FIG. 1.

(A) Purification of lymphocyte populations. To assess the purity of the indicated populations, 5 × 10⁵ unstimulated NA and DR⁻ cells were costained with MAbs against HLA-DR, CD25, CD19, and CD14 cell markers as described in Materials and Methods. The DR⁻ cell population is 99% pure in comparison to the NA population, which contains greater than 10% cells expressing HLA-DR, as well as macrophages and B cells. (B) Requirements of costimulation for proliferation of the DR⁻ population. NA and DR⁻ cells were cultured in medium alone (US), were stimulated with 1 μg of anti-CD3 MAb per ml immobilized on GAM-coated plates (αCD3), or were costimulated with anti-CD3 plus soluble anti-CD28 each at a concentration of 1 μg/ml (αCD3+αCD28) for 3 days. Cells were harvested in triplicate and assayed for thymidine incorporation as described in Materials and Methods. Results are the averages of triplicate wells. These results are representative of more than 10 experiments.