Effect of CD4+ Th1 cytokines and HER2-targeted antibodies on MHC class I restoration and HER2369–377-CD8+ T-cell targeting of HER2-expressing cancer cells.

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Effect of CD4+ Th1 cytokines and HER2-targeted antibodies on MHC class I restoration and HER2369–377-CD8+ T-cell targeting of HER2-expressing cancer cells. Effect of CD4+ Th1 cytokines and HER2-targeted antibodies on MHC class I restoration and HER2369–377-CD8+ T-cell targeting of HER2-expressing cancer cells. HER2low MDA-MB-231, HER2intermediate MCF-7 and SK-OV-3A2, and HER2high BT-474 and SK-BR-3 cells were treated with the following: no treatment (A), rhIFNγ alone (B), rhTNFα alone (C), trastuzumab alone (D), IFNγ + TNFα (E), or trastuzumab + IFNγ + TNFα (F). For each cell line, representative panels show flow cytometric analysis of APC anti–HLA-ABC expression; filled traces represent isotype-matched control staining, and open traces represent specific Ab staining. Results in adjoining histograms are representative of three experiments, and quantified as average HLA-ABC mean channel fluorescence (MCF) ± SEM. B, direct tumor recognition of HER2-expressing cells by HER2369–377-sensitized CD8+ T cells was assessed by IFNγ ELISA. HER2intermediate MCF-7, SK-OV-3A2, and HER2high SK-BR-3 cells—pretreated with trastuzumab alone, IFNγ + TNFα, or trastuzumab + IFNγ + TNFα—were cocultured 1:1 with HER2369–377-sensitized CD8+ T cells; coculture supernatant was harvested and subjected to IFNγ ELISA. Results, representative of three experiments using cells from different HER2pos-DCIS donors, are expressed as mean IFNγ (pg/mL) ± SEM. C, cytotoxicity of HER2-expressing cells induced by HER2369–377-sensitized CD8+ T cells was assessed by flow cytometry. CFSE-labeled tumor cells from cocultures in B were harvested and stained with 7-AAD and FITC:anti-CD8. As shown in representative dot-plot panels for each cell line, CSFE+ (y-axis), but not CD8+, cells were gated and proportion of 7-AAD+ (x-axis) cells was assessed. Results shown in adjoining histograms are representative of three experiments, and expressed as a percentage of apoptotic tumor cells (±SEM) in coculture minus background. ns, not statistically significant; tx, treatment. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with post hoc Tukey testing. Jashodeep Datta et al. Cancer Immunol Res 2015;3:455-463 ©2015 by American Association for Cancer Research