Human ab serum

The capacity of T cells to proliferate upon stimulation with recall antigens was assessed in a 3-d proliferation assay with memory response mix (MRM), composed of tetanus toxoid (Netherlands Vaccine Institute), tuberculin purified protein derivative (Netherlands Vaccine Institute), and Candida (HAL Allergenen Lab), as previously published by our research group.¹⁵ (link) Briefly, cryopreserved PBMCs were thawed and in triplicate wells (1.0 × 10⁵ cells/well) exposed to three conditions: medium control (90% IMDM supplemented with 10% human AB serum (PAA laboratories, Pasching, Austria)), MRM and Phytohaemagglutinin (PHA, 0.5 µg/mL), which was used as a positive control. Proliferation was measured by ³H-thymidine incorporation during the last 18 h of the assay. A positive response was defined as equal or above a stimulation index (SI) of 3. This SI was calculated by dividing the mean counts of stimulated wells by that of the medium control wells.

Naïve CD4+ T cells were isolated from PBMCs by negative selection using the naïve CD4+ T cell isolation kit II (Miltenyi Biotech; > 90% cell purity) from three healthy donors (male, age 32 years; female, age 28 years; male, age 28 years). The study was performed according to the Declaration of Helsinki and was approved by the ethics committee of the Ärztekammer Westfalen-Lippe and Medizinische Fakultät der Westfälischen Wilhelms-Universität Münster (registration number 2010262fS). The participants provided informed consent. Cells were stimulated with 0.5 μg/mL of plate-bound anti-human CD3 (clone UCHT1; Beckman Coulter) and 0.5 μg/mL of soluble anti-human CD28 antibody (clone CD28.2; eBioscience). Cells were stimulated in 24-well plates at 2 × 10⁶ cells/mL in 1 mL of X-Vivo 15 medium (Lonza). For Mock control cells, no further reagents were added, while for iTregs, 10 ng/mL of IL-2 (Peprotech), 10 ng/mL of TGF-β1 (R&D Systems), 10 nM ATRA (Sigma-Aldrich), and 10% (v/v) human serum (Human AB Serum, PAA Laboratories) were added. Samples were taken after 0.5, 1, 2, 6, 12, 24, 48, 72, 96, and 120 h, and lysates were stored at −80 °C until RNA extraction. At each time point, cells were stained by intracellular flow cytometry for FOXP3 expression.