Recombinant il 2

NK activity was evaluated in effectors recovered from PBMC/MSC mixed cultures in the presence (mixed culture) or absence (ctrl-culture) of MSCs. Briefly, 1 × 10⁶ PBMCs obtained from one healthy donor were cultured in RPMI 1640 (Euroclone, Milan, Italy), 5% autologous plasma and 100 U/ml recombinant IL-2 (Novartis, Varese, Italy) in the presence or absence of NB-MSC or BM-MSC at a 1:10 MSC:PBMC ratio, in 24-well plates for 24 h at 37 °C, 5% CO₂. Recovered effectors (E) were tested for their capacity to lyse ⁵¹Cr labelled K562 cells (T) at 100:1, 30:1, 10:1 E:T ratios. After 4 h incubation, 25 μl of the supernatant was collected from each well and counted for 1 min in a gamma-counter (Topcount, Packard, Downers Grove, Illinois). Results were expressed as the percentage of T lysis calculated with the formula: [(experimental release – spontaneous T release)/(total T release – spontaneous T release)] × 100 [20 (link)].

The PD1-CD28 fusion protein was described previously (32 (link)). The retroviral vector pMP71 (kindly provided by Christopher Baum, M.D., Institute of Experimental Hematology, Medizinische Hochschule Hannover, Germany) was utilized for all transduction experiments. Detailed protocols for murine T cell transduction have been published (34 (link)–37 (link)). In brief, pMP71 PD1-CD28 vector was transfected into Platinum-E cells and retrovirus-containing supernatants were collected for transduction of murine T cells. Primary murine T cells were first stimulated with anti-CD3e and anti-CD28 antibody (eBioscience, clones 145-2C11 and 37.51, respectively) and recombinant IL-2 (Novartis, Switzerland). Priot to transduction, anti-CD3- and anti-CD28 beads (Life technologies, USA) were added. Recombinant IL-15 (Peprotech, Germany) was used for T cell expansion. The CD4+ T cell fraction was purified on the day of spleen extraction by magnetic activated cell sorting using a CD4+ T cell isolation kit (Miltenyi Biotec, Germany).