Fig. S4.
Cell–cell contact between myeloid and tumor cells stimulates differentiation of F4/80+PD-L1+ macrophages. Gr-1+ cells were enriched using magnetic beads (Miltenyi Biotec) from BM of naïve C3/He mice. Equal numbers of Gr-1+ cells were plated in 48-well plates (4 × 105 cells per well) alone or mixed with MBT-2 tumor cells (1.5 × 105 cells per well). In some wells, Gr-1+ cells (bottom) were separated from tumor cells (insert) by 1-μM pore diameter membrane. On day 5, cells were collected and stained with PE–PD-L1 and Alexa 488-F4/80 Abs. The percentage of F4/80+PD-L1+ cells was evaluated using an immunofluorescent imaging microscope. Average means ± SD are shown. *P < 0.05.