Zohair Mishal

Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).

Early endosome membrane dynamics characterized by flow cytometry

Early endosomes are very dynamic intracellular membrane organelles that undergo multiple fusion and fission events. In this study, we developed a novel assay based on multiparametric flow cytometric analyses and early endosome sorting to characterize better the mechanisms of early endosome membrane dynamics in vitro. In particular, we have investigated the role of rab4 and rab5, two small GTPases known to regulate distinct steps of membrane traffic in the endocytic pathway. We show that early endosomes undergo homotypic fusion reactions, which lead to the formation of fusion intermediates with increased size. Fusion is efficiently stimulated by recombinant rab5 but not by recombinant rab4. Subsequently, membrane fission consumes this larger fusion compartment. This fission process is stimulated by rab4 and by the GTP hydrolysis-defective mutant rab4Q67L.

A calmodulin antagonist increases the apparent rate of endocytosis of liposomes bound to MHC molecules via monoclonal antibodies

We have investigated the molecular mechanisms required for endocytosis of MHC-encoded proteins by a cell line, TRH 42, that expresses endogenous murine and introduced human class I molecules. As probes we have used protein A-bearing liposomes which bind to cell surface determinants via monoclonal antibodies. The technique of fluorescence quenching release was used with liposome encapsulated quenched carboxyfluorescein as the marker for endocytosis. We demonstrate that the calmodulin antagonist trifluoperazine (TFP) enhances the apparent rate of endocytosis of liposomes bound to MHC class I molecules. Drugs that interfere with energy metabolism, microfilament organization, or phospholipase A2 activity all block endocytosis both in the presence and absence of TFP. The requirement of extracellular Ca2+ for endocytosis was found to be partial. The implications for the structural and enzymatic requirements of endocytosis of MHC class I molecules are discussed.

Expression of human histocompatibility antigens on the surface of murine cells transformed by cosmid clones containing HLA genes

Abstract Thymidine kinase-negative C3H mouse L fibroblasts (LMTK−) transformed with cosmid clones containing both herpes virus-derived thymidine kinase (TK) and HLA class I genes were first selected in HAT (hypoxanthine, aminopterin, thymidine) medium and subsequently analyzed for the expression of human transplantation antigens. TK+-transformed cells expressing HLA class I molecules were characterized by surface radioimmunoassay, cytofluorimetric analysis and immunoperoxidase PAP technique at the light and electron microscopic levels, using a set of monoclonal antibodies. Comparisons were made with human B (Raji) and T (1 301) lymphoblastoid cell lines which respectively express high and low levels of HLA molecules on their surface. The expression of HLA class I in association with murine β2-microglobulin on the surface of transformed cells did not reduce the level of expression of H-2 molecules.

Molar quantification by flow cytometry of fatty acid binding to cells using dipyrrometheneboron difluoride derivatives

Fatty acid analogs of a dipyrrometheneboron difluoride fluorophore (BDY-FA) have recently been developed. Relative to other fluorescent fatty acids, some of these have the advantages of excitation and emission spectra similar to those of fluorescein and of high quantum yield, which permits their use in conventional argon laser cytometry or microscopy. For the cytofluorimetric quantification of BDY-FA analogs, expressed as molecules bound per cell, we have compared the fluorescence of BDY-dodecanoic acid (BDY-C12) with that of fluorescein. Fluorescent beads with different amounts of bound fluorescein were used to calibrate a flow cytometer in order to correlate the fluorescence intensity with the number of fluorescein molecules per bead. In addition, starting from the basic equation defining the relationship between fluorescence and concentration, we have derived another equation which makes it possible to establish, for a given fluorescence, the relative molar concentration of both fluorochromes and, consequently, to express the fluorescence intensity emitted by the BDY-FA as the equivalent number of BDY-FA molecules. As an example of the potential application of this procedure, the time-course and concentration-dependent binding of BDY-C12 to quiescent and mitogen-activated human lymphocytes and to cultured human T-lymphoma cells have been studied. The method described is of general interest as it can also be applied to the flow cytometric or laser scanning microscopic quantification of other fluorescent dyes.

Dual parameter, quantitative cytofluorometric analysis of endogenous H-2Kk and foreign HLA class I molecules expressed at the surface of murine transformed L cells☆

By using a calibrated dual laser cell sorter and monoclonal antibodies directly conjugated to fluorescein and rhodamine and specific for H-2Kk and HLA class I antigens, quantitative cytofluorometric analysis was performed on individual HLA-A3 or -CW3 transformed mouse L cells (H-2k). More than 80% of these cells expressed both HLA class I and H-2Kk molecules. Their respective levels of expression were calculated: a mean of 4 X 10(5) HLA class I and 2.3 X 10(5) H-2Kk molecules per single cell. Quantitative comparison with control untransformed L cells and double fluorescence contour maps showed a positive correlation between the levels of expression of HLA class I and H-2Kk molecules suggesting that expression of foreign class I molecules did not occur at the expense of the endogenous H-2k product.