Flow cytometry enables a high-throughput homogeneous fluorescent antibody-binding assay for cytotoxic T cell lytic granule exocytosis

Abstract

We developed a homogeneous phenotypic fluorescence end-point assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay's ability to detect novel active compounds, we screened at a concentration of 50 µM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 µM were investigated further. Four reduced elevations in intracellular Ca(2+); it is likely that depolarization of the cells' membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the mitogen-activated protein (MAP) kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.

Figure 1. Detecting exocytosis via anti-LAMP antibody binding without washing

A) Histograms of anti-LAMP antibody fluorescence for cells that were washed (top) or not washed (bottom). Unstimulated cells are represented by a dashed line, while stimulated cells are denoted by a solid line. Note the shift in the fluorescence of the unwashed unstimulated cells compared to the washed unstimulated cells. B) Plot of the geometric mean of anti-LAMP antibody fluorescence from a test conducted in a 96 well plate.

Figure 2. Screening a 91-compound library in plate format

A) Histograms of anti-LAMP antibody fluorescence from a negative control well, a positive control well, and a well treated with compound 9b. Cells were gated on forward and side scatter so as to exclude dead cells; for all conditions, ~40% of cells were in the live cell gate. B) Plot of the geometric mean of anti-LAMP antibody fluorescence from one screen of the synthetic 91 compound library. Data have been rearranged from the original plate format so that compounds containing each of the 16 different scaffolds are displayed in a contiguous manner. Positive control (filled squares) and negative control (filled circles) wells are shown first, and dashed lines separate compounds containing different scaffolds. Structures of three compounds possessing scaffolds present in four or more inhibitory compounds are shown above the plot. C) Plot of the geometric mean of anti-LAMP antibody fluorescence from one screen of the 91 compound library vs. that from a second screen, demonstrating reproducibility.

Figure 3. Assessing the mechanism of action of tetracyano-hexahydroisoindole compounds

A) Representative Fura-2 ratio data for the six tetracyano-hexahydroisoindole compounds that inhibited lytic granule exocytosis by ~90% when tested at 10 μM. TG was added as indicated to trigger Ca²⁺ influx. For some traces, some symbols have been omitted for clarity. The lines connecting the symbols are drawn through those missing points. B) Representative bisoxonol data from one experiment of three. The geometric mean of bisoxonol fluorescence (Fl-1) is plotted for compounds (filled circles), Normal Ringer’s (open circles) and K⁺ Ringer’s (open squares). C) Cells treated as indicated were fixed, permeabilized and then stained with an anti-phospho ERK antibody followed by a CY5-labeled secondary antibody.

Figure 4. Screening the Prestwick Compound Library in high-throughput format

A) Left, sample data from the high-throughput cytometer system showing the acquisition of 16 negative and 16 positive control wells. Data from different wells are separated by air bubbles, allowing them to be discriminated off-line. Right, representative histograms of anti-LAMP antibody fluorescence from the experiment shown on the left. B) Plot of the geometric mean of anti-LAMP fluorescence from one run of Plate 1 of the PCL. Negative (closed triangles) and positive (closed squares) control data are indicated, and compound-containg test wells are represented by open circles. C) Heat map of the data shown in (B). The lookup table is set such that the lowest mean fluorescence intensity is represented by black, while the highest is represented by white. D) Plot of the number of hits (solid symbols, left axis) and the Jaqqard similarity index (open triangles) for two runs of the entire PCL analyzed at inhibitory thresholds of 40, 50 and 60 %.