This study was conducted at the Dohgo Spa Hospital, Toranomon Hospital, and Ehime University Graduate School of Medicine, and was approved by the clinical ethics committees of these institutions. SSc, rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM), osteoarthritis (OA) patients and healthy volunteers, who were provided adequate information and signed a written consent form, were included in this study. Patients who suffered from cutaneous cancer, hematologic cancer and any cutaneous infection were excluded. Clinical data were obtained from medical records. All SSc patients fulfilled the preliminary American College of Rheumatology (ACR) criteria for classification in 1980 . Patients with SSc were divided into those with limited and diffuse diseases according to a previous report . Interstitial pneumonia (IP) based on SSc was defined as the change in the bilateral basilar lung portions, which were detected with high-resolution computed tomography . The definition of scleroderma renal crisis (RC) was according to a previous report in 2007 . The existence of pulmonary artery hypertension (PAH) was demonstrated with echocardiography, and subsequent right heart catheterization was performed to confirm the diagnosis .
The skin specimens of SSc were collected from the forearms, and those of the control patients were collected from the forearms or lower legs. Formalin-fixed paraffin-embedded tissue sections were used for the IHC studies. The following primary antibodies were employed: anti-XIAP (rabbit polyclonal, Abcam, Tokyo, Japan), anti-cIAP (cIAP1 and cIAP2) (mouse monoclonal, clone number 681724, R&D Systems, Minneapolis, MN, USA), anti-survivin (rabbit polyclonal, R&D Systems), anti-survivin-ΔEx3 (rabbit polyclonal, Abcam), anti-survivin-2B (rabbit polyclonal, Abcam), anti-CD1a (mouse monoclonal, clone 7A7, Abcam), anti-CD4 (mouse monoclonal, clone CRRY77, eBioscience, Affymetrix Japan K.K., Tokyo, Japan) and anti-CD69 (rabbit polyclonal, Bioss, Woburn, MA, USA). Following deparaffinization and antigen retrieval in citrate buffer (pH 6.0), the specimens were incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies and then visualized with the 3-amino-9-ethylcarbazole (AEC) + kit (Agilent Technologies Dako, Tokyo, Japan). Multiple immunofluorescence staining was also conducted using an indirect method with Alexa Fluor-conjugated secondary antibodies (Invitrogen, Thermo Fisher Scientific, Yokohama, Japan). The slides were also stained with hematoxylin, Hoechst 33342 or 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI). The fluorescence was captured with an Olympus FV1000D laser scanning microscope (Olympus, Tokyo, Japan) and a Leica 6000E microscope (Leica microsystems, Tokyo, Japan).
Enzyme-linked immunosorbent assay (ELISA)
Three types of SSc-specific autoantibodies (anti-topoisomerase I (Scl-70), anti-centromere and anti-RNA polymerase III (RNAPIII)) were measured with MESACUP ELISA kits (Medical & Biological Laboratories, Nagoya, Japan). For the detection of serum anti-survivin immunoglobulin G (IgG) antibody, an ELISA was performed as previously described . Briefly, 96-well plates were coated with glutathione S-transferase (GST) fusion recombinant proteins of survivin-wild type (WT) (1,000 ng/ml), which were prepared as previously reported . The wells were blocked with Blocking One P (Nacalai Tesque, Kyoto, Japan). Serum samples were diluted 1:100 in Tris-buffered saline (TBS) containing Blocking One P and added to the wells. After incubation and washing, alkaline phosphatase-conjugated goat anti-human IgG antibody was added. Following additional washing, p-nitrophenyl phosphate (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) was added and measured at 405 nm on a spectrophometer. The mean + 2SD (standard deviation) among the healthy controls (HCs) (n = 15, data not shown) was defined as the cutoff value for anti-survivin IgG antibody.
Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR
Peripheral blood mononuclear cells (PBMCs) from SSc patients and HCs were enriched using Ficoll-Paque PLUS (GE Healthcare, Tokyo, Japan). Total RNA of the PBMCs or cultured cells was extracted and purified using TRIzol (Invitrogen) followed by cDNA synthesis using a PrimeScript RT Reagent Kit with a gDNA Eraser (Takara-Bio, Shiga, Japan). Quantitative real-time PCR using TaqMan probes (FAM/TAMRA) and SYBR Green was executed in triplicate using the ABI Prism 7500 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Yokohama, Japan). The HL-60 cell line was used as a positive control for RT-PCR. The primer pairs and probes for amplification of human BIRC5 variants and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were described previously . The upstream and downstream primer sequences used for interleukin (IL)-6 were 5′-GAGAAAGGAGACATGTAACAAGAGT-3′ and 5′-GCAAGTCTCCTCATTGAATCCA-3′, respectively. The upstream and downstream primer sequences for IL-12B were 5′-CCAGAGCAAGATGTGTCAC-3′ and 5′-CTACGACATAAACATCTTTCTTCAG-3′, respectively. The expression of human HPRT was evaluated as a housekeeping gene to normalize the ΔCt values. The relative expressions of the target genes were obtained using the difference in the comparative threshold (ΔΔCt) method.
Fluorescence-activated cell sorting (FACS)
A flow cytometric analysis was performed using the FACSCalibur and LSRFortessa platforms (BD Biosciences, Tokyo, Japan). For Ki-67 and 7-amino-actinomycinD (7-AAD) staining, the FOXP3 Staining Buffer Set (eBioscience) was used. Antibodies conjugated with FITC, PerCP or APC were used. Anti-human CD14 (clone MφP9), anti-human CD45 (clone 2D1) antibodies and 7-AAD were purchased from BD Biosciences. Anti-human CD1a (clone HI149), anti-human Ki-67 (clone Ki-67) antibodies and Fixable Viability Dye (FVD520) were obtained from eBioscience. The cells were washed and stained. For intracellular staining of survivin and its splice variants, normal rabbit IgG (Santa Cruz Biotechnology, TX, USA), anti-survivin (rabbit polyclonal, R&D Systems), anti-survivin-ΔEx3 (rabbit polyclonal, Abcam), and anti-survivin-2B (rabbit polyclonal, Abcam) were labeled using the Zenon Rabbit IgG Labeling Kit (Alexa Fluor 647, Invitrogen). The FOXP3 Staining Buffer Set (eBioscience) was used for fixation and permeabilization. A survivin-WT-specific antibody was not available, therefore, the mean fluorescence intensity (MFI) of the estimated survivin-WT expression level was calculated from other MFI parameters as follows: (survivin-WT) = (survivin (total) – normal IgG) – (survivin-ΔEx3 – normal IgG) – (survivin–2B – normal IgG).
Preparation of monocyte-derived dendritic cells (Mo-DCs)
Human monocytes were purified with biotin-conjugated anti-CD14 antibody (clone 61D3, eBioscience) and streptavidin microbeads (Miltenyi Biotec, Tokyo, Japan) from PBMCs, which were enriched using Ficoll-Paque PLUS (GE Healthcare). CD14+ monocytes isolation resulted in >95 % pure population. Mo-DCs were generated by culturing CD14+ monocytes in RPMI1640 medium supplemented with 10 % fetal bovine serum (Gibco, Thermo Fisher Scientific, Yokohama, Japan), 200 ng/ml rhIL-4 (R&D Systems) and 200 ng/ml rhGM-CSF (Wako Junyaku Kogyo, Tokyo, Japan). After 5 days of culture, CD1a+CD14− Mo-DCs (>95 % CD1a+ purity) were harvested for use.
Transfection for dendritic cells (DCs)
The transfection of in vitro transcribed (IVT) messenger RNA (mRNA) for DCs was performed using Lipofectamine MessengerMAX Transfection Reagent (Invitrogen). To generate templates for IVT mRNA, we developed the pcDNA3-A(124) vector plasmid and BIRC5-inserted vector plasmids from artificial custom genes, according to a protocol described previously [16, 17]. The transcription of pcDNA3-A(124)-based plasmids was performed with the mMESSAGE mMACHINE T7 transcription Kit (Ambion, Thermo Fisher Scientific, Yokohama, Japan). After in vitro transcription, mRNA was purified using the MEGAclear Kit (Ambion).
Cell lysates and recombinant proteins were processed using SDS-PAGE and Western blotting according to the standard procedures. The blotted membranes were incubated with anti-actin (mouse monoclonal, clone AC-15, Invitrogen) and anti-survivin antibodies followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). The GST-fusion recombinant proteins of survivin variants were prepared as previously reported .
The significance in the differences between the two groups was determined using an unpaired t test, the Mann–Whitney U test or the chi-square test. The data processing and analyses were conducted using the Microsoft Excel software program (Microsoft, Tokyo, Japan).