Sensitive detection of residual alloresponses in MLR assay. (A) Primary MLR. Serial dilution of freshly purified CD4+ T cells reveals readily detectable MLR responses with less than 500 cells. Because some wells were positive and others were completely negative at limiting dilution, there is a wide standard deviation; 5000 or less purified CD4 cells do not have auto MLR responses even in the 96-well round-bottom culture, and counts were at background. Average of 4 donors is shown from LDA experiments. (B) Secondary MLR. Sorted NA cells had no MLR reactivity at peak (day 7) in secondary MLR with 3 × 104 cells as responders (12 replicate wells); results from 8 donor combinations. In 6 of 8 of those combinations, secondary MLR with 105 cells as responders was also nonreactive (6 replicate wells). Error bars indicate 1 standard deviation from the mean.
