Journal: Signal Transduction and Targeted Therapy

Article Title: Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab

doi: 10.1038/s41392-021-00629-w

Figure Lengend Snippet: HLA-G depletion on HER2-positive breast cancer cells enhances trastuzumab-induced lysis by cocultured NK cells. a Representative immunohistochemical staining of HLA-G in a breast cancer tissue array. (Right panel) The number of HLA-G-positive and -negative normal and neoplastic tissues obtained from patients with HER2-positive or other subtypes of breast cancer were plotted. b Correlation analysis for HLA-G and HER2 expression in breast cancer tissues. c Kaplan–Meier survival analysis based on the prognosis and recurrence rates of 108 HER2-positive breast cancer patients grouped by HLA-G expression. d FCM examination of HLA-G expression in breast cancer and gastric cancer cell lines. e Western blot analysis (left) and qRT-PCR assay (right) of HLA-G expression in SK-BR-3 cells 48 h after transfection with the indicated siRNA. f NK cells prepared from PBMCs of healthy donors (effector, abbreviated as “E”) were cocultured with control or HLA-G siRNA-transfected SK-BR-3 cells (target, abbreviated as “T”) supplemented with or without the indicated antibodies. The cytotoxicity of the NK cells was measured via FCM as described in the “Materials and methods.” g NK cells in the presence of trastuzumab were cocultured with different neoplastic cell lines at the indicated E:T ratios supplemented with or without a control or HLA-G blocking antibody. The cytotoxicity of NK cells was measured via FCM. h Parental or HLA-G knockdown (siHLA-G#2) SK-BR-3 cells were cocultured with NK cells (E:T = 10:1) in the presence of trastuzumab with or without an HLA-G-blocking antibody. The cytotoxicity of NK cells was measured via FCM. All experiments were performed three times. Statistical significance was determined by Student’s t test. * P < 0.05, ** P < 0.01, and *** P < 0.001. n.s. Nonsignificant

Article Snippet: For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against HER2 (Cat. No. ab11710, Abcam) and KIR2DL4 (Cat. No. ab154386, Abcam).

Techniques: Lysis, Immunohistochemical staining, Staining, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Blocking Assay, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab

doi: 10.1038/s41392-021-00629-w

Figure Lengend Snippet: The association of HLA-G expression with trastuzumab response in patients with HER2-positive breast cancer ( n = 30)

Article Snippet: For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against HER2 (Cat. No. ab11710, Abcam) and KIR2DL4 (Cat. No. ab154386, Abcam).

Techniques: Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab

doi: 10.1038/s41392-021-00629-w

Figure Lengend Snippet: Clinical and pathological information for HER2-positive breast cancer patients treated with trastuzumab

Article Snippet: For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against HER2 (Cat. No. ab11710, Abcam) and KIR2DL4 (Cat. No. ab154386, Abcam).

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab

doi: 10.1038/s41392-021-00629-w

Figure Lengend Snippet: HLA-G engages KIR2DL4 on NK cells to suppress trastuzumab-elicited ADCC. a FCM assay of the expression of the indicated receptors on human PBMC-derived primary NK cells. b NK cells in the presence of trastuzumab were cocultured with HER2-overexpressing breast cancer cells at the indicated E:T ratios supplemented with or without the indicated blocking antibodies. The toxicity of NK cells to malignant cells was measured. c , d NK cells and SK-BR-3 cells were cocultured as described in ( b ) (E:T = 30:1). IFN-γ production was measured via ELISA ( c ), and the degranulation of NK cells was evaluated via FCM assay for CD107a expression ( d ). e Representative immunohistochemical staining of KIR2DL4 on infiltrating NK cells in clinical HER2-positive breast cancer tissues. f Representative immunofluorescent staining of HER2 and KIR2DL4 in breast cancer tissues of individual patients. All experiments were performed three times. Statistical significance was obtained by Student’s t test. * P < 0.05, ** P < 0.01, and *** P < 0.001. n.s. Nonsignificant

Article Snippet: For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against HER2 (Cat. No. ab11710, Abcam) and KIR2DL4 (Cat. No. ab154386, Abcam).

Techniques: Expressing, Derivative Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab

doi: 10.1038/s41392-021-00629-w

Figure Lengend Snippet: TGF-β and IFN-γ upregulate HLA-G on breast cancer cells and KIR2DL4 on NK cells, respectively. a NK cells were cultured alone or with SK-BR-3 cells (E:T = 1:1) supplemented with the indicated antibodies. The supernatant was harvested and measured for cytokine levels using a cytokine antibody array. b NK cells and SK-BR-3 cells were cocultured or cultured alone for 48 h with or without trastuzumab, followed by measurement of IFN-γ and TGF-β production via ELISA. c FCM assay for determining the trastuzumab-elicited cytotoxicity of NK cells to cocultured HER2-positive breast cancer cells when treated with or without TGF-β (50 ng/ml) for 6 h. d Cells were incubated with TGF-β for the indicated times and were subjected to FCM assay for determining HLA-G level. e , f FCM assay for the expression of KIR2DL4 and ILT2 on NK cells incubated with IFN-γ and different inhibitors for the indicated times. g NK cells were incubated with IFN-γ (50 ng/ml) for the indicated times and were subjected to Western blot analysis. h ChIP assay for determining the enrichment of STAT1 on the KIR2DL4 promoter using NK cells prepared from different donors. All experiments were performed three times. Statistical significance was obtained by Student’s t test. * P < 0.05, ** P < 0.01, and *** P < 0.001. n.s. Nonsignificant

Article Snippet: For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against HER2 (Cat. No. ab11710, Abcam) and KIR2DL4 (Cat. No. ab154386, Abcam).

Techniques: Cell Culture, Ab Array, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab

doi: 10.1038/s41392-021-00629-w

Figure Lengend Snippet: HLA-G/KIR2DL4 signaling impairs the in vivo antitumor activity of NK cells mediated by trastuzumab. a Nude mice were inoculated with luciferase-expressing SK-BR-3 cells. When the tumor volume reached 200 mm 3 (day 0), mice were subjected to intraperitoneal treatment with 1 × 10 7 primary NK cells, trastuzumab (5 mg/kg), and KIR2DL4/HLA-G-blocking antibodies (1 mg/kg) on days 0, 3, 6, 9, and 12. Bioluminescence imaging was performed on the indicated days. b Volumes of the tumors in the mice as described in ( a ) were monitored and plotted. Arrows indicated days when treatment was conducted. c Mice were sacrificed on day 30, and tumors were excised and weighed. d Tumors in ( b ) were sectioned and subjected to immunohistochemical staining. e Nude mice were inoculated with GKO #3 SK-BR-3 cells. When the tumor volume reached 200 mm 3 (day 0), mice were subjected to intraperitoneal treatment with 1 × 10 7 primary NK cells, trastuzumab (5 mg/kg), and KIR2DL4-blocking antibody (1 mg/kg) on days 0, 3, 6, 9, and 12. Volumes of the tumors were monitored and plotted. f , g Mice in ( e ) were sacrificed on day 30, and tumors were excised and weighed. h Representative immunohistochemical staining and IHC scores of PD-L1 in xenograft tumor tissues as shown in ( f ). i Representative immunohistochemical staining and IHC scores of HLA-G, PD-L1, KIR2DL4, and PD-1 in post-treatment breast cancer tissues collected from trastuzumab-sensitive and trastuzumab-resistant individual patients. j Immunohistochemical staining of HLA-G was performed on paired pre- and post-treatment specimens collected from HER2-positive breast cancer patients. k Schematic diagram for the regulatory roles of HLA-G and KIR2DL4 in trastuzumab-induced ADCC. In the absence of HLA-G, KIR2DL4 forms a feedback circuit with IFN-γ to activate NK cells, which is potentially compromised by IFN-γ upregulation of PD-L1 on neoplastic cells. HLA-G engagement of KIR2DL4 inactivates NK cells. Although not always occurring simultaneously, PD-1 upregulation by tumor-derived TGF-β might coordinate with HLA-G to repress the cytotoxicity of NK cells. All experiments were performed three times. Statistical significance was obtained by Student’s t test. * P < 0.05, ** P < 0.01, and *** P < 0.001. n.s. Nonsignificant

Article Snippet: For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against HER2 (Cat. No. ab11710, Abcam) and KIR2DL4 (Cat. No. ab154386, Abcam).

Techniques: In Vivo, Activity Assay, Luciferase, Expressing, Blocking Assay, Imaging, Immunohistochemical staining, Staining, Derivative Assay