Leukemic pDCs express functional CD33. (A) Leukemic pDCs from patient no. 1 (P1) and patient no. 2 (P2) were incubated for 48 hours with 5 μg/mL anti-CD33 mAb (□) or isotype control IgG1 (▧) in the presence of the indicated cytokine (IL-3, GM-CSF, or IL-3 + CD40L). Purified monocytes exposed to anti-CD33 mAb were used as positive controls for cell death.²⁸  To evaluate the effect of mAb-mediated cross-linking, goat anti–mouse IgG (GAM) antibodies were added to leukemic cells from patient no. 2. Cell viability was assessed by annexin V/PI staining and flow cytometry. In the absence of cytokine, most of the cells were dead (double positive for annexin V and PI). Results are expressed as a percentage of viable cells (ie, negative for both annexin V and PI). Apoptosis was observed only in the presence of GAM antibodies. (B) Leukemic pDCs from patient no. 2 (P2) were incubated for 48 hours with 5 μg/mL anti-CD33 mAb or isotype control IgG1 in the presence of maturation agents IL-3 plus CD40L. Then, cells were stained with either anti-CD40, anti-CD80, anti-CD86, or HLA-DR and analyzed by flow cytometry. An increased expression of HLA-DR, CD40, CD80, and CD86 was observed after culture with IL-3/CD40L even in the presence of anti-CD33 mAb. (C) Leukemic pDCs from patient no. 1 (P1) and patient no. 2 (P2) were incubated for 48 hours with 5 μg/mL anti-CD33 mAb (□) or isotype control IgG1 (▨) in the presence of IL-3. Cytokines (IL-12, TNF-α, IL-10, IL-1β, IL-6, and IL-8) present 48 hours later in the supernatant were measured by a cytokine bead assay. A production of proinflammatory cytokines was observed after anti-CD33 mAb treatment. A high spontaneous production of IL-8 (greater than 5000 pg/mL) in response to IL-3 was observed in pDCs from patient no. 2. Stimulation of pDCs from patient no. 2 with CD40L (▪) was used as control. The amount of produced cytokine from a representative experiment is indicated. When the amount is not mentioned, cytokine production was less than 40 pg/mL.