Phenotypical analysis of tolerized T-cell populations.

After a 7-day primary MLC in the absence or presence of mAbs, the responder lymphocytes were harvested, allowed to rest, and analyzed by flow cytometry. (A) Contour plots showing forward scatter (linear scale) and either CD4 (upper 2 panels) or CD8 (lower 2 panels) expression (fluorescence log scale) in the live lymphocyte gate. The left panel shows T cells from a control MLC, and the right panel shows T cells from an mAb-blocked MLC. (There were no observed differences between mAb combinations). The percentages in the upper 2 panels indicate the relative number of CD4⁺ T cells with a large blast-like appearance, while in the lower 2 panels, the total percentage of CD8⁺ T cells is indicated. (B) The upper panel shows the size difference in a forward scatter histogram. The 3 lower panels show the expression of CD4, TcR (WT31), and CD45RA on the large-sized backgated CD4⁺ T-cell population, which was derived from either a control (shaded histogram) or an mAb-blocked MLC (open histogram). The histograms show the number of events on the vertical axis (lin scale) and fluorescence intensity on the horizontal axis (log scale). (C) CD8⁺ cells from an MLC with simultaneous blocking of CD86 and CD40 showed a decrease in the number of CD45RO expressing cells and CD45RO intensity. Additional CD80 blocking had no effect. Cells were derived from either a control MLC or an mAb-blocked MLC, where the indicated mAbs were present. The contour plots show the fluorescence intensity of CD45RO on CD8-expressing T cells. Note that the Y axis shows a log scale from 10² to 10⁴, which indicates that all cells shown are CD8⁺.