Fig 2: Characterization of surface receptor expression.1 out of 4 representative FACS stainings against various inhibitory and activating NK cell markers (CD2, CD56), KIR (CD158, KIR2DL4), activating receptors (NKp30, NKp44, NKp46, NKG2D, NKG2C, DNAM-1, 2B4, NTB-A, CD84), and inhibitory receptors (TIGIT, CEACAM-1, PD-1, NKG2A) grey = background staining, red = NK-92med., blue = RPMIf, violet = RPMIp.

Fig 3: Rapid Intratumoral and Progressive Systemic Dysfunction of Neoantigen-Specific T Cells(A) Frequency of PD-1, Lag3, Tim3, and TIGIT expression by tetramer+ T cells in the spleen (Spl) and tumor (Tum) at day 22 post-tumor implantation. Each dot is an independent mouse, and data are mean ± SEM. *p < 0.05, **p < 0.05, and ***p < 0.005 (one-way ANOVA, with a post hoc test to correct for multiple comparisons).(B) Representative plots of markers quantified in (A).(C) Mean frequency of CB101–109:H-2Db-specific CD8+ T cells that express none (0), one (PD-1), or co-express 2, 3, or 4 inhibitory receptors at day 22.(D) Proportion of total CD8+ T cells producing IFN? following ex vivo re-stimulation with CB101–109 peptide at day 22.(E) Number of tetramer+ T cells producing IFN? at day 22. Data are mean ± SEM, and each dot is an independent mouse. *p < 0.05 (one-way ANOVA, with a post hoc test to correct for multiple comparisons).(F) Progressive phenotypic and functional changes in splenic tetramer+ CD8+ T cells in mice bearing KPC2a tumors ± aPD-L1. Data are mean ± SEM. n = 4 or 5 mice per group.(G) Progressive phenotypic and functional changes in intratumoral tetramer+ CD8+ T cells. Data are mean ± SEM. *p < 0.05 (unpaired two-tailed Student’s t test). n = 4 or 5 mice per group.(H) Analysis of the indicated antigens on circulating tetramer+ T cells following aPD-L1 treatment. Data are mean ± SEM. n = 4 or 5 mice per group. See also Figure S4.

Fig 4: T cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene 3 (LAG-3), and programmed cell death 1 ligand 1 (PD-L1) expression on CD4+ and CD8+ T cells in bronchoalveolar lavage fluid (BALF). (A,G) Representative flow cytometric analysis of TIGIT expression on CD4+ T cells (A) and CD8+ T cells (G) in BALF. (C,I) Representative flow cytometric analysis of LAG-3 expression on CD4+ T cells (C) and CD8+ T cells (I) in BALF. (E,K) Representative flow cytometric analysis of PD-L1 expression on CD4+ T cells (E) and CD8+ T cells (K) in BALF. (B,H) Percentages of TIGIT+ cells among CD4+ T cells (B) and CD8+ T cells (H). (D,J) Percentages of LAG-3+ cells among CD4+ T cells (D) and CD8+ T cells (J). (F,L) Percentages of PD-L1+ cells among CD4+ T cells (F) and CD8+ T cells (L). (B,D,F,H,J,L) Individual patient data as well as medians and interquartile ranges are shown. NS: not significant.

Fig 5: 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.