Ficoll pacque plus

Peripheral blood mononuclear cells were isolated from whole blood using Ficoll-Pacque Plus (GE Healthcare). CD4⁺ T cells were subsequently enriched using the EasySep human CD4⁺ T cell isolation kit (StemCell Technologies) and activated using Dynabeads human T activator CD3/CD28 microbeads (Gibco). Activated CD4⁺ T cells were cultured in RPMI 1640 (HyClone) supplemented with 20% heat-inactivated FBS (HyClone), 2 mM l-glutamine (HyClone), 100 μg/ml Primocin (Invivogen), and 30 U/ml interleukin-2 (provided by Maurice Gately, Hoffmann-La Roche Inc., through the NIH AIDS Reagent Program). Infections were performed 3 days postactivation by spinoculation in the presence of 40 μg/ml Polybrene (Millipore).

Peripheral blood from healthy donors was obtained from buffy coats provided by the Red Cross donor center (Red Cross-Flanders, Mechelen, Belgium), and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll Pacque PLUS, GE Healthcare, Amsterdam, The Netherlands). The Pan-DC enrichment kit (Miltenyi biotech) was used to isolate DCs from the PBMC. From the remaining PBMC fraction, peripheral blood lymphocytes (PBLs) were depleted from CD14+ monocytes using CD14+ immunomagnetic selection (CD14 Reagent, Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The CD14-depleted cell fraction (i.e., peripheral blood lymphocytes (PBLs)) was cryopreserved in FBS supplemented with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, Bornem, Belgium) and stored at −80 °C for later use in an allogeneic mixed leukocyte reaction (allo-MLR).

Blood samples were collected within 72 h of delivery and at three and six months postpartum. At each collection, 10 mL of maternal blood was collected by venipuncture into EDTA Vacutainers, and plasma was isolated by centrifugation (3000 rpm, room temperature, 10 min). Aliquots were stored at −80 °C for subsequent cytokine analysis in batch assays. Another 10 mL of maternal blood was collected into lithium heparin vacutainers, and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll^® Pacque Plus (GE Healthcare Life Sciences, Mississauga, ON, Canada). The PBMCs were collected and washed with 1X PBS twice. Pellets were re-suspended in 10% DMSO/FBS and stored at −150 °C until immune cell phenotyping in batch assay. Additional maternal blood was collected for C-reactive protein, fasting glucose, HbA1C, LDL, HDL, triglycerides and total cholesterol quantification, and urine was collected for albumin:creatinine ratio by the hospital laboratory.