Purification, Fractionation and Assay of Antibody‐Dependent Lymphocytic Effector Cells (K Cells) in Human Blood

Abstract

In this article we present methods for the purification and fractionation of human blood lymphocytes, which have been used in our laboratory to characterize antibody-dependent cytotoxic effector cells (K cells). The assay system consists of highly purified lymphocytes, ⁵¹Cr-labelled chicken erythrocytes (E_c) and IgG rabbit anti-E_c in high dilutions. Various ways of comparing K-cell potentials of different lymphocyte preparations in this system are discussed. When purified lymphocytes are partially depleted (60-85% depletion) of cells forming rosettes with sheep erythrocytes (E+ cells), the K-cell activity of the depleted fraction is increased, indicating that the majority of the E+ cells are inactive in this assay. Depletion of EAC-rosette-forming cells shows that most or all K cells have complement receptors. For depletion of B cells, the lymphocytes may be passed through glass bead columns, charged with F(ab')₂ fragments of human IgG and F(ab')₂ fragments of rabbit antibodies to the F(ab')₂ part of human IgG. These columns give high yields of B-cell depleted fractions. These preparations are rich in E+ cells and contain ˜80% of the Fc-receptor lymphocytes which form rosettes with bovine erythrocytes, coated with IgG antibodies. Their K-cell activity is unchanged or slightly elevated, indicating that mature B cells, i.e. SIg+ cells, have little or no K-cell activity. In contrast, passage of the lymphocytes through immune complex columns (ovalbumin/anti-ovalbumin) leads to ˜ 70% depletion of Fc receptor-bearing cells, while most of the B cells (SIg+ cells) pass through the columns. The relative frequency of E+ cells in the passed fraction frequently shows a slight reduction. These preparations have a very low K-cell activity, indicating that K cells are lymphocytes with Fc receptors of relatively strong avidity.