Naive t cell isolation beads

RosetteSep enriched APCs were stimulated with anti-CD40Ab/poly(I:C) as described above for 1 hour. Naive T cells were sorted from an allogenic donor using naive T cell isolation beads (Miltenyi) and an autoMACs (Miltenyi). T cells were then added to stimulated unwashed APCs in a 5:1 ratio. After 6 or 24 hours of culture, IL-10 was captured on the surface of producing cells for 45 minutes using the IL-10 Secretion Assay according to manufacture’s protocol (Miltenyi) and stained for flow cytometry (Table S1).

RosetteSep enriched APCs or skin isolated APCs were stimulated with anti-CD40/Poly I:C as described above for 1 hour. Naive T cells were sorted from an allogenic donor using naive T cell isolation beads (Miltenyi) and an autoMACs (Miltenyi). Naive T cells were labeled with CFSE (Life Technologies) according to standard protocols. T cells were then added to stimulated unwashed APCs in a 5:1 ratio and incubated for 6 days before staining for flow cytometry (Table S1). Alternatively, T cells were stimulated using the T cell activation/expansion kit containing beads coated with antibodies against CD2, CD3, and CD28 (Miltenyi). For transwell experiments, purified monocytes were cultured in the upper chamber and T cells stimulated with T cell activation/expansion kit in the lower chamber in a 1:5 ratio using a 24-well plate with polycarbonate membrane inserts (Corning). Where indicated, APCs were pre-incubated with a 5ug/ml neutralizing anti-IL10 (clone JES3–19F1, BD Biosciences) or isotype (clone R35–95, BD Biosciences) for 1 hour prior to anti-CD40/poly(I:C) stimulation. For IL-10 add-in experiments, recombinant IL-10 (Peprotech) was added directly to cultures. A dose of 250 pg/ml IL-10 was determined by titration.