Downregulation of HLA class I expression may contribute to a poor prognosis in cancer patients. There is limited information about epigenetic and oncogenic regulation of HLA class I, and multiple mechanisms may be involved. In the current study, we examined the relationship between the HER2-signaling pathway (MAPK and PI3K-Akt) and the expression of HLA class I and Ag-processing machinery (APM) components. A panel of gastric and esophageal cancer cell lines was treated with wortmannin as an Akt-signal inhibitor; the MAPK signal inhibitor PD98059; lapatinib, which inhibits both the epidermal growth factor receptor and HER2 tyrosine kinase; or siRNA for MAPK. The levels of HER2-signaling molecules, APM components, and HLA class I were evaluated by Western blot, quantitative PCR, and flow cytometry. Resected gastric tumor tissues (n = 102) were analyzed for p-Erk and HLA class I expression by immunohistochemistry. As a result, inhibition of the MAPK pathway induced upregulation of HLA-A02 and HLA-A24 expression in parallel with an increase in APM components and enhanced target sensitivity to tumor Ag–specific CTL lysis. HLA-A expression was predominantly regulated by the MAPK pathway, but it was also influenced, in part, by the Akt pathway. There was a strong inverse correlation between p-Erk expression and HLA class I expression in clinical tumor samples. In conclusion, HLA-A expression is predominantly regulated by the MAPK pathway in gastric and esophageal cancer.

PurposeThere is growing evidence that tumor-specific immune responses play an important role in anti-cancer therapy, including radiotherapy. Using mouse tumor models we demonstrate that irradiation-induced anti-tumor immunity is essential for the therapeutic efficacy of irradiation and can be augmented by modulation of cytotoxic T lymphocyte (CTL) activity.Methods and MaterialsC57BL/6 mice, syngeneic EL4 lymphoma cells, and Lewis lung carcinoma (LL/C) cells were used. Cells were injected into the right femurs of mice. Ten days after inoculation, tumors were treated with 30 Gy of local X-ray irradiation and their growth was subsequently measured. The effect of irradiation on tumor growth delay (TGD) was defined as the time (in days) for tumors to grow to 500 mm3 in the treated group minus that of the untreated group. Cytokine production and serum antibodies were measured by ELISA and flow cytometry.ResultsIn the EL4 tumor model, tumors were locally controlled by X-ray irradiation and re-introduced EL4 cells were completely rejected. Mouse EL4-specific systemic immunity was confirmed by splenocyte cytokine production and detection of tumor-specific IgG1 antibodies. In the LL/C tumor model, X-ray irradiation also significantly delayed tumor growth (TGD: 15.4 days) and prolonged median survival time (MST) to 59 days (versus 28 days in the non-irradiated group). CD8(+) cell depletion using an anti-CD8 antibody significantly decreased the therapeutic efficacy of irradiation (TGD, 8.7 days; MST, 49 days). Next, we examined whether T cell modulation affected the efficacy of radiotherapy. An anti-CTLA-4 antibody significantly increased the anti-tumor activity of radiotherapy (TGD was prolonged from 13.1 to 19.5 days), while anti-FR4 and anti-GITR antibodies did not affect efficacy.ConclusionsOur results indicate that tumor-specific immune responses play an important role in the therapeutic efficacy of irradiation. Immunomodulation, including CTLA-4 blockade, may be a promising treatment in combination with radiotherapy.

HER2 is a promising target for immunotherapeutic interventions with T cell-based approaches since it is amplified and overexpressed in 20–30% of breast cancers. However, several previous studies including ours showed that HER2-overexpressing tumors may escape cytotoxic T lymphocyte-mediated lysis by downregulating MHC Class I and components of the antigen-processing machinery. The aims of the present study were to analyze the relationship between HER2 and MHC Class I expression and to elucidate the mechanisms underlying MHC Class I downregulation in breast cancer. We explored expression of HER2, MHC Class I, PTEN, Ki67, estrogen and progesterone expression in 70 breast cancer patients by immunohistochemistry (IHC) and analyzed their correlation. We also explored the components of the signal transduction pathway that are involved in the regulation of MHC Class I expression using small-interfering RNAs targeting HER2 as well as an inhibitor of HER2 signaling. HER2 expression in breast cancers correlated inversely with MHC Class I expression analyzed by IHC. HER2 depletion by small-interfering RNAs resulted in MHC Class I upregulation. Moreover, MHC Class I expression on breast cancer cell lines was upregulated by PD98059, an inhibitor of mitogen-associated protein kinases, in a dose-dependent manner. Thus, agents that target the MAPK signaling pathway may increase MHC Class I expression in breast cancer cells.

It has been reported that an increased population of regulatory T cells (T-regs) is one of the reasons for impaired anti-tumor immunity. We investigated the frequency of Foxp3(+) T-regs in tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) of patients with esophageal squamous cell carcinoma (ESCC). Furthermore, in order to elucidate the mechanisms behind T-regs accumulation within tumors, we evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3(+) T-regs. CD4(+)CD25(+)Foxp3(+) T-regs as a percentage of CD4(+) cells were counted by flow cytometry. The frequency of CCL17(+) or CCL22(+) cells among CD14(+) cells in tumors was also evaluated by flow cytometry. Moreover, an in vitro migration assay using T-regs derived from ESCC was performed in the presence of CCL17 or CCL22. The frequency of Foxp3(+) T-regs in TILs was significantly higher than that in the normal esophageal mucosa (24.6 +/- 10.0 vs 7.1 +/- 5.9%, P < 0.01). The frequency of Foxp3(+) T-regs in PBLs of ESCC patients was significantly higher than that in normal healthy donors (7.0 +/- 4.2 vs 2.5 +/- 1.0%, P < 0.01). Furthermore, the frequency of CCL17(+) or CCL22(+) cells among CD14(+) cells within tumors was significantly higher than that of normal esophageal mucosa, and there was a significant correlation between the frequency of CCL17(+) or CCL22(+) cells and Foxp3(+) T-regs in TILs. In addition, the in vitro migration assay indicated that T-regs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3(+) T-regs in ESCC.

NK cells can be divided into two subsets, CD56(dim) and CD56(bright) NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56(dim) or CD56(bright) NK cells gated on CD56(+)CD3(-) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)-expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56(dim) and CD56(bright) NK cells between ESCC patients and healthy donors, down-regulated CD16 and up-regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab-mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down-regulated CD16 and up-regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF-beta1 partially contributed to down-regulation of CD16 on NK cells. Down-regulated CD16 and up-regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.