The HTLV-1-infected MT-2, MT-4, C5/MJ, SLB-1, HUT-102, MT-1, TL-OmI and ED-40515(−) T-cell lines, and the negative control uninfected human leukemia Jurkat, MOLT-4 and CCRF-CEM T-cell lines, were grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. JPX-9 cells are derivatives of Jurkat with Tax gene, which is stably integrated under the control of a metallothionein promoter (28). To induce Tax expression, JPX-9 cells were cultured in the presence of 20 μM CdCl₂. The human acute monocytic leukemia cell line, THP-1, was set as a positive control for IRF-5 expression. TY8-3/MT-2 was established from the interleukin (IL)-2-dependent human T-cell line, TY8-3, co-cultured with mitomycin C (MMC)-treated MT-2 cells, and was capable of growth completely independent of IL-2 (29). Jurkat cells were stimulated with 1,000 U/ml of recombinant human IFN-α for the indicated time intervals.

Peripheral blood mononuclear cells (PBMC) from a healthy donor were isolated from the heparinized blood sample by centrifugation over a Ficoll-Paque layer (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). MT-2 cells were pretreated with 200 μg/ml of MMC for 60 min, pipetted vigorously, and washed three times with phosphate-buffered saline. PBMC and MMC-treated MT-2 cells were co-cultured in the presence of 10 ng/ml of IL-2. The culture medium was half-changed with fresh medium supplemented with IL-2 every 3 days. Since MT-2 cells were pretreated extensively with MMC, no discernible MT-2 cells were found.

Total RNA was extracted from cells with TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the protocol provided by the manufacturer. The RNA was reverse transcribed into cDNA using a PrimeScript™ RT-PCR kit (Takara Bio Inc., Otsu, Japan). The sequences of the primers for IRF-5, IFN-α, IFN-β, IFN-γ, 2′,5′-oligoadenylate synthetase (2–5 AS), MxA, TNF-α, lymphotoxin (LT)-β, β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Tax in HTLV-1-infected T cells, and Tax in JPX-9 cells are summarized in Table I (2,30–36).

Immunoblot analysis was performed on whole cell lysate, and nuclear and cytoplasmic fractions. Each protein was extracted as previously described (37–39), and subjected to sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were then probed for IRF-5 (Abnova Corp., Taipei, Taiwan), actin (NeoMarkers Inc., Fremont, CA, USA), lamin B (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or Myc-Tag (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Mouse monoclonal antibody to Tax, Lt-4, was previously described (40). The bands were visualized using enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA).

Cells were fixed in 4% paraformaldehyde and permeabilized with Triton X-100. Then, the cells were stained with mouse anti-IRF-5 antibody (Abnova Corp.). For immunofluorescence studies, washed cells were incubated with anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen Life Technologies). The nuclei were stained with Hoechst 33342 (Wako Pure Chemical Industries, Ltd.). After final washing, the cells were examined under a Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany). Mounted coverslips were viewed through a 63× oil immersion lens (NA1.4) on a Leica TCS confocal system.

The diagnosis of ATL was based on clinical features, hematological findings and the presence of anti-HTLV-1 antibodies in the serum. Biopsy samples were taken from the lesional skin and lymph nodes of four patients with ATL. IRF-5 immunohistochemistry was performed using an anti-IRF-5 antibody (Abnova Corp.) after pretreatment of the deparafinized tissue sections with ready-to-use proteinase K (Dako, Carpinteria, CA, USA). The sections were counterstained with methyl green, hydrated in ethanol, cleaned in xylene, and mounted. The stained cells were examined under a light microscope (Axicoskop 2 Plus) with an Achroplan 40×/0.65 lens (both from Zeiss, Jena, Germany). Images were acquired with an AxioCam MRc camera and AxioVision 4.7 software (Zeiss). A signed consent form was obtained from each tissue donor.

The IRF-5 P-V3 promoter fragment was cloned into the KpnI and XhoI sites of pGL3-Basic vector (Promega Corp., Madison, WI, USA) (30). Details of the plasmid expressing the HTLV-1 Tax through β-actin promoter were published previously (41). The coding sequences of IRF-5 were cloned into the pcDNA4/myc-His A vector (Invitrogen Life Technologies).

Jurkat cells were transfected at 5×10⁶ cells with the luciferase reporter plasmid, together with 0.5–5 μg of the Tax expression vector by electroporation (250 V, 960 μF). Each transfection included the phRL-TK plasmid (Promega Corp.) as an internal control for variation in transfection efficiency. After 48 h, transfected cells were collected by centrifugation, washed with phosphate-buffered saline, and lysed in reporter lysis buffer (Promega Corp.). The luciferase activity was determined by the Dual-Luciferase Reporter system (Promega Corp.) using the protocol supplied by the manufacturer.

Jurkat cells were transfected with a control pcDNA4/myc-His A vector or IRF-5 expression vector using MicroPorator MP-100 (Digital Bio Technology Co., Ltd., Seoul, Korea), pulsed three times at 1,325 V for 10 msec each. After 24 h, total RNA samples were prepared using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) and confirmed to be of good quality by Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Waldbronn, Germany). Microarray analysis using a SurePrint G3 Human GE 8×60 K Microarray kit version 2.0 (Agilent Technologies, Inc.) was performed as previously described (42).

To investigate IRF-5 expression in both HTLV-1-infected and -uninfected T-cell lines, we collected three uninfected (Jurkat, MOLT-4 and CCRF-CEM), five HTLV-1-transformed (MT-2, MT-4, C5/MJ, SLB-1 and HUT-102) and three ATL-derived [MT-1, TL-OmI and ED-40515(−)] T-cell lines. All HTLV-1-transformed T-cell lines constitutively expressed Tax mRNA (Fig. 1A, panel 1). Expression of IRF-5 mRNA in 11 T-cell lines was analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using IRF-5 specific primers. All eight of the HTLV-1-positive T-cell lines examined (lanes 4–11) strongly expressed IRF-5 mRNA, whereas the three HTLV-1-negative T-cell lines (lanes 1–3) did not (Fig. 1A, panel 2). IRF-5 is transcribed into various distinct alternatively spliced isoforms (30), and the IRF-5 variant 1 (V1), V2 and V3 transcripts have different noncoding first exons, whereas V1 and V4 share the same first exon. Primer sets that specifically recognize exon 1 of each isoform (Ex1V1, Ex1V2 and Ex1V3) and a common region in exon 4 of IRF-5 were optimized (Table I).

PCR amplification using the exon 1-specific sense primers was isoform-specific. We next examined the levels of constitutive exon 1-specific IRF-5 isoform expression in several T-cell lines (Fig. 1A, panels 3–5). Ex1V1 transcripts were detected in all HTLV-1-infected T-cell lines (Fig. 1A, panel 3). In comparison, transcripts associated with Ex1V2 could not be detected (Fig. 1A, panel 4). IRF-5 V3 transcript levels were specifically high in Tax-positive HTLV-1-transformed T-cell lines (lanes 5–8), excluding MT-2, suggesting a possible role for Tax in enhanced IRF-5 Ex1V3 transcript levels (Fig. 1A, panel 5). High level of IRF-5 protein expression was evident in Tax-positive HTLV-1-transformed T-cell lines and ATL-derived MT-1 (Fig. 1A, panel 7, lanes 4–9). Intracellular mapping by immunoblotting and immunofluorescence staining showed high levels of IRF-5 in the nuclei of HTLV-1-infected HUT-102 cells (Fig. 1B and C). In contrast, almost no IRF-5 was detected in uninfected Jurkat cells (Fig. 1C).

Immunohistochemical staining of ATL cells in archived lymph nodes and skin tissue samples showed abundant IRF-5 in the nuclei of these cells (Fig. 2).

To examine whether HTLV-1 infection induces IRF-5 expression, studies were performed using the parental TY8-3 cell line and TY8-3 cells infected with HTLV-1 (TY8-3/MT-2). TY8-3/MT-2 cells strongly expressed Tax mRNA (Fig. 3A). RT-PCR analysis also demonstrated upregulation of IRF-5 mRNA in TY8-3/MT-2 cells. To substantiate HTLV-1 control of IRF-5 expression in PBMC, we co-cultured PBMC and MMC-treated MT-2 cells. At seven days after co-cultivation, PBMC were harvested for assessment of expression of HTLV-1 viral gene by RT-PCR. PBMC co-cultured with MMC-treated MT-2 cells expressed Tax mRNA (Fig. 3B). Furthermore, IRF-5 expression levels increased in these cells following induction of HTLV-1 gene. Noteworthy, IRF-5 expression was still detected in PBMC at 14 days after co-cultivation, which expressed Tax at a very low level (Fig. 3B). These results indicate that HTLV-1 infection induces the expression of IRF-5 in PBMC as well as T-cell line.

Tax gene product is the primary viral transactivator protein that modulates the expression of both viral and cellular genes (17,18). To test whether Tax directly induces IRF-5 expression, we used JPX-9, the Jurkat subline carrying Tax under the control of the metallothionein gene promoter (28). This cell line has been widely used to examine the effect of Tax on the expression of various cellular genes (28). The results are shown in Fig. 3C. Treatment of JPX-9 with CdCl₂ rapidly induced Tax mRNA expression (panel 1). Similarly, Tax protein expression was induced within 24 h after addition of CdCl₂ and reached a maximal level within 120 h (panel 7). Expression of the IRF-5 gene was not detected at 24 h but became evident within 48 h after addition of CdCl₂ (panel 2). A significant increase in the level of expression of IRF-5 protein (panel 8) was also noted; the protein level began to rise 96 h after the addition of CdCl₂ with the maximal level observed at 120 h.

We next examined IRF-5 isoform expression in CdCl₂-treated and -untreated JPX-9 cells. As shown in Fig. 3C, CdCl₂ upregulated Ex1 V1-associated transcript levels (panel 3), whereas V2 transcript levels were not detected in treated or untreated samples (panel 4). In comparison, CdCl₂ upregulated IRF-5 V3 transcript levels (panel 5).

We also examined the effects of Tax on IRF-5 isoform 3 promoter (P-V3). The activities of the reporter construct containing the entire P-V3 region were analyzed in transient transfection assays in Jurkat cells (Fig. 3D). Co-transfection of the P-V3 promoter construct in Jurkat cells with Tax expression plasmid enhanced promoter activity in Tax dose-dependent manner. Considered collectively, the above results indicate that Tax can activate IRF-5 P-V3 promoter.

Type I IFN is a well-known transcriptional inducer of IRF-5 (10). However, its efficacy on IRF-5 induction in T cells is still unknown. Jurkat cells were exposed to IFN-α before RT-PCR. Unexpectedly, IFN-α failed to induce IRF-5 (Fig. 4A). However, treatment of these cells with IFN-α significantly increased the levels of other IFN-stimulated genes, MxA and 2–5 AS.

To assess the potential role of IRF-5 in initiating HTLV-1-infected cell-characteristic gene expression in T cells, Jurkat cells were transfected with control or IRF-5 expression plasmid. After 24 h of transfection, RT-PCR and immunoblotting were performed using specific primers, and anti-IRF-5 and anti-Myc antibodies to confirm transgene expression (Fig. 4B). In these experiments, changes in IRF-5-induced gene expression were analyzed by microarray analysis. The analysis showed significant enrichment of the up- and downregulated genes in IRF-5-transfected T cells. Table II shows genes that were upregulated by at least 5-fold in the presence of IRF-5. Previous studies reported that HTLV-1 infection induces T-cell activation and in vitro spontaneous lymphocyte proliferation, leading to the production of high levels of TNF-α in non-stimulated PBMC (43). Typical features of HTLV-1-infected T cells were recapitulated by IRF-5 activity, such as upregulation of TNF-α and LT-β (Table II and Fig. 4B).

HTLV-1-infected T-cell lines constitutively secrete TNF-α (44). Furthermore, TNF-α expression is activated in the arthritic joints of Tax transgenic mice compared with the normal joints of non-transgenic mice (45). In the next set of experiments, we examined the effects of Tax on TNF-α expression in T cells. As expected, TNF-α was induced in Tax-expressing JPX-9 (Fig. 5A). Next, we used several T-cell lines to analyze the role of IRF-5 in the expression of type I and II IFN, and TNF-α. Compared to control T-cell lines, HTLV-1-infected T-cell lines expressed high levels of TNF-α (Fig. 5B). Similar to control T-cell lines, HTLV-1-infected T-cell lines also constitutively expressed IFN-α and -β. IFN-γ expression was not associated with HTLV-1 infection. There was no correlation between IFN and Tax expression in HTLV-1-infected T-cell lines. The constitutive expression of TNF-α tended to be associated with IRF-5 or Tax expression.