iMacs differentiate into myeloid DCs under the influence of GM-CSF and IL-4.

To assess the influence of the GM-CSF and IL-4 combination on theiMac differentiation, CD11b⁺ splenocytes from TS/A tumor-bearing mice were isolated with antibody-coated magnetic beads. Cells were then exposed to a combination of GM-CSF (20 ng/mL) and IL-4 (100 ng/mL). After 5 days of culture, the nonadherent cells (11.5% of the initial number of CD11b⁺ cells) were collected, and tested for phenotype (A, right panel) and function (B). After removal of nonadherent cells, the remaining cells were further incubated in medium containing GM-CSF and IL-4 for 5 days. The nonadherent cells recovered on day 10 constituted an additional 3.7% of the cells initially seeded. These cells were tested for phenotype (A, left panel) and morphology (C). (A) Cell surface phenotype of nonadherent cells on day 5 or 10 was determined after triple staining with anti-CD86, anti-class II MHC molecules (I-A^d/I-E^d), and anti-CD11c; percentages of I-A^d/I-E^d+/CD86⁺ also expressing CD11c marker are indicated in parentheses. (B) Cytokine-treated, nonadherent and adherent cells were irradiated and cultured in various numbers with 2 × 10⁵ allogeneic C57BL/6n splenocytes. In control wells, various numbers of γ-irradiated BALB/c splenocytes were used as stimulators. After 3.5 days of culture, cells were pulsed with ³H-TdR, and the results from triplicate wells were corrected for ³H-TdR incorporation by irradiated stimulators alone and allogeneic splenocytes alone. (C) Morphology of cells stained with MGG after the 10-day cytokine regimen. Magnification × 500.