Fig 2: Using ICAM-1 as a proxy for LMP1 expression and LMP1-mediated NF?B signaling. (A) FACS analysis of ICAM-1 as a proxy for LMP1-mediated NF?B activity of 7 dpi EBV-infected latency IIb proliferating B cells and donor matched LCLs. The plot at the right shows sorting groups of latency IIb as well as the bottom, middle, and upper 15% of ICAM-1-expressing LCLs with corresponding MFI values. (B) RT-qPCR for ICAM-1 in sorted groups. Each bar represents the average of six independent matched donors. (C) RT-qPCR for LMP1 in sorted groups. Each bar represents the average of results from six independent matched donors. (D) Correlation between LMP1 RT-qPCR expression and ICAM-1 RT-qPCR expression. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s pairwise t test). All error bars denote standard errors of the means (SEM).

Fig 3: ICAM-1 controls proinvasive ECM remodelingA. Percentage of gel contraction by hDF six days after RNAi transfection and subsequent LIF stimulation (left panel, n=2 in triplicates). Schematic representation of the experimental conditions (right panel). B. Percentage of gel contraction by CAF 6 days after RNAi transfection (left panel, n=2 in triplicates). Schematic representation of the experimental conditions (right panel). C. Immunoblot of ICAM-1 in hDF following LIF or TGFß1 stimulation, in the presence (48h) or absence of the LIF blocking antibody. Immunoblot of tubulin was the internal control. D. Immunoblot of ICAM-1 in three hDF #1, #2 and #3) and in three CAF (Head and Neck, Breast and Lung). Immunoblot as control. E. Percentage of gel contraction by CAF transfected with control RNAi (siLuc) or RNAi targeting ICAM-1 (siICAM-1#1, #2, #3 and #4) (n=3 in triplicates, mean + s.d, ***p<0.001). F. Representative images of H&E coloration of paraffin-embedded sections of SCC12 3D-cultures in response to CAF transfected with control (siLuc) or targeting ICAM-1 (siICAM-1#1, #2, #3 and #4) siRNA (n=3, I.I., invasion index, mean ± s.d., ***P<0.001). Scale bar 100µm. G. Percentage of gel contraction by LIF-stimulated or not (Veh.) HDF subsequently transfected with control (siLuc) or ICAM-1-targeting ICAM-1 (siICAM-1#1 and #2) RNAi (n=3 in triplicates, mean + s.d, ***p<0.001). H. Representative images of H&E staining of paraffin-embedded sections of SCC12 in response to control (veh) or LIF-activated hDF subsequently transfected with control (siLuc) or ICAM-1-targeting siICAM-1#1 and #2) RNAi ((n=3, I.I., invasion index, mean ± s.d., ***P<0.001). Scale bar 100µm.

Fig 4: CAR TN/SCM are less toxic, independently of the costimulation provided.Experiments were conducted as described in Figure 4A but with CAR T cells carrying the 4-1BB costimulatory domain. (A) NALM-6–derived bioluminescence signal measured at different time points after treatment and expressed as relative light units (RLU) (n = 13 for CAR TBULK, n = 12 for CAR TN/SCM, n = 6 for Mock). (B) T cell expansion in the peripheral blood of mice. (C) Weight loss evaluation at different time points after treatment. (D and E) IL-6 and other cytokine (CTK) serum levels, with their heatmap visualization, on day 4 after treatment (n = 18 for CAR TBULK, n = 19 for CAR TN/SCM, n = 6 for Mock). (F) Severe CRS–related (sCRS-related) Kaplan-Meier survival analysis of mice. (G) CRS grading. Left panel: Kaplan-Meier curves. Right panel: Histograms summarizing CRS grading. (H) Monocyte absolute number immediately before T cell infusion (n = 13 for CAR TBULK, n = 12 for CAR TN/SCM, n = 6 for Mock). (I) Percentage of activated monocytes coexpressing CD80, CD86, CD54, and HLA-DR activation receptor markers (ARs) 1 day after treatment (n = 7 for CAR TBULK and CAR TN/SCM, n = 3 for Mock). (J) Evaluation of AR upregulation on CAR T cells (CD54, CD86) and monocytes (CD54, CD86, CD163) expressed as MFI on day 1 after treatment (n = 11 for CD54 and n = 7 for CD86 evaluated on CAR TN/SCM, n = 9 for CD54 and n = 6 for CD86 evaluated on CAR TBULK, n = 6 for CD163 in the CAR TBULK cohort, n = 7 for CD163 in the CAR TN/SCM cohort). (K) Correlation between CAR T cell and monocyte activation statuses on day 1 after treatment. Data are represented as box and violin plots, mean ± SEM together with overlapping scattered values, or scaled according to a graded-color range depending on relative minimum and maximum levels, when referring to the heatmap. *P < 0.05, **P < 0.01 by 2-way ANOVA (A–C), unpaired t test (D, E, and H–J), Mantel-Cox 2-sided log-rank test (F), or Gehan-Breslow-Wilcoxon test (G).

Fig 5: Protein validation of transcriptional markers reveals nonbinary latency specificity. (A) FACS analysis of CCR6 in day 7 latency IIb proliferating B cells and latency III LCLs stratified by ICAM-1 expression. (B) Quantification of mean fluorescence intensity of CCR6 expression data from panel A. (C) Percentage of CCR6-positive cells represented in panel A. (D) FACS analysis of CCR7 in day 7 latency IIb proliferating B cells and latency III LCLs stratified by ICAM-1 expression. (E) Quantification of mean fluorescence intensity of CCR7 expression presented in panel D. (F) Percentage of CCR7-positive cells represented in panel D. Each bar represents the average of results from 3 independent donors *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s pairwise t test). All error bars denote SEM.