Article Figures & Data
Figures
- Figure 1.
Elotuzumab promoted maximal ADCC if NK cells expressed high-affinity CD16 and required SLAMF7 expression on MM cells. A, Comparison of CD16 expression (GRM1 antibody, Southern Biotech) on parental NK-92 cells (CD16−; gray) and NK-92 cells transduced to express 176V (solid line) or 176F (dashed) polymorphic variants of CD16, and mean GMFI ± SD from five experiments (bottom). B, SLAMF7 expression before and after the genetic modification of two human MM cell lines (Elo + anti-IgG1 secondary stain). MM.1R cells were transfected with SLAMF7 zinc finger nucleases (ZFN) to genetically deplete SLAMF7 (MM.1R SLAMF7KO; left), and RPMI8226 cells were stably transduced with SLAMF7 cDNA (RPMI8226+SLAMF7; right), and mean GMFI ± SD from five experiments (bottom). Cells were stained with biotinylated Elo plus secondary streptavidin–APC. C and D, Effect of SLAMF7 expression on NK cell–mediated ADCC in the presence of various concentrations of Elo (0.001–300 μg/mL). Values are mean ± S D. Asterisks indicate statistical significance comparing cytotoxicity by NK-92 cells expressing 176V or 176F variants of CD16 using Welch unequal variance t test. *, P < 0.05; n = 5 independent experiments.
- Figure 2.
Elotuzumab enhances natural cytotoxicity by NK cells in a CD16-independent manner. A, Effect of Elo (0.001–300 μg/mL) on natural cytotoxicity of parental NK-92 cells expressing SLAMF7 and lacking CD16 (SLAMF7+, CD16−), toward MM.1R (top) and RPMI8226 (bottom) cells. Gray squares are genetically modified MM.1R SLAMF7KO (top) or RPMI83226 + SLAMF7 (bottom) targets. Values are mean ±SD. Overhead bars mark statistical significance between untreated and different Elo concentration groups using paired Student t test. **, P < 0.01; *, P < 0.05; n = 5 independent experiments. B, SLAMF7 expression on parental SKOV3 cells (solid line) and SKOV3 cells that were stably transduced with SLAMF7 cDNA (dashed line) and stained with the 162.1 SLAMF7 antibody (BioLegend), and mean GMFI ± SD from five experiments (bottom). C, Kinetics of parental SKOV3 (top graph) or SKOV3(+SLAMF7; bottom graph) target cell death by parental NK-92 cells (SLAMF7+, CD16−) analyzed in xCELLigence assays. n = 4–6. D, Mean, SD, and statistical analysis for percentage target cell death (compared with detergent lysis conditions as 100%) at 10 hours of xCELLigence assays from four to six independent experiments. Overhead bars mark statistical comparison between indicated groups using paired Student t test; **, P < 0.01; *, P < 0.05. E, Schematic of Elo effect on the cytotoxicity of NK cells against SLAMF7+ and SLAMF7− targets.
- Figure 3.
Elotuzumab is unable to enhance natural cytotoxicity of NK cells lacking expression of both SLAMF7 and CD16. A, SLAMF7 expression (162.1 SLAMF7 antibody, BioLegend) on NK-92 control (GFP knockout, SLAMF7+, CD16−; solid line) and NK-92 SLAMF7 KO (CD16−; dashed line) cells generated using CRISPR/Cas9, and mean GMFI ± SD from five experiments (bottom). B, Cytotoxicity toward SKOV3(+SLAMF7) target cells by NK-92 (SLAMF7+, CD16−) and NK-92 (SLAMF7 KO, CD16−) cells studied over time in xCELLigence assays with or without Elo (10 μg/mL; top) or Elo Fc mutant (Elo Fc mut; 10 μg/mL; bottom). C, Mean ± SD target cell death at 10 hours from xCELLigence assays for Elo (top) and Elo Fc mut (bottom) toward SKOV3(+SLAMF7) target cells from four to five independent experiments. Overhead bars mark statistical comparison between indicated groups using paired Student t test. **, P < 0.01; *, P < 0.05. D, Cytotoxicity toward SKOV3(+SLAMF7) target cells by NK-92 (SLAMF7+, CD16−) cells studied over time in xCELLigence assays (top) and mean ±SD target cell death at 10 hours (bottom) for Elo versus Elo F(ab')2 versus Fab (3 and 10 μg/mL). Overhead bars mark statistics as in C. n = 4 independent experiments. E, Schematic showing requirement for SLAMF7 expression on NK cells for CD16-independent NK cell–mediated cytotoxicity.
- Figure 4.
Elotuzumab uniquely promotes SLAMF7–SLAMF7 interactions. A, SLAMF7-TCRζ reporter cell line was cultured with four different plate-bound SLAMF7 antibodies (Elo, ChuLuc90, PDL241, and 162.1; coated overnight at 10 μg/mL) and mouse IL2 (mIL2) was assayed in culture supernatant. B, Indicated SLAMF7 antibodies or isotype controls (0.01–3 μg/mL) were added to 3A9 SLAMF7-TCRζ reporter cell line in plates coated with recombinant human SLAMF7 (hSLAMF7) overnight, and mIL2 secretion was assayed. C, Soluble Elo (left) or hIgG1 isotype control (right) were added to SLAMF7-TCRζ reporter cell line on plate-bound (PB) recombinant hSLAMF7 or PB mesothelin control protein, or hSLAMF7 was added as soluble protein (+hSLAMF7; right). Plotted as mean ± SEM.
- Figure 5.
Elotuzumab promotes costimulation-mediated cytotoxicity. Impact of various SLAMF7 antibodies (10 μg/mL each) on cytotoxicity by NK-92(SLAMF7+,CD16−) cells (A) or SLAMF7-deficient NK-92 (SLAMF7 KO, CD16−) cells (B) toward SKOV3(+SLAMF7) target cells in xCELLigence assays. Mean, SD; and statistics for target cell death are shown at 10 hours from three to four independent xCELLigence assays. Overhead bars mark statistical significance between indicated groups using paired Student t test. **, P < 0.01; *, P < 0.05. ns, not significant. Elo, elotuzumab; CL90, ChLuc90; 162, 162.1; P241, PDL241. C and D, Test of the impact of blocking NKG2D (C) and DNAM1 (D) antibodies on Elo-induced cytotoxicity by NK-92(SLAMF7+, CD16−) cells toward SKOV3(+SLAMF7) target cells in xCELLigence assays. Mean, SD; statistics for target cell death at 10 hours from five independent assays using blocking antibodies at 10 μg/mL each. Overhead bars mark statistics as in A and B. Kinetic plots for all of these xCELLigence assays can be found in Supplementary Fig. S5C and S5D.
- Figure 6.
Elotuzumab enhances NK cell cytotoxicity only if SLAMF7 contains the full cytoplasmic domain. A, Representative SLAMF7 expression (162.1 antibody, BioLegend) on NK-92 SLAMF7 KO cells (gray) and reconstituted with SLAMF7-L (dashed) or SLAMF7-S (solid line) isoforms, and mean GMFI ± SD from five experiments (bottom). B, Impact of Elo (10 μg/mL) on cytotoxicity by NK-92(SLAMF7 KO, CD16−) cells reconstituted with either SLAMF7-L or SLAMF7-S toward SKOV3(+SLAMF7) target cells studied in xCELLigence assays. C, Mean, SD; statistics for target cell death at 10 hours from five independent assays. Overhead bars mark statistical comparisons between indicated groups using paired Student t test. *, P < 0.05.
- Figure 7.
Costimulation of healthy donor NK cell cytotoxicity by Elo Fc mut depends on NK cell activation status. A, Elo or Elo Fc mut (10 μg/mL) was added to SKOV3(+SLAMF7) target cells in the presence or absence of freshly purified NK cells from healthy donor (HD) #1 in xCELLigence assays. B, Compilation of cytotoxicity data for n = 7 healthy donors. Percentage target cell death at the 10-hour time point from xCELLigence assays in the absence (−) or presence (+) of Elo Fc mut by purified NK cells toward SKOV3(+SLAMF7) target cells on the day of the purification (left) or after cultured in the presence of recombinant human IL2 (100 U/mL; +) for 48 hours (right). Each patient is designated by a unique icon, and results with or without antibody addition are connected with lines. Overhead bars mark statistical comparison between indicated groups using Wilcoxon matched-pairs signed rank test. C and D, Representative time course data in which purified NK cells from HD#3 and HD#8 were assayed fresh (left) or after cultured in the presence of recombinant human IL2 (100 U/mL) for 48 hours (right).
Additional Files
Supplementary Data
- Supplementary Data - Supplementary Figures 1-9
Volume 7, Issue 10
