CD4⁺ T cells induce airway myocyte proliferation, dependent on T cell activation and direct T cell–myocyte contact. (A) Ninety-nine percent of the cells in primary ASM cell cultures expressed α-SMA. (B) CD4⁺ T cells (purity ≥ 98%) were cocultured with the ASM cells for 48 hours, in the presence of BrdU during the last 24 hours. Cells were immunostained for CD4, and BrdU incorporation by the ASM cells was quantified. (C) The CD4⁺ T cells and the smooth muscle cells (SMCs) were resolved with at least 99% specificity, as calculated from samples containing ASM cells only (upper plot) or T cells only recovered from Transwells (lower plot). In the lower plot, the left-shifted tail of the T cell population reflects some loss of CD4 expression from apoptosis. (D) BrdU incorporation by ASM cells. In each panel, BrdU incorporation is represented as a density plot (upper plots) and as a histogram (lower plots, thick line) overlaid on the baseline histogram (thin lines). The percentages of BrdU⁺ cells were calculated by subtraction. Myocytes incubated with 10% FBS served as a positive control for proliferation. The baseline BrdU incorporation was calculated from myocytes cultured in 0.5% FBS and 20 U/ml IL-2. The ASM cells were cocultured with: CD4⁺ T cells activated with OVA, either in direct contact (CD4⁺, OVA, direct) or separated by a Transwell membrane (CD4⁺, OVA, TransW); and with nonstimulated CD4⁺ T cells in direct contact (CD4⁺, no-OVA, direct). Coculture in direct contact with OVA-activated T cells elicited a significant increase in BrdU incorporation. (E) Dose-response curve of T cell effect on myocyte BrdU incorporation. Data normalized as percentage of baseline. n = 3–6 independent experiments per data point. *P < 0.05 versus baseline; ^#P < 0.05 versus Transwell and nonactivated T cells.