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. 2023 Oct 5;12(1):2265703. doi: 10.1080/2162402X.2023.2265703

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© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

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Figure 1. — Design, signal activation, and functional validation of the PD1-TIGIT CISR. (a) schematic representation of the CISR. The extracellular domain (ECD) of the CISR consists of the TIGIT ECD and the PD1 ECD, connected by a linker. The newly designed extracellular recognition region is fused to the transmembrane (TM) region and intracellular domain (ICD) of CD28. The CISR binds to PDL1 and CD155 molecules on tumor cells, delivering a positive signal to activate intracellular CD28 signaling. (b) schematic representation of the anti-EGFR CAR (EGFRz-CAR) and the EGFRz-CAR linked to the CISR (Ez.CISR). (c, d) coculture of EGFRz-CAR T cells or Ez.CISR T cells with target cells (c, Daudi-E and Daudi-E.P cells; d, Daudi-E cells and Daudi-E.155 cells) at E:T ratios of 1:1, 5:1, and 10:1. After 24 hours, culture supernatants were collected, and levels of IL-2 were measured by enzyme-linked immunosorbent assays (means ± SDs, n = 3). Statistical significance in panels (c) and (d) was analyzed using two-way analysis of variance (ANOVA); *P < .1, **P < .01, ***P < .001, ****P < .0001. (e) schematic representation of the anti-EGFR CAR with the 4-1BB co-stimulatory molecule (EGFRBBz-CAR) and the EGFRBBz-CAR linked to the CISR (EBBz.CISR). (f, g) coculture of EGFRBBz-CAR T cells or EBBz.CISR T cells with target cells (f, Daudi-E and Daudi-E.P cells; g, Daudi-E cells and Daudi-E.155 cells) at E:T ratios of 1:1, 5:1, and 10:1. Culture supernatants were collected after 24 hours to measure levels of IL-2 (means ± SDs, n = 3). Data were analyzed using two-way ANOVA; *P < .1, **P < .01, ****P < .0001. (h) coculture of T cells with EGFR⁺PDL1⁺CD155⁺ HCT116-P cells at E:T ratios of 1:1 and 5:1. After 24 hours, culture supernatants were collected, and levels of IL-2 and IFN-γ were measured (means ± SDs, n = 3). Data were analyzed using two-way ANOVA; **P < .01, ***P < .001, ****P < .0001.