Figure 1.

In vitro T-cell responses in mc²6030-immunized mice. (a) T-cell responses to infected macrophages or crude mycobacterial antigen preparations. IFN-γ production by purified splenic T cells from a CD4^–/– mouse that was primed and boosted with mc²6030. T cells were stimulated for 4·5 days by coculture with syngeneic bone marrow-derived macrophages (BMM) which had been infected for 2 hr with mc²6030 at an MOI of 10 : 1 (mc²6030 INF), or pulsed for 10 hr with sonicates of either mc²6030 (mc²6030 SON) or H37Rv bacilli (H37Rv SON) or PPD. IFN-γ levels were assayed in culture supernatants by ELISA. For this experiment, priming was performed 13 months before killing by s.c. injection in the flank of 10⁶ live mc²6030 in 200 μl vehicle (PBS + 0·05% Tween-80), and boosting was 2 weeks before killing by i.v. injection of 10⁶ live mc²6030 in vehicle. The concentration of mc²6030 sonicate used for pulsing BMM was adjusted to give the equivalent of a bacterial load corresponding to MOI of 10 : 1. H37Rv sonicate was used at 1·0 mg/ml based on dry weight of sonicated bacteria, and PPD was used at a total protein concentration of 100 μg/ml. IFN-γ production was below the level of detection (∼0·01 ng/ml, indicated by dotted line) in the absence of infection or antigen pulsing of BMM. Asterisks indicate statistically significant increases in IFN-γ production over the background levels observed with T cells from naive mice (*P < 0·05; **P < 0·01; ***P < 0·001; one-way anova, Bonferroni post-test).(b) Responses to purified and recombinant M. tuberculosis protein antigens. T cells purified from spleens of a naive CD4^–/– mouse (open bars) or an mc²6030-primed and boosted CD4^–/– mouse (filled bars) were assayed for responses to the indicated antigens by measurement of IFN-γ secretion. Levels of IFN-γ are shown for T cells stimulated for 5 days by BMM in the presence of the indicated antigens at 10 μg/ml. For this experiment, priming was performed by s.c. injection of 10⁶ live mc²6030 in vehicle (PBS + 0·05% Tween-80), and 6 months later boosting was by i.v. injection of 10⁶ live mc²6030. Naive mice were sham primed and boosted on the same schedule with vehicle only. Mice were killed and splenic T cells were isolated 2 weeks after boosting. ICL, isocitrate lyase; MS, malate synthase; ND, not determined. *P < 0·05 compared to no antigen control (one-way anova, Bonferroni post-test).