IL-23 promotes the production of IL-17 by antigen-specific CD8 T cells in the absence of IL-12 and type-I interferons

Abstract

In contrast to CD4 T cells, CD8 T cells inherently differentiate into IFN-gamma-producing effectors. Accordingly, while generation of IFN-gamma-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-gamma-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-gamma/IL-17 dual-producing cells and was associated with increased expression of ROR-gammat and ROR-alpha. Addition of IL-6 and TGF-beta to IL-23 further augmented ROR-gammat and ROR-alpha 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-beta 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.

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 post-infection. 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 OVA_257–264 peptide for 72 hours. 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 OVA_257–264. 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 antigen stimulation with OVA_257–264 The graphs indicate the mean ± SD from three independent experiments with 4–5 mice/group. Statistics indicate unpaired Student’s t-test p values where ** p<0.01; *** p<0.001, ns not significant.

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 post-infection. 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 OVA_257–264 peptide for 72 hours. 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 post-infection and in vitro stimulated with OVA_257–264. Plots are gated on the total CD8⁺ population. C. Percent IFN-γ-producing or percent IL-17-producing CD8 T cells in response to antigen stimulation with OVA_257–264 The graphs indicate the mean ± SD from two independent experiments with 2–4 mice/group. Statistics indicate unpaired Student’s t-test p values where * p<0.05; ** p<0.01; *** p<0.001; ns not significant.

Figure 3

IL-23 enables the development of antigen-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 GP_33–41 peptide in various polarizing conditions for five 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 antigen stimulation with OVA_257–264 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.

Figure 4

IL-23, but not p40 homodimer, induces co-production 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 GP_33–41 peptide in various polarizing conditions for five 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 five days. Experiments were performed twice and representative plots from one of two experiments are shown.

Figure 5

IL-23 induces the expression of IL-23R on naïve CD8 T cells. P14 Tg+ CD8 T cells stimulated with GP_33–41 in indicated conditions for either 48 hours or 5 days were purified and re-stimulated with PMA and ionomycin for 4.5 hours. RNA was extracted, cDNA was generated, and abundance of A. IL-12Rβ1 and B. IL-23R was analyzed by quantitative PCR. All samples are calibrated to the naïve CD8 sample and internally normalized based on expression of eukaryotic 18s. Experiments were performed twice and bars represent the mean ± SD

Figure 6

IL-23 and IL-6 plus TGF-β independently and synergistically induce Rorc and Il17 and dampen Eomes expression as naïve CD8 T cells differentiate into effectors. A. P14 Tg+ CD8 T cells stimulated with GP_33–41 in various polarizing conditions, as described in figure 3, were purified and re-stimulated with PMA and ionomycin for 4.5 hours. 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.

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 GP_33–41 peptide in various polarizing conditions for five 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 naïve CD8 sample and internally normalized based on expression of eukaryotic 18s. Bars represent the mean ± SD from three independent experiments.