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

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.

Fig 1. Subsets of CD4 and CD8 memory T cells produce IFNγ in response to TLR7 simulation in vitro.

Splenocytes from C57Bl/6 mice were cultured in vitro for 18h in the presence of different TLR agonists (as indicated). Cells were stained for surface markers and intracellular IFNγ. (A) Bars represent percentage of total splenocytes positive for intracellular IFNγ. (B) Gating strategy for IFNγ+ splenocytes upon TLR7 simulation for 18h (Representative FACS plots) and quantification of CD4 and CD8-positive cells among IFNγ+ splenocytes. (C) Bar graphs represent percentages of IFNγ+ among CD4 or CD8 T cells after 18h of splenocytes stimulation with different TLR ligands as indicated. (D, E) Percantages of IFNγ+ CD4 and CD8 T cells in response to different doses (D) or different time (E) of stimulation with R848. Bars represent the means +/- SEM (n = 3). All data are representative of three or more independent experiments. Statistics is shown for each condition over R848 stimulated cultures.

Fig 2. TLR7 agonist in the presence of IL-12 stimulates T cells directly leading to IFNγ production.

(A) Spleen cells obtained from C57BL/6, MyD88^fl/flxLCK^CRE and MyD88^fl/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 MyD88^fl/fl xWT or MyD88^fl/flxLCK^CRE 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.

Fig 3. IFNγ produced by T cells in response to TLR7/IL-12 simulation is required for T-bet induction in B cells.

(A) Sorted memory or naïve CD4 T cells were mixed with purified naïve B cells and incubated in the presence of anti-BCR, anti-BCR and R848, or combination of anti-BCR, R848 and IL-12 for 48h. Cells were stained for surface markers and intracellular T-bet. Bar graph represents gMFI of T-bet expression in B cells (gated as live, B220+, CD19+, CD4-, CD8-). Bars represent the means +/- SEM. (B) WT or TLR7-/- sorted CD4 T cells were mixed with purified naïve WT B cells and incubated for 48h with indicated stimuli. Cells were stained for surface markers and intracellular T-bet. Bar graph represents gMFI of T-bet expression in B cells (gated as live, B220+, CD19+, CD4-, CD8-). Bars represent the means +/- SEM (C) WT or TLR7-/- sorted CD4 T cells were mixed with purified naïve WT B cells and incubated in the presence of indicated stimuli for 7 days. Culture supernatants were assessed for the presence IgG2a by ELISA. (D) Sorted memory or naïve CD4 T cells were mixed with purified naïve B cells and incubated in the presence of indicated stimuli for 7 days. Supernatants were analyzed for the presence of IgG2a by ELISA. Bars represent the means +/- SEM. Data are representative of three or more independent experiments.

Fig 4. TLR7 expression in T cells is required for the accumulation of T-bet+ B cells and effective production of anti-viral IgG2a during Friend virus infection.

(A) C57BL/6 mice (n = 5 per time point) were infected with 10⁴ SFFU of Friend virus (FV). Spleens were harvested on day 7, 14, 21 or 28 post infection. The presence of T-bet+ cells among B cells was assessed by FACS. Bar graphs represent percentage of T-bet+ cells among B cells. (B, C) Bone marrow chimera were constructed as described in Methods, such as in (TLR7^-/- + TCR^-/-) mice only T cells completely lack TLR7 expression and the rest of the hematopoetic cells were 80% WT and 20% TLR7-/-. Bone marrow chimeras were infected with FV (as in (A)) and spleens and serum were harvested on 14 dpi percentage and numbers of T-bet+ or CD11c+ B cells is shown (B). Serum collected at 14dpi was assessed for the presence of anti-FV IgG2a by ELSIA (C). (D) T-bet^fl/fl, and T-bet^fl/flxCD19^CRE mice (n = 4 mice per group) were infected with FV as in (A). Serum was collected at 15 dpi and the presence of anti-FV IgG2a was assessed by ELISA. Bars represent the means +/- SEM. Data are representative of three or more independent experiments.