Fty720

EPOC was a 6-month, randomized, open-label, multicenter, phase 4 study conducted in the USA and Canada. Patients were randomized 3:1 to switch to fingolimod (FTY720; Gilenya®, Novartis Pharma AG, Basel, Switzerland) 0.5 mg or remain on/switch to an iDMT for 6 months with no intervening washout period. The primary analysis evaluated two groups, namely fingolimod versus any iDMT. Patients randomized to the iDMT group either remained on the same therapy or, following consultation with a physician, were switched immediately to another approved iDMT. The four iDMTs were subcutaneous (SC) IFN beta-1b (Extavia®, Novartis Pharma AG, Basel, Switzerland, or Betaseron®, Bayer AG, Leverkusen, Germany) 0.25 mg every other day, IM IFN beta-1a (Avonex®, Biogen Idec, Cambridge, MA, USA) 30 μg once weekly, SC IFN beta-1a (Rebif®, Merck Serono, Darmstadt, Germany, and Pfizer Inc., New York City, NY, USA) 22 or 44 μg three times weekly, or SC GA (Copaxone®, Teva Pharmaceutical Industries Ltd, Petah Tikva, Israel) 20 mg once daily. The protocol and informed consent form were reviewed and approved by an institutional review board (Quorum Review) at each study center, and every patient provided written informed consent.

FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol) and FTY720-P (2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, mono dihydrogen phosphate ester) were provided by Novartis. For in vitro experiments, powdered FTY720 and FTY720-P were reconstituted in dimethyl sulfoxide hydrochloric acid (DMSO)/50 mM HCl; for the intraperitoneal (ip) and icv injections, FTY720 was diluted in vehicle (saline solution). For the ip injection, FTY720 was freshly prepared every day. KA was from Abcam (Cambridge, USA); MK801 was from Tocris (Bristol, UK). NMDA, propidium iodide (PI) and LPS (Escherichia coli O11:B4) were from Sigma-Aldrich (St. Louis, USA). All cell culture supplies, if not otherwise indicated, were purchased from Gibco (Life Technologies, Madrid, Spain). Cytotoxicity 96 colorimetric assay for the quantification of lactate dehydrogenase (LDH) release was purchased from Promega Corporation (Madison, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ). Naïve T cells from PBMCs were isolated using a Miltenyi Biotec (Alburn, CA) negative selection kit. Purified naïve CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/ml, BD Bioscience, San Jose CA), soluble anti-human CD28 (1 μg/ml, BD Bioscience), and IL-2 (20 ng/ml, R&D Systems) with or without FTY720 (100 ng/ml, Novartis). After 6 days, cell-free culture supernatants were collected for cytokine analysis by Luminex assay (Miltenyi Biotec), and cells were harvested for RNA extraction and intracellular staining. Naïve T cells were stimulated with PMA (Sigma), ionomycin (Sigma), and Golgistop for 4 h. Cells were stained for anti-human CD4 APC (BD Bioscience) and violet fluorescent reactive dye VVD (Life Technologies), and then cells were fixed and permeabilized with BD fixation and permeabilization buffer and stained for IFN-γ FITC and GZMB FITC (BD Bioscience). For surface staining, the following antibodies were used: anti-human CD4 pacific blue, anti-human CCR7 PE, and anti-human CD45RA APC, IgG2a PE isotype control, IgG2b, and k APC isotype control (all from BD Bioscience). All antibodies were titrated for flow cytometry, which was performed on a BD LSR II (BD Bioscience) and analyzed using Flowjo software.

To inhibit the circulation of memory T cells, 1 mg/kg of FTY720 (Sigma, St. Louis, MO, USA) in PBS was intraperitoneally (i.p.) administered daily for 10 days. In addition, to assess the protective efficacy of the vaccines, mice were immunized with the same vaccine. Intravascular staining was performed by injecting mice i.v. with FITC-conjugated anti-mouse CD45 antibody (5 μg) for 8–10 min before euthanasia (Beckman Coulter, Villepinte, France). After immunization and FTY720 treatment (Novartis Pharma AG, Basel, Switzerland), intestinal tissue was collected and collagenase-digested, and cells were isolated for flow cytometry analysis as described previously. The lymphocytes were isolated from the intestine of mice 6 weeks after immunization. Antigen-loaded DCs treated or not with ICAM1 inhibitor were incubated with lymphocytes isolated from immunized mouse intestine at 37 °C for 3 days. Collected cells were stained with anti-CD3 antibody for 20 min and with HA_518–526⁺ tetramer for 1 h. Moreover, to assess whether HA-tetramer⁺-specific T cells were present in the intestine at 6 weeks after immunization, antigen-loaded DCs treated or not with ICAM1 inhibitor were incubated with IMALs isolated from immunized mice at 37 °C for 3 days.