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. 2020 May 18;4(10):2143–2157. doi: 10.1182/bloodadvances.2019001091

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Figure 6. — Daratumumab induces expansion of activated and patient-tumor specific cTLs. (A-B) cTLs were sorted from PBMCs of healthy donors (n = 6) and patients with CLL (n = 6), with CD38 levels determined as percentage expression (A) and MFI (B). (C) Flow-sorted cTLs were labeled with carboxyfluorescein succinimidyl ester followed by treatment with daratumumab for 72 hours. cTL proliferation was measured and data were represented as multicolor cell proliferation histograms. (D) PBMCs from patients with CLL were incubated with IgG1 control Ab or daratumumab (1 µg/mL) for 72 hours and CD3⁺CD8⁺ T cells were gated to determine percentage of cTLs. (E) Cell surface markers of T-cell activation (CD69, GITR, CD137) and (F) exhaustion (TIGIT, Lag3, CD244, PD1, CTLA4) were measured on CD3⁺CD8⁺ gated cTLs from PBMCs of patients with CLL, which were ex vivo treated with daratumumab (D, 1 µg/mL) or kuromanin (30 µM) for 72 hours. Contour plots are representative and compiled data are presented as mean ± SEM with individual data points overlaid. (G) The presence of CD38⁺PD1⁺ co-expressing cTLs was measured in CD3⁺CD8⁺ gated T cells from PBMCs from patients with CLL treated with daratumumab (1 µg/mL, 72 hours) or kuromanin (30 µM, 72 hours). Data are represented as scatter dot plots of percentage PD1⁺ cells or percentage CD38⁺PD1⁺ cells ± SEM. (H) An illustration of the experimental design in testing daratumumab-primed or unprimed cTL cytolytic response to either untreated CLL cells (−D) or daratumumab-treated CLL cells (+D) from 9 patients is presented. Notably, 7 of these patients were considered as having CD38⁻ disease. (I) Untreated CLL cells (−D) or daratumumab-treated CD19⁺ CLL cells (+D) were labeled with Calcein-AM and cocultured with daratumumab-primed patient autologous cTLs or unprimed cTLs (exposed to IgG1-isotype control Ab) for 6 hours. Percentage specific lysis of CLL cells was measured as percentage calcein release in supernatant. Calcein-AM released from Triton X-100 treated cells (%) was used as maximum lysis and that from untreated CLL cells was used as spontaneous lysis. (J) Similarly, patient-specific tumor specificity of daratumumab-primed cTLs was measured using Calcein-AM labeled autologous/allogeneic CLL cells. Data are represented as scatter dot plots of percentage mean specific lysis of tumor cells ± SEM. Each experiment was performed at least twice in duplicate. *P < .05.

Figure 6. — Daratumumab induces expansion of activated and patient-tumor specific cTLs. (A-B) cTLs were sorted from PBMCs of healthy donors (n = 6) and patients with CLL (n = 6), with CD38 levels determined as percentage expression (A) and MFI (B). (C) Flow-sorted cTLs were labeled with carboxyfluorescein succinimidyl ester followed by treatment with daratumumab for 72 hours. cTL proliferation was measured and data were represented as multicolor cell proliferation histograms. (D) PBMCs from patients with CLL were incubated with IgG1 control Ab or daratumumab (1 µg/mL) for 72 hours and CD3⁺CD8⁺ T cells were gated to determine percentage of cTLs. (E) Cell surface markers of T-cell activation (CD69, GITR, CD137) and (F) exhaustion (TIGIT, Lag3, CD244, PD1, CTLA4) were measured on CD3⁺CD8⁺ gated cTLs from PBMCs of patients with CLL, which were ex vivo treated with daratumumab (D, 1 µg/mL) or kuromanin (30 µM) for 72 hours. Contour plots are representative and compiled data are presented as mean ± SEM with individual data points overlaid. (G) The presence of CD38⁺PD1⁺ co-expressing cTLs was measured in CD3⁺CD8⁺ gated T cells from PBMCs from patients with CLL treated with daratumumab (1 µg/mL, 72 hours) or kuromanin (30 µM, 72 hours). Data are represented as scatter dot plots of percentage PD1⁺ cells or percentage CD38⁺PD1⁺ cells ± SEM. (H) An illustration of the experimental design in testing daratumumab-primed or unprimed cTL cytolytic response to either untreated CLL cells (−D) or daratumumab-treated CLL cells (+D) from 9 patients is presented. Notably, 7 of these patients were considered as having CD38⁻ disease. (I) Untreated CLL cells (−D) or daratumumab-treated CD19⁺ CLL cells (+D) were labeled with Calcein-AM and cocultured with daratumumab-primed patient autologous cTLs or unprimed cTLs (exposed to IgG1-isotype control Ab) for 6 hours. Percentage specific lysis of CLL cells was measured as percentage calcein release in supernatant. Calcein-AM released from Triton X-100 treated cells (%) was used as maximum lysis and that from untreated CLL cells was used as spontaneous lysis. (J) Similarly, patient-specific tumor specificity of daratumumab-primed cTLs was measured using Calcein-AM labeled autologous/allogeneic CLL cells. Data are represented as scatter dot plots of percentage mean specific lysis of tumor cells ± SEM. Each experiment was performed at least twice in duplicate. *P < .05.