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. 2018 Oct;103(10):e458-e461.
doi: 10.3324/haematol.2018.188680. Epub 2018 May 10.

Autologous T-cell activation fosters ABT-199 resistance in chronic lymphocytic leukemia: rationale for a combined therapy with SYK inhibitors and anti-CD20 monoclonal antibodies

Esteban Enrique Elías  1 , María Belén Almejún  1   2 , Ana Colado  1 , Gregorio Cordini  1   3 , Maricef Vergara-Rubio  1 , Enrique Podaza  1 , Denise Risnik  1   4 , María Cabrejo  5 , Horacio Fernández-Grecco  5 , Raimundo Fernando Bezares  6 , María Del Rosario Custidiano  7 , Julio César Sánchez-Ávalos  7 , Ángeles Vicente  8 , Gonzalo Martín Garate  8 , Mercedes Borge  1   4 , Mirta Giordano  1   4 , Romina Gamberale  9   4
Affiliations

Autologous T-cell activation fosters ABT-199 resistance in chronic lymphocytic leukemia: rationale for a combined therapy with SYK inhibitors and anti-CD20 monoclonal antibodies

Esteban Enrique Elías et al. Haematologica. 2018 Oct.
No abstract available

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Figures

Figure 1.
Figure 1.
The activation of autologous T lymphocytes induces ABT-199 resistance in CLL which is overcome by GS-9973. A) PBMC from CLL patients (n= 30, 4 × 106 cells/mL) were cultured with DMSO (vehicle) or different doses of ABT-199. The survival of CD19+, CD3+CD56−, CD3−CD56+and CD14+ cells were evaluated daily by flow cytometric alterations of light-scattering properties (Online Supplementary Figure S1A) and confirmed by Annexin V-FITC assay (Online Supplementary Figure S1B). The figure shows the mean ± SEM of the percentage of cells within the gate of viable cells at 24 hours in control and ABT-199 cultures. Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test. *P<0.05, ****P<0.0001, treated vs. control. B) PBMC from CLL patients (n=15, 4×106 cells/mL) were cultured with aCD3 (50 ng/well, 48-well plate) or the corresponding isotype control (data not shown) for 24 hours with or without ABT-199. Then, the expression of CD69 and CD25 on CD4+ and CD8+ T cells were evaluated by flow cytometry. The figure shows the mean ± SEM of the percentage of CD4+ or CD8+ cells expressing CD25 or CD69 in each condition. Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test,*P<0.05, treated vs. control. C) PBMC from CLL patients (4 × 106 cells/mL) were cultured with aCD3 (50 ng/well, 48-well plate) or the corresponding isotype control for 48 hours. Then, ABT-199 or DMSO were added to the cultures for another 24 hours. The survival of CD19+ cells was evaluated as mentioned above. The graph shows the mean ± SEM of the percentage of CD19+ cells within the gate of viable cells and the results obtained with each CLL patient (n=18) are also shown. Open circles highlight CLL patients with less than 1% of T cells within PBMC (Patients #8, #15 and #17 of Online Supplementary Table S1). Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test, **P<0.01. D) PBMC from CLL patients (n=19, 4×106 cells/mL) were cultured with aCD3 or the isotype control (control cultures) for 48 hours. The expression of the activation marker CD86 on CLL cells (CD19+) was evaluated by flow cytometry. The figure shows the percentage of CD19+CD86+ cells of each patient in control and aCD3 cultures. Statistical analysis was performed using the Wilcoxon matched pair test, ****P<0.0001. E) PBMC from CLL patients (n=8, 4×106 cells/mL) were cultured with or without aCD3 for 48 hours. Next, BCL-XL and MCL-1 expression on purified CLL cells were evaluated by Western Blot as detailed in the Online Supplementary Materials and Methods. The figure shows the expression of BCL-XL, MCL-1 and ACTIN in control and aCD3 cultures. F) PBMC from CLL patients (4×106 cells/mL) were cultured with or without aCD3, in the presence or absence of GS-9973 (1 μM) for 48 hours. Then, ABT-199 was added to the cultures for another 24 hours. CD19+ cell survival was evaluated as mentioned above. The figure shows the mean ± SEM of the percentage of CD19+ cells within the gate of viable cells, and the results obtained with each CLL patient (n=18) are also shown. Statistical analysis was performed, using the Friedman test followed by Dunn’s multiple comparison test, **P<0.01. CLL: chronic lymphocytic leukemia.
Figure 2.
Figure 2.
ABT-199 enhances CLL cells phagocytosis by macrophages. A-B) Purified CFSE-labeled CLL cells (n=20) were cultured for 3 hours with ABT-199 or DMSO and then coated or not with rituximab (50 μg/mL). The phagocytosis assay was performed as detailed in the Online Supplementary Materials and Methods with macrophages obtained by culturing monocytes from healthy donors PBMC in complete medium with MCSF (50 ng/mL) for 5 days. The phagocytosis was evaluated by flow cytometry after 1 hour of culture when macrophages were trypsinized. The bars in Figure A show the percentage of macrophages (determined by morphology in the FSC-H and SSC-H dot plot) that have taken up CFSE-labeled CLL cells. Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test.*P<0.05, **P<0.01, ****P<0.0001. Representative dot plots showing FSC-H and CFSE with the percentage of macrophages in each quadrant are shown in Figure B. C-D) PBMC from CLL patients were cultured with ABT-199 or DMSO for 3 hours. PtdSer exposure induced by ABT-199 was evaluated by Annexin-V binding using Annexin-V FITC and PI. Figure C shows the mean ± SEM of the percentage of Annexin V+ PI-cells in each condition, and the results obtained with each patient (n=9) is also shown. Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test, ***P<0.001. Representative dot plots showing the Annexin V-FITC binding and PI staining of control and ABT-199 cultures, while the percentages of cells in each quadrant are shown in Figure D. E-F) PBMC from CLL patients treated or not with ABT-199 (1 μM) for 3 hours were then coated, or not, with rituximab and the antibody binding was evaluated by flow cytometry using anti-human IgG FITC. Figure E shows the mean ± SEM of the mean fluorescence intensity (MFI) of anti-human IgG FITC in control and ABT-199 cultures. The results obtained from each patient (n=9) is also shown. Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test. Representative histogram showing rituximab binding on CLL cells treated or not with ABT-199 for 3 hours are depicted in Figure F. G) Purified CFSE-labeled CLL cells were cultured for 3hours with ABT-199 or DMSO and then coated, or not, with rituximab. The phagocytosis assay was performed as mentioned above, with macrophages pre-incubated 30 min with or without GS-9973 (n=13). Statistical analysis was performed using the Friedman test followed by Dunn’s multiple comparison test, *P<0.05. CLL: chronic lymphocytic leukemia; DMSO: dimethyl sulfoxide.
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References

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