Monocyte-derived and primary DCs costimulate CD40L expression. (A) Purified CD14^hiCD16⁻ and CD14^loCD16⁺ monocytes were cultured for 5 days in granulocyte-macrophage colony-stimulating factor and IL-4 to generate CD14^hi monocyte-derived DCs (CD14 MDDC) and CD14^lo CD16⁺ monocyte-derived DCs (CD16 MDDC). Purified CD4⁺ T cells were stimulated with anti-TCR mAb for 6 hours without monocytes (None) or after reconstitution (1:1) with CD14^hiCD16⁻ monocytes, CD14 MDDC, or CD16 MDDC. Cells were harvested at 6 hours and stained for CD40L expression. Data are mean ± SE (n = 3). (B) Purified CD4⁺ T cells were stimulated with anti-TCR mAb for 6 hours without antigen-presenting cells (0.0) or after reconstitution with increasing numbers of CD14^hi monocytes (CD14 Monos), myeloid DCs (mDC), or plasmacytoid DCs (pDC). The ratio of antigen-presenting cells to CD4⁺ T cells is shown along the x-axis. Monocytes are present in PBMCs at approximately a 1:1 ratio with CD4⁺ T cells, whereas circulating DCs constitute less than 2% (0.02) of PBMCs. Cells were harvested at 6 hours and stained for CD40L expression. Data are mean ± SE (n = 3).