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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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Combined treatment with IL-12 and IL-27 modulates polarization of Th2 and enhances IFN-γ production by pre-existing Th1 anti-CEA CD4+ T cells.CD4+ T cells from pt#43 and ND#11 were cultured in five replicates, as described in Materials and Methods, with the responsive CEA peptides, in the absence (grey bars) or in the presence (black bars) of the combined treatment with IL-12 (5 ng/ml) plus IL-27 (100 ng/ml). After 14 days, IL-5, IL-13, GM-CSF and IFN-γ release was tested by ELISA. Data reported are means of duplicate determination±SD. Dashed lines identify the basal level of cytokine secretion in the presence of APC only. n.d. = not determined.
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pone-0007234-g005: Combined treatment with IL-12 and IL-27 modulates polarization of Th2 and enhances IFN-γ production by pre-existing Th1 anti-CEA CD4+ T cells.CD4+ T cells from pt#43 and ND#11 were cultured in five replicates, as described in Materials and Methods, with the responsive CEA peptides, in the absence (grey bars) or in the presence (black bars) of the combined treatment with IL-12 (5 ng/ml) plus IL-27 (100 ng/ml). After 14 days, IL-5, IL-13, GM-CSF and IFN-γ release was tested by ELISA. Data reported are means of duplicate determination±SD. Dashed lines identify the basal level of cytokine secretion in the presence of APC only. n.d. = not determined.

Mentions: To verify the effect of combined IL-12 and IL-27 treatment on the polarization of CEA-specific CD4+ T cells in a more physiologic environment and at polyclonal level, CD4+ T cells from patient (pt#43) and normal donor (ND#11) were tested in a 14-days in vitro re-stimulation assay for the recognition of the relevant peptides in the absence or in the presence of the two immunomodulatory cytokines (Figure 5). As previously reported in [3], in the absence of the immunomodulatory cytokines CD4+ T cells from pt#43 produced IL-5 in the presence of CEA425–437; IL-13 in the presence of CEA568–582; GM-CSF in the presence of CEA568–582 and IFN-γ in the presence of CEA568–582 and, although at a much lower but significant level, of CEA177–189/355–367 (Fig. 5, left panels, grey bars). The combination of IL-12 and IL-27 almost abolished IL-5 (99% inhibition) and IL-13 (76% inhibition) while induced de novo IFN-γ secretion in the presence of CEA425–437, enhanced (9-fold increase) IFN-γ production in the presence of CEA177–189/355–367 and confirmed IFN-γ while strongly reduced IL-13 (98% inhibition) and GM-CSF (80% inhibition) production in the presence of CEA568–582 (Fig. 5, left panels, black bars). As previously reported [3], in the absence of the immunomodulatory cytokines CD4+ T cells from ND#11 showed significant proliferation [3] and very little amount of IFN-γ secretion in the presence of CEA99–111, while no significant production of IL-5 and GM-CSF was observed in the presence of any peptide (Fig. 5, right panels, grey bars). IL-13 release was not tested. Cytokine treatment strongly augmented (23-fold increase) IFN-γ release against CEA99–111, while, as expected, no effect on IL-5 and GM-CSF production was observed (Fig. 5, right panels, black bars). In both cases, the two cytokines induced modulation of Th2 or amplification of Th1 responses that were already present, although at very low level, in the absence of the cytokines combination, while there was no evidence of de-novo specific recognition of CEA peptides.


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)

Combined treatment with IL-12 and IL-27 modulates polarization of Th2 and enhances IFN-γ production by pre-existing Th1 anti-CEA CD4+ T cells.CD4+ T cells from pt#43 and ND#11 were cultured in five replicates, as described in Materials and Methods, with the responsive CEA peptides, in the absence (grey bars) or in the presence (black bars) of the combined treatment with IL-12 (5 ng/ml) plus IL-27 (100 ng/ml). After 14 days, IL-5, IL-13, GM-CSF and IFN-γ release was tested by ELISA. Data reported are means of duplicate determination±SD. Dashed lines identify the basal level of cytokine secretion in the presence of APC only. n.d. = not determined.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749205&req=5

pone-0007234-g005: Combined treatment with IL-12 and IL-27 modulates polarization of Th2 and enhances IFN-γ production by pre-existing Th1 anti-CEA CD4+ T cells.CD4+ T cells from pt#43 and ND#11 were cultured in five replicates, as described in Materials and Methods, with the responsive CEA peptides, in the absence (grey bars) or in the presence (black bars) of the combined treatment with IL-12 (5 ng/ml) plus IL-27 (100 ng/ml). After 14 days, IL-5, IL-13, GM-CSF and IFN-γ release was tested by ELISA. Data reported are means of duplicate determination±SD. Dashed lines identify the basal level of cytokine secretion in the presence of APC only. n.d. = not determined.
Mentions: To verify the effect of combined IL-12 and IL-27 treatment on the polarization of CEA-specific CD4+ T cells in a more physiologic environment and at polyclonal level, CD4+ T cells from patient (pt#43) and normal donor (ND#11) were tested in a 14-days in vitro re-stimulation assay for the recognition of the relevant peptides in the absence or in the presence of the two immunomodulatory cytokines (Figure 5). As previously reported in [3], in the absence of the immunomodulatory cytokines CD4+ T cells from pt#43 produced IL-5 in the presence of CEA425–437; IL-13 in the presence of CEA568–582; GM-CSF in the presence of CEA568–582 and IFN-γ in the presence of CEA568–582 and, although at a much lower but significant level, of CEA177–189/355–367 (Fig. 5, left panels, grey bars). The combination of IL-12 and IL-27 almost abolished IL-5 (99% inhibition) and IL-13 (76% inhibition) while induced de novo IFN-γ secretion in the presence of CEA425–437, enhanced (9-fold increase) IFN-γ production in the presence of CEA177–189/355–367 and confirmed IFN-γ while strongly reduced IL-13 (98% inhibition) and GM-CSF (80% inhibition) production in the presence of CEA568–582 (Fig. 5, left panels, black bars). As previously reported [3], in the absence of the immunomodulatory cytokines CD4+ T cells from ND#11 showed significant proliferation [3] and very little amount of IFN-γ secretion in the presence of CEA99–111, while no significant production of IL-5 and GM-CSF was observed in the presence of any peptide (Fig. 5, right panels, grey bars). IL-13 release was not tested. Cytokine treatment strongly augmented (23-fold increase) IFN-γ release against CEA99–111, while, as expected, no effect on IL-5 and GM-CSF production was observed (Fig. 5, right panels, black bars). In both cases, the two cytokines induced modulation of Th2 or amplification of Th1 responses that were already present, although at very low level, in the absence of the cytokines combination, while there was no evidence of de-novo specific recognition of CEA peptides.

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