ng/ml rIL-12/23p40 homodimer, 5 ng/ml rhTGF-β1, 5 ng/ml rIL-12, all from R&D Systems; 10 μg/ml anti-mouse/human TGF-β1 (clone 1D11.16.8) and 10 μg/ml anti-mouse IL-6R (clone 15A7), both from Bio-X-Cell. Cells were split on day 2 in fresh complete Iscove’s modified Dulbecco’s medium plus cytokines. Intracellular cytokine staining was performed after 5 days in culture by stimulating cells with phorbol 12-myristate 13-acetate (5 ng/ml) and ionomycin (0.7 μM) for a total of 4.5 h in the presence of Brefeldin A (Golgi Plug, BD Biosciences). Abs for flow cytometry were purchased from either BD Pharmingen or eBioscience, except for anti-human granzyme B-PE that was purchased from Caltag Laboratories. IL-17 and IFN-γ concentration within the harvested supernatants were analyzed using Luminex Beadlyte Mouse MultiCytokine Flex Kit (Upstate Biotechnology) on the Bio-Plex System (Bio-Rad). Alternatively, cultured CD8 T cells (CD8+Thy1.1+) were purified on the FACS Aria, stimulated with PMA/ionomycin for 4.5 h, and total cellular RNA was extracted (Qiagen). SuperScript II RNase H Reverse Transcriptase (Invitrogen) was used to create cDNA and transcript abundance was analyzed by RT-PCR using TaqMan probes (Applied Biosystems) and the following primer sets: Tbx21 (Mm00450960_m1); Eomes (Mm01351985_m1); Rorc (Mm01261019_g1); Rora (Mm00443103_m1); Ifng (Mm00801778_m1); Il17a (Mm00439619_m1); Il21 (Mm00517640_m1); Il22 (Mm00444241_m1); Gzmb (Mm00442834_m1); Prf1 (Mm00812512_m1); Il12rb1 (Mm00434189_m1); Il12rb2 (Mm00434200_m1); Il23r (Mm00519942_m1); eukaryotic 18s ribosomal RNA.

Statistics

Differences in supernatant cytokine concentrations, percentage cytokine producing cells, and relative transcript levels were evaluated by Student’s unpaired t test (Graph Pad, Prism Software).

Results

IFN-γ/IL-17 dual-producing CD8 T cells emerge in response to Lm in the absence of type-I IFN and IL-12 signaling

We previously reported that IL-12 and type-I IFNs collaborate to prime the differentiation of Ag-specific IFN-γ-producing Th1 CD4 T cells after Lm-OVAΔactA infection (3) and that this Th1 response was abolished in IFNAR × IL12/23p40 double-knockout (DKO) mice. Despite this defect, these mice mounted a robust, protective IFN-γ-producing CD8 T cell response comparable to that of wild-type (WT) mice. Because the IL-12p40 subunit is also shared by IL-23, we sought to explore further the Ag-specific T cell response after Lm infection using IL-12 receptor β2-deficient (IL12Rβ2 knockout (KO)) mice, which have a more specific defect. Unexpectedly, we found that while the capacity of CD8 T cells from IFNAR × IL12Rβ2 DKO mice to produce IFN-γ after in vitro Ag restimulation was fully maintained, these cells also secreted substantial amounts of IL-17 (Fig. 1⇓A). Consistent with this observation, intracellular cytokine staining revealed a modest, but consistently reproducible population of IFN-γ/IL-17 dual-producing CD8 T cells in addition to cells producing only IFN-γ in the IFNAR × IL12Rβ2 DKO mice (Fig. 1⇓, B and C). The IFNAR × IL12/23p40 DKO mice had ∼3-fold fewer ΙFN-γ/IL-17 dual-producing CD8 T cells than the IFNAR × IL12Rβ2 DKO mice (0.04% vs 0.12%). No ΙFN-γ/IL-17 dual-producing CD8 T cells were detected in the IFNAR KO and IL-12Rβ2 KO mice using intracellular staining (Fig. 1⇓, B and C), but low amounts of IL-17 were detected in culture supernatants (Fig. 1⇓A). By contrast, IL-17 production by CD8 T cells from IL-12/23p40 KO and WT mice was not detected by either method. These findings indicate that IL-23 and/or p40 homodimers (15) support the generation of Ag-specific, IFN-γ/IL-17 dual-producing CD8 T cells when both type-I IFN and IL-12 signals are absent, and that these specific cytokines play important roles in regulating IL-17 production by CD8 T cells.

FIGURE 1.
FIGURE 1.

IFN-γ/IL-17 dual-producing CD8 T cells emerge in response to Lm in the absence of type-I IFN and IL-12 signaling. A, WT or immunodeficient mice were infected with 106 CFU Lm-OVAΔactA, and splenocytes were harvested at day 7 postinfection. Bar graphs represent the concentration of IFN-γ and IL-17 within the culture supernatants of splenocytes from Lm-infected mice that have been stimulated with OVA257–264 peptide for 72 h. B, FACS plots indicating IFN-γ and IL-17 production by CD8 T cells in WT or immune-deficient mice after in vitro stimulation with OVA257–264. Plots are gated on the total CD8+ population. C, Percent IFN-γ-producing or percent IL-17/IFN-γ dual-producing CD8 T cells in response to Ag stimulation with OVA257–264. The graphs indicate the mean ± SD from three independent experiments with four to five mice/group. Statistics indicate unpaired Student’s t test p values where **, p < 0.01; ***, p < 0.001; ns, not significant.

IFN-γ also inhibits the development of IL-17-producing Lm-specific CD8 T cells

During Lm infection, type-I IFNs and IL-12 synergize to prime the production of IFN-γ by NK cells (3). IFN-γ, in turn, antagonizes the development of CD4 Th17 cells both in vitro and in vivo (3, 16, 17, 18). To determine the contribution of IFN-γ to the development of Ag-specific IL-17-producing CD8 T cells that developed in the absence of type-I IFN and IL-12 signaling, we administered IFN-γ neutralizing Ab or IgG1 isotype control Ab 1 day before Lm infection to WT and IFNAR-deficient mice.

Administration of anti-IFN-γ to IFNAR KO mice resulted in a 5-fold increase compared with the isotype control in Ag-specific IL-17 production (2.4 ng/ml vs 0.5 ng/ml) by CD8 T cells (Fig. 2⇓A). This increase did not come at the expense of Ag-specific IFN-γ production, which was also increased, but rather paralleled the emergence of an IFN-γ/IL-17 dual-producing CD8 T cell population (Fig. 2⇓, B and C); however, this dual-producing population was less robust than in the IFNAR × IL12Rβ2 DKO mice (Fig. 1⇑, B and C). These results, with those shown in Fig. 1⇑, indicate that type-I IFNs, IL-12, and IFN-γ together inhibit the development of IL-17-producing CD8 T cells during Lm infection, and suggest that the increased IL-17 production observed in IFNAR × IL12Rβ2 DKO mice may be due in part to reduced innate immune production of IFN-γ.

FIGURE 2.
FIGURE 2.

IFN-γ and type-I IFNs inhibit development of Lm-specific IL-17-producing CD8 T cells. A, WT or IFNAR-deficient mice were treated with either 1 mg α-IFN-γ or isotype IgG1, infected with 106 CFU Lm-OVAΔactA, and splenocytes were harvested at day 7 postinfection. Bar graphs represent the concentration of IFN-γ and IL-17 within the culture supernatants of splenocytes from Lm-infected mice that have been stimulated with OVA257–264 peptide for 72 h. B, FACS plots indicating the IFN-γ and IL-17 production by CD8 T cells in WT or IFNAR KO mice. Splenocytes were harvested at day 7 postinfection and in vitro stimulated with OVA257–264. Plots are gated on the total CD8+ population. C, Percent IFN-γ-producing or percent IL-17-producing CD8 T cells in response to Ag stimulation with OVA257–264. The graphs indicate the mean ± SD from two independent experiments with two to four mice/group. Statistics indicate unpaired Student’s t test p values where *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

IL-23 enables the development of Ag-specific IFN-γ/IL-17 dual-producing CD8 T cells in vitro

To more precisely evaluate the specific cytokine signals required for the generation of IFN-γ/IL-17 dual-producing CD8 T cells, we isolated naive CD8 and CD4 T cells and stimulated them in vitro with antigenic-peptide presented by APCs from IL-12/23p40 KO mice. These APCs allow the effects of IL-12 and IL-23 on IL-17 and IFN-γ production by T cells to be more precisely addressed.

In the absence of added cytokines, nearly all naive CD8 T cells differentiated into effectors that produced IFN-γ and none produced detectable IL-17 (Fig. 3⇓). When IL-23 alone was added, a small but distinct population of IFN-γ/IL-17 dual-producing CD8 T cells was consistently observed. IL-6 and TGF-β dampened IFN-γ production and induced substantial numbers of IL-17-producing and smaller numbers of IFN-γ/IL-17 dual-producing cells; in these conditions IL-17 production was augmented only slightly by the further addition of IL-23.

FIGURE 3.
FIGURE 3.

IL-23 enables the development of Ag-specific IFN-γ/IL-17 dual-producing CD8 T cells in vitro. A, FACS plots indicating the IFN-γ and IL-17 production by P14 Tg+ CD8 T cells after stimulation with GP33–41 peptide in various polarizing conditions for 5 days. Plots are gated on the CD8+Thy1.1+ P14 population. Experiments were performed four times and representative plots from one experiment are shown. B, Bar graphs representing percent IFN-γ-producing or percent IL-17-producing CD8 T cells in response to Ag stimulation with OVA257–264. The graphs indicate the mean ± SD from four independent experiments. Statistics indicate unpaired Student’s t test p values where *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Consistent with the inhibitory effect of IL-12 on IL-17 production in vivo, addition of IL-12 abolished the induction by IL-23 of IFN-γ/IL-17 dual-producing CD8 T cells and antagonized the inhibitory effects of IL-6 and TGF-β on IFN-γ production (Fig. 3⇑). These findings indicate that in the absence of IL-12, IL-23 supports the differentiation of a fraction of naive CD8 T cells into effectors that produce both IFN-γ and IL-17 in vitro. This effect was specific to the IL-23 p40/p19 heterodimer, because like IL-12, p40 homodimer induced only IFN-γ-producing cells (Fig. 4⇓A). Moreover, increasing the concentration of IL-23 10-fold augmented the percentage of IL-17-producing cells, while a similar increase in p40 homodimer did not (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

IL-23, but not p40 homodimer, induces coproduction of IFN-γ and IL-17 by CD8 T cells in an IL-6/TGF-β-dependent manner. A, FACS plots indicating the IFN-γ and IL-17 production by P14 Tg+ CD8 T cells after stimulation with GP33–41 peptide in various polarizing conditions for 5 days. Plots are gated on the CD8+Thy1.1+ P14 population. B, FACS plots indicating the IFN-γ and IL-17 production by P14 Tg+ CD8 T cells after stimulation with antigenic peptide in the presence of 100 ng/ml IL-23 or p40 homodimers for 5 days. Experiments were performed twice and representative plots from one of two experiments are shown.

IL-23 induces IFN-γ/IL-17 dual-producing CD8 T cells in concert with endogenous IL-6 and TGF-β

The ability of exogenous IL-23 to support the generation of IFN-γ/IL-17 dual-producing CD8 T cells in the absence of IL-12 was surprising. To determine whether IL-23 alone was sufficient or if it was acting in concert with IL-6 and TGF-β produced by the cultured cells, we added blocking Abs to the IL-6R and TGF-β. Addition of these Abs greatly reduced the numbers of IFN-γ/IL-17 dual-producing CD8 T cells in cells cultured in the presence of exogenous IL-23 or in Tc17 (IL-6, TGF-β, IL-23, anti-IL-4, and anti-IFN-γ) conditions (Fig. 4⇑A). These findings indicate that IL-23 is not sufficient to induce IL-17-producing CD8 T cells, but, in the absence of IL-12, can do so in concert with the amounts of IL-6 and TGF-β present in our cultures.

The induction by exogenous IL-23 of IFN-γ/IL-17 dual-producing CD8 T cells, suggested that these conditions were also sufficient to induce IL-23 receptor expression on these cells. To address this prediction, we assessed expression of mRNA encoding the two chains of the IL-23 receptor. Resting naive CD8 T cells did not express these receptor chains (Fig. 5⇓). IL-12Rβ1 mRNA was induced when cells were activated under all conditions evaluated. In contrast, IL-23R expression was not induced by stimulation with Ag alone, but was markedly up-regulated within 48 h of activation in the presence of exogenous IL-23 or in Tc17 conditions.

FIGURE 5.
FIGURE 5.

IL-23 induces the expression of IL-23R on naive CD8 T cells. P14 Tg+ CD8 T cells stimulated with GP33–41 in indicated conditions for either 48 h or 5 days were purified and restimulated with PMA and ionomycin for 4.5 h. RNA was extracted, cDNA was generated, and abundance of IL-12Rβ1 (A) and IL-23R (B) was analyzed by quantitative PCR. All samples are calibrated to the naive CD8 sample and internally normalized based on expression of eukaryotic 18s. Experiments were performed twice and bars represent the mean ± SD.

IL-23 and IL-6 plus TGF-β independently and synergistically induce Rorc and Il17a and dampen Eomes expression

T cell effector differentiation is controlled by transcription factors. The transcription factors T-bet and Eomes collaborate to sustain IFN-γ production and cytotoxic function and to repress IL-17 production by CD8 T cells (4, 19, 20). Both ROR-γt and ROR-α influence the development of CD4 T cells into Th17 cells (21, 22), but their involvement in IL-17 expression by CD8 T cells is less well understood. To explore the role of these transcriptional regulators in the development of IFN-γ/IL-17 dual-producing CD8 T cells, we evaluated their expression under the conditions described above. Expression of Rorc (ROR-γt), Rora (ROR-α), Il17a, and Il21 increased modestly when naive CD8 T cells were activated in the presence of IL-23 alone, more strongly in the presence of IL-6 plus TGF-β, and maximally in Tc17 conditions. Reciprocally, IL-23 alone did not substantially alter Eomes or Ifng expression, whereas both were repressed by IL-6 plus TGF-β and in Tc17 conditions. IL-12 blocked this induction of Rorc and Il17a and up-regulated Ifng expression, whereas expression of Rora (ROR-α) and Il21 were not affected (Fig. 6⇓, A and B). Levels of Tbx21 mRNA, the transcript for T-bet, did not vary substantially under these conditions (data not shown). Under none of the polarizing conditions was Il22 mRNA detected. Thus, expression of Il17a by CD8 T cells correlated directly with Rorc expression and inversely with Eomes expression. Conversely, Ifng expression correlated inversely with the expression of Rorc.

FIGURE 6.
FIGURE 6.

IL-23 and IL-6 plus TGF-β independently and synergistically induce Rorc and Il17 and dampen Eomes expression as naive CD8 T cells differentiate into effectors. A, P14 Tg+ CD8 T cells stimulated with GP33–41 in various polarizing conditions, as described in Fig. 3, were purified and restimulated with PMA and ionomycin for 4.5 h. RNA was extracted, cDNA was generated, and abundance of transcription factors was analyzed by quantitative PCR. All samples are calibrated to the “No Added Cytokines” CD8 sample and internally normalized based on expression of eukaryotic 18s. B, Expression of Ifng, Il17a, and Il21 in P14 Tg+ CD8 T cells stimulated in various polarizing conditions. All samples are calibrated to the “No Added Cytokines” CD8 sample and internally normalized based on expression of eukaryotic 18s. Bars represent the mean ± SD from three independent experiments.

IL-6 and TGF-β but not IL-23 diminish the expression of the cytolytic molecules granzyme B and perforin

A key function of effector CD8 T cells resides in their ability to produce granzyme B and perforin to exert cytotoxic activity on infected cells, the expression of which is up-regulated by T-bet and Eomes (4). Production of IFN-γ and cytotoxic activity in CD8 T cells is not always coordinate, as IL-21 can repress the production of IFN-γ while maintaining cytotoxicity (10). When we assessed these mediators of cytotoxicity, we found that IL-12 enhanced, TGF-β plus IL-6 and Tc17 conditions inhibited, and exogenous IL-23 alone had no effect on granzyme B abundance (Fig. 7⇓A) and granzyme B and perforin mRNA expression (Fig. 7⇓B). Together, these results indicate that IL-6 and TGF-β are sufficient to dramatically reprogram CD8 T cell function by inducing robust IL-17 production and inhibiting their canonical functions of cytotoxicity and IFN-γ production, whereas IL-23 preserved these canonical functions while inducing low-level IL-17 production.

FIGURE 7.
FIGURE 7.

IL-6 and TGF-β diminish CD8 expression of granzyme B and perforin. A, Histogram indicating granzyme B production by P14 Tg+ CD8 T cells after stimulation with GP33–41 peptide in various polarizing conditions for 5 days. Plots are gated on the CD8+Thy1.1+ P14 population. B, Bar graphs representing the mean fluorescence intensity (MFI) of granzyme B in P14 Tg+ CD8 T cells cultured in indicated polarizing conditions. The graphs indicate the mean ± SD from two independent experiments. C, Expression of Gzmb and Prf1 in P14 Tg+ CD8 T cells stimulated in various polarizing conditions. All samples are calibrated to a naive CD8 sample and internally normalized based on expression of eukaryotic 18s. Bars represent the mean ± SD from three independent experiments.

Discussion

Taken together, these findings demonstrate that Ag-specific CD8 effector T cells that produce both IL-17 and IFN-γ can arise in response to infection in vivo. These findings corroborate those of Hamada et al. (12), who identified the emergence of an IFN-γ/IL-17 dual-producing CD8 T cell population in the lungs of influenza-infected mice. During Lm infection, IL-17A produced by γδ T cells helps to promote bacterial clearance and the formation of organized granulomas within the liver (23). Our studies indicate that in the absence of inhibitory signals, IL-17-producing adaptive immune cells can also be generated during Lm infection. We show in this study that the emergence of these IFN-γ/IL-17 dual-producing CD8 T cells is inhibited by IL-12, type-I IFNs and IFN-γ, which instead promote the canonical cytotoxic, IFN-γ-producing CD8 T cell phenotype. Furthermore, we also show that emergence of these IL-17-producing CD8 T cells in vivo was also dependent on IL-23, because it was markedly reduced in IFNAR × 40 DKO mice, which lack IL-23, IL-12, and type-I IFN signaling, while IL-17-producing CD8 T cells were most pronounced in IFNAR × IL-12Rβ2 DKO mice, in which IL-23 signaling is retained. When we tested the ability of p40 homodimers to induce IL-17 production in vitro, we found that they were unable to do so, indicating that IL-23 and not p40 homodimers was required.

IL-6 and TGF-β have been clearly implicated in the induction of CD4 Th17 cells, while IL-23 is believed to be more important in the terminal differentiation and survival of Th17 cells (24, 25). We found that IL-6 and TGF-β were also essential for IL-17 production by CD8 T cells, and previous reports have demonstrated that IL-23 can stimulate the production of IL-17 by memory CD8 T cells (7, 8). However, to our knowledge, this is the first demonstration that IL-23, when type I promoting cytokines are blocked, is sufficient to promote the development of naive CD8 T cells into IFN-γ/IL-17 dual-producers. These effects of IL-23 were dependent on endogenous IL-6 and TGF-β and could be amplified by the further addition of exogenous IL-6 and TGF-β. Weaver and colleagues (26) showed that IL-23 could support IL-17 production by CD4 T cells in the absence of exogenous IL-6 and TGF-β when inhibitory signals, such as IL-12 and IFN-γ, were eliminated. Our results show that this is also true for CD8 T cells.

Our report also directly links cell-extrinsic cytokine-triggered IL-17 and IFN-γ production with levels of cell-intrinsic lineage-defining transcription factors. The importance of T-bet and Eomes in CD8 effector function has been appreciated, and gradations of T-bet and Eomes modulate CD8 function and survival (27, 28, 29). Our studies implicate Eomes as an important regulator of CD8 T cell cytotoxic activity, as the repression of Eomes upon the addition of IL-6 and TGF-β paralleled the suppression of granzyme B and perforin. Conversely, inhibition of these cytotoxic proteins and IFN-γ correlated directly with the increased expression of ROR-γt.

In summary, our findings show that, depending on the cytokine milieu, CD8 T cells have the ability to differentiate into one of several populations: canonical CD8 effectors producing only IFN-γ, and subsets producing IFN-γ and IL-17, or, at least in vitro, only IL-17. The diversity of populations indicates that although CD8 T cell differentiation is less plastic than for CD4 T cells, CD8 effector function is nonetheless clearly affected by the overall cytokine milieu.

Acknowledgments

We thank Michael Bevan and Murali Krishna-Kaja for providing TCR transgenic mice.

Disclosures

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 in part by an Infectious Diseases Society of America Career Development Award (to S.S.W.), a March of Dimes Basil O'Connor Research Award (to S.S.W.), and National Institutes of Health Grants T32 CA009537 and T32 GM07270 (to M.M.C.), K08HD51584 (to S.S.W.), and R01 HD18184 (to C.B.W.).

  • ↵2 Address correspondence and reprint requests to Dr. Christopher B. Wilson, Department of Immunology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-7650. E-mail address: cbwilson{at}u.washington.edu

  • ↵3 Abbreviations used in this paper: Lm, Listeria monocytogenes; DKO, double knockout; WT, wild type; KO, knockout; Tg, transgenic; CFU, colony forming unit.

  • Received March 24, 2009.
  • Accepted May 4, 2009.

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