Abrogation of secondary MLR after sorting NA cells from DC MLR. Purified CD4⁺ T cells were stimulated with DCs for primary MLR for 7 days. CFSE^bright/NA or CFSE^dim primed sorted cells were recultured with mononuclear cell (MNC) stimulators. (A) Shown is kinetic analysis of proliferation in secondary MLR. The sorted NA cells did not respond at any time point in a secondary MLR. The counts are at background. Primed cultured cells responded vigorously with sustained responses. Sorted primed cells mounted short 1000 to 2000 cpm responses without stimulators, which were subtracted from MLR curves. Results are representative of 8 donor combinations. Error bars indicate 1 standard deviation from the mean. (B) Limiting dilution analysis reveals 2-log depletion of alloreactive precursors. Populations freshly purified or sorted CD4⁺ T cells were tested for their ability to proliferate on serial dilution to limiting numbers. Fresh cells had mean LDA of approximately 1/350, and primed cells of approximately 1/50. Sorted NA cells had no detectable alloreactivity at 3 × 10⁴ cells/well, so LDA was undetermined (< 1/30 000). The same NA cells, however, did react to third-party stimulators with approximately 1/900 frequency. Comparison is of fresh cells versus NA cells (P < .01) and fresh cells versus NA-third party (P = .05). Four donor combinations were evaluated.