(A) Kinetics of IL-4Rα expression at the cell surface of tumor-induced CD11b⁺ splenocytes. CD11b⁺ splenocytes from tumor-free (normal) or tumor-bearing mice (tumor) were incubated for 72 hours alone, with IFN-γ, with a neutralizing antibody against mouse IL-13, or with both. Isotype-matched antibodies were included as negative controls. IL-4Rα protein expression on the cell surface was analyzed at various time points by flow cytofluorimetry and reported as the mean percentage of positive cells ± SEM of 3 separate determinations. Differences between diverging kinetics values were statistically significant (P < 0.05). (B–D) Effect of CD11b⁺ cell exposure to IL-13 and IFN-γ. CD11b⁺ cells were sorted from the spleens of tumor-free and tumor-bearing mice. These last cells were analyzed immediately (Fresh) or cultured for 48 hours in medium alone (No cytokine) or in the presence of IFN-γ, IL-13, or both. Some samples were treated either with IFN-γ for 24 hours followed by addition of IL-13 for a further 24 hours (IFN-γ → IL-13) or with the inverse cytokine combination (IL-13 → IFN-γ). Cells were then analyzed for expression of Arg1 and Nos2 mRNA by real-time PCR (B), for Arg1 and Nos2 protein levels by Western blotting (C), and for relative enzyme activity (D). Log₂FC is the log-transformed gene expression value of the target gene, normalized to an endogenous reference and relative to a calibrator sample. ND, not done. Data for real-time-PCR, Western blot, and enzyme activity are from the same experiment representative of 3.