Separation of human T lymphocyte subpopulations (Tmu, Tgamma) by density gradient electrophoresis

Abstract

Purified human peripheral blood T lymphocytes were fractionated by density gradient electrophoresis on the basis of their surface charge. The high mobility cell fractions were found to be enriched in T cells having receptors for IgM (Tmu cells) with only minor proportions of T cells having receptors for IgG (Tgamma cells). In contrast, the low mobility cell fractions were enriched in Tgamma cells with very low numbers of contaminating Tmu cells. Both populations were of higher mobility than human peripheral blood B lymphocytes. High-affinity sheep erythrocyte resette-forming cells (E-RFC) were relatively enriched in the high and intermediate mobility fractions and appear to include the Tmu cells and the T cells without receptors for IgG or IgM (Tvarphi). The low affinity E-RFC were found only in the lower mobility fractions that included the Tgamma cell population. A direct correlation was observed between the number of Tgamma lymphocytes and the low affinity E-RFC in all the fractions. The separated cell fractions were treated in vitro with different concentrations of concanavalin A (Con A) and examined for the numbers of Tmu and Tgamma cells. Low concentrations of Con A (2.5 mug/ml) significantly increased the number of Tmu cells, whereas high concentrations of Con A (20 mug/ml and 40 mug/ml) markedly reduced the number of Tmu cells and increased the number of Tgamma cells. Furthermore, all fractions (both Tmu and Tgamma cell enriched) responded by proliferation to Con A, whereas only the high and intermediate mobility fractions (enriched in Tmu cells) responded to phytohemagglutinin. Fractions enriched in Tgamma cells responded very poorly to phytohemagglutinin. This method provides another technique for separating human Tmu- and Tgamma-enriched lymphocyte subpopulations and does not modulate the Fc receptors of the cells, in contrast to the rosetting techniques currently in use for the separation of these lymphocytes.