Extended Data Fig. 3: In silico design of the prototype and derivative HS molecules, their forced expression and detection on T cells, and studies of their in vitro dynamic interactions with endothelial cells under shear stress.
From: A homing system targets therapeutic T cells to brain cancer
a, The potential interaction between ALCAM V1 (grey ribbon) and CD6 from computational docking. D1 of CD6 is coloured blue, D2 green and D3 orange. b, Details of the potential interaction interface between ALCAM V1 (grey ribbon) and CD6 D3 (orange ribbon). A rendering of the electrostatic surface of ALCAM V1 (grey ribbon) with the D3 domain of CD6 (orange ribbon) in the same orientation. Potential interacting residues are highlighted in the models and in a diagram generated from PDBe PISA and PDBSum (c). A small region of positively charged residues in ALCAM V1 appears to interact with a negatively charged patch of residues on CD6 D3. d, Structure of the prototype HS molecule. e, HS multimers 3HS and 5HS. f, HS molecules with non-signalling endodomains, HSΔ, 3HSΔ and 5HSΔ. g, Strategy used for surface detection of the HS exodomain using a D3-specific antibody and specific binding of the HS exodomain to soluble ALCAM. h, Flow cytometry confirming HS surface expression (using D3 monoclonal antibody) on T cells. i, Design of the HS–ALCAM PLA experiment. j, Digital rendition of PLA using ImageTool. The ALCAM probe (–) binds to the D3 probe (+) to trigger the PCR generating the red fluorescent signal that is quantified as total signal per region (TSR) in Fig. 2f. k, l, Dynamic microfluidic studies showing still image from Supplementary Video 1 of Bioflux channels with non-transduced control (NT) T cells (top) versus 1 × 106 HS T cells interrogated under shear force over an ALCAM-expressing endothelium (k), and still image from MJtracker demonstrating various T cells under interrogation for various TEM dynamic measures, the standard grid used and the equation used for calculations (l). m, Dynamic adhesion of T cells to endothelial cells per field of view. n, Average dynamic rolling velocity against time; *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA with Tukey’s test for multiple-comparisons (compared to NT cells).
