Isolation and activation of human CD4+ T cell

Human CD4⁺T Cells were isolated from the umbilical cord blood collected from healthy neonates in Turku University Hospital, Turku Finland with approval from the Finnish Ethics Committee. Participants provided their verbal informed consent to participate in this study. This was considered more feasible and practical than a written consent. However, midwifes kept records of this consent. Moreover, this consent procedure has been approved by the Finnish Ethics Committee.

Briefly, mononuclear cells were isolated with Ficoll-Paque gradient centrifugation (GE Healthcare Biosciences AB, Uppsala, Sweden). CD4⁺ T cells were separated by using magnetic beads (Dynal CD4 Positive Isolation Kit, Invitrogen, Oslo, Norway).

All human CD4⁺T cells used in this study were pooled samples from 5–6 individuals. Each pool of cord blood CD4+ T cells samples were divided into two aliquots. Down-regulation of CIP2A expression in human CD4⁺ T cells were carried out by introducing CIP2A specific siRNAs in one aliquot (siRNA-1, 5’-CUGUGGUUGUGUUUGCACU-3; siRNA-2, 5’-GCACGGACACUUGCUAGUA-3; siRNA-3, 5’-GUACCACUCUUAUAGAACA-3’) and control non-targeting scramble siRNA into another aliquot of CD4⁺ T cells (4.5 μg siRNA/4x10⁶ cells) using Nucleofector (Amaxa Biosystems, Lonza), after which cells were rested for 48 hr before culturing. Cells were activated with plate-bound anti-CD3 (500 ng/24well culture plate, Immunotech, Marseille, France) and soluble anti-CD28 (1 μg/ml, Immunotech) in Yssel's medium [36] supplemented with 1% human AB serum (Red Cross Finland Blood Service). At 24 hr, cells were collected and CD69-FITC or isotype control (both from BD Biosciences) staining were performed. Expression of CD69 was detected by flow cytometer (FACS LSRII or FACSCalibur both from BD Biosciences) and analyzed by using flowing software. CFSE dilution experiment was performed as previously described [35]. For Western blot detections, cells were collected after 48 hr of activation with anti-CD3/anti-CD28 and were probed with the following antibodies: CIP2A [31], ETS1 (Santa Cruz Biotechnology), MYC (Covance, New Jersey), IRF4 (Santa Cruz Biotechnology), ERK1,2 (Cell Signaling Technology), and ß-actin (Sigma).