Fig 2: ICOS+TIGIT+CD4+T cells enriched in TILs are functionally impaired. (A) tSNE map of CD4+ T cells in TILs (yellow dots), NTILs (purple dots), and TILs (green dots) of the second HCC cohort (n = 13). (B) tSNE maps showing the expression level and the distribution of each indicated marker. The color scale indicates the relative expression levels. (C) tSNE map with each cluster of CD4+ T cells colored differently. (D) Box plots showing the frequency of CD4+ T cells within cluster CD4-6 in TILs, NTILs, and PBMCs. (E) Heatmap showing the normalized expression of each marker in the indicated clusters. The color scale indicates the relative expression levels. * indicates cluster CD4-6, which is increased significantly in TILs vs NTILs. (F) Box plots showing the expression of indicated transcription factors on ICOS+ TIGIT+ CD4+ and ICOS- TIGIT- CD4+ TILs. *P = .05 and **P = .01, Wilcoxon signed-rank test, adjusted with the Benjamini and Hochberg method. ns, not significant.

Fig 3: ICOS+TIGIT+CD4+T cells are enriched in TILs. (A) tSNE map of CD4+ T cells in TILs (yellow dots), NTILs (purple dots), and PBMCs (green dots) of the first HCC cohort (n = 15). (B) tSNE maps showing the expression level and the distribution of each indicated marker. The color scale indicates the relative expression levels. (C) tSNE map of CD4+ T cells with each cluster colored differently. (D) Box plots showing the frequency of CD4+ T cells in each significantly enriched cluster in TILs vs NTILs. (E) Heatmap showing the normalized expression of the markers for selected clusters. The color scale indicates the relative expression levels. *P = .05 and **P = .001, Wilcoxon signed-rank test, adjusted with the Benjamini and Hochberg method.

Fig 4: CD19-CAR T cells demonstrate impaired recall potential in patients with antigen-positive cells(A) Schematic depiction of a CD19-CAR T cell quantity relative to antigen-positive cells during a primary and secondary immune response.(B) Top panel: longitudinal graph of CD19-CAR T cell quantity and antigen-positive B cells from representative patient 5. Bottom panel: summary graph of CD19-CAR T cell expansion during a primary response to antigen (initial infusion) versus during endogenous CD19+ B cell recovery. n = 5 patient samples.(C) Patients #4 and #6 CAR T cell vector copy is calculated at the given time points relative to the overall quantity of DNA from the PBMCs. DNA methylation profiles at LEF1, CX3CR1, BATF, PDCD1, TIGIT, and TOX loci from patient 6 GMP and week 2 CD8+ CD19-CAR T cells as well as a Tpex sample are shown. Individual CpG sites are represented by vertical lines, with red indicating methylation and blue indicating a lack of methylation. DMRs are represented by a green box, and enhancers are shown with a purple line.

Fig 5: The PTG-T16R-scFV-CAR T cells exerted potent antitumor activity in vitro, (A) siRNA mediated immunosuppressive factor receptors knockdown efficiency screening in HEK293T cells by qPT-PCR: including targeted TGFßRII, IL-10RA and IL-6R genes. The red boxes represented the siRNA with the highest knockdown efficiency. (B) Left schematic representation of the lentiviral vectors carrying an EGFR-CAR moiety and a cluster of PTG-shRNA or T16R-shRNA, or both. Right schematic representation showed the same modification of the lentiviral vectors carrying B7H3-CAR. The PTG-shRNA cluster can target the knockdown of PD-1, Tim-3, and Tigit genes and the T16R-shRNA cluster can target the knockdown of TGFßRII, IL-10RA and IL-6R genes. These cluster skeletons were derived from the miR-106b cluster in human genome. (C) The expressions of EGFR-CAR and B7H3-CAR after shRNA clusters modification were analyzed by immunoblotting with anti-flag tag. The positive control was GP120-CAR. (D) The cytotoxic activities of the PTG-T16R-EGFR-CAR-T cells, the PTG-EGFR-CAR-T cells, the T16R-EGFR-CAR-T cells, the EGFR-CAR-T cells and the GP120-CAR-T cells co-cultured with TFK-1 or Hucct1 cells were compared by LDH assay. The negative control was the GP120-CAR-T cells group (n = 3, healthy donors). (E) The cytotoxic activities of the PTG-T16R-B7H3-CAR-T cells, the PTG-B7H3-CAR-T cells, the T16R-B7H3-CAR-T cells, the B7H3-CAR-T cells and the GP120-CAR-T cells co-cultured with TFK-1 or Hucct1 cells were compared by LDH assay. The negative control was the GP120-CAR-T cells group (n = 3, healthy donors). Experiments were performed independently at least 3 times. One-way ANOVA was used in Tukey's multiple comparison test. Experimental data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.Abbreviations: siRNA, small interfering RNA; TGFßR, transforming growth factor ß receptor; IL-10R, interleukin 10 receptor; IL-6R, interleukin 6 receptor; PD-1, programmed death-1; Tim-3, T-cell immunoglobulin and mucin domain-3; Tigit, T cell immunoglobulin and ITIM domain; CAR, chimeric antigen receptor T; EGFR, epidermal growth factor receptor; B7H3, B7 homolog 3; GP120, gp120 antigen; LDH, lactate dehydrogenase; SEM, standard error of the mean.