Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients.

Impaired T-cell function in patients with advanced cancer has been a widely acknowledged finding, but mechanisms reported thus far are those primarily operating in the tumor microenvironment. Very few mechanisms have been put forth to explain several well-described defects in peripheral blood T cells, such as reduction in expression of signaling molecules, decreased production of cytokines, or increased apoptosis. We have closely examined the peripheral blood mononuclear cell (PBMC) samples derived from patients and healthy individuals, and we have observed an important difference that may underlie the majority of reported defects. We observed that in samples from patients only, an unusually large number of granulocytes copurify with low density PBMCs on a density gradient rather than sediment, as expected, to the bottom of the gradient. We also show that activating granulocytes from a healthy donor with N-formyl-L-methionyl-L-leucyl-L-phenylalanine could also cause them to sediment aberrantly and copurify with PBMCs, suggesting that density change is a marker of their activation. To confirm this, we looked for other evidence of in vivo granulocyte activation and found it in drastically elevated plasma levels of 8-isoprostane, a product of lipid peroxidation and a marker of oxidative stress. Reduced T-cell receptor zeta chain expression and decreased cytokine production by patients' T cells correlated with the presence of activated granulocytes in their PBMCs. We showed that freshly obtained granulocytes from healthy donors, if activated, can also inhibit cytokine production by T cells. This action is abrogated by the addition of the hydrogen peroxide (H(2)O(2)) scavenger, catalase, implicating H(2)O(2) as the effector molecule. Indeed, when added alone, H(2)O(2) could suppress cytokine production of normal T cells. These findings indicate that granulocytes are activated in advanced cancer patients and that granulocyte-derived H(2)O(2) is the major cause of severe systemic T-cell suppression.

Suppressed T-cell receptor zeta chain expression and cytokine production in pancreatic cancer patients.

Suppression of various functions of T cells derived from cancer patients has been linked previously to changes in the T-cell receptor (TCR)-associated signal transduction molecules, in particular the zeta chain of the TCR complex. In this study, we have examined the TCRzeta chain expression and cytokine production in vivo and in vitro in T cells of patients with metastatic adenocarcinomas of the pancreas that participated in a Phase I clinical trial of the MUC1 peptide plus bacillus Calmette-Guerin cancer vaccine. A majority of the patients had reduced TCRzeta chain expression and interleukin 4 production by T cells, and all of the patients showed decreased production of IFN-gamma of their peripheral T cells when compared with healthy individuals. Peripheral blood T cells were activated with the phorbol ester phorbol myrisate acetate and ionomycin to show that although aberrant TCRzeta chain expression and decreased cytokine production were often correlated, the reduced cytokine production was not simply a consequence of an impaired TCRzeta chain expression. Rather, these are two separate but parallel defects in signal transduction in T cells, which are potentially modulated by the same mechanisms. Half of the patients showed an improvement for TCRzeta chain or IFN-gamma expression after vaccination.

Tumor necrosis factor alpha induces the expression of transforming growth factor alpha and the epidermal growth factor receptor in human pancreatic cancer cells.

Recombinant human tumor necrosis factor (TNF)-alpha increased the expression of epidermal growth factor receptor (EGFR) mRNA and protein in all of six human pancreatic carcinoma cell lines tested. In addition, TNF-alpha increased the expression of an EGFR ligand, transforming growth factor (TGF)-alpha, at the mRNA and protein level in all cell lines. Increased expression of EGFR protein was associated with elevated steady-state EGFR mRNA levels. Nuclear run-on analysis showed that increase in EGFR mRNA was due to an increased rate of transcription. Induction of EGFR mRNA expression by TNF-alpha was abrogated by cycloheximide but occurred independently of TNF-alpha-induced production of TGF-alpha protein. Protein kinase A or Gi-type guanine nucleotide-binding proteins were not involved in this process as assessed by using appropriate stimulators and inhibitors of these signal transduction pathways. By contrast, staurosporine, an inhibitor of protein kinase C, partially inhibited, and 4-bromophenacyl bromide, a phospholipase inhibitor, completely inhibited TNF-alpha-dependent EGFR mRNA expression. The phospholipase C-specific inhibitor tricyclodecan-9-yl xanthogenate did not alter TNF-alpha-dependent EGFR mRNA expression, suggesting that phospholipase A2 is involved in the modulation of EGFR expression by TNF-alpha. The simultaneous induction of a ligand/receptor system by TNF-alpha suggests that this cytokine modulates autocrine growth-regulatory pathways in pancreatic cancer cells.

Cytokine-mediated enhancement of epidermal growth factor receptor expression provides an immunological approach to the therapy of pancreatic cancer.

The increased expression of epidermal growth factor receptor induced by tumor necrosis factor alpha renders pancreatic cancer cells more susceptible to antibody-dependent cellular cytotoxicity by a mAb specific for this receptor. Laboratory studies with athymic mice bearing xenografts of human pancreatic cancer cells demonstrated a cytokine-induced ability of the mAb to cause significant tumor regression. In a phase I/II clinical trial, 26 patients with unresectable pancreatic cancer were enrolled into three cohorts receiving variable amounts of the antibody together with a constant amount of tumor necrosis factor alpha. With increasing doses of antibody, the growth of the primary tumor was significantly inhibited. This was reflected by a longer median survival, with one complete remission lasting for 3 years obtained with the highest dose of antibody employed. Thus, a combination of the cytokine, tumor necrosis factor alpha, with a mAb to the epidermal growth factor receptor offers a potentially useful approach for the treatment of pancreatic cancer.