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. 2023 May 16;31(7):2132–2153. doi: 10.1016/j.ymthe.2023.05.009

Figure 1.

Figure 1

Generation of CER-T cells, TIM-4-expressing T cells, activated in response to PS, can phagocytose targets, and upregulate phagocytic gene modules

(A) Schematic of CER-1236 expression construct and the CER-1251 PS binding site mutant variant. Expression of CER-1236 allows for target-dependent T cell activation and phagocytosis of antigen-positive target. (B) Transduction of transduced T cells, as measured by expression of the TIM-4 extracellular domain, was determined at the end of production by flow cytometry. A representative histogram is shown, and average transduction ± SEM is shown (n = 4 donors). Significance was determined using a one-way ANOVA with Geisser-Greenhouse correction with Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C) IFN-γ secretion in response to immobilized PS. IFN-γ secretion was measured in the supernatant of cells 24 h after plating on the indicated amount of PS or PE by automated ELISA. Average ± SEM is shown (n = 3 donors). ∗p < 0.05. (D and E) CER-1236 phagocytosed PS-coated beads. CER-1236 T cells were incubated with PS-coated agarose beads labeled with the pH-sensitive dye pHrodo Red. After 16 h, phagocytosis was measured by flow cytometry, as determined by pHrodo Red+ T cells. The average ± SEM is shown (n = 4 for untransduced, 6 for CER-1236, and 2 for CER-1251). Parallel samples were imaged by fluorescence microscopy at 40×. Representative images of CER-1236 or untransduced cells at 40× are shown in (D). Representative images at 4× are shown in (E). (F) CER-1236 upregulate both T cell activation and pro-phagocytic genes. RNA sequencing was performed on CER-1236 T cells, TIM-4 mutant 1251 T cells, or untransduced control T cells stimulated with plate bound PS for 24 h. A volcano plot of genes with an absolute fold change > 4, FDR < 0.05 are shown. (G) IPA pathway gene sets (FDR < 0.05, absolute fold change > 4) comparing 1236 with TIM-4 mutant 1251 from three donors 24 h post-stimulation. CER, chimeric engulfment receptor; TIM-4, T cell immunoglobulin and mucin domain containing 4; TLR, Toll-like receptor; LTR, long terminal repeat; MHC, main histocompatibility complex; IFN, interferon; PS, phosphatidylserine; PE, phosphatidylethanolamine.