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Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines.

Tran E, Nielsen JS, Wick DA, Ng AV, Johnson LD, Nesslinger NJ, McMurtrie E, Webb JR, Nelson BH - PLoS ONE (2010)

Bottom Line: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients.The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects.Our results may explain the limited efficacy of cytokine therapies for EOC to date.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Biochemistry, University of Victoria, Victoria, British Columbia, Canada.

ABSTRACT

Background: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/principal findings: Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/significance: The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.

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Effects of IL-18 alone, or in combination with IL-2 and IL-12, on CD8+ T-cell proliferation and function.Bulk ascites cells were stimulated with plate bound α-CD3 in media or 100% autologous ascites fluid for 48 h. Proliferation and function of CD8+ T cells in the presence of media or autologous ascites fluid (top panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. (A) Effect of IL-18 (middle panel) and IL-2+ IL-12+ IL-18 (bottom panel) on CD8+ T-cell proliferation and various functions in the presence of ascites fluid. Data has been normalized to α-CD3 stimulated cells in media (i.e., media values were subtracted from each stimulation condition). *There was a significant effect of IL-2+ IL-12+ IL-18 for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05). (B) Mean fluorescence intensity (MFI) of IFN-γ positive CD8+ T cells was determined by intracellular IFN-γ staining. *The effect of IL-2+ IL-12+ IL-18 was significantly greater than the other two cytokine combinations (Wilcoxon matched pairs t test, p<0.05). (C) Representative flow cytometry plots demonstrating the effect of cytokine combinations on IFN-γ production on a per-cell basis. Shown are the mean flourescence intensity (Y axis) of IFN-γ production and the percentage of CD8+ cells expressing IFN-γ (numbers). All data are gated on CD8+ lymphocytes.
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pone-0015625-g003: Effects of IL-18 alone, or in combination with IL-2 and IL-12, on CD8+ T-cell proliferation and function.Bulk ascites cells were stimulated with plate bound α-CD3 in media or 100% autologous ascites fluid for 48 h. Proliferation and function of CD8+ T cells in the presence of media or autologous ascites fluid (top panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. (A) Effect of IL-18 (middle panel) and IL-2+ IL-12+ IL-18 (bottom panel) on CD8+ T-cell proliferation and various functions in the presence of ascites fluid. Data has been normalized to α-CD3 stimulated cells in media (i.e., media values were subtracted from each stimulation condition). *There was a significant effect of IL-2+ IL-12+ IL-18 for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05). (B) Mean fluorescence intensity (MFI) of IFN-γ positive CD8+ T cells was determined by intracellular IFN-γ staining. *The effect of IL-2+ IL-12+ IL-18 was significantly greater than the other two cytokine combinations (Wilcoxon matched pairs t test, p<0.05). (C) Representative flow cytometry plots demonstrating the effect of cytokine combinations on IFN-γ production on a per-cell basis. Shown are the mean flourescence intensity (Y axis) of IFN-γ production and the percentage of CD8+ cells expressing IFN-γ (numbers). All data are gated on CD8+ lymphocytes.

Mentions: Finally, we assessed whether activation of non-STAT signaling pathways could further enhance T-cell function. To this end, we evaluated the cytokine IL-18, which belongs to the IL-1 cytokine family, activates the MyD88/NFκb pathway, and can directly enhance CD8+ T-cell responses in a variety of settings [39], [40], [41]. IL-18 alone had weak effects on T-cell proliferation and function (Fig. 3A). However, when combined with IL-2+ IL-12, IL-18 significantly enhanced expression of IFN-γ and, to a lesser extent, TNF-α (Fig. 3A). Moreover, the combination of IL-2+ IL-12+ IL-18 significantly increased the amount of IFN-γ produced on a per-cell basis, as evidenced by increased mean fluorescence intensity (MFI) (Fig. 3B, C). Nonetheless, the combination of IL-2+ IL-12+ IL-18 was unable to significantly enhance expression of CD107a or IL-2 (Fig. 3A) and only modestly enhanced granzyme B release (Supp. Fig. S2A). Likewise, this combination failed to restore expression of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, or bFGF in culture supernatants (Supp. Fig. S2B). This combination did, however, weakly increase the levels of IL-5 and IL-10 in culture supernatants (Supp. Fig. S2C). Thus, the combination of IL-2+ IL-12+ IL-18 only partially restores T-cell functionality in the EOC environment.


Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines.

Tran E, Nielsen JS, Wick DA, Ng AV, Johnson LD, Nesslinger NJ, McMurtrie E, Webb JR, Nelson BH - PLoS ONE (2010)

Effects of IL-18 alone, or in combination with IL-2 and IL-12, on CD8+ T-cell proliferation and function.Bulk ascites cells were stimulated with plate bound α-CD3 in media or 100% autologous ascites fluid for 48 h. Proliferation and function of CD8+ T cells in the presence of media or autologous ascites fluid (top panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. (A) Effect of IL-18 (middle panel) and IL-2+ IL-12+ IL-18 (bottom panel) on CD8+ T-cell proliferation and various functions in the presence of ascites fluid. Data has been normalized to α-CD3 stimulated cells in media (i.e., media values were subtracted from each stimulation condition). *There was a significant effect of IL-2+ IL-12+ IL-18 for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05). (B) Mean fluorescence intensity (MFI) of IFN-γ positive CD8+ T cells was determined by intracellular IFN-γ staining. *The effect of IL-2+ IL-12+ IL-18 was significantly greater than the other two cytokine combinations (Wilcoxon matched pairs t test, p<0.05). (C) Representative flow cytometry plots demonstrating the effect of cytokine combinations on IFN-γ production on a per-cell basis. Shown are the mean flourescence intensity (Y axis) of IFN-γ production and the percentage of CD8+ cells expressing IFN-γ (numbers). All data are gated on CD8+ lymphocytes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008736&req=5

pone-0015625-g003: Effects of IL-18 alone, or in combination with IL-2 and IL-12, on CD8+ T-cell proliferation and function.Bulk ascites cells were stimulated with plate bound α-CD3 in media or 100% autologous ascites fluid for 48 h. Proliferation and function of CD8+ T cells in the presence of media or autologous ascites fluid (top panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. (A) Effect of IL-18 (middle panel) and IL-2+ IL-12+ IL-18 (bottom panel) on CD8+ T-cell proliferation and various functions in the presence of ascites fluid. Data has been normalized to α-CD3 stimulated cells in media (i.e., media values were subtracted from each stimulation condition). *There was a significant effect of IL-2+ IL-12+ IL-18 for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05). (B) Mean fluorescence intensity (MFI) of IFN-γ positive CD8+ T cells was determined by intracellular IFN-γ staining. *The effect of IL-2+ IL-12+ IL-18 was significantly greater than the other two cytokine combinations (Wilcoxon matched pairs t test, p<0.05). (C) Representative flow cytometry plots demonstrating the effect of cytokine combinations on IFN-γ production on a per-cell basis. Shown are the mean flourescence intensity (Y axis) of IFN-γ production and the percentage of CD8+ cells expressing IFN-γ (numbers). All data are gated on CD8+ lymphocytes.
Mentions: Finally, we assessed whether activation of non-STAT signaling pathways could further enhance T-cell function. To this end, we evaluated the cytokine IL-18, which belongs to the IL-1 cytokine family, activates the MyD88/NFκb pathway, and can directly enhance CD8+ T-cell responses in a variety of settings [39], [40], [41]. IL-18 alone had weak effects on T-cell proliferation and function (Fig. 3A). However, when combined with IL-2+ IL-12, IL-18 significantly enhanced expression of IFN-γ and, to a lesser extent, TNF-α (Fig. 3A). Moreover, the combination of IL-2+ IL-12+ IL-18 significantly increased the amount of IFN-γ produced on a per-cell basis, as evidenced by increased mean fluorescence intensity (MFI) (Fig. 3B, C). Nonetheless, the combination of IL-2+ IL-12+ IL-18 was unable to significantly enhance expression of CD107a or IL-2 (Fig. 3A) and only modestly enhanced granzyme B release (Supp. Fig. S2A). Likewise, this combination failed to restore expression of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, or bFGF in culture supernatants (Supp. Fig. S2B). This combination did, however, weakly increase the levels of IL-5 and IL-10 in culture supernatants (Supp. Fig. S2C). Thus, the combination of IL-2+ IL-12+ IL-18 only partially restores T-cell functionality in the EOC environment.

Bottom Line: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients.The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects.Our results may explain the limited efficacy of cytokine therapies for EOC to date.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Biochemistry, University of Victoria, Victoria, British Columbia, Canada.

ABSTRACT

Background: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/principal findings: Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/significance: The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.

Show MeSH
Related in: MedlinePlus