Fumiko Ichihara

Inhibition of Activated/Memory (CD45RO+) T Cells by Oxidative Stress Associated with Block of NF-κB Activation

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-γ release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO+) T cells and correlated with a block in NF-κB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-γ, TNF-α, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO+, T cells found in the tumor milieu are functionally suppressed.

Identification of HER2/neu-derived peptide epitopes recognized by gastric cancer-specific cytotoxic T lymphocytes

We have derived HLA‐A2.1‐restricted, gastric cancer‐specific cytotoxic T lymphocyte (CTL) lines by repetitive in vitro stimulation of tumor‐associated lymphocytes (TAL) with autologous tumor cells. The HER2/neu specificity of these gastric cancer‐specific CTLs was demonstrated using HER2/neu‐transfected cell lines and HER2/neu‐expressing tumors, and with a set of HER2/neu‐derived peptide epitopes. Gastric cancer‐specific CTLs specifically lysed autologous and allogeneic HLA‐A2.1+, HER2/neu+ gastric cancer cells, HER2/neu‐transfected C1R/A2 cell lines (HLA‐A2.1+, HER2+) and HLA‐A2.1‐transfected SW626 tumor cell lines (HLA‐A2.1+, HER2+). This recognition could be inhibited by anti‐HLA‐A2 antibody or by cold target HER2/neu‐transfected C1R/A2 cells. Our results demonstrate that the HER2/neu‐encoded HLA‐A2.1‐associated epitopes recognized by CTLs are presented as naturally processed peptides on gastric cancer lines. Furthermore, 3 of 19 tested HER2/neu‐derived peptide epitopes [HER2(9106), HER2(9369), HER2(9689)], which all bound HLA‐A2.1 with high (IC50 < 50 nM) affinity, were able to sensitize HLA‐A2+ C1R/A2 cells to be recognized by the gastric cancer‐specific CTLs, demonstrating the immunodominance of these epitopes. In conclusion, our findings implicate HER2/neu‐derived epitopes as potential candidates for novel immunotherapy and vaccine strategies against gastric cancer. Int. J. Cancer 78:202–208, 1998.

Vascular endothelial growth factor inhibits maturation of dendritic cells induced by lipopolysaccharide, but not by proinflammatory cytokines

Purpose: Dendritic cells (DCs) play an important role in the host’s immunosurveillance against cancer. It has been shown that the function of DCs is impaired and their population decreased in a cancer-bearing host. In the present study, we investigated the mechanism of down-regulation of DCs in a cancer-bearing host. Methods: We evaluated the relationship between DC infiltration and production of vascular endothelial growth factor (VEGF) in carcinoma tissue by immunohistochemistry. Furthermore, functional and phenotypical alterations of DCs were evaluated when monocyte-derived, mature DCs were treated with VEGF in vitro. Monocyte-derived DCs were generated in a culture of monocyte with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and the maturation of DCs was induced by either lipopolysaccharide (LPS) or a proinflammatory cytokine cocktail: tumor-necrosis factor α, prostaglandin E2, IL-6, and IL-1β. Results: A significant inverse correlation was found between the density of DCs and the quantity of VEGF production in gastric carcinoma tissue (r=−0.39, p<0.05). In LPS-induced maturation, the ability of mature DCs to stimulate allogenic T cells and produce IL-12 (p70 heterodimer) was suppressed by the addition of VEGF in a dose-dependent manner. A lesser expression of costimulatory molecules (CD80 and CD86) was seen in DCs treated with exogenous VEGF than in DCs not treated with VEGF. The population of dead DCs (early and late apoptosis) treated with VEGF increased more than that without VEGF treatment, using the annexin V and propidium iodide evaluation in DCs matured by LPS. In contrast, in DCs matured by the proinflammatory cytokine cocktail, the down-regulation of costimulatory molecules and induction of DC apoptosis was not seen. Conclusions: These findings show that the inhibition of DC maturation by VEGF differs depending on the maturation status of the DCs.

High frequency of c-Met expression in gastric cancers producing alpha- fetoprotein.

Gastric cancers producing α-fetoprotein (AFP) have a poor prognosis and a high incidence of liver metastasis. Hepatocyte growth factor (HGF) and its receptor, c-Met, are known to induce mitosis and cell movement and to promote tumor progression. In the present study, c-Met and HGF expression in AFP-producing gastric cancer was compared with those gastric cancers that do not produce AFP. Twenty-six patients with AFP-producing gastric cancers [AFP(+)] and 26 patients stage-matched gastric cancers without AFP production [AFP(–)] were evaluated for c-Met and HGF expression and proliferating cell nuclear antigen-labelling index using immunohistochemical analysis. A higher frequency of c-Met expression was observed in the AFP(+) group than in the AFP(–) group (p < 0.01). A higher expression of c-Met might be one explanation for the poorer prognosis of AFP-producing gastric cancers.

Evaluation of VEGF and VEGF-C expression in gastric cancer cells producing α-fetoprotein

Background. Gastric cancers producing α-fetoprotein (AFP) are known to have a poor prognosis and to show a high incidence of liver metastasis. Vascular endothelial growth factor (VEGF) and its isoform VEGF-C are reported to be associated with tumor progression through an angiogenic or lymphangiogenic function. In the present study, to clarify the cellular characteristics of AFP-producing gastric cancers, the expression of VEGF and that of VEGF-C in AFP-producing gastric cancer was compared with their expression in gastric cancers that do not produce AFP. Methods. Twenty-six patients with AFP-producing gastric cancers [AFP(+)] and 26 patients with stage-matched gastric cancers that did not produce AFP [AFP(−)] were evaluated for VEGF and VEGF-C expression and vessel density, using immunohistochemical analysis. Results. The survival rate of the AFP(+) group was significantly worse than that of the stage-matched AFP(−) group (P < 0.05). The frequency of VEGF-C expression was significantly higher in the AFP(+) group than in the AFP(−) group (P < 0.01). There was no significant difference in VEGF expression between the AFP(+) and AFP(+) groups. The microvessel density was higher in the AFP(+) group than in the AFP(−) group (P < 0.05). Conclusions. A higher expression of VEGF-C might be one explanation for the poorer prognosis of AFP-producing gastric cancers.

Expression of signal transducing T‐cell receptor ζ molecules after adoptive immunotherapy in patients with gastric and colon cancer

We and others have shown decreased expression of T‐cell receptor‐CD3‐associated signal transducing ζ molecules (TCRζ) in tumor infiltrating and peripheral T cells of patients with advanced cancer. In the present study, we performed adoptive immunotherapy (AIT) with tumor‐associated lymphocytes (TAL) in patients with gastric (n = 11) and colon (n = 3) cancer with stage IV and investigated whether the alteration of signal transducing molecules was observed with AIT, compared to an untreated control group (n = 13). Autologous TALs isolated from malignant ascites or pleural effusion were cultured with stimulation of autologous tumor in the presence of interleukin‐2 (IL‐2) and were transferred to the patients. TCR ζ expression in peripheral T cells was measured by flow cytometric analysis of permeabilized cells with anti‐ζ monoclonal antibody (MAb) (TIA‐2) before and after AIT. We confirmed the down‐regulation of TCR ζ expression in peripheral blood lymphocytes (PBL) of patients with gastric and colon cancer with stage IV compared to healthy donors (n = 15). AIT induced up‐regulation of TCR ζ expression in 2 of 14 treated patients, caused no significant change of TCR ζ expression in 7 patients and induced further down‐regulation in 5 patients. The patients who achieved clinical responses (n = 3) with AIT showed no significant change of TCR ζ expression. On the other hand, in the control group without adoptive transfer, further down‐regulation of TCR ζ expression was observed during the corresponding periods, paralleling disease progression. Taken together, TCR ζ expression in the patients was further down‐regulated, corresponding to disease progression in individual cancer patients. In some patients, AIT could induce increased or stable TCR ζ expression. The quantitative analysis of TCR ζ expression might provide vital information that can be used to optimize therapy by preserving immune functions within cancer patients. Int. J. Cancer 78:301–305, 1998.© 1998 Wiley‐Liss, Inc.

Frequencies of HER-2/neu overexpression relating to HLA haplotype in patients with gastric cancer.

We have identified that HER‐2/neu‐derived peptides are naturally processed as tumor rejection antigens recognized by tumor‐specific, HLA‐A2‐restricted cytotoxic T lymphocytes in gastric cancer. To evaluate candidates for immunotherapy using HER‐2/neu‐derived, HLA‐A2‐restricted peptides, we examined the frequency of HLA‐A2 relating to HER‐2/neu overexpression or the infiltrating grade of tumor‐infiltrating lymphocytes (TILs) in Japanese patients with gastric cancer. HER‐2/neu‐overexpressing tumors detected by immunohistochemistry amounted to 19% of primary gastric cancers and HLA‐A2‐positive patients with gastric cancer were 31% of primary gastric‐cancer cases. Finally, gastric‐cancer patients with both HLA‐A2‐positive and HER‐2/neu‐overexpressing tumors amounted to 6.6% of these cases. There was no significant difference in the infiltrating grade of TILs between gastric cancers overexpressing HER‐2/neu and those that did not. The candidate for HER‐2/neu‐based immunotherapy with HLA‐A2‐restricted peptides represent a very limited population of Japanese patients.

Macrophages in tumor‐draining lymph node with different characteristics induce T‐cell apoptosis in patients with advanced stage‐gastric cancer

A hosts immune‐defense system is suppressive by many factors in patients with cancer. We have previously shown one possible mechanism behind the T‐cell dysfunction, whereby H2O2 secreted from macrophages in tumor‐draining lymph node (MTDL) induced T‐cell dysfunction with down‐regulation of TCR ζ molecules. In the present study, we analyzed how MTDL affect T cells, with a particular focus on T‐cell apoptosis, by co‐culturing MTDL with autologous peripheral blood T cells in gastric cancer. Moreover, we characterized the MTDL according to surface marker, oxygen‐burst capacity and intracellular cytokine status. T‐cell apoptosis was significantly induced in comparison to T‐cell alone control in patients with advanced disease, concomitant to the elevated caspase activity and following impaired T‐cell function. In patients with early disease, no significant difference was seen in the proportions of T cells that underwent apoptosis between T cells plus MTDL and T cells alone. Moreover, the addition of a selective scavenger of H2O2, catalase inhibited the apoptosis of T cells co‐cultured with MTDL in patients with advanced disease. In the characterization of MTDL, the production of H2O2 in MTDL from advanced disease was significantly higher than that in early disease. The amounts of intracellular IL‐10 and IL‐12 in MTDL in advanced disease were significantly higher than those in early disease. These results indicated that MTDL induced apoptosis of autologous T cells and this T‐cell dysfunction was mediated by H2O2 derived from MTDL. Furthermore, the characteristics of MTDL including the capacity of oxygen‐burst and intracellular cytokine production were different depending on the disease progression.

Differences in the recognition of tumor-specific CD8+ T cells derived from solid tumor, metastatic lymph nodes and ascites in patients with gastric cancer

We established gastric cancer‐specific CD8+ T‐cell (TCD8+) lines derived from different lymphocyte sources in the same patients by repeated stimulation with mitomycin‐C‐treated autologous tumor cells with low‐dose interleukin‐2, and we compared recognition patterns among the TCD8+ derived from solid tumor, lymph node metastasis and ascites in the same patient (n = 3) to determine their similarities and differences for therapeutic purposes. We confirmed that gastric cancer‐specific TCD8+ lines can be isolated, in a MHC class I‐restricted manner, from solid tumors, metastatic lymph nodes and malignant ascites. TCD8+ lines derived from tumor‐infiltrating lymphocytes (TIL) in solid tumor recognized autologous tumor cells derived from solid tumor, but not autologous tumor cells derived from ascites or metastatic lymph node, while TCD8+ lines derived from tumor‐associated lymphocytes (TAL) in malignant ascites recognized autologous tumor cells derived from ascites, but not tumor cells from solid tumor or metastatic lymph node. Furthermore, TCD8+ lines derived from regional lymph node lymphocytes (RLNL) recognized autologous tumor cells derived from metastatic lymph nodes, but not tumor cells derived from ascites. No significant differences were seen in MHC class I expression among the tumors derived from solid tumor, lymph node metastasis or ascites in the same patient. This suggests that there are differences of recognition patterns among the TILs, TALs and RLNLs in the same patient and that it is important to consider the source of lymphocytes, e.g., a combination of TILs, TALs and RLNLs, for adoptive immunotherapy in gastric cancer patients. Int. J. Cancer 71: 978‐981, 1997.

T-Cell Dysfunction in a Patient with Short Bowel Syndrome: Report of a Case

We describe herein the case of a 48-year-old man who underwent emergency massive resection of the small intestine due to strangulated ileus, which led to short bowel syndrome (SBS) as he was left with 7 cm of jejunum and 8 cm of ileum. We evaluated the immune function in this patient, focusing particular attention on T-cell-mediated immunity. Biochemical and nutritional parameters, including minerals and trace elements, indicated that the patient was in relatively good health; however, the proliferative response to mitogen and tumor necrosis factor-α production in response to the anti-CD3 monoclonal antibody in peripheral blood lymphocytes (PBL) were impaired compared with age-matched postgastrectomy patients and healthy donors. Moreover, the expression of T-cell receptor (TCR) ζ, which is involved in signal transduction and the subsequent activation of T cells, was downregulated in this patient compared with that in the age-matched postgastrectomy patients and healthy donors. These observations suggest that T-cell function was disturbed in our patient, and that this dysfunction was associated with the decreased expression of TCR ζ molecules.