Phytohemagglutinin p pha

Whole-blood samples were cultured overnight in RPMI medium supplemented with 20 U/mL IL-2 (Shionogi) and 100 ng/mL IL-4 (R&D Systems) for samples from CM 005 and CM 006 and with 20 U/mL IL-2 and 10 μg/mL phytohemagglutinin-P (PHA; Sigma) for samples from CM 003 and CM 007. Then, Tax antigen-expressing cells were analyzed by FCM. Whole-blood samples obtained at 4 weeks for CM 005 and CM 006 and at 52 weeks for CM 003 and CM 007 were used.

Human PBMCs from normal donors and PBMCs derived from a cynomolgus monkey were used as indicator cells. These indicator cells (2 × 10⁶ cells) were cocultured with mitomycin C (MMC)-treated HTLV-1-producing cells (2 × 10⁵ cells) in a volume of 0.5 mL/well in a 48-well plate (Falcon) in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 U/mL human IL-2 (Miltenyi), and 10 μg/mL phytohemagglutinin-P (PHA; Sigma) at 37°C for 2 weeks. The medium was changed for fresh medium without PHA two times per week. Then, the cells were stained for cell surface CD4 and HTLV-1 Tax antigen as reported previously (51 (link)). A syncytium formation assay was performed by coculturing HTLV-1-producing cells and HTLV-1-negative Jurkat cells. Equal numbers of cells were cultivated for the indicated time periods, and then CPE was confirmed by microscopy.

Human peripheral blood samples were obtained from healthy donors upon signature of an informed consent and following approval by the Ethic sub-commission of the Puerto Real University Hospital (dependent from the Central Quality Commission), in accordance to Spanish and European Union Regulations. PBMC were isolated by density gradient centrifugation using Lymphocyte separation medium (Eurobio™, Montpellier, France). Cells were washed three times with PBS, subsequently stimulated with 1 µg/ml phytohemagglutinin-P (PHA) (Sigma™, Saint Louis, Missouri, USA) and cultured in DMEM supplemented with 1% (v/v) sodium pyruvate, non essential aminoacids, vitamins, L-arginin, L-asparragin, folic acid, 10 mM Hepes, 50 µM 2-mercaptoethanol, 100 mg/ml streptomycin, 100 U/ml penicillin (Life Technologies, Carlsbad, CA, USA) and 10% heat-inactivated FBS (Gibco) at 37°C in a 10% CO₂ atmosphere. 40 U/ml IL-2 was added to the cultures every 48 hours, for a total of six days. Unless indicated otherwise cells were collected at day 7, washed with PBS and resuspended at 10⁶ cells/ml without IL-2.

phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni²+-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-Gel^TM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific. The source of antibodies and their catalog numbers were listed in Table 1.

Lymphocytes were isolated from lymph nodes (LN) of adult C57BL/6J mice (H2b haplotype). Cervical, axillary, brachial, inguinal, lumbar, renal, pancreatic, and mesenteric lymph nodes were collected and gently disrupted into cold PBS + BSA between frosted margins of glass slides. Cells were then filtered through a 40 µm cell strainer, centrifuged, and incubated into red blood cells lysis buffer (Sigma-Aldrich). Freshly harvested mouse LN lymphocytes were characterized by IF and FC. Additionally, LN lymphocytes from adult FVB/N mice (H2q haplotype) were also isolated to perform a two-way mixed lymphocyte reaction (MLR) with LN lymphocytes from C57BL/6J mice. In some experiments, mouse lymphocyte MLR cultures were additionally stimulated with 25 µg/mL phytohemagglutinin (PHA-P; Sigma-Aldrich) to promote a strong proliferative response as indicated in initial lymphocytes cultures settings performed by our laboratory. For proliferation assays, mouse lymphocytes were stained for 8 min with 8 µm CFSE (formally known as 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester of CFDA SE) (Biotium, Fremont, CA, USA) diluted into a DMEM low glucose basal media (Gibco, 21885) and used after extensive washing. Additionally, peritoneal fluid cells (principally macrophages and B cells) were isolated from adult C57BL/6J mice and used to test the immunosuppressive activity of OMC-S and MSC-S.

EZN-2968 (anti-HIF-1α) and EZN-3088 (negative control or Scrambled) were obtained from Enzon Pharmaceuticals Inc. (Piscataway, New Jersey) [35 (link)]. Recombinant human interleukin 6 (IL-6) and insulin like growth factor 1 (IGF-1) were purchased from R&D System (Minneapolis, MN, USA), whereas phytohemagglutinin (PHA-P) and cobalt chloride (CoCl₂) from Sigma-Aldrich (St. Louis, MO, USA).