Multiple Integrations of Human T-Lymphotropic Virus Type 1 Proviruses in the Engrafted Cells from the Asymptomatic Carriers in NOD/SCID/cnull Mice

Objectives: Successful engraftment of human T-lymphotropic virus type 1 (HTLV-1)-infected cells and a marked increase of proviral DNA loads (PVLs) in non-obese diabetic/ severe combined immunodeficient (NOD/SCID)/ c null (NOG) mice have been reported. Whether the increased PVL in transplanted mice is due to the new infection of HTLV-1 was examined. Methods: Mononuclear cells from 3 NOG mice with primary engraftment from asymptomatic HTLV-1 carriers were transplanted into a second group of NOG mice. HTLV-1 PVL, proviral integration by fluorescence in situ hybridization assay, expression of viral antigen, and T-cell clonality were analyzed. Results: The PVLs in the secondarily transplanted NOG mice were significantly higher than those of primarily transplanted NOG mice. Multiple signals of HTLV-1 proviruses in the nucleus of the infected cells were revealed by fluorescence in situ hybridization analysis. Expression of HTLV-1 tax/rex mRNA and antigen was observed. The variety of T-cell clones was limited in the transplanted NOG mice. Conclusions: Multiple proviral integrations were Received: September 2, 2009 Accepted after revision: December 9, 2009 Published online: March 30, 2010 Akihiko Okayama, MD, PhD Department of Rheumatology, Infectious Diseases and Laboratory Medicine Faculty of Medicine, University of Miyazaki 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan) Tel. +81 985 85 7284, Fax +81 985 85 4709, E-Mail okayama @ med.miyazaki-u.ac.jp © 2010 S. Karger AG, Basel 0300–5526/10/0534–0229$26.00/0 Accessible online at: www.karger.com/int Yamamoto /Takajo /Umeki /Morishita / Hatakeyama /Kataoka /Nomura /Okayama Intervirology 2010;53:229–239 230 vivo. Non-obese diabetic (NOD)/severe combined immunodeficient (SCID) mice, such as the NOD/LtSzscid and the NOD/Shiscid mouse, have been considered appropriate models for this purpose [11–14] . We have shown the successful engraftment of HTLV-1-infected cells from asymptomatic carriers in NOD/SCID/ c null (NOG) mice [15] . In this previous study, HTLV-1 provirus was recognized only in the human mononuclear cells, which infiltrated into the various organs of transplanted NOG mice. The increased proviral DNA loads 6 100,000 copies per 100,000 human cells were shown. This proviral DNA load increasing may be due to clonal expansion of HTLV1-infected cells directly derived from the peripheral blood of asymptomatic carriers. Alternatively, transmission of HTLV-1 from the infected human cells to other human cells might occur in vivo. To clarify whether these speculations are true or not, we isolated the human cells in NOG mice with primary engraftment from HTLV-1 asymptomatic carriers, and inoculated them into the other NOG mice. The integration of HTLV-1 provirus into the chromosome of human cells was examined by fluorescence in situ hybridization (FISH) assay. To evaluate whether there is an active replication of HTLV-1 or not, the expression of tax/rex mRNA and HTLV-1 antigen was also tested. In addition, the rearrangement of the T-cell receptor(TCR) gene that reflects the clonality of T cells [16] was compared between mice with primary and secondary engraftment. The results suggested that the transmission of HTLV-1 from infected cells to other cells occurred efficiently in NOG mice and only limited fraction of T cells seemed to have selectively survived. Materials and Methods Subjects Three asymptomatic HTLV-1 carriers (carrier A, B, and C) were recruited into this study after obtaining written informed consent. The white blood cell counts of the HTLV-1-infected carriers were within the reference value without any abnormal cells. The peripheral blood mononuclear cells (PBMCs) were isolated from carriers A, B, and C by density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich, St. Louis, Mo., USA), washed three times with phosphate-buffered saline (PBS) and preserved with Cell banker  (JUJI Inc., Tokyo, Japan) at –80 ° until use. The study protocol was approved by the Institutional Review Board of University of Miyazaki. Inoculation of Human PBMCs from HTLV-1 Carriers into the NOG Mice NOG mice were purchased from the Central Institute of Experimental Animals (Kawasaki, Japan). All mice were bred and maintained under specific pathogen-free conditions in the Department of Bioresources, Division of Biotechnology, Frontier Science Research Center, University of Miyazaki (Miyazaki, Japan). Female NOG mice aged from 8 to 10 weeks were used for the experiments. A total of 5 ! 10 6 human PBMCs from carriers A, B, and C were injected intraperitoneally into one half of them and intravenously into the other half (primary transplantation, NOG A-1, B-1, and C-1, respectively). The mice were sacrificed 4 weeks after inoculation. Mononuclear cells were isolated from the livers of NOG A-1, B-1, and C-1 using density gradient centrifugation. More than 99% of these mononuclear cells were T cells (data not shown). A total of 3 ! 10 6 cells were inoculated into new NOG mice (secondary transplantation, NOG A-2, B-2, and C-2, respectively). The secondarily transplanted NOG mice were sacrificed 4 weeks after inoculation. The experimental protocol was approved by the University of Miyazaki ethics review committee for animal experimentation. Quantification of Human Cells and HTLV-1 Provirus Chromosomal DNA was isolated from the PBMCs of carriers and mononuclear cells obtained from the livers of NOG mice by sodium dodecyl sulfate proteinase K digestion at 56 ° , followed by phenol-chloroform extraction and ethanol precipitation. HTLV-1 proviral copy number (i.e. proviral DNA load) was measured by real-time polymerase chain reaction (PCR) using LightCycler DX 400 (Roche Diagnostics, Mannheim, Germany) as described previously [15] . In brief, quantitative real-time PCR was performed for human albumin DNA, mouse GAPDH DNA, and HTLV-1 provirus. The primers and probe for the human albumin gene were as follows: the forward primer Alb-S (5 -GCTGTCATCTCTTGTGGGCTGT-3 ), the reverse primer Alb-AS (5 -AAACTCATGGGAGCTGCTGGT-3 ), and the FAM-labeled albumin TaqMan probe (5 -FAM-CCTGTCATGCCCACACAAATCTCTCC-TAMRA-3 ). TaqMan Gene Expression Assays (Applied Biosystems Japan, Tokyo, Japan) were used for the primers and the probe for the mouse GAPDH gene. The primers and the probe for the pX region of HTLV-1 were as follows: the forward primer pX2-S (5 -CGGATACCCAGTCTACGTGTT-3 : positions 2359–2379), the reverse primer pX2-AS (5 -CAGTAGGGCGTGACGATGTA-3 : positions 7458–7439), and the FAM-labeled pX2 probe (FAM-CTGTGTACAAGGCGACTGGTGCC-TAMRA : positions 7386–7408). Quantitative PCR was performed in a duplicate manner. FISH Analysis of HTLV-1-Infected Cells FISH analysis was performed to detect HTLV-1 proviral signals in the mononuclear cells isolated from the livers of NOG mice according to a slightly modified version of the method reported by Taniwaki et al. [17] . For FISH assay probes, PCR products prepared using four different primer sets for HTLV-1, which cover almost the entire genome of HTLV-1, were used. Primer sets used for PCR were as follows: region 1: the forward primer (HTLV23F 5 -TGACAATGACCATGAGCCCCAA-3 : positions 23–44), and the reverse primer (HTLV2060R 5 -CTAATAGGAGGGCATCTTCCTC-3 : positions 2060–2039), region 2: the forward primer (HTLV2013F 5 -AGCCCACTATCCCAGAACCAGA-3 : positions 2013–2034), and the reverse primer (HTLV4741R 5 -CGAATAGCAAGGGAGGTACACA-3 : positions 4741–4720), region 3: the forward primer (HTLV4562F 5 AGACACCTTTTCAGGAGCCATC-3 : positions 4562–4583), and the reverse primer (HTLV7292R 5 -AGGGCTGTTTCGAT-