Differentiation and function of iMacs can be regulated in opposite ways by Th1- and Th2-type cytokines.

To assess the influences of various cytokines on the differentiation ofiMacs, we isolated Gr-1⁺ splenocytes from mice bearing a TS/A tumor. Inhibitory cells were then exposed to either no cytokines, IL-4 (100 ng/mL), IL-12 (20 ng/mL), or a combination of IFN-γ (50 ng/mL) and TNF-α (5 U/mL). After 6 days of culture, the adherent cells were tested for the phenotype (A) and function (B, C). (A) Phenotypic characterization of cells after the 6-day cytokine regimen. Isotype-matched controls are shaded. (B) Cytokine-treated inhibitory cells were added at a final concentration of 6 × 10⁴ cells (1%) to cultures consisting of 6 × 10⁶ BALB/c splenocytes of mice previously immunized with rAd-β-gal and stimulated with the β-gal peptide. Cytolytic activity was assessed by standard ⁵¹Cr release assay after an additional 5 days of culture using β-gal peptide pulsed (■) or unpulsed (●) CT26 cells either at E:T ratios starting at 100:1, followed by 3-fold dilutions (100:1, 33:1, 11:1, 3:1, 1:1, 0.3:1). (C) Suppressor cells (iMacs) derived from cultures of Gr-1⁺ cells in the presence or absence of IL-4 were added to allo-MLC consisting of 5 × 10⁶ BALB/c splenocytes (H-2^d) and 10⁵ γ-irradiated C57BL/6n DC (H-2^b). The iMacs-to-DC ratio is indicated at the bottom of the panel. The MLC was assessed by standard⁵¹Cr release assay for activity after an additional 5 days of culture using an H-2^b target, MBL-2 (●), and a control H-2^d target, CT26 (▾), at E:T ratios starting at 100:1, followed by 3-fold dilutions (33:1, 11:1, 3:1).