Fig 2: Lysis of RMS cell lines by resting NK cells is dependent on NKG2D and DNAM-1-mediated pathways. a Histograms of expression levels (gray area isotype control thin line) of NKG2D (MIC A/AB, ULBP1-3), DNAM-1 ligands (CD112 and CD155) and HLA-1 for the cell line TE671 measured by flow cytometry. b Representative specific lysis of the cell line TE671 by resting (left panel) and IL-15-activated NK cells (right panel) in the presence or absence (open circle) of blocking antibodies to NKG2D (square), DNAM-1 (triangle) or the combination of these two receptors (closed circle). Error bars represent the SEM of triplicates. c Combined data for the lysis of the RMS cell lines by resting (5 donors, E:T ratio 25:1, left panel) and IL-15-activated (6 donors, E:T ratio 10:1, right panel) NK cells in the presence of blocking antibodies to NKG2D (dark gray bars), DNAM-1 (light gray bars) and the combined antibodies (white bars). Data are depicted as percentage of the specific lysis obtained in the absence of blocking antibody. Error bars represent the SEM. Statistical analyses were performed using one-way ANOVA, followed by the Dunnett’s multiple comparisons test: p value <0.05 is indicated by *; <0.01 by **)

Fig 3: NECTIN2 in HCC cells suppresses T cell activity.a CellTrace Violet (CTV)-labeled mouse splenic T cells were isolated and co-cultured with Hepa1–6 cells in the presence or absence of anti-Nectin2 neutralizing antibody (15 µg/mL). Mean ± SD is presented. b CTV-labeled T cells were cocultured with Hepa1–6 (WT), -Nectin2-KO1, -Nectin2-KO2, -Nectin2-KO3 cells. Mean ± SD is presented. c Representative picture and weight of Nectin2 WT (Nectin2WT:Tp53KO:c-MycOE), and Nectin2 KO (Nectin2KO:Tp53KO:c-MycOE) HCC tumors. Scale bar = 1 cm. d–f Numbers of tumor-infiltrating lymphocytes were analyzed by flow cytometry. g, h Representative pictures, and quantification of CD4 + T cells and CD8 + T cells in HCC tumors by IHC staining. Scale bar = 100µm in IHC representative pictures. a–h Student’s t test. The experiment was performed with a variable number of biologically independent samples (n number) (a n = 6, n = 3, and n = 3 for T cells only Ctrl and anti-Nectin2 respectively in CD4+ cells, and n = 3 for all groups in CD8+ cells; b n = 10, n = 6, n = 4, and n = 4 for WT, Nectin2-KO1, Nectin2-KO2, and Nectin2-KO3, respectively in both CD4+ and CD8+ cells; c–e, g: n = 7 per group; f, h: n = 21 per group). Source data are provided as a Source Data file.

Fig 4: Immune checkpoint analysis in HCC implicates TIGIT–NECTIN2 interaction.a We examined the immune checkpoint interactions between lymphocytes and APCs (tumor cells and TAMs) and identified the prominent interaction via the TIGIT–NECTIN2 axis (circle size indicates the statistical significance and circle color indicates the level of interaction). The empirical P value was estimated by 1000 imputations. b The expression of TIGIT and NECTIN2 was respectively enriched in T cells and APCs. c Upregulation of NECTIN2 was detected in HCC tumors, as compared to non-tumorous livers in both in-house and TCGA datasets. Student’s t test (2-sided). Source data are provided as a Source Data file.