Tumor-associated transforming growth factor-beta and interleukin-10 contribute to a systemic Th2 immune phenotype in pancreatic carcinoma patients

Abstract

In this study, we report coexpression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) in pancreatic carcinoma tissue associated with significantly elevated levels of both cytokines in the sera of pancreatic carcinoma patients. Using conditioned media (CM) of pancreatic carcinoma cells, we further demonstrate that tumor cell-derived TGF-beta and IL-10 inhibited in an additive fashion both proliferation and the development of Th1-like responses in peripheral blood mononuclear cell (PBMC) preparations derived from normal donors. The antiproliferative and Th1-suppressive activities contained in CM of pancreatic carcinoma cells were due primarily to IL-10 and/or TGF-beta, as shown by the capacity of cytokine-specific neutralizing antibodies to reverse these effects. Finally, as compared to normal controls, PBMC derived from pancreatic carcinoma patients displayed a Th2-like cytokine expression pattern upon activation with either anti-CD3 antibody or Staphylococcus aureus strain Cowan I. Taken together, these results suggest that aberrant production of TGF-beta and IL-10 in pancreatic tumor patients skews T-cell cytokine production patterns in favor of a Th2 immunophenotype.

Figure 1.

Elevated TGF-β1, TGF-β2, and IL-10 concentrations in the sera of pancreatic carcinoma patients (tumor) as compared to sera of donors free of inflammatory or neoplastic diseases (normal). Bars represent median values; as determined by Student’s t-test, TGF-β1, TGF-β2, and IL-10 levels were significantly higher (P < 0.05) in sera from pancreatic carcinoma patients as compared to normal controls.

Figure 2.

Expression of TGF-β1, -β2, -β3, and IL-10 transcripts in pancreatic carcinoma tissues (Lanes 2–8) and cell lines (Lane 10, BxPC3; Lane 11, Capan2; Lane 12, PT45) when compared to normal pancreatic tissue (Lane 1) . Ethidium bromide-stained RT-PCR products generated by using cytokine-specific primers and separated by agarose gel electrophoresis are shown. Lanes 9 and 13 show lack of amplification products in the absence of mRNA template. To control for RNA integrity and quantity, RT-PCR products generated by using β-actin primers are shown in the bottom panel. The specificity of TGF-β primers was assessed by diagnostic enzyme digestion of RT-PCR products.

Figure 3.

Representative immunohistochemical analysis of TGF-β1, -2, -3, and IL-10 expression in pancreatic carcinoma tissue. Antibodies reacting specifically with TGF-β1 (A), TGF-β2 (C), TGF-β3 (E), or IL-10 (G) were used. B, D, F, and H show negative controls stained with antibodies preincubated with saturating amounts of the appropriate recombinant proteins. Magnification, ×400.

Figure 4.

Inhibition of PBMC DNA synthesis by CM derived from pancreatic carcinoma cell lines Capan2, BxPC3, and PT45. The effects of 5× concentrated CM at different concentrations ranging from 5 to 20 vol% on [³H]TdR uptake of PBMCs derived from three normal donors are shown.

Figure 5.

Effects of neutralizing antibodies to IL-10 and TGF-β on the inhibition of PBMC proliferation caused by CM of pancreatic carcinoma cell lines Capan2, BxPC3, and PT45. The effects of anti-IL-10, anti-TGF-β, and anti-IL-10 plus anti-TGF-β are shown on anti-CD3-induced PBMC proliferation measured in the absence (control) and in the presence of 20 vol% CM derived from pancreatic carcinoma cell lines (all other conditions). Control cultures also received anti-IL-10 and anti-TGF-β antibodies. Column 1 (□) of all experimental groups shows PBMC proliferation in the presence of Capan2 CM; column 2 () shows proliferation in the presence of BxPC3 CM; and column 3 (▨) shows proliferation in the presence of PT45 CM. Each column shows the mean ± SD of three independent experiments using three different normal donors. Results are expressed as a percentage of [³H]TdR uptake relative to that of cells grown in the absence of either neutralizing antibodies or CM. No effect was observed in the presence of a nonimmune rabbit antiserum used as the control (data not shown). Asterisks indicate statistically significant differences (P < 0.05 in Student’s t-tests) of data sets when compared to controls in the presence of anti-IL-10 and anti-TGF-β neutralizing antibodies.

Figure 6.

Effects of neutralizing antibodies to IL-10 and TGF-β on the inhibition of IFN-γ (A) and IL-12p40 (B) production of normal PBMCs by pancreatic carcinoma CM. CMs were derived from Capan2, BxPC3, and PT45 cells, as indicated. Experimental conditions were as described in the legend to Figure 5▶ . No effect was observed in the presence of a nonimmune rabbit antiserum used as the control (data not shown). Each column shows the mean ± SD of three independent experiments using three different normal donors. Results are expressed as a percentage of cytokine production relative to that of cells grown in the absence of neutralizing antibodies (▨) or CM (▪).