A total of 5 patients with HAM and 5 healthy donors were included in the present study between April 2013 and December 2016 at Kansai Medical University Hospital (Hirakata, Japan). All patients with HAM were of Japan. Another 5 healthy subjects were included in the normal control group. The age and sex composition were not significantly different (P>0.05; Table I). Human leukocytes (derived from patients with HAM and normal donors) were separated from the peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation using Ficoll Histopaque. The cells were maintained in a RPMI-1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. All procedures were approved by the Ethics Committee of Kansai Medical University. Written informed consent was obtained from all patients or their families.

Female 4-week-old NOD/Shi-scid/IL-2Rγc null (NOG) mice were purchased from the Central Institute of Experimental Animals (Kawasaki, Japan). Mice were handled under sterile conditions and maintained in germ-free isolators. The Animal Care Committees of Kansai Medical University approved all animal experiments. Five-week-old NOG mice were sublethally irradiated with 250 cGy from a 137Cs source (Gammacell 40 exactor; Nordion International). Within 24 h of irradiation, each mouse was injected with 5×10⁴ human CD133⁺ cells by intrabone marrow injection. After 5–6 months, the HTLV-1-infected T-cell line JEX28, HTLV-1 molecular clone pX1 MT-M was prepared by electroporation into jurkat cells, was irradiated with 10 Gy from a 137Cs source irradiator. Then, the irradiated JEX28 cells (2.5×10⁶) or phosphate-buffered saline were intraperitoneally inoculated (Table II). All infections were performed in a bio-safety P2A Laboratory level in accordance with the guidelines of Kansai Medical University.

The RNeasy kit (Qiagen, Inc., Valencia, CA, USA) was employed for the isolation of human total RNA. A concentration of 0.5 µg of total RNA was used for the synthesis of complementary DNA (cDNA) samples by the reverse transcriptase ReverTra Ace (MMLV Reverse Transcriptase RNase H-; Toyobo Co., Ltd., Osaka, Japan). The RNA concentration was determined by using an IMPLEN NanoPhotometer. Further, humanized mouse RNA was isolated by using the ZR-Duet™ DNA/RNA MiniPrep kit, and humanized mouse cDNA samples were synthesized using the same method as in human cDNA synthesis.

The primers used for RT-PCR are listed in Table III. cDNA levels were quantified by using the RT-PCR method (MyiQ Single color real-time PCR detection system; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reactions were performed in 96-well plates each of which containing 10 µl of SYBR premix Ex Taq GC (Takara Bio, Inc., Otsu, Japan). The cycling conditions used were as follows: initial denaturation at 95°C for 10 min, followed by 15 cycles at 95°C for 10 sec and 60°C for 30 sec, and 35 cycles at 95°C for 10 sec and 59°C for 30 sec. The relative RNA expression levels were calculated by the MyiQ system (Bio-Rad Laboratories, Inc.).

Genomic DNA was extracted from splenocyte or peripheral blood by using ZR-Duet™ DNA/RNA MiniPrep kit (Zymo Research Corp., Irvine, CA, USA). Proviral loads (PVLs) were measured by real-time PCR using the previously described protocol with some modifications (TaqMan Fast Advanced Master Mix; Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) (MyiQ or CFX96 real-time PCR system; Bio-Rad Laboratories, Inc.) (13). The primers and probes targeting HTLV-1 pX and human β-globin (HBB; as an internal control) are listed in Table III. PVLs were calculated by the following formula: [(copy number of pX)/(copy number of HBB/2)] × 100.

RNA was extracted from each sample and purified by using an RNeasy mini kit (Qiagen, Inc.) following the manufacturer's instructions. RNA labeling and hybridization were performed using a commercial kit from Hokkaido System Science Co., Ltd., Sapporo, Japan. A 200-ng aliquot of total RNA was converted to double-stranded cDNA, and Cyanie3-labeled cRNA was produced using the Low Input Quick Amp Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The resulting labeled cRNA was purified by using the RNeasy mini kit (Qiagen, Inc.) and fragmented into strands in length in accordance with the protocols of the Agilent Technologies, Inc. These strands were then hybridized to GeneChip Array, and the hybridization data were analyzed using SurePrint G3 Human GE 8×60k 1color (Agilent Technologies, Inc.).

Mice with a body weight of below 17 g were euthanized. Single-cell spleen suspensions were prepared as described previously (14). To stain surface markers, anti-human CD45-PerCP or APC-Cy7, CD3-fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-Cy7, CD4-PE, CD25-FITC antibodies were used, along with mouse immunoglobulin G1 and FITC as isotype controls (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometric analysis was performed on a BD FACSCan for 3-color staining and a BD FACSCant II (BD Biosciences) for 7-color staining. The CellQuest and Diva software programs were used for data acquisition (BD Biosciences), and the collected data were analyzed by using FACS express 3 (De Novo Software, Piscataway, NJ, USA). T cells expressing human CD4⁺, CD8⁻, and CD25 were sorted from splenic mononuclear cells by using FACSAria or FACSAria III (BD Biosciences).

Mann-Whitney U-test and Wilcoxon's signed-rank test were used to determine the significance of differences and data comparison. P<0.05 was considered to indicate a statistically significant difference.

The APOBEC superfamily proteins has several and important functions in human health and disease (15). We first analyzed the mRNA expression of the APOBEC3 superfamily of in vivo samples from healthy donors and patients with HAM. However, we found no significant differences between these groups (Fig. 1A). HTLV-1 tax is known as a transactivator that can activate many genes (16) and its expression is suppressed in vivo (17). Therefore, we hypothesized that the tax might influence the expression of the APOBEC3 superfamily. As the HTLV-1 tax expression increased after 24 h of ex vivo culture, we compared the mRNA expression of the APOBEC3 superfamily in the samples of patients with HAM before and after the ex vivo culture. After culture, tax expression was apparently increased as well as that of A3C, A3D, A3F, and A3G. On the other hand, there was a tendency of decreasing A3B expression (Fig. 1B). As a negative control, we conducted the same experiment by using samples from healthy donors. We found that after 24 h of ex vivo culture, A3A expression was decreased; a tendency of decrease was also exhibited by the level of A3B expression. However, no changes were observed in the expression of A3C, A3D, A3F, A3G, and A3H (Fig. 1C). There are reports that A3B is a mutagen (8,9), whereas ATL is known as a malignant disease that is caused by HTLV-1 infection. Therefore, A3B expression might be increased in HTLV-1-infected individuals. However, these results did not show the tendency. HTLV-1 infection might not influence A3B expression because we used samples from the patients with HAM in the first experiment, which is an inflammatory disease not a malignant disease. Recently, we developed a humanized mouse model similar to ATL (13). For further research, we used PBMCs that were obtained from uninfected humanized mouse and 4–6 weeks postinfected humanized mouse and analyzed the mRNA expression of the APOBEC3 superfamily in these humanized mouse models. Although no significant changes in the expression of A3C, A3D, A3F, and A3G were observed, an increase in the level of expression of A3B and a decrease in the level of expression of A3A was noted. In contrast, the level of expression of A3H was significantly increased (Fig. 2A). As it is difficult to extract sufficient qualities of PBMCs from the animals in humanized mouse model, we used splenocytes instead of PBMCs for ex vivo culture experiments. After 24-h ex vivo culture, the levels of the expression of tax and A3A apparently increased. There was a tendency of decreasing A3B expression and increasing A3C, A3D, A3F, A3G, and A3H expression (Fig. 2B). Almost the data, except A3A, showed the same tendency with the samples from the patients with HAM.

The levels of the expression of A3B did not change in patients with HAM (Fig. 1A). In contrast, it decreased after ex vivo culture in both the samples from patients with HAM and infected humanized mouse model (Figs. 1B and 2B). On the other hand, there was a tendency of increasing A3B expression in the in vivo humanized mouse samples (Fig. 2A). It was too short to investigate the change in A3B expression, because we used the ATL-like humanized mouse model samples with 4–6 weeks post infection. Therefore, we analyzed the long-term humanized mouse model 18 weeks after HTLV-1 infection. Initially, we checked the phenotype of the humanized mouse splenocytes by using FACS. In the infected mice, the expression of CD4⁺ CD25⁺ cells was higher compared with that in the uninfected (Fig. 3). When the ATL-like humanized mouse model was developed, CD4⁺ CD25⁺ cells was quite increased (13). It is hard to collect sufficient quantities of CD4⁺ CD25⁺ cells from uninfected control mice because this population is exceedingly small. There is a report that CD4⁺ CD25⁻ cells are also infected with HTLV-1 in HTLV-1-infected individual (18). Therefore, instead of CD4⁺ CD25⁺ cells, we analyzed and compared CD4⁺ CD25⁻ cells. Gene expression array of CD4⁺ CD25⁻ splenocytes showed a 16.3-fold increase of CADM1 and a 12.6-fold increase of APOBEC3B levels in the long-term humanized mouse model after HTLV-1 infection compared with the uninfected one (Table IV). In the humanized mouse model, both CADM1 and APOBEC3B were increased in CD4⁺ CD25⁺ cells compared with those in CD4⁺ CD25⁻ cells. On the other hand, APOBEC3 superfamily expressions except APOBEC3B were not so increased in this humanized mouse model (Table IV). These data showed that APOBEC3B was increased in the ATL-like humanized mouse model as well as such as CADM1, which are ATL indicators (19,20).