Fig. 8: Addition of ICAM-1 Knockout (KO) to First Generation Hypoimmune Pluripotent Stem Cell (PSCs).

A First generation β2M-/-, CIITA-/-, CD47⁺⁺ (DKO) PSCs were edited to introduce the ICAM-1-KO, making a triple KO (TKO) cell line. PSCs were differentiated into highly pure ( > 98% CD31⁺CD144⁺) endothelial cells (ECs) and stimulated with 10 ng/ml TNFα and 50 ng/ml IFNγ for 48 h and assessed for HLA (human leukocyte antigen) class I (left) and ICAM-1 (right) surface protein expression. Data representative of n = 3 independent experiments. MFI Median Fluorescence Intensity. The red and the blue lines indicate the MFI of the unstimulated and the stimulated ECs respectively. Analysis was performed via FlowJo v10 software. B Western Blot was performed using the total protein extracted from wild type (WT), DKO, and TKO PSCs stimulated as above for 44 h. The H9 WT protein sample consisted of n = 5 biological replicates (BR) from different cell preparations, H9 DKO consisted of n = 5 BR, and H9 TKO consisted of n = 5 BR. C H9 WT, DKO, and TKO PSCs were differentiated into highly pure ( > 98% CD31⁺CD144⁺) ECs and stimulated as above, then assessed by Luminex for secreted soluble ICAM-1, as in Fig. 5. Error bars = standard deviation. N = 2 BR, each dot representing the mean of two technical replicates per BR. D Similar to Figs. 1 and 6, adhesion assays were performed (top plot) using U937s as immune effector cells, with each point representing a region of interest (ROI), and each plot being representative of > 3 independent experiments. U937s were stained with violet proliferation dye 450 (VPD450). Adhesion assays were also conducted with peripheral blood mononuclear cells (PBMCs; bottom plot). Images were acquired on ECHO Revolve | R4 microscope and analyzed using ImageJ version 1.54 g. E To test in vitro immune responses to edited cells, highly-pure ( > 99% CD31⁺ CD144⁺) PSC-derived ECs differentiated from WT, ICAM-1 KO, DKO, and TKO PSCs were co-cultured with HLA-mismatched PBMCs (1:6 T:E ratio) labeled with VPD450 dye in mixed lymphocyte reactions. Alloreactivity was assessed against two PBMC donors (#3 and #5) separately via CD8⁺CD3⁺ T cell proliferation (VPD450 dye dilution) and CD8⁺CD38⁺ activated T cell subpopulation by flow cytometry, representative data shown from 3 replicate assays. Error bars = standard deviation, analysis in D and E via one-way ANOVA with Tukey’s multiple comparisons test. WT = H9 PSC line with ALG (Akaluc + GFP) reporter construct; DKO = H9 PSC line with KO of β2M and CIITA, with ALG reporter construct; TKO = DKO line with addition of ICAM-1 KO. Statistical analysis was performed via Prism 10.2.2 software. Source data are provided as a Source Data file.