Alloreactivity assessed by CFSE dilution and FACS sorting after MLR culture. After culture of purified CD4⁺ T cells in MLR for 7 days, all cells were collected and analyzed on a FACS Caliber. Viable cells were analyzed, and the histogram for CFSE (FL1) is shown. (A) Responder CD4⁺ cells were cultured with PBMCs at an R/S ratio of 1:2. Approximately 33% of the cells are proliferating blasts, and 60% of the cells are CFSE^bright and nondivided. Quantitative evaluation of the exponential expansion of blast number, estimates an alloreactive precursor frequency of approximately 1% in the starting CD4 T-cell population (B). DCs used at 15 000 cells/well generate the most aggressive MLR, with 67% of resulting cells blasts and less than 30% undivided. Alloreactive precursor frequency is estimated to be approximately 5%. Shown are representative examples of MLRs of each type (of 6 donor combinations each). Bars indicate population gates as percent of total cells shown. (C) PBMC-stimulated MLR culture evaluated by 2-color plot of CD25 expression versus CFSE. Quadrant gates and population statistics are shown. Also shown is FACS sorting gate, including all CFSE^bright cells. (D) Contour plot of same cells as panel C, showing the nonactivated (NA cells) CFSE^bright and CD25^- sorting gate. Arrows indicate sort gates.