Easysep t cell enrichment kit

Human DCs transfected with antigen (or control)-encoding RNA were used to stimulate T cell responses in vitro. PBMCs were thawed, washed, resuspended in PBS and treated with DNase I (Sigma) at 200 U/ml for 20 min at 37°C. The EasySep T cell Enrichment Kit (StemCell Technologies, Vancouver, Canada) was used to negatively isolate CD3+ T cells from DNase I-treated PBMCs. The isolated CD3+ T cells were stimulated with RNA-transfected, matured DCs at a responder to stimulator ratio (R:S) of 10:1 in the presence of 25 ng/ml IL-7 at 37°C. Stimulations were done in RPMI 1640 with 10% FCS, 2 mM L-glutamine, 20 mM HEPES, 1 mM Na-pyruvate, 0.1 mM MEM non-essential amino acids, MEM amino acids (50× solution), 100 IU/ml penicillin, 100 μg/ml streptomycin and 5 × 10⁻⁵ M β-mercaptoethanol (CTL stimulation medium). The responder T cell concentration was 2–2.5 × 10⁶ cells/ml. IL-2 was added on day 3 at 50–100 U/ml. On day 7, the T cells were restimulated with RNA-electroporated DC at a R:S ratio of 10:1 in complete RPMI-10% FCS in the presence of 50–100 U/ml IL-2, with the T cells at 1–2 × 10⁶ cells/ml in CTL stimulation medium. After 5–7 days the T cells were harvested, counted and used as effector cells in a europium-based CTL assay50 (link).

Atherosclerotic plaques were obtained from patients who were undergoing carotid endarterectomy at the Department of Surgery, Vascular Surgery, Södersjukhuset, Stockholm, Sweden. This study was pre-approved by the research ethics committee of Karolinska Institutet and in accordance with the Declaration of Helsinki. All subjects gave their written informed consent before entering the study.
Cells were isolated from atherosclerotic plaques as described earlier (22) . In brief, the plaques were first dissected into small pieces, which were then incubated with 1.25 mg/ml collagenase IV (Life Technologies Europe BV, Stockholm, Sweden), 25 μg/ml Liberase DL (Roche Applied Science, Stockholm, Sweden), and 0.2 mg/ml DNase I (Roche Applied Science, Stockholm, Sweden) for 1 h at 37°C. The dissociated plaque cells were then passed through a 100-μm Celltrics filter (Millipore AB, Stockholm, Sweden) to remove unwanted fat and debris, and T cells were purified with the EasySep T-cell enrichment kit (STEMCELL Technologies). DCs obtained from the peripheral blood of the plaque donors were treated with or without MDA-HSA as described above and thereafter co-cultured with plaque T cells for 48 h. In addition, plaque T cells were cultured in the presence or absence of MDA-HSA for 24 h.