Direct Cell Death Induced by CD20 Monoclonal Antibodies on B Cell Lymphoma Cells Revealed by New Protocols of Analysis

Abstract

CD20 monoclonal antibodies (mAbs) eliminate B cells in several clinical contexts. At least two of these Abs, obinutuzumab (OBI) and rituximab (RTX), induce quick elimination of targets and put cancer patients at risk of tumor lysis syndrome (TLS) within 12-24 h of the first dose. The mechanisms of killing can require the recruiting of effector mechanisms from the patient's immune system, but they can induce direct killing as well. This can be more rapid than recruiting cellular effectors and/or complement. We showed here that OBI and RTX induce quick (<1 h) and high (up to 60% for OBI) killing of two different B cell lines. This was unveiled by using two different techniques that circumvent cell centrifugation steps: a Muse^® Cell Analyzer-based approach and a direct examination of the cells' physical properties by using forward scatter (FS) area and side scatter (SS) area by flow cytometry. These results excluded the presence of aggregates and were also confirmed by developing a normalized survival ratio based on the co-incubation of RTX- and OBI-sensitive cells with MOLM-13, an insensitive cell line. Finally, this normalized survival ratio protocol confirmed the RTX- and OBI-direct killing on primary tumor B cells from B cell chronic lymphocytic leukemia (B-CLL) and Non-Hodgkin's lymphoma (NHL) patients. Moreover, we unveiled that direct killing is higher than previously expected and absent in patients' samples at relapse. We also observed that these mAbs, prior to increasing intracellular calcium levels, decrease calcium entry, although manipulating calcium levels did not affect their cytotoxicity. Altogether, our results show that direct killing is a major mechanism to induce cell death by RTX and OBI mAbs.

Keywords: B cell lymphoma; CD20 mAbs; LLC; direct killing; obinutuzumab; rituximab.

Figure 1

RTX and OBI induce quick direct cell death measured by Muse^® Cell Analyzer. The depicted cell lines (1 million/mL) were incubated with RTX or OBI (10 µg/mL) for the indicated times. Next, cells were incubated with the Muse^TM Count & Viability Assay kit and cell viability was directly analyzed in a Muse^® Cell Analyzer. The graphs display the percentage of viability after normalizing the viability at time 0 to 100%. The graphs display the mean ± SD of 3 independent experiments with biological replicates; * p < 0.05, ** p < 0.01, **** p < 0.0001; Tukey’s multiple comparisons test compared to control or as depicted in the graphics.

Figure 2

RTX and OBI induce quick direct cell death measured by 7AAD and Viobility^TM and analyzed by FACs. The depicted cell lines (1 million/mL) were incubated with RTX or OBI (10 µg/mL) for the indicated times. Then, cells were incubated with 7-AAD or Viobility^TM 405/452, following the provider’s instructions. Cells were washed and cell viability analyzed by FACs, using a Beckman Coulter Gallios cytometer. The graphs display the percentage of dead cells after normalizing the death at time 0 to 0%. Graphs show mean +/− SD of 3 (Raji and Daudi) or 2 (K-562) experiments performed in duplicate; * p < 0.05, ** p < 0.01, **** p < 0.0001; Tukey’s multiple comparisons test compared to control or as depicted in the graphics comparing groups to each other when using the same label.

Figure 3

RTX and OBI induce a quick cell death measured by changes in cell morphology. The depicted cell lines (1 million/mL) were incubated with RTX or OBI (10 µg/mL) for the indicated times. Then, cells were directly analyzed by FACs with neither washing nor centrifugation steps. Doublets and very small debris were removed as described in Figure S1. (a) One hour post-treatment; (b) four hours post-treatment. Each graph displays 20,000 events.

Figure 4

Using CD20 mAb-resistant MOLM-13 cells unveiled a high killing of target cells by RTX and OBI eight hours after treatment. Daudi, Raji, or K-562 cells were labeled with CTFR, whereas resistant MOLM-13 cells were with CFSE, following the provider’s instructions. Both cells were mixed at a 1-to-1 ratio and treated with RTX or OBI (10 µg/mL) before analyzing the number of cells in the mixed and the viability of the remaining cells by 7-AAD or Viobility^TM 405/452 staining (left graphs). The normalized survival ratio (right graphs) was calculated as depicted in Figure S2. Graphs show mean +/− SD of at least 5 experiments performed in duplicate; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Tukey’s multiple comparisons test compared to control or as depicted in the graphics comparing groups to each other when using the same label.

Figure 5

RTX and OBI induced cell aggregation and direct cell death on PBMCs from B-CLL patients. B-CLL PBMCs (1 million/mL) were thawed and directly incubated with RTX or OBI (10 µg/mL) for the indicated times. Next, cells were directly analyzed by FACs with neither washing nor centrifugation steps. (a) The percentage of singlets were quantified by FACs as described in Figure S1b,c. After treatment, cells were washed and stained with an CD19 mAb to select B cells and Viobility^TM 405/452 to stain dead events. Cells were washed and analyzed by FACs using Beckman Coulter Gallios 3 Lasers. The graphs display the raw percentage of viability (b) or the treatment-specific cell death, which was obtained after subtracting the percentage of cell death from the control 1 h after incubation (c). Each graph represents mean +/− SD of 4 patients with measurements performed in duplicate. * p < 0.05, *** p < 0.001, **** p < 0.0001; Tukey’s multiple comparisons test compared to control or as depicted in the graphic.

Figure 6

CD20 mAbs induced direct cell death on tumor cells from NHL and B-CLL patients. PBMCs from NHL (a) or B-CLL (b) patients at diagnosis were treated for 8 h with the depicted mAbs and tumor B cell death was examined by the method of cell viability (left) or normalized survival ratio (right) described in Figure S5. Each point represents one patient at diagnosis and performed in duplicate. Graphs show mean +/− SD, of 21 and 10 patients for NHL or B-CLL, respectively; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Tukey’s multiple comparisons test compared to control or as depicted in the graphics.

Figure 7

Tumor B cells from NHL and B-CLL patients at relapse are resistant to CD20 mAb-induced direct cell death. PBMCs from patients at diagnosis (top graphs) and after relapse (bottom graphs) were treated for 8 h with the depicted mAbs, and tumor B cell death was examined by the method of cell viability (left) or normalized survival ratio (right) described in Figure S5. Each point represents results for one patient at one time point (diagnosis or relapse) and was performed in duplicate. Graphs show mean +/− SD of 3 experiments; * p < 0.05; Tukey’s multiple comparisons test compared to control or as depicted in the graphics.

Figure 8

CD20 mAbs show antagonist effects on CCE depending on treatment duration. (a) Cells were treated with 10 µg/mL of RTX or OBI for 45 min and CCE was measured after addition of 5 mM of calcium. (b) RTX or OBI was applied just before adding 5 mM of extracellular calcium. Intracellular Ca2+ variations compared to the control condition were evaluated using the fluorescence emission of the Fura2 probe measured at 510 nm with an excitation light at 340 and 380 nm. Each graphic represents mean +/− SD of the number of experiments depicted in the graphs. Mann–Whitney was used to compare treatments. * p < 0.05; ** p< 0.01.