Supplementary Materials and methods

Patient samples

Peripheral blood samples were collected from HTLV-1 carriers (n=72) and patients with smoldering-type (n=29), chronic-type (n=14), acute-type (n=27) and lymphoma-type ATLL (n=11). Blood samples were also obtained from 10 healthy volunteers as controls. A total of 90 lymph nodes were obtained from 36 patients with lymphoma-type ATLL, 15 patients with T-cell or natural killer (NK)-cell lymphomas, 37 patients with B-cell lymphomas and 2 patients with null-cell lymphomas. These samples were collected at the Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, as a collaboration with the Miyazaki University Hospital, National Miyakonojo Hospital, Kagoshima University Hospital and Imamura Bun-in Hospital. Informed consent was obtained from all of the patients. This study was approved by the Institutional Review Board at Faculty of Medicine, University of Miyazaki, in accordance with the Declaration of Helsinki. The diagnosis of ATLL was based on clinical features, hematological characteristics and monoclonal integration of the HTLV-1 provirus according to a Southern blot analysis. Peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol.

Cell lines

MOLT4, Jurkat, KOPT-K1, KAWAI and HUT78 are HTLV-1-negative human T-cell acute lymphoblastic leukemia (T-ALL) cell lines. BALM1 and BALL1 are B-cell acute lymphoblastic leukemia (B-ALL) cell lines. MT2 and HUT102 are human T-cell lines transformed by HTLV-1 infection. KOB, SO4 and KK1 are interleukin 2 (IL-2)-dependent ATLL cell lines. ED, Su9T and S1T are IL-2-independent ATLL cell lines. The IL-2-dependent ATLL cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 JRU/ml recombinant human IL-2. HTLV-1-negative cell lines, cell lines transformed with HTLV-1 and IL-2-independent ATLL cell lines were maintained in the same medium without IL-2. Human embryonic kidney (HEK) 293 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS.

Reagents

PE-conjugated anti-human CD4 and FITC-conjugated anti-human CD25/IL2RA antibodies were purchased from Becton Dickinson (San Jose, CA, USA). PE-conjugated anti-FoxP3 antibody was purchased from Miltenyi Biotec (Bergich Gladbach, Germany). The chicken anti-CADM1 monoclonal antibodies 3E1 and 9D2 were from MBL (Nagoya, Japan). The anti-β-actin (AC-15) antibody was from Sigma-Aldrich.

Cell separation

To purify CADM1+ T cells from PBMCs, magnetic cell sorting using positive selection was used according to the manufacturer's protocol (Miltenyi Biotec). Briefly, PBMCs were isolated by density gradient centrifugation over Histopaque. The cells were resuspended in MACS buffer (phosphate-buffered saline (PBS), 0.5% BSA and 2 mM EDTA), pre-incubated with 6.25 µg/µl of human immunoglobulin (Kenketsu glovenin-I-Nichiyaku, Nihon Pharmaceutical, Tokyo, Japan) in MACS buffer for 15 min at 4°C and then reacted with the biotin-conjugated anti-CADM1 antibody (035-212) for 15 min at 4°C. After the cells were washed, they were incubated with 10 µl of Anti-Biotin microbeads per 107 cells for 15 min at 4°C, washed again and passed through magnetic separation columns (MACS, Miltenyi Biotec). The labeled cells were collected from the magnetic positive fraction.

Fluorescence activated cell sorting (FACS) analysis

A total of 5 x 105 cells were washed with MACS buffer and incubated with the anti-CADM1 antibody (9D2, MBL) for 15 min on ice. After washing, the cells were incubated with the Alexa 488-conjugated goat anti-chicken IgY antibody for 15 min on ice. The cells were then washed, incubated with a PE-conjugated anti-CD4 antibody for 15 min on ice and immediately analyzed by FACS (FACScan, Becton-Dickinson) with the Cellquest software. The blood samples were also analyzed by SRL Inc. (Tokyo, Japan).

Preparation of recombinant CADM1 protein

The DNA fragment encoding human CADM1 including the extracellular domain (amino acid resides; 45-373) was amplified by polymerase chain reaction (PCR) from the plasmid pMSCV-CADM117 using the following primers: forward, 5’- GCCATATGCAGAATCTGTTTACGAAAGAC-3’; and reverse, 5’- CGGAGCTCTCCACTGCCCTGATCGAGCCTTT-3’. The amplified product was digested with NdeI and XhoI and cloned into the corresponding sites of the pET28a (+) vector (Novagen, Madison, WI, USA) to give an N- and C-terminal His-tagged CADM1 expression plasmid (pCADM1/N). The construct was sequenced to ensure that no errors had been introduced. The recombinant CADM1 protein was expressed in the E. coli strain BL21 (DE3) pLysS (Novagen). Protein expression was induced by the addition of 0.6 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the recombinant protein was purified using Ni-NTA agarose resin (Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer's protocol.

Preparation of the CADM1 monoclonal antibody

Recombinant His-tagged CADM1 protein was used to immunize mice for the production of hybridomas. Twelve hybridomas that produced monoclonal antibodies against CADM1 protein were selected by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Clone 1-10C was used in immunohistochemical studies.

Preparation of a soluble form of CADM1

To construct a mammalian secretion expression plasmid for soluble CADM1 with a C-terminal His-tag, a cDNA encoding amino acids 1-374 of human CADM1 that lacks the transmembrane and cytoplasmic domains was amplified by PCR from KK1 cDNA with the following primers: 5′-CCGCTCGAGACATGGCGAGTGTAGTGC-3′ and 5′- GGAATTCTCAGTGATGATGATGATGATGATCCACTGCCCTGATCGAGCC-3′. The PCR product was digested with XhoI and EcoRI and inserted into the corresponding sites of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) to make psCADM1-His. HEK293 cells were transfected with the psCADM1-His construct using a transfection reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. After transfection, the cells were cultured in the presence of 400 µg/ml G418 for 3-4 weeks to select stable clones. Positive clones derived from single G418-resistant cells were then isolated by cloning rings and further grown in the same conditions. After incubation with CD hybridoma medium for 48 h, the conditioned medium was collected, and His6-tagged CADM1 in the supernatant was purified using a Ni-NTA resin.

Determination of HTLV-1 proviral DNA by semiquantitative PCR

HTLV-1 HBZ gene amplification was carried out in a 20 μl PCR mixture consisting of 12.5 pmol of each primer, 200 µmol dNTPs, 2 ml 10X PCR buffer, 0.2 ml ExTaq Polymerase (Takara, Tokyo, Japan) and 100 ng of DNA extracted from cell lines and clinical samples. The PCR cycles consisted of an initial denaturation of 96°C for 2 min, 35 cycles of 96°C for 30 s, 57°C for 30 s and 72°C for 100 s, followed by 72°C for 5 min. The PCR products were run on 2% agarose gels and visualized by ethidium bromide staining. The b-actin gene (ACTB) was used as an internal reference control. The primer sequences used were as follows: HBZ forward, 5'-CGCTGCCGATCACGATG-3'; HBZ reverse, 5'-GGAGGAATTGGTGGACG-3'; ACTB forward, 5'-CGTGCTCAGGGCTTCTT-3'; and ACTB reverse, 5'-tgaaggtctcaaacatgatctg-3'. Amplifications with these pairs of oligonucleotides yielded 177 bp HBZ and 731 bp ACTB fragments. The S1T and KK1 cell lines were used as positive controls when the clinical samples were assayed. Jurkat cell lines were used as a negative reference control.

Western blot analysis

Cell pellet (5 x 104 cells) and blood serum (12.5 µg) samples were separated electrophoretically in an 8% SDS polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes. The membranes were then hybridized with anti-CADM1 monoclonal antibody (3E1, MBL), followed by horseradish peroxidase (HRP)-conjugated anti-chicken secondary antibody and visualized by chemiluminescence (Lumi-light Plus, Roche, Indianapolis, IN, USA).

Immunocytochemistry

Cells that were collected onto glass slides after the cytospin were fixed in methanol and treated with 1.5% H2O2 in Tris-buffered saline (TBS) for 10 min. The slides were pre-incubated in 1% bovine serum albumin (BSA) in TBS containing 0.1% Tween 20 (TBS-T) for 30 min at 37°C and then incubated with the anti-human CADM1 antibody (051-054IgG) in TBS-T for 1 h at room temperature. After washing with TBS-T and incubation with the HRP-conjugated anti-human IgG (H+L) secondary antibody (100x dilution in TBST) for 1 h at room temperature, visualization was performed using the DAB staining kit (Dako, Tokyo, Japan).

Immunohistochemistry

Tissues were fixed in 10% buffered formalin solution and embedded in paraffin blocks. Two-micrometer-thick sections were prepared. The slides were deparaffinized with xylene, rehydrated with solutions of ethanol at decreasing concentrations and rinsed once with PBS. Antigen retrieval was performed using a microwave, and then endogenous peroxidase activity was blocked in 3% hydrogen peroxide in methanol for 20 min. After blocking in 5% skim milk in PBS for 60 min, sections were incubated with the anti-human CADM1 monoclonal (1-10C) antibody for 30 min at 37°C, washed three times with PBS and incubated with the Dako Envision anti-mouse secondary antibody (Dako) for 30 min at 37°C. Horseradish peroxidase activity was visualized with 3, 3′-diaminobenzidine containing hydrogen peroxide. The negative control contained a normal mouse IgG instead of the primary antibody. The slides were counter-stained with Mayer's hematoxylin, dehydrated and cleared, followed by the application of a coverslip. The slides were evaluated by an experienced, board-certified pathologist on a four-tiered scale (0–3) for intensity and percentage of viable tumor cells that were positive (staining in more than 30% of cells was scored as 3+; staining in more than 5% but fewer than 30% of cells was scored as 2+; staining in fewer than 5% of cells was scored as 1+; and a lack of staining was scored as 0).

Statistical analysis

Student’s t-test and the Mann-Whitney U test were used to compare differences between the two groups. Spearman correlation coefficients were calculated to assess the association between two continuous variables.