MOESM1 of Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Additional file 1: Table S1. Antibodies for flow cytometry. Figure S1. Design of the in vivo study. A. Steady state leukapheresis from 2 patients (1 KT and 1 LT patient) were processed using GMP-compliant devices and reagents. Tregs positive fraction (CD8−CD25+ cells) was purified using the CliniMACS System. Forty millions of CD8−CD25 + cells were expanded in vitro for 3 weeks. At day 21 all the cultured cells were collected and the beads were removed using the CliniMACS device, according to manufacturer’s instructions. Negative fraction (CD8−CD25− T cells) after GMP selection at day 0 and the final product after GMP expansion at day 21 were cryopreserved and thawed as described in “Materials and methods” section. B. Irradiated NSG mice were infused with the KT or the LT CD8−CD25− T cells, either alone or in combination with autologous expanded Tregs at 1:1 ratio, to assess their ability to ameliorate GVHD. C. Mice were bled 4/7 weeks after transplantation and sacrificed 7 weeks after transplantation. FACS analysis of the injected cells (day 1), of PB (4 weeks ± 3 days after transplantation) and of PB and spleen (7 weeks ± 3 days after transplantation) was performed. Figure S2. Circulating Tregs in KT and LT patients. Mean absolute number of circulating CD4+CD25+CD127−FoxP3+ Tregs from healthy controls and selected LT and KT patients (p = NS).