Stromal cell-mediated suppression of human T-cell leukemia virus type 1 expression in vitro and in vivo by type I interferon

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis, and other inflammatory diseases. Despite such severe outcomes of HTLV-1 infection, the level of HTLV-1 expression in vivo is very low and rapidly increases after transfer of cells to culture conditions. The mechanisms of this phenomenon have remained obscure. In the present study, we found that human and mouse stromal cells, such as epithelial cells and fibroblasts, suppressed HTLV-1 expression in ATL and non-ATL HTLV-1-infected cells. HTLV-1 mRNA and proteins in HTLV-1-infected cells markedly decreased upon coculture with human epithelial-like cells (HEK293T) or mouse embryo fibroblasts (NIH 3T3). When infected cells were reisolated from the cocultures, viral expression was restored to the original level over the following 48 h. Spontaneous induction of HTLV-1 expression in primary ATL cells in the first 24 h of culture was also inhibited by coculture with HEK293T cells. Coculture of HTLV-1-infected cells and HEK293T cells induced type I interferon responses, as detected by beta interferon (IFN-beta) promoter activation and IFN-stimulated gene upregulation. HEK293T-mediated suppression of HTLV-1 expression was partly inhibited by antibodies to human IFN-alpha/beta receptor. NIH 3T3-mediated suppression was markedly abrogated by neutralizing antibodies to mouse IFN-beta. Furthermore, viral expression in HTLV-1-infected cells was significantly suppressed when the infected cells were intraperitoneally injected into wild-type mice but not IFN regulatory factor 7 knockout mice that are deficient of type I IFN responses. These findings indicate that the innate immune system suppresses HTLV-1 expression in vivo, at least through type I IFN.

FIG. 1.

Suppression of HTLV-1 expression by coculture with stromal cells. (A) ILT-Hod cells (left panel) or primary PBMC from a chronic ATL patient (right panel) were cultured with or without HEK293T cells for 72 h or 24 h, respectively, and the HTLV-1 p19 concentrations in the supernatants were measured by ELISA. The data are the means and standard deviations (SD) of duplicate wells. (B) ILT-Hod, ILT-Myj, or MT-2 cells were cultured with or without HEK293T for 24 h, and the HTLV-1 gag mRNA levels in floating cells were measured by quantitative RT-PCR. The HTLV-1 gag mRNA copy numbers were standardized by the human GAPDH mRNA copy numbers and are presented as the fold changes compared to each HTLV-1-infected cell line cultured in medium alone. (C) ILT-Hod cells were cultured with or without HEK293T, HeLa, or NIH 3T3 cells for 24 h, and the HTLV-1 gag mRNA levels in floating cells were measured by quantitative RT-PCR as described above. The results shown are representatives of several independent experiments for each cell line. (D) Intracellular HTLV-1 Gag proteins in floating ILT-Hod cells were stained before coculture (green line) and after 24 h (blue line) and 48 h (red line) of coculture with NIH 3T3 cells and analyzed by using a flow cytometer. The closed histogram indicates control staining with ascites and a FITC-labeled anti-mouse IgG+IgM antibody. (E) ILT-Hod cells were cocultured with (right panel) or without (left panel) HEK293T cells in the presence of 10 μM BrdU for 24 h, and the floating cells were stained for surface CD25 (y axis) and intracellular BrdU (x axis) and then analyzed by a flow cytometer. The proportions of CD25⁺ BrdU⁺ cells are indicated. All culture media used contained 10 U of rhIL-2/ml.

FIG. 2.

Recovery of HTLV-1 expression in infected cells after separation from stromal cells. (A) ILT-Hod (○, •) or ILT-#29 (▵, ▴) cells were cultured with (•, ▴) or without (○, ▵) NIH 3T3 cells for 24 h, and then nonadherent cells were isolated and cultured in new wells with fresh medium. The HTLV-1 gag mRNA levels in floating cells were measured before coculture (control), at 24 h after coculture (day 0), and at 1 and 3 days after separation. The HTLV-1 gag mRNA copy numbers were standardized by the human GAPDH mRNA copy numbers and are presented as the fold changes compared to ILT-Hod cells before coculture. The results represent the means and SD of duplicate wells. (B and C) ILT-Hod cells cocultured with NIH 3T3 (B) or 293T (C) cells for 24 h were isolated and cultured in new wells with fresh medium. Flow cytometric analyses for intracellular HTLV-1 Gag proteins were performed at the indicated time points after separation. The green histogram indicates ILT-Hod cells without coculture, and the closed histogram indicates control staining with ascites and an FITC-labeled anti-mouse IgG+IgM antibody. All culture media used contained 10 U of rhIL-2/ml.

FIG. 3.

Involvement of type I IFNs in suppression of HTLV-1 expression by stromal cells. (A and B) ILT-Hod cells were cultured in the presence of rhIFN-α, rhIFN-β, or rhIL-10 at the indicated concentrations for 72 h, and the concentrations of HTLV-1 p19 in the supernatants (A) and viable cell numbers (B) were evaluated by ELISA or using Cell Counting Kit-8, respectively. The results indicate means and SD of duplicate wells. (C) The HTLV-1 gag mRNA expression levels in ILT-Hod cells were measured after a 24-h coculture without (open bars) or with (closed bars) NIH 3T3 cells in the presence of anti-mouse IFN-α or IFN-β neutralizing antibodies (4 × 10³ neutralizing units/ml). (D) ILT-Hod cells were pretreated with or without 1 μg of anti-human IFN α/β receptor monoclonal antibody or control mouse IgG2a/ml for 1 h prior to coculture with HEK293T cells for 2 h, and then the HTLV-1 gag mRNA expression levels in ILT-Hod cells were evaluated. The data in panels C and D are the fold changes compared to control cells cultured in medium alone and represent the means and SD of duplicate wells. (E) Reporter 293T cells containing an IFN-β reporter plasmid (293T/IFN-β-luc) were cultured with or without ILT-Hod cells or ILT-Hod cells fixed with 1% formaldehyde, and the luciferase activities in the whole-cell lysates were measured after coculture for 24 h. The results are means and SD of duplicate samples. (F) The MxA (○) and HTLV-1 gag (•) mRNA levels in ILT-Hod cells were measured after coculture with HEK293T cells for the indicated periods. The data represent the fold changes compared to control ILT-Hod cells cultured alone for 1 h.

FIG. 4.

Suppression of HTLV-1 expression in wild-type but not IRF-7^−/− mice. (A) ILT-Hod cells were cocultured with or without MEFs isolated from WT or IRF-7^−/− C57BL/6J mice as indicated for 24 h, and the HTLV-1 gag mRNA levels in floating ILT-Hod cells were measured. The data are the fold changes compared to control wells cultured in medium alone, and represent the means and SD of duplicate wells. Similar results were obtained in two other independent experiments. (B) WT and IRF-7^−/− C57BL/6J mice were i.p. injected with an anti-asialo-GM1 antibody on the day before an ILT-Hod injection and then i.p. injected with ILT-Hod cells (2 × 10⁷ cells/head). The cells in the peritoneal cavity were harvested with 10 ml of PBS at 16 h after the ILT-Hod injection and analyzed by quantitative RT-PCR. The HTLV-1 gag mRNA levels standardized by the human GAPDH mRNA levels are indicated as the fold changes compared to the control value of ILT-Hod cells cultured in IL-2-containing medium. The HTLV-1 gag mRNA expression level in ILT-Hod cells cultured without IL-2 in vitro is also indicated. The data represent the means and SD of duplicate wells. A representative result of at least three independent experiments is shown.