Differential effects of monocyte- and tissue macrophage-derived AAMs on naïve CD4⁺ T cells. (A) RT-PCR analysis of Aldh1a2/Raldh2 expression in peritoneal macrophages normalized to expression of GAPDH. Graphs depict mean ± standard error of the mean of individual mice pooled from 5 to 6 independent experiments. (B) FACS analysis of Aldh activity gated on CD11b⁺ cells from the peritoneal cavity. Peritoneal cells were stained with aldefluor to detect Aldh activity for 2 hours prior to staining with cell surface antibodies antibodies. (C) Graph depicts the proportion of CD11b⁺ cells that are ALD⁺ from individual mice. Results are pooled from 4 independent experiments. (D) Flow cytometry contour plots showing the percentage of CD25⁺, Foxp3⁺CD4⁺ T cells after 6 days of coculture with peritoneal macrophages either with or without the RA inhibitor LE540 (1 µM). (E) Quantitation of the percentage of CD25⁺, Foxp3⁺ cells from the CD4⁺ compartment after coculture. Data are shown from 3 independent experiments. (F) Inhibition of CD4⁺ T-cell proliferation by IL-4c- and Thio+IL-4c-induced AAMs. FACS analysis of activated (anti-CD3⁺IL-2) Cell tracer-labeled naïve CD4⁺ cells cultured with peritoneal macrophages (ratio of 2:1) after 3 days of coculture. Results are representative of 3 independent experiments.