Fig. 4: D4-IgG4H-CD28TM CAR T cells exhibit robust effector functions in vitro and in vivo. | Nature Communications

Fig. 4: D4-IgG4H-CD28TM CAR T cells exhibit robust effector functions in vitro and in vivo.

From: The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer

Fig. 4

a Schematics of D4 CAR constructs differed in H and TM domains. The illustration was created with BioRender.com. b Transduction efficiency of four D4 CAR constructs as measured by hEGFRt expression and their binding to antigen GPC1. Data are representative of two independent experiments. c D4-IgG4H-CD28TM CAR T cells showed the best cytolytic activity among all four D4 CAR T cells when co-cultured with low GPC1-expressing T3M4 cells, but not GPC1 KO T3M4 cells. n = 3 independent experiments. ns = p > 0.05, *p = 0.022, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test. d D4-IgG4H-CD28TM CAR T cells induced the most secretion of IFN-γ upon stimulation with T3M4 cells at E:T ratio of 6:1. n = 3 independent experiments. *p = 0.032, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test. The CAR T cells used in (b–d) were produced using the donor 2’s PBMCs. e Cell surface expression of GPC1 on indicated pancreatic cancer cell lines. GPC1 molecules per cell as determined by QuantiBRITE phycoerythrin (PE) assay. n = 1 independent experiment. f D4-IgG4H-CD28TM CAR T cells potently lysed Bxpc3 (dotted lines) and Panc-1 (solid lines) cells after 24 h of co-culture. n = 3 independent experiments. *p = 0.034, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test. g Experiment schematic (created with BioRender.com). NSG mice were surgically implanted with Panc-1 cells in pancreas and treated with 5 million mock, D4-CD8H-CD8TM CAR, and D4-IgG4H-CD28TM CAR T cells on day 7 via tail vein injection. n = 5 mice/group. h Mice receiving D4-IgG4H-CD28TM CAR T cells were 100% tumor-free, whereas D4-CD8H-CD8TM CAR T group still carried small tumors. n = 5 mice/group. *p < 0.05, two-tailed unpaired Student’s t test. i Analysis of T-cell exhaustion in each group at different time points post-infusion. n = 3 mice/group. ns = p > 0.05, two-tailed unpaired Student’s t test. j Memory T-cell phenotypes of D4-CD8H-CD8TM and D4-IgG4H-CD28TM CAR T cells retrieved from mouse blood. n = 3 mice/group. Two-tailed unpaired Student’s t test. Relative proportion of stem cell-like memory (Tscm CD62L+CD45RA+CD95+), central memory (Tcm CD62L+CD45RA−CD95+), effector memory (Tem CD62L−CD45RA−CD95+), and terminally differentiated effector memory (Temra CD62L−CD45RA+CD95+) phenotypes were defined by CD62L, CD45RA and CD95 expression. The CAR T cells used in (f–j) were produced using the donor 3’s PBMCs. Values represent mean ± SEM. Source data and exact p values are provided in the Source data file.

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