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. 2023 Apr 20;24(5):869–883. doi: 10.1038/s41590-023-01477-2

Extended Data Fig. 1. OT1 T cells can be effectively transduced to secrete PD1d, IL-2v and IL-33.

Extended Data Fig. 1

a left: Intracellular expression of PD1d by FACS. Right Top: Secretion into the SN determined by ELISA (n = 3 independent experiments, 2 technical replicate/experiment). Right Bottom: Binding to plate-coated mPD-L1. SN from OT-1 T cells gene-engineered to secrete TIM-3.IgG4 decoy was used as negative control. (n = 4 independent experiments, 2 technical replicate/experiment). b) Anti-tumor activity of OT-1 T cells gene-engineered for secreting the PD-1_IgG4 decoy in small B16-OVA tumors (~ 30 mm3). Effect on tumor growth (left) and on overall survival (right) of ACT using OT1 cells gene-engineered to secrete the PD-1.IgG4 decoy. A representative experiment out of two independent experiments using 6 mice per group is shown. Survival curves were compared using a log-rank Mantel−Cox test. Tumor growth comparison at day 35 post tumor inoculation was done using a Mann–Whitney Test. ***P < 0.001. c, d OT-1 CD8 T cells can be gene-engineered to secrete PD1d in combination with either IL-2V or IL-33. c Left: intracellular expression of PD1d in PD1d/2V gene-engineered OT1 cells (there are no commercially available antibodies for detecting the IL-2V using Flow Cytometry). Right: secretion of PD1d and IL-2V into the supernatant by ELISA, (n = 2 independent experiments, 2 technical replicate/experiment). d Left: Intracellular expression of PD1d and IL-33 in PD1d/33 gene-engineered OT1 cells. Right: secretion of PD1d and IL-33 into the supernatant by ELISA. OT1 cells gene-engineered to secrete TIM-3.IgG4 decoy were used as negative control to claim specificity in the PD-1_IgG4 ELISA, (n = 2 independent experiments, 2 technical replicate/experiment). Data are presented as mean values ± s.d. d Phenotypic characterization of gene-engineered OT1 cells before ACT. Top: schematic showing the transduction and expansion protocol of gene-engineered OT1 cells. Bottom: expression of CD44, CD62L, TCF1 and PD1 in expanded OT1 cells on day 10 post viral transduction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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