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. Author manuscript; available in PMC: 2023 Aug 3.
Published in final edited form as: Cancer Immunol Res. 2023 Feb 3;11(2):171–183. doi: 10.1158/2326-6066.CIR-22-0333

Figure 5: Incorporation of LEF1 into CAR.GD2 construct mitigates exhaustion and suppresses immediate effector function of transduced NKTs.

Figure 5:

A, CAR constructs containing the anti-GD2 14g2a scFv, CD8 hinge and transmembrane domains, 4-1BB co-stimulatory domain, and CD3 zeta domain, with LEF1 following a 2A sequence (CAR-LEF1) or without (CAR). B–G, Two days after secondary stimulation with αGalCer-pulsed aAPCs, NKTs were transduced with parental or LEF1-containing CAR.GD2 constructs and phenotypes were studied eight days after. (B) Surface CAR and intracellular LEF1 expression were determined by flow cytometry. Representative dot plots show LEF1 expression relative to CAR expression from one of two donors. (C) NKT cell number was determined by trypan blue exclusion assay. Mean cell count ± SEM (n = 11 donors) is shown. (D) CD62L, (E) CD27, and (F) TIM-3 expression were assessed by flow cytometry in CAR+-gated NKTs. Representative histograms and mean ± SEM of CD62L, CD27 percentage or TIM-3 MFI (n = 11 donors) are shown. *P < 0.05, ***P < 0.001, ****P < 0.0001, paired Student’s t test for paired result from each donor. (G) Luciferase-transduced GD2+ CHLA-255 cells were co-cultured with CAR or CAR-LEF1 NKTs for four hours. Cytotoxicity was calculated from luminescence intensity using non-transduced (NT) NKTs as control. Results are from five donors tested in two independent experiments. Mean ± SEM is shown. ***P < 0.001 for AUC analysis between CAR and CAR-LEF1 groups. H, CAR and CAR-LEF1 NKTs were stimulated with GD2+ CHLA-255 cells, and supernatants were collected at 24 hours. GM-CSF, IFNγ, TNFα, and IL4 levels were measured by Luminex assay. Results are from one of two donors tested with similar results. ***P < 0.001, ****P < 0.0001, unpaired Student’s t test.