Macs gmp expact treg kit

In the BRC GMP Facility, cells were seeded in MACS GMP Cell Differentiation/Expansion Bags at 0.5 × 10⁶ cells/mL in TexMACS GMP Medium (Miltenyi Biotec, Germany), supplemented with 5% human serum containing 100 nM rapamycin (Rapamune) and activated with anti-CD3- and anti-CD28-coated beads (4:1 bead:cell ratio, MACS GMP ExpAct Treg Kit, Miltenyi Biotec, Germany). Human recombinant IL-2 (500 IU/mL; Proleukin) was added at day 4–6 and replenished every 2 to 3 days. The cells were rested for 4 days before restimulation. Stimulation occurred on days 12 and 24, during which time cells were pooled, fresh beads (1:1), rapamycin and IL-2 were added, and the suspension was re-seeded at 0.5 × 10⁶ cells/mL into new bags (250, 500, or 1,000 mL) For a schematic representation of the process see Figure 5. Expanded cells were harvested on day 36 and pooled. The ExpAct Treg expansion beads were depleted using the CliniMACS Plus System (Miltenyi Biotec) to form a bead-depleted cell population. A small aliquot of the cells was then taken for safety and functional analysis.

CD25⁺ Treg cells were isolated from leukapheresis product of healthy volunteers using the CliniMACS Plus system (Miltenyi) as described^{23 (link)}. Treg cells were identified as CD4⁺/CD25⁺/FoxP3⁺/CD127⁻ and isolated with a purity above 85% throughout the study. Conventional, non Treg-Tcells (Tconv) were sorted from PBMCs with the human CD4⁺ T cell isolation kit (StemCell Technologies, Cambridge, MA). For initial stimulation of cells, Treg and/or Tconv cells were placed in culture at a concentration of 10⁶ cells/mL in Treg cell culture medium: TexMACS GMP medium (Miltenyi) supplemented with MACS GMP ExpAct Treg kit (Miltenyi) at a bead-to-cell ratio of 2:1 and recombinant human IL2 (rhIL-2 MACS GMP, Miltenyi, at 500 IU/mL). Rapa (100 nM), FH535 (1 µM) or Y3 (1 µM) were added where indicated. To assess the sequential effects of treatment with NABs and then Rapa (abbreviated as FH535 → Rapa or Y3 → Rapa), Rapa was added to the cells after initial 48-h in culture with FH535 or Y3 alone.