Fig. 6. Although PD-1⁺, C5 T_SE cells are not functionally restrained by PD-1.

a, Experimental design to assess the functional restriction of OT1 TILs by plate-bound PD-L1 (ref. ³⁷) upon TCR restimulation ex vivo. Genetically engineered IL-2v/IL-33 OT1 TILs were adoptively transferred into C57BL/6 mice bearing B16-OVA tumors. Total CD45⁺ TILs were then magnetically isolated (Miltenyi Biotech) from tumors 12 d after the first ACT infusion and stimulated for 48 h ex vivo with plate-bound anti-CD3 (3C11, eBiosciences) alone or in the presence of chimeric mouse PD-L1/Fc (1 or 3 µg ml⁻¹). In addition, 20 µg ml⁻¹ of anti-mouse PD-L1 monoclonal antibody (clone 10F.9G2, BioXcell) was added to the cultures as a control. Moreover, naive OT1 cells were cultured under identical conditions as the control for PD-1 inhibition of T-cell activation. s.c., subcutaneous. b–d, Normalized number of cells (b), and normalized expression of activation (c) and effector molecules (d) relative to anti-CD3-mediated stimulation. Data are from three independent experiments (n = 3 internal replicates per experiment). A Brown–Forsythe and Welch ANOVA test combined with Dunnet’s test were used to correct for multiple comparisons relative to anti-CD3 stimulation alone; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. mAB, monoclonal antibody; MFI, mean fluorescence intensity. FC, fold change.

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