Analysis of T cell subpopulations by laser Doppler spectroscopy and the association of electrophoretic mobility differences with differences in rosette-forming affinity

Abstract

Rosetting procedures were used to identify and isolate human T lymphocytes having different affinities for sheep red blood cells (SRBC). High-affinity E rosette-forming cells (E-RFC) were obtained by rosetting at 29°C with limited numbers of SRBC. Low-affinity and total E-RFC were obtained by rosetting at 4°C with an excess of SRBC. The E-RFC isolated from these fractions on Ficoll-Hypaque gradients exhibited different electrophoretic mobilities as measured by laser Doppler spectroscopy. The high-affinity E-RFC mobility was centered at 2.35 μm/sec/V/cm (for 25°C, 0.28 M sucrose medium of 0.005 ionic strength), whereas the low-affinity E-RFC mobility was centered at 2.15 μm/ sec/V/cm. The total E-RFC fractions consisted of cells of both mobilities. When IgG-sensitized E monolayers were used to remove cells bearing Fc receptors from mononuclear cell preparations, the non-adherent and the adherent fraction were each found to contain a different subpopulation of T cells having mobilities centered at 2.35 and 2.15 μm/sec V/cm, respectively. Rosetting assays and mobility measurements were performed simultaneously with the mononuclear cell preparations and the non-adherent fraction from IgG-sensitized E monolayers. In both cases, there was quantitative agreement between the number of high-affinity E-RFC and the number of high-mobility cells. In contrast, there were consistently more low-mobility cells than could be accounted for by low-affinity E-RFC. The determination of cell electrophoretic mobility is a direct physical measurement and its association with such surface markers as different affinity receptors for SRBC and Fc receptors for IgG shows that it should be useful parameter in the characterization of lymphocytes.