PMC

(A,C) Representative histograms of the flow cytometry data of CD8⁺ PD-1⁺ Tdx (A) and EP (C) HBV-specific TCR T cells at 24 h are shown. Tinted and non-tinted histograms, respectively, represent the stained sample or matched isotype control, where a horizontal line is drawn based on the isotype control to demarcate for PD-1⁺ cells. Percentage values of CD8⁺ PD-1⁺ cells are indicated. (B,D) Bar plots show the mean ± SEM of CD8⁺ PD-1⁺ TCR T cells of three donors for Tdx (B) or EP (D) HBV-specific TCR T cells; bars are drawn where a comparison was made with *P ≤ 0.05, **P ≤ 0.01 and ****P ≤ 0.0001. Statistical significance was evaluated by a one-way ANOVA with Holm-Sidak’s multiple comparisons test. EP, mRNA-electroporated; HBV, hepatitis B virus; Hep, HepG2-preS1-GFP; Mo, monocyte; Tc, control T cell; TCR T cells, T cell receptor-redirected T cells; Tdx, transduced; Ts, HBV-specific T cell.

(A,C) Representative histograms of the flow cytometry data of CD14⁺ PD-L1⁺ monocytes for co-cultures with either Tdx (A) or EP (C) HBV-specific TCR T cells at 24 h are shown. Tinted and non-tinted histograms, respectively, represent the stained sample or matched isotype control, where a horizontal line is drawn based on the isotype control to demarcate for PD-L1⁺ cells. Percentage values of CD14⁺ PD-L1⁺ cells are indicated. (B,D) Bar plots show the mean ± SEM of PD-L1⁺ monocytes for co-cultures involving Tdx (B) or EP (D) HBV-specific TCR T cells; bars are drawn where a comparison was made with *P ≤ 0.05, ***P ≤ 0.001, and ****P ≤ 0.0001. Statistical significance was evaluated by a one-way ANOVA with Holm-Sidak’s multiple comparisons test. EP, mRNA-electroporated; HBV, hepatitis B virus; Hep, HepG2-preS1-GFP; Mo, monocyte; Tc, control T cell; TCR T cells, T cell receptor-redirected T cells; Tdx, transduced; Ts, HBV-specific T cell.

(A) A 3D multicellular tumor microenvironment microfluidic model consisting of a middle hydrogel channel (2) flanked by two media channels (1, 3) for the mechanistic study of the effect of monocytes on T cell receptor-redirected T cell (TCR T cell) killing of tumor cell aggregates. Human monocytes were inserted together with target HepG2-preS1-GFP cell aggregates in collagen gel in the central hydrogel region (2), while hepatitis B virus (HBV)-specific TCR T cells were added into one fluidic channel (1) to mimic the intrahepatic carcinoma environment. (B) Representative confocal image of a target cell aggregate (in green) surrounded by monocytes (in blue) and HBV-specific TCR T cells (in white), in which the presence of dead target cells is DRAQ7⁺ (in red). Target cell death is quantified as shown based on the DRAQ7⁺ volumetric portion in the total volume of each GFP-labeled aggregate.

(A,B) Results from the impedance assay plotted as growth of the target cells over time for up to 24 h, measured as a NCI for Tns (top lines) and Tdx (A) or EP (B) HBV-specific TCR T cells (bottom lines). Each row represents a different donor. The numbers in each plot indicate the percentage difference of the AUC of target cell killing in HepG2-T cells co-cultures either in the absence (blue) or presence (red) of monocytes. Every curve is the average of at least two measurements for each condition and at least three experiments were performed. (C) Bar plots representing the average of all differential AUC measured in the experiments for Tdx (left panel) and EP (right panel) HBV-specific TCR T cells. Statistical significance was evaluated by a two-tailed t-test, with P < 0.05 taken as evidence of statistical significance. AUC, area under the curve; EP, mRNA-electroporated; HBV, hepatitis B virus; Hep, HepG2.2.15; Mo, monocyte; NCI, normalized cell index; n.s., not significant; TCR T cells, T cell receptor-redirected T cells; Tdx, transduced; Tns, non-specific T cells; Ts, HBV-specific T cell.

(A–D) Representative target cell aggregates of the conditions Hep (A), Hep Mo (B), Hep Ts (C), and Hep Ts Mo (D), in which the presence of dead target cells is DRAQ7⁺ (in red), HBV-specific TCR T cells are labeled with Cell tracker violet dye (in white), while monocytes are unlabeled. (E) Box plot of the percentage of dead target (HepG2-Pres1-GFP) volume after 24 h of co-culture with Tdx HBV-specific TCR T cells. Data in terms of a “Dead Target Index” are also shown where the percentage of dead target volume is normalized to the percentage of TCR⁺ T cells. (F) Box plot of the percentage of dead target volume after 24 h of treatment with Tdx HBV-specific TCR T cells with or without PD-L1- or PD-1-blocking antibody or their respective isotype (IgG) controls. (G) Box plot of the percentage of dead target (HepG2-Pres1-GFP) volume after 24 h of co-culture with EP HBV-specific TCR T cells. Data in terms of a “Dead Target Index” are also shown. Data points reflect the measured values of individual target cell aggregates and collectively represent the pooled results of three donors, where the 25th, 50th, and 75th percentiles as well as minimum and maximum values are indicated. Bars are drawn where a comparison was made with *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Statistical significance was evaluated by a one-way ANOVA with Holm-Sidak’s multiple comparisons test. EP, mRNA-electroporated; HBV, hepatitis B virus; Hep, HepG2-preS1-GFP; Mo, monocyte; n.s., not significant; Tc, control T cell; TCR T cells, T cell receptor-redirected T cells; Tdx, transduced; Ts, HBV-specific T cell.