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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.

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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.
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pone.0166322.g001: 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.

Mentions: We recently demonstrated that, of all the TLRs, engagement of TLR7 induced the highest production of IFNγ by mouse spleen cells [13]. At that time we showed that B cells were not the origin of the cytokine. Here we studied this issue in greater depth. First, we confirmed that an agonist for TLR7, compared with agonists for other TLRs, is indeed the most potent inducer of IFNγ by spleen cells. Thus, the percentage of IFNγ+ splenocytes was highest in TLR7 stimulated cultures compared to splenocytes stimulated with other TLR ligands (Fig 1A). To characterize the IFNγ producing cells, we stimulated the cells with R848 (TLR7 agonist) for 18h and then stained them as described for their surface markers and intracellular IFNγ (Fig 1B). Very few B cells and no NK cells were IFNγ+ (data not shown). However, more than 60% of the IFNγ+ cells were CD4 or CD8 positive, suggesting that T cells were the major source of the cytokine (Fig 1B). The IFNγ+ T cells did not bear Tfh or Treg markers (data not shown). However, all the IFNγ+ T cells expressed high levels of CD44, indicating a memory phenotype (Fig 1B).


T Cell Production of IFN γ in Response to TLR7/IL-12 Stimulates Optimal B Cell Responses to Viruses
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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5120817&req=5

pone.0166322.g001: 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.
Mentions: We recently demonstrated that, of all the TLRs, engagement of TLR7 induced the highest production of IFNγ by mouse spleen cells [13]. At that time we showed that B cells were not the origin of the cytokine. Here we studied this issue in greater depth. First, we confirmed that an agonist for TLR7, compared with agonists for other TLRs, is indeed the most potent inducer of IFNγ by spleen cells. Thus, the percentage of IFNγ+ splenocytes was highest in TLR7 stimulated cultures compared to splenocytes stimulated with other TLR ligands (Fig 1A). To characterize the IFNγ producing cells, we stimulated the cells with R848 (TLR7 agonist) for 18h and then stained them as described for their surface markers and intracellular IFNγ (Fig 1B). Very few B cells and no NK cells were IFNγ+ (data not shown). However, more than 60% of the IFNγ+ cells were CD4 or CD8 positive, suggesting that T cells were the major source of the cytokine (Fig 1B). The IFNγ+ T cells did not bear Tfh or Treg markers (data not shown). However, all the IFNγ+ T cells expressed high levels of CD44, indicating a memory phenotype (Fig 1B).

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