FIGURE 3.

Effect of Cat-S inhibitor on MLR and cytotoxicity. (A) Experiment design of mixed lymphocyte reaction for proliferation and LDH assay. LPS-primed BMDCs (Balb/c) and CFSE-stained T cells (C57BL/6J) were used as stimulator and responder, respectively. Mixed cells were cocultured for 4 days and analyzed by flow cytometry or LDH assay. (B) BMDC stimulation assays. BMDCs were stimulated with 500 ng/ml LPS, Cat-S inhibitor, or vehicle for 24 h and analyzed for MHC-II expression among CD11c + cells. (C) CD4 + T cell proliferation monitored using CFSE labeling. Mixed cells were in the presence of 0.2, 2, 20 μg/ml, or vehicle, and proliferation was analyzed by flow cytometry at 4 days of culture. As controls, mixed cells with no CFSE were treated as the same way as vehicle but without CFSE staining; single T cells were treated with vehicle but without adding BMDCs. After gating in CD4 + cells, divided cells were gated in the histograms of the CFSE channel. A representative experiment from two separate experiments is shown. For each experiment, one mouse was used for BMDC differentiation and two mice were used for T cell isolation. Two or three replications was made from each T cell host for MLR. (D) Division index of CD4 + T cells. Based on the deconvoluted histograms of the CFSE channel, the division index was calculated by the ratio of the total number of divisions over the number of cells at start of culture. (E) The division index of CD8 + T cells. (F) LDH assay for Cat-s inhibitor–treated cells. Supernatant of mixed cell culture was analyzed at day 4 by LDH. *p < 0.05, **p < 0.01, ****p < 0.0001.