Fig 2: The blockage of tumor-derived sNKG2DLs results in the recovery of the antitumor function of T cells. (A and B) Human T cells derived from healthy donors were activated and treated with tumor cell culture supernatant for 12 h. Then, these T cells were treated with neutralizing antibodies against a single or several sNKG2DLs (anti-MICA/B and/or ULBP1~3). After 2 h of treatment, cell-surface expression of NKG2D was analyzed on CD8+ T cells by FACS. (C and D) After an additional 10 h of treatment, intracellular IFN-? production, Ki67 expression, and CD107a translocation of CD8+ T cells were detected by flow cytometry. Data are presented as the mean ± SEM. P-values were determined using a one-way ANOVA in (B and D). *P<0.05, **P<0.01 and ****P<0.0001. s, soluble; MICA, major histocompatibility complex (MHC) class-I-related chain A; ULBP, UL-16 binding protein.

Fig 3: sNKG2DLs impair CD8+ T cell function and memory formation. (A) Human T cells derived from healthy donors were activated and treated with different concentrations of the tumor cell culture supernatant for 12 h. NKG2D expression was detected using FACS. (B) Intracellular IFN-? production, Ki67 expression and CD107a translocation of CD8+ T cells were detected by flow cytometry. (C and D) Surface markers (CD25, CD69, CD45RO, and CCR7) of CD8+ T cells were also analyzed by FACS. Data are presented as the mean ± SEM. P-values were determined using a one-way ANOVA in (A-D). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. s, soluble.

Fig 4: Characterization of mRNA-driven Nb-CAR.BiTE-?dT cells. A) Nb-CAR expression was assessed after lentiviral transduction or mRNA electroporation. ?dT cells (1 × 106) were electroporated with 2 µg Nb-CAR/CAR.BiTE IVT mRNA or transduced with lentiviral Nb-CAR/CAR.BiTE particles (MOI = 3). The Nb-CAR expression on the indicated days was determined through flow cytometry using a specific antibody against VHH. B,C) Superior cell growth and Nb-BiTE secretion using the mRNA delivery strategy. B) Cell number and viability were recorded during 7 d after transfection of the mRNA or lentiviral Nb-CAR.BiTE transgene. C) Contents of PD-L1-targeted Nb-BiTE in the supernatants from mRNA- or lentiviral-driven Nb-CAR.BiTE-?dT cell cultures were detected by an ELISA-based coating with recombinant full-length PD-L1. D) Phenotype and purity of mRNA-driven Nb-CAR.BiTE-?dT cells. T effector and central memory phenotype, activating marker, and purity of Nb-CAR.BiTE mRNA-electroporated ?dT cells were analyzed through flow cytometry using specific antibodies against CD27, CD45RA, CD3, NKG2D, Vd2, V?9, aßTCR, ?dTCR, CD4, CD8, CD19, CD14, CD66b, or CD56. Isotype staining was used as a gating control for positive staining. E) Increased secretion of granzyme B and IFN-?, but not IL-17A, from Nb-CAR.BiTE-?dT cells after coculture with A549 or MDA-MB-231 cells. The contents of granzyme B (upper panel), IFN-? (middle panel), and IL-17A (bottom panel) in culture supernatants were measured using ELISA. These results showed that mRNA-driven Nb-CAR.BiTE-?dT cells would be an effective strategy for the manufacture of Nb-CAR.BiTE-?dT cells. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3–4, *p < 0.05; **p < 0.01; and ***p < 0.001 based on paired Student's t-tests.