FIGURE 1 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2
Authors: Ciudad M. Teresa , Quevedo Rene , Lamorte Sara , Jin Robbie , Nzirorera Nadine , Koritzinsky Marianne , McGaha Tracy L. 
Published: March 2024

<p>Dabrafenib (DAB) impairs MDSC proliferation and differentiation. <b>A,</b> Representative flow cytometry histograms showing antigen-specific proliferation of CD8<sup>+</sup> T cells from OTI mice modulated by bone marrow–derived MDSC. T cells were cocultured with OVA<sub>257–264</sub> peptide-pulsed DCs and MDSC at ratio DC: T cell: MDSC 1:10:30, where 1 = 10,000 cells, in 96-well plates for 60 hours. Experiment repeated three times with similar results. DAB = dabrafenib. <b>B,</b> Frequency of OTI CD8<sup>+</sup> T undergoing one (D1), two (D2), three (D3), four (D4), or more than five (D5) cycles of division from plot A. NP = nonproliferative cells. Data are expressed as mean value. Experiment repeated three times with similar results. <b>C,</b> Mean <i>Arg1</i>, <i>Nos2, and Eif2ak4</i> expression relative to 18s RNA, assessed by qPCR (<i>n</i> = 4). MDSCs were generated in presence ± 1.5 µmol/L DAB for 4 days. <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. Experiment repeated three times with similar results. <b>D,</b> Representative flow cytometry histograms showing antigen-specific proliferation of CD8<sup>+</sup> T cells from OTI mice modulated by mature bone marrow–derived MDSC. T cells were cocultured with OVA<sub>257–264</sub> peptide-pulsed DCs and MDSC at ratio DC: T cell: MDSC 1:10:30, where 1 = 10,000 cells, in 96-well plates for 60 hours in presence of ± 1.5 µmol/L DAB. Experiment repeated three times with similar results. DAB = dabrafenib. <b>E,</b> Frequency of OTI CD8<sup>+</sup> T undergoing one (D1), two (D2), three (D3), four (D4), or more than five (D5) cycles of division from D. NP = nonproliferative cells. Data are expressed as mean value. Experiment repeated three times with similar results. <b>F,</b> Number of MDSCs generated <i>in vitro</i> in the presence of ± 1.5 µmol/L DAB after 4 days in culture (<i>n</i> = 4). <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. Experiment repeated three times with similar results. <b>G,</b> Dose–response for inhibition of proliferation after 4 days in culture. DNA fluorescence was measured for cultures with the indicated concentration of dabrafenib. IC<sub>50</sub> value was calculated at 1.59 µmol/L. Lines represent the mean ± SEM of four independent experiments. <b>H,</b> Dose–response for viability after 4 days in culture in presence of 1.5 µmol/L DAB. Each datapoint indicates the percentage of total live cells assessed by flow cytometry, normalized to control samples. Lines represent the mean ± SEM of four independent experiments. <b>I,</b> Percentage of live cells in MDSC <i>in vitro</i> cultures ± 1.5 µmol/L DAB assessed by flow cytometry. Error bars indicate ± SD (<i>n</i> = 4). <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. Experiment repeated four times with similar results. <b>J,</b> Mean frequency and total cell number of different subsets of immune cells in fresh bone marrow (BM) and MDSC. A total of 1 × 10<sup>6</sup> BM cells were seeded at the start of the experiment and differentiated for 4 days ± 1.5 µmol/L DAB (<i>n</i> = 4). Color coding represents each subtype. Experiment repeated two times with similar results. <b>K,</b> Representative flow cytometry contour plots of MDSC surface phenotype when expanded for 4 days with ± 1.5 µmol/L DAB compared with the ones found in fresh bone marrow. Gates show mean frequency (<i>n</i> = 4). Experiment repeated two times with similar results. <b>L</b> and <b>M,</b> Mean frequency of the PMN population CD11b<sup>+</sup>MHCII<sup>neg</sup>Ly6C<sup>+</sup>Ly6G<sup>+</sup> in MDSC ± 1.5 µmol/L DAB (<b>J</b>) or increasing doses of DAB (K) (<i>n</i> = 4). Experiment repeated three times with similar results. In <b>J</b>, <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. <b>N,</b> Representative flow cytometry histograms showing antigen-specific proliferation of CD8<sup>+</sup> T cells from OTI mice modulated by FACS sorted m-MDSC generated in presence of ± 1.5 µmol/L DAB. T cells were cocultured with OVA<sub>257–264</sub> peptide-pulsed DCs and MDSC at ratio DC: T cell: MDSC 1:10:20, where 1 = 5,000 cells, in 96-well plates for 60 hours. Experiment repeated three times with similar results. DAB = dabrafenib.</p>