Possible misinterpretation of the mode of action of therapeutic antibodies in vitro: homotypic adhesion and flow cytometry result in artefactual direct cell death

To the editor:

Two groups have recently proposed that type II anti-CD20 monoclonal antibodies (mAbs) B1 (tositumomab) and GA101, as well as anti–HLA-DR antibody (L243), induce strong homotypic adhesion and direct cell death. They claim, on the basis of annexin V, propidium iodide (PI), and flow cytometric analysis, that up to 80% of cell death occurs after 24 hours of treatment with these mAbs.^1-3 

We have also observed, after treatment of B lymphoma cells in vitro with either anti–HLA-DR BK267 or anti-CD20 antibody B1 (BioGenex), an apparent 30% to 70% increase in cell death of different human B lymphoma cell lines by fluorescence-activated cell sorting (FACS) analysis of either 7-AAD–stained cells (Figure 1A and data not shown). However, in parallel observations in Bürker chambers, we found that only 5% of antibody-treated cells were stained with Trypan blue and most appeared alive (data not shown). We then observed in alamarBlue vital dye (AbD Serotec) assays that only 2% to 12% cell death could be measured after 24 hours of treatment of the same cell lines with anti–HLA-DR or B1 antibody (Figure 1B and data not shown). Seeing that the cells formed large and tight aggregates in the presence of the antibodies, which were resistant to vigorous pipetting, we thought that this aggregation could have significantly altered the flow cytometric data. We therefore performed cytospins of cells treated with mAbs and stained with 7-AAD and DAPI. Counting 7-AAD⁺ cells in cytospins showed very low mortality, with about 5% increased dead cells in antibody compared with controls (Figure 1C). This was markedly different from parallel flow cytometry (Figure 1A). Indeed, with Raji cells, 83% and 48.6% 7-AAD⁺ cells were revealed by FACS analysis after HLA-DR and B1 treatment, respectively, compared with only 7% in cytospins (Figure 1A,C). As positive control, B-CLL treated with alemtuzumab and human serum showed more than 70% cell death in cytospins (Figure 1C), consistent with the results obtained by FACS.⁴ 

We conclude that flow cytometry results with anti–HLA-DR or type II anti-CD20 antibodies are largely due to cell aggregation. Large aggregates containing 30 to 100 cells are probably mostly excluded from the analysis, falling outside the gate of scatter plots. Dead cells are in contrast enriched within the single-cell population because aggregation is an energy-dependent process. An apparent rapid loss of cells due to aggregation was confirmed in flow cytometry by counting the absolute number of events using calibrated beads. Indeed, 87% and 70% apparent decrease in total cell number was observed at 24 hours in anti–HLA-DR– and B1-treated samples, respectively (Figure 1D).

Altogether, these observations suggest that FACS analysis should be interpreted with caution in the functional study of antibodies that cause strong homotypic adhesion, unless means are found to disaggregate cells without causing cell death. They also suggest that anti–HLA-DR and anti-CD20 B1 antibody may induce much less direct cell death than previously suggested and the reason for their efficacy in vivo may reside in other properties, such as homotypic adhesion itself inducing opsonization or immune mediated mechanisms, as recently suggested for type II anti-CD20 mAbs.⁵