Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB 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-gamma 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-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, 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.

Soluble MHC class I molecules induce cellular death in a CD8+ T-cell hybridoma tumor model.

T cells undergo activation-induced cell death (AICD) after T-cell receptor cross-linking in the absence of co-stimulation. In this study, we examined whether AICD induced by purified MHC class I molecules could be used to selectively eliminate tumor cells in T-cell malignancies. As a model, soluble H-2K(b) molecules refolded with the chicken ovalbumin SIINFEKL peptide (K(b)-OVA) and a CD8+ T-cell hybridoma (CD8-OVA) specific for this peptide were used. Addition of CD8-OVA hybridoma cells to plastic plates adsorbed with K(b)-OVA molecules resulted in a concentration-dependent decrease in cellular proliferation. Exogenous IL-4 further depressed the proliferation of CD8-OVA cells in a dose-dependent manner in the presence, but not in the absence, of K(b)-OVA. Staining of these cells with propidium iodide confirmed that the decrease in cellular proliferation was due to apoptosis. The cytotoxic effect of plastic-immobilized K(b)-OVA could be mimicked by soluble K(b)-OVA tetramers. Furthermore, co-injection of K(b)-OVA tetramers and CD8-OVA cells into mice suppressed the tumorigenicity of CD8-OVA cells. In conclusion, we describe a system whereby soluble MHC class I molecules can be used to selectively induce cellular death in a monoclonal T-cell tumor model. With future development, the use of MHC molecules may help to eliminate specific T cells in cases of T-cell malignancy and auto-immunity.

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

Gastric cancers producing alpha-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.

Surgical stress induces decreased expression of signal-transducing zeta molecules in T cells.

Surgical stress is known to induce immunosuppression of T cell functions, but the mechanism behind this phenomenon is unclear. The purpose of this study was to determine whether surgical stress affects the expression of signal-transducing zeta molecules in peripheral T cells. In the present study, the expression of signal-transducing zeta molecules was studied by flow-cytometric analysis of permeabilized cells in peripheral blood mononuclear cells from 28 patients before and after surgery. The results demonstrate that T cell receptor (TCR) zeta levels in the peripheral T cells were lower on the 2nd or 3rd postoperative days (POD) compared to the preoperative period and recovered on the 7th POD in patients who received major surgery. Surface CD3 epsilon levels were also decreased after surgery but did not recover on the 7th POD. Culturing of T cells from the preoperative period with autologous monocytes from the 2nd POD induced a decreased expression of their TCR zeta. The reduction was prevented by the addition of catalase, a selective scavenger of hydrogen peroxide, indicating that this phenomenon was mediated by hydrogen peroxide. These results suggest that the decreased expression of TCR zeta molecules in peripheral T cells was induced by surgical stress, and was mediated by hydrogen peroxide derived from monocytes.

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-alpha 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) zeta, 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 zeta molecules.

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(9(106)), HER2(9(369)), HER2(9(689))], 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.

Expression of signal transducing T-cell receptor zeta 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 zeta molecules (TCRzeta) 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 zeta expression in peripheral T cells was measured by flow cytometric analysis of permeabilized cells with anti-zeta monoclonal antibody (MAb) (TIA-2) before and after AIT. We confirmed the down-regulation of TCR zeta 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 zeta expression in 2 of 14 treated patients, caused no significant change of TCR zeta 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 zeta expression. On the other hand, in the control group without adoptive transfer, further down-regulation of TCR zeta expression was observed during the corresponding periods, paralleling disease progression. Taken together, TCR zeta 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 zeta expression. The quantitative analysis of TCR zeta expression might provide vital information that can be used to optimize therapy by preserving immune functions within cancer patients.

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 (T(CD8+)) 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 T(CD8+) 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 T(CD8+) lines can be isolated, in a MHC class I-restricted manner, from solid tumors, metastatic lymph nodes and malignant ascites. T(CD8+) 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 T(CD8+) 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, T(CD8+) 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.