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Non-redundant role for IL-12 and IL-27 in modulating Th2 polarization of carcinoembryonic antigen specific CD4 T cells from pancreatic cancer patients.

Tassi E, Braga M, Longhi R, Gavazzi F, Parmiani G, Di Carlo V, Protti MP - PLoS ONE (2009)

Bottom Line: This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis.We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal.In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type immunity.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology Unit, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT

Background: Pancreatic cancer is a very aggressive disease with dismal prognosis; peculiar is the tumor microenvironment characterized by an extensive fibrotic stroma, which favors rapid tumor progression. We previously reported that pancreatic cancer patients have a selective Th2 skew in the anti-carcinoembryonic antigen (CEA) CD4(+) T cell immunity, which correlates with the presence of a predominant GATA-3(+) tumor lymphoid infiltrate. This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis. Aim of this study was to evaluate whether the Th2 polarization of CEA-specific CD4(+) T cells from pancreatic cancer patients is stable or can be reverted by immunomodulating cytokines.

Methodology/principal findings: We first evaluated the influence of IL-12 and IL-27, as single agents and in association, on the polarization of CEA-specific Th2 CD4(+) T cell clones from a pancreatic cancer patient. We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal. Second, we evaluated the effect of the combined treatment on polyclonal CEA-specific CD4(+) T cells in short-time re-stimulation assays. In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type immunity.

Conclusions/significance: Collectively, our results demonstrate that tumor antigen specific Th2 CD4(+) T cells in pancreatic cancer are endowed with functional plasticity. Hence, loco-regional cytokines delivery or targeted therapy based on antibodies or molecules directed to the tumor stroma might improve anti-tumor immunity and ameliorate fibrosis, without systemic toxicity.

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Related in: MedlinePlus

Effects of combined IL-12 and IL-27 given as a single shot or continuously in culture.(A). CD4+ T cells were cultured with the relevant peptide and either left untreated (peptide) or treated for 2 days with combined IL-12 (5 ng/ml) and IL-27 (100 ng/ml) and then washed and left untreated (IL-12+IL-27 (2-days)) or treated continuously with IL-12 and IL-27 at the same doses for 2 weeks (IL-12+IL-27 (14 days)). At day 14, cells were tested in the presence or the absence of the relevant peptide and the specific IL-5, IL-13 and IFN-γ release in the supernatant tested by ELISA. The data are means of duplicate determination±SD. The basal level of cytokines secretion of CD4+ T cells in the presence of LCL only was subtracted from the sample values and was as follows: IL-5 (0,093±0,006 ng/ml), IL-13 (0,468±0,028 ng/ml) and IFN-γ (0 ng/ml). The data are representative of four experiments. (B). Surface expression of CRTH2, CCR4 CCR5 by CD4+ T cells after treatment with combined IL-12+IL-27. Analysis was performed on cells treated for 2 days and then left untreated for a further week. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.
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pone-0007234-g004: Effects of combined IL-12 and IL-27 given as a single shot or continuously in culture.(A). CD4+ T cells were cultured with the relevant peptide and either left untreated (peptide) or treated for 2 days with combined IL-12 (5 ng/ml) and IL-27 (100 ng/ml) and then washed and left untreated (IL-12+IL-27 (2-days)) or treated continuously with IL-12 and IL-27 at the same doses for 2 weeks (IL-12+IL-27 (14 days)). At day 14, cells were tested in the presence or the absence of the relevant peptide and the specific IL-5, IL-13 and IFN-γ release in the supernatant tested by ELISA. The data are means of duplicate determination±SD. The basal level of cytokines secretion of CD4+ T cells in the presence of LCL only was subtracted from the sample values and was as follows: IL-5 (0,093±0,006 ng/ml), IL-13 (0,468±0,028 ng/ml) and IFN-γ (0 ng/ml). The data are representative of four experiments. (B). Surface expression of CRTH2, CCR4 CCR5 by CD4+ T cells after treatment with combined IL-12+IL-27. Analysis was performed on cells treated for 2 days and then left untreated for a further week. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.

Mentions: To verify whether the modulation of Th2 polarization obtained by the combined treatment with IL-12 and IL-27 was stable, we designed an experiment in which CEA-specific CD4+ T cells were first stimulated with the relevant peptide in the absence or in the presence of IL-12 and IL-27 (5 and 100 ng/ml, respectively). Second, cells stimulated in the absence of the cytokines were kept in culture for 14 days in TCM plus IL-2 and used as controls. Cells treated with the cytokines were divided in two aliquots and cultured under different conditions for the following 14 days: in one condition the cytokines were removed after 2 days and the cells cultured as the controls in TCM plus IL-2, in the other condition cytokines were kept in culture all the time and replaced every two to three days. At day 14, cells from each of the three conditions were tested in a 2-day stimulation assay with the relevant peptide and cytokine release tested. The results obtained are shown in Figure 4. Control cells confirmed production of IL-5, IL-13 and no IFN-γ, while cells kept in culture continuously with the cytokines confirmed 98% and 74% inhibition of IL-5 and IL-13 production, respectively, and a 11-fold increase for IFN-γ. Importantly, cells in which cytokines were removed after 2 days of culture still showed 70% and 47% inhibition in IL-5 and IL-13 production, respectively, and a 5-fold increase in IFN-γ release.


Non-redundant role for IL-12 and IL-27 in modulating Th2 polarization of carcinoembryonic antigen specific CD4 T cells from pancreatic cancer patients.

Tassi E, Braga M, Longhi R, Gavazzi F, Parmiani G, Di Carlo V, Protti MP - PLoS ONE (2009)

Effects of combined IL-12 and IL-27 given as a single shot or continuously in culture.(A). CD4+ T cells were cultured with the relevant peptide and either left untreated (peptide) or treated for 2 days with combined IL-12 (5 ng/ml) and IL-27 (100 ng/ml) and then washed and left untreated (IL-12+IL-27 (2-days)) or treated continuously with IL-12 and IL-27 at the same doses for 2 weeks (IL-12+IL-27 (14 days)). At day 14, cells were tested in the presence or the absence of the relevant peptide and the specific IL-5, IL-13 and IFN-γ release in the supernatant tested by ELISA. The data are means of duplicate determination±SD. The basal level of cytokines secretion of CD4+ T cells in the presence of LCL only was subtracted from the sample values and was as follows: IL-5 (0,093±0,006 ng/ml), IL-13 (0,468±0,028 ng/ml) and IFN-γ (0 ng/ml). The data are representative of four experiments. (B). Surface expression of CRTH2, CCR4 CCR5 by CD4+ T cells after treatment with combined IL-12+IL-27. Analysis was performed on cells treated for 2 days and then left untreated for a further week. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749205&req=5

pone-0007234-g004: Effects of combined IL-12 and IL-27 given as a single shot or continuously in culture.(A). CD4+ T cells were cultured with the relevant peptide and either left untreated (peptide) or treated for 2 days with combined IL-12 (5 ng/ml) and IL-27 (100 ng/ml) and then washed and left untreated (IL-12+IL-27 (2-days)) or treated continuously with IL-12 and IL-27 at the same doses for 2 weeks (IL-12+IL-27 (14 days)). At day 14, cells were tested in the presence or the absence of the relevant peptide and the specific IL-5, IL-13 and IFN-γ release in the supernatant tested by ELISA. The data are means of duplicate determination±SD. The basal level of cytokines secretion of CD4+ T cells in the presence of LCL only was subtracted from the sample values and was as follows: IL-5 (0,093±0,006 ng/ml), IL-13 (0,468±0,028 ng/ml) and IFN-γ (0 ng/ml). The data are representative of four experiments. (B). Surface expression of CRTH2, CCR4 CCR5 by CD4+ T cells after treatment with combined IL-12+IL-27. Analysis was performed on cells treated for 2 days and then left untreated for a further week. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.
Mentions: To verify whether the modulation of Th2 polarization obtained by the combined treatment with IL-12 and IL-27 was stable, we designed an experiment in which CEA-specific CD4+ T cells were first stimulated with the relevant peptide in the absence or in the presence of IL-12 and IL-27 (5 and 100 ng/ml, respectively). Second, cells stimulated in the absence of the cytokines were kept in culture for 14 days in TCM plus IL-2 and used as controls. Cells treated with the cytokines were divided in two aliquots and cultured under different conditions for the following 14 days: in one condition the cytokines were removed after 2 days and the cells cultured as the controls in TCM plus IL-2, in the other condition cytokines were kept in culture all the time and replaced every two to three days. At day 14, cells from each of the three conditions were tested in a 2-day stimulation assay with the relevant peptide and cytokine release tested. The results obtained are shown in Figure 4. Control cells confirmed production of IL-5, IL-13 and no IFN-γ, while cells kept in culture continuously with the cytokines confirmed 98% and 74% inhibition of IL-5 and IL-13 production, respectively, and a 11-fold increase for IFN-γ. Importantly, cells in which cytokines were removed after 2 days of culture still showed 70% and 47% inhibition in IL-5 and IL-13 production, respectively, and a 5-fold increase in IFN-γ release.

Bottom Line: This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis.We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal.In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type immunity.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology Unit, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT

Background: Pancreatic cancer is a very aggressive disease with dismal prognosis; peculiar is the tumor microenvironment characterized by an extensive fibrotic stroma, which favors rapid tumor progression. We previously reported that pancreatic cancer patients have a selective Th2 skew in the anti-carcinoembryonic antigen (CEA) CD4(+) T cell immunity, which correlates with the presence of a predominant GATA-3(+) tumor lymphoid infiltrate. This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis. Aim of this study was to evaluate whether the Th2 polarization of CEA-specific CD4(+) T cells from pancreatic cancer patients is stable or can be reverted by immunomodulating cytokines.

Methodology/principal findings: We first evaluated the influence of IL-12 and IL-27, as single agents and in association, on the polarization of CEA-specific Th2 CD4(+) T cell clones from a pancreatic cancer patient. We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal. Second, we evaluated the effect of the combined treatment on polyclonal CEA-specific CD4(+) T cells in short-time re-stimulation assays. In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type immunity.

Conclusions/significance: Collectively, our results demonstrate that tumor antigen specific Th2 CD4(+) T cells in pancreatic cancer are endowed with functional plasticity. Hence, loco-regional cytokines delivery or targeted therapy based on antibodies or molecules directed to the tumor stroma might improve anti-tumor immunity and ameliorate fibrosis, without systemic toxicity.

Show MeSH
Related in: MedlinePlus