IL-10–dominated regulatory responses are seen when autoreactive Tr cells are activated by cognate autoantigen but not high-avidity stimulation. Clones or cell lines (1 × 10⁵/well) were incubated with irradiated (30 Gy [3000 rad]) autologous CD4-depleted antigen-presenting cells (1 × 10⁶/well) for 96 hours in 1-mL cultures at 37°C, 5% CO₂ in the presence or absence of antigen (Rh) or nonspecific stimulus (2 μg anti-CD3/CD28). Tr clone P8(1) and P8 Tr cell line (A) secrete predominantly IL-10 and little IFN-γ or IL-4, characteristic of a Tr response, when stimulated specifically by presentation of the Rh protein autoantigen. In contrast, the cells mount Th0-like effector responses, with secretion of IFN-γ and IL-4 in addition to IL-10, and proliferation, after nonspecific high-affinity activation with anti-CD3 and anti-CD28. Data are presented as mean plus or minus standard error. (B) Number of IL-10–secreting CD4⁺ T cells are increased on stimulation with Rh autoantigen and nonspecific stimulation with anti-CD3/CD28 T-cell stimulatory antibody. Experimental detail as above. CD4⁺ clones actively secreting IL-10 were detected on day 4 by flow cytometry using anti-CD4-FITC antibody with an IL-10 cell enrichment and detection kit (PE-label). Number of IL-10 positive cells are represented in upper right quadrant as a percentage of the total Cd4⁺ T-cell population. The results shown are representative of 3 separate experiments.