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T Cell Production of IFN γ in Response to TLR7/IL-12 Stimulates Optimal B Cell Responses to Viruses

View Article: PubMed Central - PubMed

ABSTRACT

Knowledge of the processes that underlie IgG subclass switching could inform strategies designed to counteract infections and autoimmunity. Here we show that TLR7 ligands induce subsets of memory CD4 and CD8 T cells to secrete interferon γ (IFNγ) in the absence of antigen receptor stimulation. In turn, TLR ligation and IFNγ cause B cells to express the transcription factor, T-bet, and to switch immunoglobulin production to IgG2a/c. Absence of TLR7 in T cells leads to the impaired T-bet expression in B cells and subsequent inefficient IgG2a isotype switching both in vitro and during the infection with Friend virus in vivo. Our results reveal a surprising mechanism of antiviral IgG subclass switching through T-cell intrinsic TLR7/IL-12 signaling.

No MeSH data available.


Related in: MedlinePlus

TLR7 agonist in the presence of IL-12 stimulates T cells directly leading to IFNγ production.(A) Spleen cells obtained from C57BL/6, MyD88fl/flxLCKCRE and MyD88fl/fl x WTmice were incubated in the presence of different TLR agonists as indicated for 5 days. Supernatants were analyzed for the presence of IFNγ by ELISA. Bar graphs represent concentration of IFNγ in the culture supernatants. (B) Spleen cells from MyD88fl/fl xWT or MyD88fl/flxLCKCRE mice were incubated in the presence of different TLR agonists as indicated for 18h. Cells were surface stained and intacellularly stained for IFNγ. Bar graphs represent percentage of CD4 or CD8 T cells which are positive for IFNγ. (C) TLR7-/- or conjenically marked WT (B6.SJL) splenocytes were incubated either separately (black bars) or mixed at 1:1 ratio in the presence (R848) or absence (media) of TLR7 agonist. IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM. (D) Splenocytes were incubated as indicated for 18h, IFNγ production by CD4 and CD8 T cells in response to indicated stimulations was assessed by intracellular staining. Bar graphs indicate percentage of IFNγ+ cells among CD4 and CD8 T cells. (E) WT or IL-18-/- splenocytes were incubated with R848 for 18h, IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM (similar results were obtained for CD8 T cells—not shown). (F) Naïve and memory CD4 and CD8 T cells were flow sorted as CD4 (or CD8) positive, CD19-, CD44+ (for memory) and CD44- (for naïve). IFNγ production by sorted T cells in response to indicated stimulations was assessed by intracellular staining. Bar graph represent percentage of IFNγ+ sorted memory of naïve CD4 T cells (similar data was obtained for CD8 T cells—not shown). All data are representative of three or more independent experiments.
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pone.0166322.g002: TLR7 agonist in the presence of IL-12 stimulates T cells directly leading to IFNγ production.(A) Spleen cells obtained from C57BL/6, MyD88fl/flxLCKCRE and MyD88fl/fl x WTmice were incubated in the presence of different TLR agonists as indicated for 5 days. Supernatants were analyzed for the presence of IFNγ by ELISA. Bar graphs represent concentration of IFNγ in the culture supernatants. (B) Spleen cells from MyD88fl/fl xWT or MyD88fl/flxLCKCRE mice were incubated in the presence of different TLR agonists as indicated for 18h. Cells were surface stained and intacellularly stained for IFNγ. Bar graphs represent percentage of CD4 or CD8 T cells which are positive for IFNγ. (C) TLR7-/- or conjenically marked WT (B6.SJL) splenocytes were incubated either separately (black bars) or mixed at 1:1 ratio in the presence (R848) or absence (media) of TLR7 agonist. IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM. (D) Splenocytes were incubated as indicated for 18h, IFNγ production by CD4 and CD8 T cells in response to indicated stimulations was assessed by intracellular staining. Bar graphs indicate percentage of IFNγ+ cells among CD4 and CD8 T cells. (E) WT or IL-18-/- splenocytes were incubated with R848 for 18h, IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM (similar results were obtained for CD8 T cells—not shown). (F) Naïve and memory CD4 and CD8 T cells were flow sorted as CD4 (or CD8) positive, CD19-, CD44+ (for memory) and CD44- (for naïve). IFNγ production by sorted T cells in response to indicated stimulations was assessed by intracellular staining. Bar graph represent percentage of IFNγ+ sorted memory of naïve CD4 T cells (similar data was obtained for CD8 T cells—not shown). All data are representative of three or more independent experiments.

Mentions: All the experiments described above involved cultures of unseparated spleen cells, therefore there are two possible explanations for the ability of T cells to produce IFNγ in response to TLR7 agonists: either T cells respond directly to the agonists, or some other splenic cell detects the TLR7 agonist and produces material that subsequently acts on the T cells (a bystander effect). To distinguish between these two possibilities, we used spleen cells from mice with a T cell-specific MyD88 deletion (generated by intercrossing MyD88fl/fl and LCKCre mice) (S1 Fig). We stimulated splenocytes from C57Bl/6, MyD88fl/fl x LCKCre mice or MyD88fl/fl littermate controls with R848 (TLR7 agonist), ODN1668 (TZLR9 agonist) or LPS (TLR4 agonist) and measured the levels of IFNγ in the culture supernatants by ELISA and in T cells by intracellular cytokine staining. The cultures that contained MyD88 deficient T cells did not produce IFNγ in response to TLR7 triggering (Fig 2A) and the T cells in these cultures did not stain intracellularly for IFNγ (Fig 2B). These data indicate that memory T cells produce IFNγ following TLR7 stimulation via T cell intrinsic MyD88 signaling.


T Cell Production of IFN γ in Response to TLR7/IL-12 Stimulates Optimal B Cell Responses to Viruses
TLR7 agonist in the presence of IL-12 stimulates T cells directly leading to IFNγ production.(A) Spleen cells obtained from C57BL/6, MyD88fl/flxLCKCRE and MyD88fl/fl x WTmice were incubated in the presence of different TLR agonists as indicated for 5 days. Supernatants were analyzed for the presence of IFNγ by ELISA. Bar graphs represent concentration of IFNγ in the culture supernatants. (B) Spleen cells from MyD88fl/fl xWT or MyD88fl/flxLCKCRE mice were incubated in the presence of different TLR agonists as indicated for 18h. Cells were surface stained and intacellularly stained for IFNγ. Bar graphs represent percentage of CD4 or CD8 T cells which are positive for IFNγ. (C) TLR7-/- or conjenically marked WT (B6.SJL) splenocytes were incubated either separately (black bars) or mixed at 1:1 ratio in the presence (R848) or absence (media) of TLR7 agonist. IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM. (D) Splenocytes were incubated as indicated for 18h, IFNγ production by CD4 and CD8 T cells in response to indicated stimulations was assessed by intracellular staining. Bar graphs indicate percentage of IFNγ+ cells among CD4 and CD8 T cells. (E) WT or IL-18-/- splenocytes were incubated with R848 for 18h, IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM (similar results were obtained for CD8 T cells—not shown). (F) Naïve and memory CD4 and CD8 T cells were flow sorted as CD4 (or CD8) positive, CD19-, CD44+ (for memory) and CD44- (for naïve). IFNγ production by sorted T cells in response to indicated stimulations was assessed by intracellular staining. Bar graph represent percentage of IFNγ+ sorted memory of naïve CD4 T cells (similar data was obtained for CD8 T cells—not shown). All data are representative of three or more independent experiments.
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pone.0166322.g002: TLR7 agonist in the presence of IL-12 stimulates T cells directly leading to IFNγ production.(A) Spleen cells obtained from C57BL/6, MyD88fl/flxLCKCRE and MyD88fl/fl x WTmice were incubated in the presence of different TLR agonists as indicated for 5 days. Supernatants were analyzed for the presence of IFNγ by ELISA. Bar graphs represent concentration of IFNγ in the culture supernatants. (B) Spleen cells from MyD88fl/fl xWT or MyD88fl/flxLCKCRE mice were incubated in the presence of different TLR agonists as indicated for 18h. Cells were surface stained and intacellularly stained for IFNγ. Bar graphs represent percentage of CD4 or CD8 T cells which are positive for IFNγ. (C) TLR7-/- or conjenically marked WT (B6.SJL) splenocytes were incubated either separately (black bars) or mixed at 1:1 ratio in the presence (R848) or absence (media) of TLR7 agonist. IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM. (D) Splenocytes were incubated as indicated for 18h, IFNγ production by CD4 and CD8 T cells in response to indicated stimulations was assessed by intracellular staining. Bar graphs indicate percentage of IFNγ+ cells among CD4 and CD8 T cells. (E) WT or IL-18-/- splenocytes were incubated with R848 for 18h, IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM (similar results were obtained for CD8 T cells—not shown). (F) Naïve and memory CD4 and CD8 T cells were flow sorted as CD4 (or CD8) positive, CD19-, CD44+ (for memory) and CD44- (for naïve). IFNγ production by sorted T cells in response to indicated stimulations was assessed by intracellular staining. Bar graph represent percentage of IFNγ+ sorted memory of naïve CD4 T cells (similar data was obtained for CD8 T cells—not shown). All data are representative of three or more independent experiments.
Mentions: All the experiments described above involved cultures of unseparated spleen cells, therefore there are two possible explanations for the ability of T cells to produce IFNγ in response to TLR7 agonists: either T cells respond directly to the agonists, or some other splenic cell detects the TLR7 agonist and produces material that subsequently acts on the T cells (a bystander effect). To distinguish between these two possibilities, we used spleen cells from mice with a T cell-specific MyD88 deletion (generated by intercrossing MyD88fl/fl and LCKCre mice) (S1 Fig). We stimulated splenocytes from C57Bl/6, MyD88fl/fl x LCKCre mice or MyD88fl/fl littermate controls with R848 (TLR7 agonist), ODN1668 (TZLR9 agonist) or LPS (TLR4 agonist) and measured the levels of IFNγ in the culture supernatants by ELISA and in T cells by intracellular cytokine staining. The cultures that contained MyD88 deficient T cells did not produce IFNγ in response to TLR7 triggering (Fig 2A) and the T cells in these cultures did not stain intracellularly for IFNγ (Fig 2B). These data indicate that memory T cells produce IFNγ following TLR7 stimulation via T cell intrinsic MyD88 signaling.

View Article: PubMed Central - PubMed

ABSTRACT

Knowledge of the processes that underlie IgG subclass switching could inform strategies designed to counteract infections and autoimmunity. Here we show that TLR7 ligands induce subsets of memory CD4 and CD8 T cells to secrete interferon γ (IFNγ) in the absence of antigen receptor stimulation. In turn, TLR ligation and IFNγ cause B cells to express the transcription factor, T-bet, and to switch immunoglobulin production to IgG2a/c. Absence of TLR7 in T cells leads to the impaired T-bet expression in B cells and subsequent inefficient IgG2a isotype switching both in vitro and during the infection with Friend virus in vivo. Our results reveal a surprising mechanism of antiviral IgG subclass switching through T-cell intrinsic TLR7/IL-12 signaling.

No MeSH data available.


Related in: MedlinePlus