T cell enrichment and depletion of human peripheral blood mononuclear cell preparations. Unexpected findings in the study of the functional activities of the separated populations

Abstract

In the present communication we present the results of a study undertaken to assess the effectiveness of several methods of depleting human peripheral blood of thymus-derived (T) cells, and to assess the purity of the T cell-enriched populations obtained. The methods of T cell depletion employed included rosetting with unmodified sheep red blood cells (SRBC) in the cold at high erythrocyte to lymphocyte ratios; rosetting with S-2-aminoethylisothiouronium bromide hydrobromide (AET)-treated SRBC; and cytotoxic depletion using anti-Leu-1, a monoclonal antibody directed toward a pan-T cell antigen, and rabbit complement. The methods used to enrich for T cells included rosetting with unmodified or AET-treated SRBC, and passage through a nylon wool column. • The purity of the T-enriched and T-depleted populations obtained was assessed by (a) repeat rosetting with unmodified SRBC, (b) staining for cell surface immunoglobulin (sIg), (c) quantitating the number of Ig secreting cells (ISC) in a reverse hemolytic plaque assay, (d) quantitating the proliferative responses to the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) and to the B cell mitogen Staphylococcus aureus Cowan (SAC), and (e) quantitating the development of ISC following culture with PWM, with or without the addition of an irradiated T cell-enriched population. • By all parameters investigated, the depletion of T cells from peripheral blood by rosetting with AET-treated SRBC or by treatment with anti-Leu-1 and complement was far more effective than by rosetting with unmodified SRBC under optimal conditions. Of particular note was the finding that functional assays (ISC at time zero (T 0), proliferative response to SAC, the development of ISC on culture with PWM) revealed a much higher degree of B cell contamination in all of the T cell-enriched populations than would have been suspected from the extent of the depletion of sIg staining cells.