ECFC and T cell isolation
Human samples were used in compliance with the declaration of Helsinki. CB samples from healthy full term newborns were obtained from the CB Bank of St Louis Hospital (Paris, France) which is authorized by the French Regulatory Authority (no. PPC51). Human APB from healthy adults was obtained from the French Establishment of Blood (EFS, authorization 14/5/011). Legal age to give blood ranges from 18 to 70 years. This activity was declared to and authorized by the French Ministry of Research under number AC- 2008-376, and to the French Organization for standardization under number 201/51848.1. Mononuclear cells (MNCs) were obtained by density gradient centrifugation using Pancoll human solution (Pan-Biothech).
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described.28 ECFC colonies appeared after 7–20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm2 and grew in EGM-2MV medium (Lonza).
Pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3+ T cells from MNCs. Furthermore, CD25+ cells were depleted from the CD3+ T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations. The resulting CD3+CD25- T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.
Co-Culture of ECs and T cells
Human CB-ECFCs (P3 to 7) or ABP-ECFCs (P3 to 5) were seeded into 6 or 12 well plates (Falcon) and incubated at least for 3h in EGM2 medium. Then, freshly isolated human T cells were added to ECFCs at different doses depending on experimental conditions in RPMI medium (containing 10% FBS, 1% HEPES buffer, 5x10−5 M β-mercaptoethanol and 1% penicillin/streptomycin/neomycin (Gibco)). All co-culture and control experiments were performed in 50% EGM2 and 50% RPMI media at 37°C in 5% CO2.
To prime ECFCs, we pre-treated them for 24 hours (day -1) with 0.01, 0.1, 1, 10, 50 and 100 ng/ml of premium grade recombinant-human-TNFα (Miltenyi-Biotec). In order to block TNFR2, we used 2 µg/ml human-TNFR2/CD120b/TNFRSF1B neutralizing antibody (Sino Biological), 24 hours prior TNFα addition (day -2).
T cell proliferation assay
3x104 ECs (APB- ECFCs, CB- ECFCs) were co-cultured with 6 increasing doses of human CD3+CD25-responder T cells in a total volume of 1 ml. The doses were 1/1, 1/2, 1/4, 1/8, 1/16, 1/32 (ECFCs/T cells). T cells were stained with Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen™) and polyclonaly stimulated by Dynabeads human T-activator CD3/CD28 according to supplier’s protocol (Gibco). 1x105 CFSE labelled, activated or non-activated T cells alone were used as controls. After 3 days, T cells were collected and immunostained and the percentage of proliferating cells among CD4+ and CD8+ T cells was analysed by flow cytometric measurements. Divided cells were identified by the decrease in CFSE expression due to its dilution after each division. Events acquired on a LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using FlowJo software v10 (FlowJo-LLC).
T lymphocytes activation assay
3x104 CB or APB-ECFCs were seeded in 12-well plates and co-cultured with 1.8x105 activated human T cells (1/6 ECFC/T cell, fixed intermediate ratio) in a total volume of 1 ml. 1.8x105 activated and non-activated human T cells were used as controls. After 3 days, T cells were collected by gentle aspiration and immunostained with mixes of following antibodies: VioBlue-anti-CD4, Biotin-anti-CD8 or PE-Vio770-anti-CD8, APC-anti-GITR, Biotin-anti-CD25 or PE-anti-CD25, PE-Vio770-anti-ICOS, PE-anti-TNFR2 or FITC-anti-TNFR2 (Miltenyi-Biotec) and Streptavidin-PE-Cy5 (eBioscience) antibodies (Abs). Events acquired on a LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using FlowJo software v10 (FlowJo-LLC).
Apoptosis measurement assay
CB-ECFCs were pre-treated for 24 hours with 0.01, 0.1, 1, 10, 50 and 100 ng/ml of TNFα. Untreated CB-ECFCs from the same donors and passages were used as control group. Then, cells were detached using cell dissociation reagent TrypLE (Gibco). Percentage of apoptotic cells were evaluated after immunostaining by annexin V and popidium iodide (PI) antibodies using Annexin V-FITC kit (Miltenyi-Biotec) according to the supplier’s protocol. Events acquired on a LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using FlowJo software v10 (FlowJo-LLC).
Endothelial principle and injury marker measurement
CB-ECFCs were pre-treated for 24 hours with 0.01, 0.1, 1, 10, 50 and 100 ng/ml of TNFα. Untreated CB-ECFCs from the same donors and passages were used as control group. Then, cells were detached using cell dissociation reagent TrypLE (Gibco) and proceeded to immunostaining using mixes of following antibodies: FITC-anti-CD31, VioBlue-anti-CD144, PE-Vio770-anti-VEGFR2 (KDR), APC-anti-TNFR1, PE-anti-TNFR2, APC-anti-ICAM (CD54), PE-anti-VCAM (CD106), APC-anti-TIE2 (CD202b), REA control (s)-APC, REA control (s)-PE (Miltenyi-Biotec). Events acquired on a LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using FlowJo software v10 (FlowJo-LLC).
Statistical analysis
Prism (GraphPad) was used for statistical analysis. Shapiro-Wilk normality test was performed to assess the normal distribution of data. Student t test or 1-way ANOVA with post hoc analysis was performed depending on the number of comparatives. For cytometry analysis, we have normalized the MFI values with T-cell alone control group. Then we used unpaired, 2-tailed Student t tests or one way ANOVA for P value generation