Fig 2: Differential gene expression analysis between CARpos and CARneg/vectorpos cellsVolcano plots of DEGs (Wilcoxon rank-sum test) between CARpos T cells and CARneg/vectorpos T cells in the (A) CD3-LV-, (B) CD4-LV-, (C) CD8-LV-, and (D) VSV-LV-treated samples from two healthy donors (D#1 & D#2). The CAR values were excluded from the graphs for better visualization. Negative fold changes indicate genes upregulated in CARpos T cells. Positive fold changes indicate genes upregulated in CARneg/vectorpos T cells. ns, non-significant; FDR, false discovery rate.

Fig 3: ERK2 inhibition promotes T cell transduction with LVs and AAVs(A) Percentages of CAR expression 72 h after transduction with CD4-LV (left) (one donor, D#7) or (CD8-LV) (one donor, D#8) in the presence of different doses of ERK2 inhibitors. 6,000 particles per cell were used.(B) Fold change differences in CAR T cell percentages 72 h after transduction using different LVs (three donors, D#4, D#5, and D#6, mean of two technical replicates) with or without 5 μΜ ulixertinib or 20 μM VX-11e. 2,000 particles per cell for CD3-LV, CD4-LV, and CD8-LV and 500 particles per cell for VSV-LV were used.(C) Representative flow cytometry plots of T cells 72 h after transduction with different LVs, with or without 20 μM VX-11e. Depending on the vector type, CAR expression was measured on total CD3 T cells (CD3-LV and VSV-LV), on CD4 T cells (CD4-LV), or on CD8 T cells (CD8-LV). V, VX-11e; U, ulixertinib; R, ravoxertinib; S, selumetinib. See also Figure S14A.(D) Fold change differences in the percentage of GFP-positive cells 72 h after transduction with sc-AAV6 (top) (five donors, D#4, D#5, D#6, D#12, and D#13, mean of two technical replicates) or ss-AAV6 (bottom) (three donors, D#4, D#5, and D#6, mean of two technical replicates) in the absence (Ctrl) or presence (V) of Vx-11e 20 μM.(E) Representative flow cytometry plots of GFP expression in T cells with sc-AAV6 (top) or ss-AAV6 (bottom) in the absence (Ctrl) or presence (V) of 20 μM Vx-11e. Ctrl, control; V, Vx-11e.

Fig 4: Expression pattern of interferon-stimulated genes among different T cell populations(A) T cells were classified into three distinct populations based on the presence of vector signal and CARp detected by AbSeq. a: CARpneg/vectorneg, b: CARpneg/vectorpos, and c: CARppos T cells.(B) Violin plots showing the expression of ISGs from CD4-LV and CD8-LV samples across the three defined cell populations from two healthy donors (D#1 & D#2). Statistics show the FDR corrected values from differential expression analysis (Wilcoxon rank-sum test). FDR, false discovery rate; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 5: StCl‐MI and StCl‐MI_GII impair tumor growth by potentiating anti‐tumor immunity. a) Timeline (days) for tumor inoculation and immunization (s.c. = subcutaneous). b) Representative images at day twenty‐one post‐tumor inoculation. c) Tumor growth curve. Data presented as mean ± SEM (6 < N < 9 animals). Two‐way ANOVA. P values correspond to tumor volume at day twenty‐one after tumor inoculation. d–g) Tumor‐infiltrating immune cell populations for d) CD3+, e) MFI PD1+CD3+, f) CTL, and g) PD1+CD8+. Tumors isolated on day twenty‐one after tumor inoculation, and quantification performed by flow cytometry. Data presented as mean ± SD, n ≥ 3 animals. Statistics: ANOVA followed by Tukey post‐hoc test. h) Antigen‐specific CD8+ T cells and IFN‐γ expression in the spleens of StCl‐MI_GII‐treated mice after incubation with Melan‐A/MART‐1 or non‐relevant CEA peptide and CD28 in solution for six days. Data presented as mean ± SD, n = 3. Statistics: One‐way ANOVA with Tukey post‐hoc test.