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. Author manuscript; available in PMC: 2020 Jun 1.

Published in final edited form as: Exp Hematol. 2019 May 25;74:52–63.e3. doi: 10.1016/j.exphem.2019.05.002

Figure 3. — (A) Peripheral blood T cells from 4 high-risk MDS patients (pt#1, #2, #5, #6) were transduced with CD123 CAR. CD123 CAR T cells (red) or mock T cells (blue) were analyzed by flow cytometry. Successful >50% transduction (%EGFRt+ cells) was obtained in 3 patients (pt#2, #5, #6) derived T cells. One patient (pt#1) had 9% transduction efficiency. CD123 CAR T cells from pt#2, #5, #6 were further analyzed for CD4 and CD8. Both CD4 and CD8 populations expressed CD123 CAR. (B) Representative flow cytometry showing transduction efficiency of CD4+ and CD8+ subpopulations in CAR T cells (EGFRt+) and non-transduced mock T cells for pt#2. Red box denotes populations further gated for CD4 and CD8 expression. (C) Histograms of CD123+ cells in pt #2, #5, #6 MDS bone marrow (CD3-) and MDS stem cells (CD3−/CD34+/CD38−) after coculture with autologous CD123 CAR T cells (red) or mock T cells (blue) for 48hrs. (D) Primary MDS cells from pt #2, #5, #6 were cocultured with autologous mock T cells (blue) or CD123 CAR T cells (red) for 48hrs at E:T ratio of 1:1. MDS stem cells (CD34+/CD38−/CD123+) in bone marrow (CD3-) are significantly decreased after coculture with autologous CD123 CAR T cells but not mock T cells. Percentage normalized to mock T cell treatment (n=3, mean ± SD). (E) Degranulation as detected by cell surface CD107A is significantly higher in CD123 CAR T cells (red) compared to mock T cells (blue) after coculture with autologous bone marrow sample at E:T ratio 1:1 for 5hrs (n=3, mean ± SD) in pt#2. (F-G) TNF-α production as detected by intracellular flow cytometry is significantly higher in CD123 CAR T cells (red) compared to mock T cells (blue) after coculture with autologous bone marrow sample in the total CD3+ T cell population (F) and both CD4+ and CD8+ subpopulations (G) at E:T ratio 1:1 for 5hrs (n=3, mean ± SD) in pt#2, #8. (H) Secretion of TNF-α, IFN-γ, and IL-2 in culture supernatant of MDS bone marrow sample with autologous CD123 CAR T cells (red) is significantly higher compared to mock T cells (blue) when cocultured for 48hrs at E:T ratio of 1:1 for pt#2 and pt#6, and 72hrs and E:T ratio 2:1 for pt#5 (n=3, mean ± SD, unpaired parametric t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 3. — (A) Peripheral blood T cells from 4 high-risk MDS patients (pt#1, #2, #5, #6) were transduced with CD123 CAR. CD123 CAR T cells (red) or mock T cells (blue) were analyzed by flow cytometry. Successful >50% transduction (%EGFRt+ cells) was obtained in 3 patients (pt#2, #5, #6) derived T cells. One patient (pt#1) had 9% transduction efficiency. CD123 CAR T cells from pt#2, #5, #6 were further analyzed for CD4 and CD8. Both CD4 and CD8 populations expressed CD123 CAR. (B) Representative flow cytometry showing transduction efficiency of CD4+ and CD8+ subpopulations in CAR T cells (EGFRt+) and non-transduced mock T cells for pt#2. Red box denotes populations further gated for CD4 and CD8 expression. (C) Histograms of CD123+ cells in pt #2, #5, #6 MDS bone marrow (CD3-) and MDS stem cells (CD3−/CD34+/CD38−) after coculture with autologous CD123 CAR T cells (red) or mock T cells (blue) for 48hrs. (D) Primary MDS cells from pt #2, #5, #6 were cocultured with autologous mock T cells (blue) or CD123 CAR T cells (red) for 48hrs at E:T ratio of 1:1. MDS stem cells (CD34+/CD38−/CD123+) in bone marrow (CD3-) are significantly decreased after coculture with autologous CD123 CAR T cells but not mock T cells. Percentage normalized to mock T cell treatment (n=3, mean ± SD). (E) Degranulation as detected by cell surface CD107A is significantly higher in CD123 CAR T cells (red) compared to mock T cells (blue) after coculture with autologous bone marrow sample at E:T ratio 1:1 for 5hrs (n=3, mean ± SD) in pt#2. (F-G) TNF-α production as detected by intracellular flow cytometry is significantly higher in CD123 CAR T cells (red) compared to mock T cells (blue) after coculture with autologous bone marrow sample in the total CD3+ T cell population (F) and both CD4+ and CD8+ subpopulations (G) at E:T ratio 1:1 for 5hrs (n=3, mean ± SD) in pt#2, #8. (H) Secretion of TNF-α, IFN-γ, and IL-2 in culture supernatant of MDS bone marrow sample with autologous CD123 CAR T cells (red) is significantly higher compared to mock T cells (blue) when cocultured for 48hrs at E:T ratio of 1:1 for pt#2 and pt#6, and 72hrs and E:T ratio 2:1 for pt#5 (n=3, mean ± SD, unpaired parametric t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).