Joel H. Kaplan

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.

The detection of phytomitogen-induced changes in human lymphocyte surfaces by laser doppler spectroscopy☆

The changes in electrophoretic mobility and isoelectric point produced by incubating human peripheral blood lymphocytes with phytohemagglutinin (PHA) and concanavalin A (Con A) have been characterized by laser Doppler spectroscopy. The results extend and partially confirm older observations made by classical procedures. Incubation with either agent for 90 min at 37 degrees C resulted in stable and reproducible decreases in electrophoretic mobility, and increases in the isoelectric point. The incubation conditions used are known to permit primary attachment of the phytomitogen, capping and endocytosis; nevertheless, at least in the case of Con A, washing the cells with a specific inhibitor for Con A binding, methyl-alpha-D-glucoside (MAG), resulted in complete reversal of the electrokinetic changes, showing that the underlying changes in cell surface constitution detected under these conditions are solely due to reversibly bound Con A. The results suggest that laser Doppler spectroscopic changes could provide a direct assay for specific binding to immunocompetent cell surfaces.

Association of electrophoretic mobility with other cell surface markers of T cell subpopulations in normal individuals and cancer patients.

Abstract Two major electrophoretically distinct T cell components were observed in peripheral blood of normal donors. The cells comprising these components differed in their binding capacity 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 and belonged to the high-mobility component. Low-affinity E-RFC belonged to the low-mobility component. In cancer patients the fraction of cells falling in the high-mobility class decreased by 20 ± 11% of total cells, but no significant decrease of high-affinity E-RFC was observed. Thus, the correspondence between high-affinity and high-mobility found for normal donor lymphocytes may not hold for a subset of lymphocytes found in individuals with cancer.

A Miniature Phytohemagglutinin Assay Using Disposable Microtiter Plates

A simplified microassay for the transformation of lymphocytes by phytohemagglutinin (PHA) has been developed. Whole blood cultures are treated with PHA, incubated in microtiter plate wells, precipitated on glass fiber filters, and assayed for new DNA synthesis via thymidine-2-14C uptake using a multiple sample precipitator. This method uses only 7μl of blood per culture and is sufficiently rapid to permit use on large numbers of replicate samples.

Early membrane events monitored by changes in cell surface charge following specific antigen-lymphocyte binding

Abstract Peripheral blood lymphocytes isolated from donors sensitized to the tuberculin antigen-purified protein derivative (PPD) were altered in electrophoretic mobility when incubated in vitro with the antigen. Initial exposure to PPD caused no measurable alteration of the electrophoretic mobility profile, but after a period of about 3.5 hr the mean mobility of approximately 50% of the cells was shifted by as much as 10% to a higher value (increased negative charge). This change in mobility was not observed for skin test-negative donors or for skin test-positive donors whose antigen-binding lymphocytes had been removed by an affinity chromatography column of PPD. The time lag of the mobility alteration as well as the large number of cells responding suggested that a factor(s) released into the cell culture medium was probably responsible for the shift of the mobility profile. In support of this hypothesis were the observations that the addition of either the protein synthesis inhibitor, puromycin, or cycloheximide during incubation with the antigen prevented these mobility changes; and supernatants from antigen incubations involving PPD skin test-positive donor lymphocytes were capable of causing mobility increases of PPD skin test-negative donor lymphocytes. Cell fractionation experiments indicated that T cells rather than B cells respond to the released factor(s). Additional kinetic studies on unfractionated lymphocytes showed that the mobility response was modulated in time with an oscillation period of about 2 hr. These results suggest that cell surface charge changes may require the involvement of some cyclic metabolic process at the lymphocyte surface or perhaps formation and regeneration of receptor—factor complexes.

Identification by monoclonal antibodies of T-cell subpopulations activated by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Two major T-cell subpopulations obtained from human peripheral blood were studied their capacity to be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). The subpopulations were identified and separated from each other by their reactivity either to the monoclonal antibody, OKT4 or OKT8, followed by complement-dependent lysis. The fraction enriched in T8+ cells showed approximately a 1.2-2.0-fold greater proliferative response to TPA (20 ng/ml) than did the T4+ cell subset. Optimal activation required the presence of monocytes. Comparison of the mitotic activity showed that the sum of each T-cell subset was less than a mixture of the two. This indicates either a unilateral or cooperative interaction of mitogenesis in the presence of TPA.

Reduction of non-specific radioactive counts in a whole blood microtiter plate assay of mitogenesis

Non-specific radioactive counts retained on the glass fiber filters from a multiple automatic sample harvester were reduced markedly by 3 simple changes in the procedure for harvesting thymidine-labeled whole blood cultured from microtiter plates. These modifications increased the sensitivity, precision and accuracy of the lymphocyte transformation assay.