(A) CD86 expression on infected AML cells. Spleen AML cells were infected with either CD86-sense (a) or CD86-antisense (b) producer clones as described in the Materials and Methods. (□) Control IgG (rat IgG-PE); (▩) CD86-PE (anti-B7.2) MoAb. The hidden portion of the control curve in (a) drops monotonically as a function of fluorescence intensity. (B) Cytokine production by transduced AML cells. Levels of GM-CSF, IL-4, and TNF-α secreted by retrovirally transduced, unselected AML cells cultured for 48 hours at 10⁶ cells/mL were determined by sandwich ELISA using specific antimurine MoAbs for capture and detection. Data are shown as the mean ± SD of six independent experiments. (C) AML cells express GM-CSF receptor. Total cell lysates (40 μg of protein/lane) from spleen AML cells from two different mice (lanes 3 and 4) or control cells (lanes 1 and 5) were fractionated by electrophoresis on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed with polyclonal antibody against mouse GM-CSFRα as described in the Materials and Methods. AML cells cultured for 24 hours in the presence of IL-3 downregulate GM-CSFR expression (lane 2).