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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 cytokine combinations 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, autologous ascites fluid (top panel), or ascites fluid in the presence of IL-2+ IL-12 (middle panel), or IL-2+ IL-12+ IL-21 (bottom panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. 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 the cytokine combination for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05).
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pone-0015625-g002: Effects of cytokine combinations 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, autologous ascites fluid (top panel), or ascites fluid in the presence of IL-2+ IL-12 (middle panel), or IL-2+ IL-12+ IL-21 (bottom panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. 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 the cytokine combination for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05).

Mentions: Reasoning that a broader degree of functional enhancement might be achieved by simultaneously activating multiple STAT pathways, we assessed two combinations of cytokines: a) IL-2+ IL-12, and b) IL-2+ IL-12+ IL-21. Importantly, the latter combination would theoretically activate all STAT pathways relevant to CD8+ T-cell responses (i.e., STAT1, STAT3, STAT4, and STAT5). In general, both cytokine combinations induced T-cell proliferation similar to that seen with IL-2 (compare Fig. 2 with Fig. 1B). Notably however, CD8+ T cells from IROC028, which failed to proliferate in response to any single cytokine (Fig. 1B), proliferated in response to both cytokine combinations (Fig. 2). Moreover, both cytokine combinations were modestly more effective than single cytokines at enhancing IFN-γ and TNF-α production (Fig. 2). Despite this, neither cytokine combination enhanced expression of CD107a or IL-2 (Fig. 2). Similar results were seen with CD4+ T cells, although the effects of cytokines were generally weaker (data not shown). In summary, the addition of cytokine combinations engaging a wide range of STAT signaling pathways failed to fully restore T-cell function in the EOC ascites 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 cytokine combinations 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, autologous ascites fluid (top panel), or ascites fluid in the presence of IL-2+ IL-12 (middle panel), or IL-2+ IL-12+ IL-21 (bottom panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. 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 the cytokine combination for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008736&req=5

pone-0015625-g002: Effects of cytokine combinations 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, autologous ascites fluid (top panel), or ascites fluid in the presence of IL-2+ IL-12 (middle panel), or IL-2+ IL-12+ IL-21 (bottom panel) was assessed by measuring expression of Ki-67, CD107a, CCL4, IFN-γ, TNF-α, and IL-2 by flow cytometry. 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 the cytokine combination for enhancing the indicated function compared to cells stimulated in media (Wilcoxon matched pairs t test, p<0.05).
Mentions: Reasoning that a broader degree of functional enhancement might be achieved by simultaneously activating multiple STAT pathways, we assessed two combinations of cytokines: a) IL-2+ IL-12, and b) IL-2+ IL-12+ IL-21. Importantly, the latter combination would theoretically activate all STAT pathways relevant to CD8+ T-cell responses (i.e., STAT1, STAT3, STAT4, and STAT5). In general, both cytokine combinations induced T-cell proliferation similar to that seen with IL-2 (compare Fig. 2 with Fig. 1B). Notably however, CD8+ T cells from IROC028, which failed to proliferate in response to any single cytokine (Fig. 1B), proliferated in response to both cytokine combinations (Fig. 2). Moreover, both cytokine combinations were modestly more effective than single cytokines at enhancing IFN-γ and TNF-α production (Fig. 2). Despite this, neither cytokine combination enhanced expression of CD107a or IL-2 (Fig. 2). Similar results were seen with CD4+ T cells, although the effects of cytokines were generally weaker (data not shown). In summary, the addition of cytokine combinations engaging a wide range of STAT signaling pathways failed to fully restore T-cell function in the EOC ascites 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