Pancoll density gradient centrifugation

CD34⁺ hematopoietic stem cells (HSC) were isolated from the umbilical cord blood based on the procedure described the first time in 2011 [35 (link)]. Briefly, umbilical cord blood was collected postpartum in appropriate blood bags (Macopharma, Langen, Germany). Mononuclear cells (MNCs) were separated from cord blood by Pancoll density gradient centrifugation (PAN Biotech GmbH, Aidenbach, Germany) and washed twice in EDTA-PBS solution. CD34⁺ cell isolation was performed using immunomagnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. If the cells were not used immediately after separation, the CD34⁺ cell fractions were cryopreserved in medium containing 45% FCS and 10% DMSO until further use.

Murine splenocytes were isolated using Pancoll density-gradient centrifugation (Pan-Biotech). B cells were enriched using positive immunomagnetic selection with murine CD19 microbeads (Miltenyi Biotec) according to manufacturer's protocol. Murine T cells were purified by negative selection using EasySep Mouse T Cell Enrichment Kit (Stem Cell Technologies) according to manufacturer's protocol.

All tumors included in this study, obtained from French and Portuguese institutions, were classified according to the criteria of the World Health Organization–European Organization for Research and Treatment of Cancer (WHO-EORTC) [37 ].
Tumor DNA and peripheral blood samples from 61 patients were collected from the dermatology department at Bordeaux University Hospital Center (CHU) (France) as well as 33 representative formalin-fixed paraffin-embedded (FFPE) samples from the pathology archives at Instituto Português de Oncologia de Lisboa (IPO-L) and Centro Hospitalar Vila Nova de Gaia/Espinho (Portugal). A total of 94 tumor samples were analyzed (29 ≤ age ≤ 87, mean age 65), including 22 LPDs (14 cALCL and 8 LyP), 39 MF (24 classic MF and 15 T-MF), and 33 SS. The institutional review board approved the manipulation of the CTCL patients’ samples (DC-2015-412). Peripheral blood from 101 healthy donors (24 ≤ age ≤ 85, mean age 60) was obtained from the Etablissement Français du Sang, Bordeaux (DC 2015 2412-18PLER012), and CHU of Bordeaux (France). Peripheral blood mononuclear cells (PBMC) from patients and healthy donors were isolated by PANCOLL^® density gradient centrifugation (PAN-Biotech, Aidenbach, Germany).

The study used 9 blood samples from 8 healthy donors (HDs; 4 men and 4 women; mean age, 39 years) and 25 blood samples from 21 patients with HAM (5 men and 16 women; mean age, 69 years). Patients with HAM were diagnosed according to World Health Organization guidelines (Osame, 1990 ). PBMCs were separated using Pancoll^® density gradient centrifugation (density: 1.077 g/mL; PANBiotech GmbH, Aidenbach, Germany). The separated PBMCs were frozen in cryopreserving fluid (Cell Banker 1; Mitsubishi Chemical Medience Corporation, Tokyo, Japan) and stored in liquid nitrogen. This study was approved by the Bioethics Committee of the St. Marianna University School of Medicine (Approval ID No. 1646). All participants gave their written informed consent.

PBMCs were isolated by Pancoll density gradient centrifugation (PANbiotech, Aidenbach, Deutschland). CD14⁺ monocytes were isolated by magnetic labeling followed by negative magnetic selection (Pan Monocyte Isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the obtained monocyte fraction was verified by immunostaining. Monocytes were then cultured at 37 °C in AIMV-Albumax medium (Thermofisher Scientific, Walltam, Massachusetts, USA) at the concentration of 1 × 10⁶ cells/mL, supplemented with human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (1000 UI/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) and human Interleukin 4 (IL-4) (400 UI/mL; Miltenyi Biotec) (=control condition) or GM-CSF, IL-4 and human HGF (=HGF condition) (20 ng/mL; Miltenyi Biotec). On day 3, the medium was changed, and fresh cytokines were added at the same concentrations. On day 6, culture supernatants and obtained cells were collected. Cytokine dosage and cellular immunostaining were then performed (see corresponding paragraphs). In 7 independent experiments, to provide a maturation signal to DCs, Lipopolysaccharide (LPS) (1 μg/mL; Sigma–Aldrich, Saint-Louis, MI, USA) or control PBS was added to the culture. After 24 h, culture supernatants and cells were collected.

Peripheral blood mononuclear cells, serum and CSF samples were prepared as described previously (Sato et al., 2013 (link)). Briefly, PBMCs were isolated with standard procedures using Pancoll^® density gradient centrifugation (density 1.077 g/mL; PAN-Biotech GmbH, Aidenbach, Germany). Serum was obtained from venous blood samples by centrifugation after clotting. CSF was obtained by lumbar puncture. A small amount of CSF was used for routine laboratory tests, which included total protein, glucose, and cell counts. The remaining CSF was aliquoted into cryotubes and stored at -80°C until undergoing further analysis. The serum concentration of soluble IL-2 receptor (sIL-2R) was determined using a chemiluminescent enzyme immunoassay (LSI Medience Corporation, Tokyo, Japan). HTLV-1 proviral load was measured using real-time PCR, following DNA extraction from PBMCs, as previously described (Yamano et al., 2002 (link)). The anti-HTLV-1 antibody titer in CSF was determined using the gelatin particle agglutination test (Serodia-HTLV-1; Fujirebio, Tokyo, Japan). CSF neopterin level was measured using high-performance liquid chromatography at a commercial laboratory (SRL Inc., Tokyo, Japan). CXCL10 in CSF was measured using a cytometric bead array (BD Biosciences, Franklin Lakes, NJ, United States).