Separation of human lymphocyte subpopulations. I. Transformation characteristics of rosette-forming cells

Lymphocytes were isolated from human peripheral blood by carbonyl-iron treatment and Ficoll-Isopaque centrifugation. The lymphocytes were allowed to form rosettes with sheep erythrocytes, either uncoated (E) or coated with antibody and complement (EAC).In 32 experiments E rosettes were separated from free lymphocytes on a Ficoll density gradient. Thus, depleted (non-E) and enriched (E) fractions were obtained. It was found that in the original suspension 24 ± 7.2% of the lymphocytes formed rosettes with EAC and 56 ± 8% with E. In fraction non-E these values were 56 ± 11.4 and 22 ± 8.9%, respectively; in fraction E 8 ± 3.8 and 79 ± 8.8%. Moreover, the percentages of Ig-bearing cells among the fractions were found to follow closely those of CRL.In a series of lymphocyte culture experiments these fractions were compared with the original suspension and a control suspension (rosetted, not separated), as well as with a recombined population (non-E + E). It was found that fraction non-E showed an increased response to PHA and PWM, and an enhanced MLC stimulatory capacity, whereas fraction E was decreased in these respects. Moreover, fraction E displayed significantly reduced spontaneous DNA synthesis.It is concluded that the responses to PHA or PWM, as well as the capacity to stimulate allogeneic cells, are not solely dependent on the cells forming rosettes with E.