Enrichment of MLR-activated human suppressor cells by discontinuous density-gradient centrifugation

6-day allogeneic mixed lymphocyte cultures (MLR) were fractionated on a four-step discontinuous Percoll^R gradient. The various fractions were added as regulatory cells to freshly established micro-MLRs between the original or third-party cell donors. Unspecific suppressor activity was highly enriched in gradient fraction III (density 1.069?1.062 g/ml), consisting mainly of blast-like cells. Suppressor cells were non-adherent T cells as determined by their spontaneous rosette formation with sheep erythrocytes and their susceptibility to lysis by anti-T-cell globulin plus complement. Mitomycin C treatment diminished their inhibitory capacity. In addition, gradient fraction III was also enriched with alloantigen-activated memory cells. By contrast, unprimed alloreactive cells were recovered from gradient fraction IV (density ≥ 1.070 g/ml), consisting mainly of small, resting lymphocytes.