Extended Data Fig. 4: Functional effects of elimination of ALCAM on endothelial cells and knockout of human ALCAM using CRISPR–Cas9 technology and its effect on T cell migration across the BBB.
From: A homing system targets therapeutic T cells to brain cancer
a, Flow cytometry of ALCAM expression on 1 × 106 wild-type pTECs at base-line, after TGFβ induction of ALCAM and after being transfected with 25 nM ALCAM siRNA for 48 h to knock down (KD) ALCAM. Transmigration assay using pTECs to simulate a cancerous BBB showing percentage of migrant T cells compared with ALCAM-KD is shown in Fig. 2k. b, Highest three scoring guide RNA designs (sgRNA-44, sgRNA-45 and sgRNA-49) as seen on the SnapGene software intended to disrupt ALCAM exons for the extracellular and transmembrane moiety. c, d, CD5-KO (c) and CD19-KO (d) were used as positive and negative experimental controls, respectively. e, Flow cytometry of ALCAM expression on wild-type HUVECs and HUVEC ALCAM-KO using CRISPR–Cas9 (using the guide sgRNA-45) assessed at baseline and after TGFβ incubation. Isotype was used as control. f, Transmigration assay showing percentage of 2 × 106 migrating T cells on wild-type HUVECs before and after ALCAM induction compared to ALCAM-KO HUVECs. Both experiments were done at baseline then after ALCAM induction was confirmed. Data shown as mean ± s.d. (n ≥ 3 experiments; donor T cells n = 3), **P < 0.01, ***P < 0.001. Tukey’s test (compared to wild-type pTECs). g, RT–PCR analysis of representative of 1 × 106 ALCAM-KO HS T cells in comparison to wild-type normal T cells. GAPDH was used as an internal control. h, Flow cytometry showing >90% knockout efficiency of the three sgRNAs on 1 × 105 T cells in comparison to wild-type normal T cells; CRISPR–Cas9 only and isotype were used as experimental controls. i, Sorted ALCAM-negative KO T cells were then successfully transduced with the six HS constructs. j, Transmigration assay showing percentage of 2 × 105 migrating T cells on a cBBB model to compare wild-type with ALCAM-KO T cells in four conditions (ALCAM–, ALCAM+ conditioned with TGFβ, after blocking ALCAM, after washing the blocking away). Data in f and j are shown as mean ± s.d. (n ≥ 3 experiments; donors n = 3), *P < 0.05, **P < 0.01. Tukey’s test (compared to ALCAM+ T cells).
