Fractionation of Human Blood Lymphocytes on Helix pomatia A Haemagglutinin Coupled to Sepharose Beads

Abstract

Treatment of human blood lymphocytes with neuraminidase has previously been shown to uncover receptors for the A haemagglutinin of the snail Helix pomatia (HP). Neuraminidase-treated lymphocytes were now fractionated on columns charged with large Sepharose particles to which HP had been coupled covalently. HP-receptor negative (HP-) lymphocytes passed the columns while HP-receptor positive (HP+) lymphocytes were retained. The latter cells were eluted by addition of the competitive hapten N-acetyl-D-galactosamine (D-GalNAc). The total yield of cells recovered after fractional was 60-80% Surface marker studies indicated that there was no selective loss of any of the major lymphocyte subpopulations. The fraction that passed the columns (fraction I) consisted of approximately 10% of all lymphocytes. It contained approximately 1% HP+cells and approximately 3% of all lymphocytes forming rosettes which sheep erythrocytes (E+ cells) present before fractionation. 50-55% of the lymphocytes in this fraction had surface-bound immunoglobulin (SIg+ cells) and complement receptors (EAC+ cells). Of the SIg+ cells, approximately 60% were true B cells while the remaining 40% had IgG adsorbed to their surface. The majority of the B cells were recovered in this fraction. The lymphocytes of this fraction responded poorly to T-cell mitogen but had an enhanced K-cell activity to chicken erythrocytes. Elution of the cells retained on the column with 0.1 mg/ml D-GalNAc gave a fraction II, consisting of approximately 15% of all lymphocytes. This fraction had a mixed composition. The majority of the cells (approximately 45%) were recovered by subsequent elution with 1.0 mg/ml D-GalNAc. This fraction III was strongly enriched with HP+ and E+ cells (T cells). About 10% of the HP+ cells in this fraction were SIg+. However, on the majority of these cells this surface-bound immunoglobulin was probably externally absorbed IgG. These HP+-SIg" cells were also EAC+ and had Fc receptors, as shown by rosette formation with IgG-coated bovine erythrocytes. The lymphocytes of fraction III responded most strongly to T-cell mitogen while their K-cell activity was weak.