Fig 2: Decreased expression of NK cell-activating receptors on NK cells in JORRP patients. (A) Percentage analysis of NKp30, NKp46, NKG2D, and NKp44 expression on NK cells as determined by flow cytometry of HCs (n = 35) and JORRP patients (n = 35) or different subgroups of JORRP patients. Agg, aggressive (n = 11); Non-agg, non-aggressive (n = 19); 6, HPV6 (n = 14); 11, HPV11 (n = 16); 6&11, HPV6&HPV11 (n = 3). (B) Correlation of CD107a expression on NK cells with NKp30, NKp46, NKG2D, and NKp44 expression on NK cells. Spearman’s correlation coefficients are shown. (C) Correlation of NKp30, NKp46, NKG2D, and NKp44 expression on NK cells with surgical times. Spearman’s correlation coefficients are shown. (D) Correlation of NKp30, NKp46, NKG2D, and NKp44 expression on NK cells with interval time of surgical reoccurrence. Spearman’s correlation coefficients are shown. Data are presented as median with interquartile range. Each dot represents a single patient. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: TGF-ß1 mediates decreased NK cell cytotoxicity. (A) NK cells purified from PBMCs of JORRP patients were preincubated with LY364947 for 3 days in the presence of 100 U/ml IL-2, then stimulated with K562 target cells for an additional 6 h at the ratio = 1:1. (A, B) Representative paragraph and percentage analysis of K562 cell apoptosis (the percentage of Annexin V on K562 cells) was determined by flow cytometry. Blank, K562 cells alone; NC, NK cells only; DMSO, NK cells + DMSO; LY364947, NK cells + LY364947. (C, D) Representative histograms and percentage analysis of CD107a on NK cells. (E) NKp30, NKG2D, and NKp46 expression on NK cells. MFI, mean fluorescent intensity. (F, G) PBMCs from HCs, PBMCs and TILs were isolated from JORRP patients. Representative paragraph and percentage analysis of CXCR6 gated on CD56+CD3- cells. TIL, tumor-infiltrating lymphocyte. Data are presented as median with interquartile range. Each dot represents a single individual. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig 4: The Human papillomavirus (HPV)6 and HPV11 E6 and E7 oncogenes affect NK cell cumulation and NK cell-activating receptor expression. PBMCs of HCs were cultured for 6 days with 100 U/ml IL-2 either in the absence (blank) or in the presence of the indicated SNU-1076 HNSCC cell lines. (A, B) Representative plots and percentage analysis of CD56+CD3- NK cells. (C, E) NKp30, NKG2D, and NKp46 expression on NK cells. (D) Relative expression of TGFB1 by RT-PCR. MFI, mean fluorescent intensity. Blank, PBMCs alone; SNU-1076, PBMCs cocultured with SNU-1076 cell line; SNU-1076 + HPV6-E6-E7, PBMCs cocultured with HPV6-E6-E7-overexpressing SNU-1076 cell line; SNU-1076 + HPV11-E6-E7, PBMCs cocultured with HPV11-E6-E7-overexpressing SNU-1076 cell line. Each dot represents a single individual. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.