Type I interferon-mediated stimulation of T cells by CpG DNA - PubMed (original) (raw)

Type I interferon-mediated stimulation of T cells by CpG DNA

S Sun et al. J Exp Med. 1998.

Abstract

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR-/- mice. Second, in contrast to normal T cells, the failure of purified IFN-IR-/- T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs. Third, IFN-I (but not IFN-gamma) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR-/-) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.

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Figures

Figure 1

Figure 1

CD69 upregulation on T cells in response to insect DNA and synthetic ODNs. (A) B6 mice were injected subcutaneously with 100 μg/mouse D. melanogaster DNA or 50 μg/mouse ODNs. Draining LN cells were removed 18 h later and double-stained for CD4 or CD8 and also for CD44 and CD69 expression. (B) 5 × 106 unseparated spleen cells/ml were cultured in vitro in the presence of D. melanogaster DNA (100 μg/ml) or ODNs (1 μM) overnight. Cells were then harvested, purified with Ficoll gradients, and stained for CD4 or CD8, CD44, and CD69 expression. For C and D, see B, except that spleen cells were cultured with various concentrations of D. melanogaster DNA or ODNs.

Figure 2

Figure 2

DNA-induced upregulation of CD69 on spleen CD8+ cells from LPS-nonresponder, μMT, and beige mice. As in Fig. 1_B_, spleen cells from C3H/HeJ versus C3H/HeOuJ mice (A), B6 versus μMT mice (B), or B6 versus beige mice (C) were cultured overnight with D. melanogaster DNA (100 μg/ml) or LPS (1 μg/ml) and then stained for CD8 and CD69 expression. The data show the percentage of CD8+ cells that were positive for CD69 relative to unstimulated CD8+ cells.

Figure 3

Figure 3

DNA-induced upregulation of CD69 on T cells requires APCs. Purified CD4+ or CD8+ cells (106 cells/well) were cultured overnight in the presence or absence of D. melanogaster DNA (100 μg/ml), either alone or in the presence of RAG-1−/− spleen cells (A) or B cell– depleted spleen cells from MHC I−/−II−/− mice (panel B; B− MHC KO spl.); spleen APCs were added at 2 × 106 cells/well. In C, purified CD8+ CD44lo/int cells (106 cells/well) ± DNA were cultured overnight with titrated numbers of RAG-1 KO spleen cells. For A–C, cells were stained for CD8 and CD69 expression. The data show the percentage of gated CD8+ cells that were positive for CD69 expression relative to unstimulated CD8+ cells.

Figure 4

Figure 4

DNA-induced upregulation of various cell surface molecules on lymphoid subsets from normal versus IFN-IR−/− mice. (A and B) Normal B6 mice (A) or 129 versus IFN-IR−/− mice (B) were injected subcutaneously with D. melanogaster DNA (100 μg/mouse); 18 h later, draining LN cells were double-stained for expression of the markers shown and also for B220, CD4, and CD8 expression. The data are expressed as the percentage increase in the mean fluorescence intensity of staining for cells from DNA-injected mice versus PBS-injected mice. In C, purified CD8+ cells from 129 or IFN-IR−/− mice (106 cells/well) were cultured in vitro with or without D. melanogaster DNA (100 μg/ml), either alone or with RAG-1−/− spleen APCs (2 × 106 cells/well), and then stained for CD8 and the markers shown 18 h later. The data are expressed as the mean fluorescence intensity of staining for the marker shown, gating on CD8+ cells.

Figure 5

Figure 5

Expression of various cell surface markers on purified B (B220+) and CD8+ cells after incubation in vitro with IFN-I or IFN-γ. Cells (2 × 106/ well) were cultured with titrated concentrations of IFN-I (IFN-β) or IFN-γ overnight and then double-stained for B220 or CD8 and the markers shown.

Figure 6

Figure 6

Prior exposure to Drosophila DNA in vivo inhibits proliferation of CD8+ cells in vitro. CD8+ cells were purified from the LNs of the indicated mice 18 h after injection of either PBS or D. melanogaster DNA (200 μg/mouse for 2C mice, 500 μg/mouse for 129 or IFN-IR KO mice). (A) Purified CD8+ cells from 129 or IFN-IR KO mice were cultured for 2 or 3 d in 200 μl wells precoated with a mixture of anti-TCR and anti-CD28 mAbs. The data show means and SDs of duplicate cultures. (B) Purified 2C CD8+ cells (2.5 × 104 cells/well) were cultured with Drosophila APCs (Ld/B7.1/ ICAM-1) and a titrated dose of QL9 peptide for the periods shown. Drosophila APCs were used at 2 × 105 cells/well. The data show means and SDs of triplicate cultures; for many of the data points, SDs were too small to be illustrated.

Figure 6

Figure 6

Prior exposure to Drosophila DNA in vivo inhibits proliferation of CD8+ cells in vitro. CD8+ cells were purified from the LNs of the indicated mice 18 h after injection of either PBS or D. melanogaster DNA (200 μg/mouse for 2C mice, 500 μg/mouse for 129 or IFN-IR KO mice). (A) Purified CD8+ cells from 129 or IFN-IR KO mice were cultured for 2 or 3 d in 200 μl wells precoated with a mixture of anti-TCR and anti-CD28 mAbs. The data show means and SDs of duplicate cultures. (B) Purified 2C CD8+ cells (2.5 × 104 cells/well) were cultured with Drosophila APCs (Ld/B7.1/ ICAM-1) and a titrated dose of QL9 peptide for the periods shown. Drosophila APCs were used at 2 × 105 cells/well. The data show means and SDs of triplicate cultures; for many of the data points, SDs were too small to be illustrated.

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