Expression of EphA2 by CD34⁺-derived DCs. (A) EphA2 expression was analyzed by flow cytometry with anti-EphA2 polyclonal antibody directed against an intracytoplasmic epitope after DC permeabilization with saponin, as described in “Materials and methods.” Cord blood CD34⁺ progenitors were cultured in the presence of GM-CSF and TNF-α for 6 to 12 days. Dotted-line overlay histograms represent staining in the presence of the EphA2 peptide used for immunization, and bold dotted-line overlay histograms represent the negative control with an antibody of unrelated specificity. (B) EphA2 expression was analyzed by flow cytometry with an anti-EphA2 mAb on nonpermeabilized DCs (day 11 with GM-CSF and TNF-α. Bold dotted-line overlay histogram shows the staining of an isotype-matched control. (C) Double staining of day-11 CD34⁺-derived DCs cultured with GM-CSF and TNF-α with surface FITC-CD1a, FITC-HLA-DR, or FITC-CD86 antibodies, and intracellular anti-EphA2 polyclonal antibody revealed with PE-antirabbit IgG. Quadrant limits were set up on the isotype-matched control dot plot. The percentage value of control used to establish the crosshairs were 92% in the lower left quadrant for the double color-negative control. Numbers in dot plots indicate percentages of cells in the relevant quadrant. (D) Immunocytochemistry analysis of EphA2 on day-11 CD34⁺-derived DC cytospins. Staining was performed with anti-EphA2 polyclonal antibody or with anti-EphA2 polyclonal antibody plus the peptide used for immunization (blocking peptide), as described in “Materials and methods.” The negative control represents a polyclonal rabbit antibody of unrelated specificity. Staining was revealed using a biotinylated goat antirabbit IgG followed by alkaline phosphatase streptavidin. Original magnification × 200.