Evaluation of sorted cell population alloreactivity by secondary MLR and LDA. (A) Secondary MLR evaluation of sorted cell populations. Cells (10⁴) of each population were stimulated with 10⁵ frozen/thawed PBMCs. Stimulator cells were derived from same donor as original stimulator. Assays were done in 96-well round-bottom plates for increased sensitivity to low precursor frequencies. Blasts (or unsorted cells) mounted quick and vigorous responses. They also had short (1-2 day) background responses (500-1000 cpm) when cultured without stimulators. These have been subtracted out of the MLR data shown. Both sorted populations had no background response without stimulators. The NA cells were the least responsive, with an undetectable response for the first 4 to 5 days. The CFSE^bright cells had weak stuttering responses. A representative example is shown of 4 experiments with 8 donor combinations. Day-4 comparison of CFSE^bright versus NA cells was statistically significant (P < .018). Error bars indicate 1 standard deviation from the mean. (B) Limiting dilution analysis of alloreactive precursor frequencies in the sorted cell populations. Fresh and sorted cell populations were evaluated by serial dilution to determine frequency of alloreactive cells. Data are plotted on log scale. Fresh cells (approximately 1/800), primed cells (approximately 1/110), CFSE^bright cells (approximately 1/7500), NA cells (approximately 1/12 500), and CFSE^bright CD25⁺ cells (approximately 1/1700). Comparison is of fresh versus NA (P < .0002) and CFSE bright versus NA (P < .04). Data are from 8 donor combinations.