Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. : II. Separation into subsets of lymphocytes with distinctive functions

Lymphocytes from human peripheral blood were separated by Countercurrent distribution (CCD) in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000. Maximal responses to the T-cell mitogens phytohemagglutinin and concanavalin A were detected in a part of the distribution corresponding to the area where the highest percentage of E rosetting cells were seen. Antibody-dependent cellular cytotoxicity was mediated by lymphocytes located in a restricted part of the distribution separate from the majority of T lymphocytes. Lymphocytes with natural killer activity against K 562, adherent melanoma cells, and fibroblasts were detected in the same region. This area of the distribution also contained the peak of lymphocytes with high-affinity Fc receptors. It was further investigated whether affinity separation may be possible in two-phase systems on the basis of cell to cell interaction. Affinity CCD utilizing the interaction between sheep erythrocytes (SRBC) and T lymphocytes was shown to redistribute the majority of T cells into the area in which SRBC were located, while other lymphocytes were not affected. Lymphocytes mediating natural killing to adherent target cells were not redistributed, indicating that they lack high-affinity receptors for SRBC

Countercurrent Distribution of Lymphocytes from Human Peripheral Blood in an Aqueous Two-Phase System : I. Separation into Subsets of Lymphocytes Bearing Distinctive Markers’

Lymphocytes from human peripheral blood have been separated by countercurrent distribution in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000 with a cell yield of 59–88% and viability above 90%. A highly reproducible partition pattern was seen with four distinct peaks. Lymphocytes with surface membrane immunoglobulin (SmIg) were located in the first part of the distribution corresponding mainly to peak I. T lymphocytes as detected by E rosetting and α-naphthyl acetate esterase (ANAE) staining showed a broad distribution with a maximum in peaks II and III. ANAE-negative lymphocytes were seen in both extremes of the distribution, corresponding to B cells in the first part and to a population of E− and SmIg− lymphocytes in the last part. Monocytes were present in all fractions with some enrichment in peaks II–IV. Lymphocytes with low-affinity Fc receptors were found in B-cell-containing fractions in the first part of the distribution, but also in the last part. Lymphocytes with high-affinity Fc receptors were detected mainly in peak IV. It is thus demonstrated that peripheral blood lymphocytes can be fractionated into subpopulations enriched in cells with characteristic markers.markers