Epitope EGFR87589 functions as a promiscuous MHC-II HTL epitope that can elicit efficient antitumor responses towards tumors expressing several HER family members member proteins which have a high degree of homology in the peptide sequence [8]. In these studies we proposed that EGFR inhibitors could be helpful adjuncts for cancer immunotherapy for the reason that these drugs enhanced MHC-II expression levels on tumor cells, which must enhance CD4+ T cell recognition. As shown in Figure 1A, the expression of HLA-DR in the SCC cell lines HSC-4 and Sa-3 was enhanced following erlotinib treatment. Moreover, the effect of erlotinib was more prominent ( 2-fold larger) around the Sa-3 cells as when compared with HSC-4 (Figure 1B). Mainly because Th1 response has been reported as a important subset in antitumor T cell responses [14], we utilized Th1 cytokine IFN- as a read out for HTL reactions toward tumor or peptide stimulation. As predicted, erlotinib pretreatment enhanced HTL responses against HSC-4 (Figure 2A). In contrast, erlotinib decreased the T cell response to Sa-3 cells despite the dramatic upregulation of HLA-DR (Figure 2B). These benefits indicate that in addition to increasing MHC-II levels on tumor cells, EGFR inhibitors can regulate T cell recognition via other mechanisms.Treatment of HNSCC cells with EGFR inhibitor downregulates the expression of HSP90 but not EGFRAll data are presented as mean normal deviation. In all experiments, group differences had been analyzed by using the two-tailed Student’s t test and p 0.05 was viewed as as statistically significant.Effective HTL responses are mediated by the interaction of T cell receptors with MHC-II molecules presenting the cognate peptide antigen on tumorFigure 1 Alteration of HLA-DR expression in HNSCCs by EGFR inhibitor. (A) HLA-DR expression of HNSCC cell lines HSC-4 and Sa-3 was examined by flow cytometry. HNSCC cells were treated with IFN- (50U/ml) alone or with IFN- and erlotinib (1 M) for 48 h. Purple graph: isotype control antibody, Green graph: anti HLA-DR antibody. (B) Imply fluorescence intensity (MFI) of HLA-DR expression was shown. Columns: implies of triplicate determinations, bars: SD. Benefits are representative of at least two separate experiments. *p 0.05.Kumai et al. Journal of Translational Medicine 2014, 12:265 http://www.translational-medicine/content/12/1/Page 4 ofFigure two EGFR inhibitor attenuated anti-tumor responses of EGFR87589-reactive CD4+ T cell clones against Sa-3. (A) (B) EGFR87589-reactive CD4+ T cell clones T8 (DR53-restricted) and M8 (DR53-restricted) have been tested for their capacity to recognize antigen straight on EGFR-positive, HLA-DR matched HNSCC cell line HSC-4 or Sa-3 by IFN- production.Odevixibat Tumor cells have been pretreated 48 h with or with no erlotinib (1 M).Tusamitamab DMSO was applied as adverse manage.PMID:24257686 Columns with no bars had SD of 10 of imply values. Results are representative of 3 separate experiments. *p 0.05.cells. In turn, the levels of protein antigen produced by the tumor cell sand the antigen-processing machinery will establish the production on the cognate peptide antigen. Thus, we evaluated the impact of EGFR inhibition around the levels of EGFR protein in tumor cells. As shown in Figure 3A, unlike phosphorylated EGFR, the total EGFR protein levels in HSC-4 and Sa-3 cells remained unchanged soon after erlotinib remedy. Meantime, we examined the expression levels of HSP70 and HSP90, that are recognized to be vital elements for antigen processing [15]. Expression of HSP70 remained unchanged.