FIG. 4.

(A) IFN-γ production by memory-type CD8⁺ T cells by stimulation with recombinant protein. Monocyte-derived DC were pulsed with MMP-MTB or Fusion-MTB at 10 or 20 μg/ml, costimulated with or without CD40L (1.0 μg/ml), and used to stimulate memory-type CD8⁺ T cells in a 4-day culture. Responder CD8⁺ T cells (1 × 10⁵) were stimulated with the Ag-pulsed DC at a T cell/DC ratio of 10:1. (B) IFN-γ production by naïve CD8⁺ T cells stimulated with recombinant proteins. Monocyte-derived DC were pulsed with the indicated recombinant protein at 10 or 20 μg/ml, further costimulated with or without CD40L (1.0 μg/ml), and used to stimulate naïve CD8⁺ T cells in a 4-day culture. Responder CD8⁺ T cells (1 × 10⁵) were stimulated with the Ag-pulsed DC at a T cell/DC ratio of 10:1. (C) Inhibition of naïve CD8⁺ T cell activation by treatment of Fusion-MTB-pulsed DC with MAb. Monocyte-derived DC were pulsed with MMP-MTB at 20 μg/ml, costimulated with CD40L (1.0 μg/ml), and subsequently treated at 10 μg/ml with MAb to HLA-ABC, CD86, or normal murine IgG. These DC were used to stimulate naïve CD8⁺ T cells (1 × 10⁵) at a T cell/DC ratio of 10:1. IFN-γ produced by T cells was measured by the ELISA method. A representative of three separate experiments is shown. Assays were performed in triplicate, and the results are expressed as means ± standard deviations. Titers were statistically compared by Student's t test. (D) Intracellular production of perforin by CD8⁺ T cells. Monocyte-derived DC were pulsed at 10 μg/ml with either MMP-MTB or Fusion-MTB and cultured with unseparated memory-type T cells (T cell/DC ratio, 40:1) for 5 days. The stimulated CD8⁺ T cells were gated and analyzed for perforin production. Values are the mean percentages of the CD8⁺ T cell population that were perforin positive in three independent experiments and the standard deviations. Titers were statistically compared using Student's t test. A representative of three separate experiments is shown.