Figure 2.

CER-1236 mediates cytotoxicity, cytokine secretion, and phagocytosis against PS-positive mantle cell leukemia cells

(A) JeKo-1 mCherry⁺ TMEM30A KO cells have high constitutive expression of PS. PS exposure on JeKo-1 mCherry⁺ TMEM30A KO or JeKo-1 mCherry⁺ parental cells was measured by flow cytometry. Cells were stained with recombinant human TIM-4-His, then stained with secondary anti-HIS antibody. A representative histogram of PS staining is shown. (B) CER-1236 T cells potently kill PS⁺ target cells. CER-1236 T cells were co-cultured with JeKo-1 mCherry⁺ TMEM30A KO cells at a 1:1 ratio in medium supplemented with 200 IU/mL IL-2. Untransduced cells were included as controls. Cytotoxicity was measured by reduction in mCherry integrated intensity using the IncuCyte Live-Cell Analysis System. Average cytotoxicity ± SEM is shown (n = 4 donors). Statistics were analyzed at each time point using a two-way ANOVA with Tukey’s multiple comparisons test. Results for 120 h are shown. ∗∗∗∗p < 0.0001. (C) Representative IncuCyte image overlays of phase/contrast and red channel taken at 120 h post co-culture. mCherry⁺ cells are false colored red. (D) CER-1236 secrete effector cytokines in response to PS⁺ target cells. CER-1236 were co-cultured with JeKo-1 mCherry⁺ TMEM30A KO cells at a 1:1 ratio for 120 h. Untransduced cells were included as controls. After co-culture, cytokine secretion was measured by automated ELISA. Average cytokine secretion ± SEM is shown (n = 3 donors for untransduced, 4 donors for CER-1236, 2 donors for CER-1251). Statistics were analyzed using a one-way ANOVA with Tukey’s multiple comparisons test. ∗∗p < 0.01. (E) Schematic of phagocytosis assay setup. CER-1236 T cells were stained with CTV, and JeKo-1 mCherry⁺ TMEM30A KO cells were stained with pHrodo Red. T cells were co-cultured with target cells at a 1:2 ratio in the presence or absence of the phagocytosis inhibitors cytochalasin D or bafilomycin A, and incubated for 40 h in cytokine-free medium. At the end of co-culture, phagocytosis was measured by flow cytometry. (F) CER-1236 phagocytosis of JeKo-1 mCherry⁺ TMEM30A KO cells is impaired by cytochalasin D. Phagocytosis assays were performed with 5, 0.5, or 0.05 μM of cytochalasin D, an actin polymerization inhibitor. The average phagocytosis ± SEM is shown (n = 1 donor). (G) CER-1236 phagocytosis of JeKo-1 mCherry⁺ TMEM30A KO cells is impaired by bafilomycin A. Phagocytosis assays were performed with 10, 1, or 0.1 nM of bafilomycin A, an inhibitor of lysosome-phagosome fusion. The average phagocytosis ± SEM is shown (n = 1 donor). (H) Representative flow plot of phagocytosis in the presence or absence of cytochalasin D. SEM, standard error of the mean; TMEM30A, transmembrane protein 30A; KO, knockout; CER, chimeric engulfment receptor; IFN, interferon; TNF, tumor necrosis factor; CTV, CellTrace Violet.