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The Nuclear Orphan Receptor NR2F6 Is a Central Checkpoint for Cancer Immune Surveillance.

Hermann-Kleiter N, Klepsch V, Wallner S, Siegmund K, Klepsch S, Tuzlak S, Villunger A, Kaminski S, Pfeifhofer-Obermair C, Gruber T, Wolf D, Baier G - Cell Rep (2015)

Bottom Line: Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is an orphan member of the nuclear receptor superfamily.Mechanistically, CD4(+) and CD8(+) T cell-intrinsic NR2F6 acts as a direct repressor of the NFAT/AP-1 complex on both the interleukin 2 and the interferon γ cytokine promoters, attenuating their transcriptional thresholds.Altogether, this defines NR2F6 as an intracellular immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity.

View Article: PubMed Central - PubMed

Affiliation: Translational Cell Genetics, Department for Pharmacology and Genetics, Medical University of Innsbruck, 6020 Innsbruck, Austria. Electronic address: natascha.kleiter@i-med.ac.at.

No MeSH data available.


Related in: MedlinePlus

Nr2f6 Suppresses Th1 CD4+ T Cell Activation(A) In vitro qRT-PCR analysis of Nr2f6 mRNA in wild-type CD4+ T cells during Th1 differentiation activated with anti-CD3 mAb (5 μg) and anti-CD28 mAb (1 μg) at the indicated time points (n = 3).(B) Bioplex technology was used to demonstrate significantly increased secretion of the pro-inflammatory cytokines IL-2 (p = 0.045), IFN-γ (p = 0.047), and TNF-α (p = 0.046) in the supernatant of in-vitro-activated Nr2f6−/− versus wild-type CD4+ T cells at day 1 and day 2 of differentiation under Th1-polarizing conditions (n = 3).(C) In vitro qRT-PCR analysis similarly detected enhanced transcript expression levels of Il2 (p = 0.003), Ifng (p = 0.044), Tnfa (p = 0.017), but not Tbx21 (p = 0.17) mRNA in Nr2f6−/− CD4+ Th1 cells compared to Nr2f6+/+ cells upon activation with anti-CD3 (5 μg) and anti-CD28 (1 μg) at the indicated time points (n = 3). Expression was normalized to the housekeeping gene GAPDH and presented as fold induction of unstimulated cells. Summary graphs represent the mean ± SD, data are representative for at least two independent experiments, and statistical differences were evaluated by applying two-way ANOVA.(D and E) (D) Analysis of IL-2 and IFN-γ producing CD4+ Th1 T Nr2f6+/+ or Nr2f6−/− cells by flow cytometry after 3 days (3d) of Th1 driving conditions and (E) followed by a restimulation with anti-CD3 (5 μg) overnight (d4/re). Numbers within outlined areas indicate the percentage of cytokine-expressing cells, and one out of three representative experiments is shown.
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fig6: Nr2f6 Suppresses Th1 CD4+ T Cell Activation(A) In vitro qRT-PCR analysis of Nr2f6 mRNA in wild-type CD4+ T cells during Th1 differentiation activated with anti-CD3 mAb (5 μg) and anti-CD28 mAb (1 μg) at the indicated time points (n = 3).(B) Bioplex technology was used to demonstrate significantly increased secretion of the pro-inflammatory cytokines IL-2 (p = 0.045), IFN-γ (p = 0.047), and TNF-α (p = 0.046) in the supernatant of in-vitro-activated Nr2f6−/− versus wild-type CD4+ T cells at day 1 and day 2 of differentiation under Th1-polarizing conditions (n = 3).(C) In vitro qRT-PCR analysis similarly detected enhanced transcript expression levels of Il2 (p = 0.003), Ifng (p = 0.044), Tnfa (p = 0.017), but not Tbx21 (p = 0.17) mRNA in Nr2f6−/− CD4+ Th1 cells compared to Nr2f6+/+ cells upon activation with anti-CD3 (5 μg) and anti-CD28 (1 μg) at the indicated time points (n = 3). Expression was normalized to the housekeeping gene GAPDH and presented as fold induction of unstimulated cells. Summary graphs represent the mean ± SD, data are representative for at least two independent experiments, and statistical differences were evaluated by applying two-way ANOVA.(D and E) (D) Analysis of IL-2 and IFN-γ producing CD4+ Th1 T Nr2f6+/+ or Nr2f6−/− cells by flow cytometry after 3 days (3d) of Th1 driving conditions and (E) followed by a restimulation with anti-CD3 (5 μg) overnight (d4/re). Numbers within outlined areas indicate the percentage of cytokine-expressing cells, and one out of three representative experiments is shown.

Mentions: Next, we investigate the underlying molecular mechanisms mediating enhanced anti-tumor immune reactivity in Nr2f6−/− mice and particularly increased cytokine production in Nr2f6−/− CD4+ T cells in vitro. Intriguingly, the established repressor of the antigen receptor activation threshold NR2F6 in Th17 cells (Hermann-Kleiter et al., 2008) is substantially upregulated upon in vitro CD3/CD28 stimulation in CD4+ T cells (Figure 6A), indicating a dynamic regulation of Nr2f6 expression as a potential negative feedback loop limiting CD4+ T cell activation. When culturing wild-type and Nr2f6-deficient naive CD4+ T cells under Th1 conditions, cytokine expression pattern analyses confirmed enhanced cytokine responses for IL-2, IFN-γ and TNF-α (Figures 6B–6E). Of note, in contrast to its negative regulatory role for effector T cell biology, NR2F6 is not required for CD4+ Treg cell function (Figures S6A–S6F).


The Nuclear Orphan Receptor NR2F6 Is a Central Checkpoint for Cancer Immune Surveillance.

Hermann-Kleiter N, Klepsch V, Wallner S, Siegmund K, Klepsch S, Tuzlak S, Villunger A, Kaminski S, Pfeifhofer-Obermair C, Gruber T, Wolf D, Baier G - Cell Rep (2015)

Nr2f6 Suppresses Th1 CD4+ T Cell Activation(A) In vitro qRT-PCR analysis of Nr2f6 mRNA in wild-type CD4+ T cells during Th1 differentiation activated with anti-CD3 mAb (5 μg) and anti-CD28 mAb (1 μg) at the indicated time points (n = 3).(B) Bioplex technology was used to demonstrate significantly increased secretion of the pro-inflammatory cytokines IL-2 (p = 0.045), IFN-γ (p = 0.047), and TNF-α (p = 0.046) in the supernatant of in-vitro-activated Nr2f6−/− versus wild-type CD4+ T cells at day 1 and day 2 of differentiation under Th1-polarizing conditions (n = 3).(C) In vitro qRT-PCR analysis similarly detected enhanced transcript expression levels of Il2 (p = 0.003), Ifng (p = 0.044), Tnfa (p = 0.017), but not Tbx21 (p = 0.17) mRNA in Nr2f6−/− CD4+ Th1 cells compared to Nr2f6+/+ cells upon activation with anti-CD3 (5 μg) and anti-CD28 (1 μg) at the indicated time points (n = 3). Expression was normalized to the housekeeping gene GAPDH and presented as fold induction of unstimulated cells. Summary graphs represent the mean ± SD, data are representative for at least two independent experiments, and statistical differences were evaluated by applying two-way ANOVA.(D and E) (D) Analysis of IL-2 and IFN-γ producing CD4+ Th1 T Nr2f6+/+ or Nr2f6−/− cells by flow cytometry after 3 days (3d) of Th1 driving conditions and (E) followed by a restimulation with anti-CD3 (5 μg) overnight (d4/re). Numbers within outlined areas indicate the percentage of cytokine-expressing cells, and one out of three representative experiments is shown.
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fig6: Nr2f6 Suppresses Th1 CD4+ T Cell Activation(A) In vitro qRT-PCR analysis of Nr2f6 mRNA in wild-type CD4+ T cells during Th1 differentiation activated with anti-CD3 mAb (5 μg) and anti-CD28 mAb (1 μg) at the indicated time points (n = 3).(B) Bioplex technology was used to demonstrate significantly increased secretion of the pro-inflammatory cytokines IL-2 (p = 0.045), IFN-γ (p = 0.047), and TNF-α (p = 0.046) in the supernatant of in-vitro-activated Nr2f6−/− versus wild-type CD4+ T cells at day 1 and day 2 of differentiation under Th1-polarizing conditions (n = 3).(C) In vitro qRT-PCR analysis similarly detected enhanced transcript expression levels of Il2 (p = 0.003), Ifng (p = 0.044), Tnfa (p = 0.017), but not Tbx21 (p = 0.17) mRNA in Nr2f6−/− CD4+ Th1 cells compared to Nr2f6+/+ cells upon activation with anti-CD3 (5 μg) and anti-CD28 (1 μg) at the indicated time points (n = 3). Expression was normalized to the housekeeping gene GAPDH and presented as fold induction of unstimulated cells. Summary graphs represent the mean ± SD, data are representative for at least two independent experiments, and statistical differences were evaluated by applying two-way ANOVA.(D and E) (D) Analysis of IL-2 and IFN-γ producing CD4+ Th1 T Nr2f6+/+ or Nr2f6−/− cells by flow cytometry after 3 days (3d) of Th1 driving conditions and (E) followed by a restimulation with anti-CD3 (5 μg) overnight (d4/re). Numbers within outlined areas indicate the percentage of cytokine-expressing cells, and one out of three representative experiments is shown.
Mentions: Next, we investigate the underlying molecular mechanisms mediating enhanced anti-tumor immune reactivity in Nr2f6−/− mice and particularly increased cytokine production in Nr2f6−/− CD4+ T cells in vitro. Intriguingly, the established repressor of the antigen receptor activation threshold NR2F6 in Th17 cells (Hermann-Kleiter et al., 2008) is substantially upregulated upon in vitro CD3/CD28 stimulation in CD4+ T cells (Figure 6A), indicating a dynamic regulation of Nr2f6 expression as a potential negative feedback loop limiting CD4+ T cell activation. When culturing wild-type and Nr2f6-deficient naive CD4+ T cells under Th1 conditions, cytokine expression pattern analyses confirmed enhanced cytokine responses for IL-2, IFN-γ and TNF-α (Figures 6B–6E). Of note, in contrast to its negative regulatory role for effector T cell biology, NR2F6 is not required for CD4+ Treg cell function (Figures S6A–S6F).

Bottom Line: Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is an orphan member of the nuclear receptor superfamily.Mechanistically, CD4(+) and CD8(+) T cell-intrinsic NR2F6 acts as a direct repressor of the NFAT/AP-1 complex on both the interleukin 2 and the interferon γ cytokine promoters, attenuating their transcriptional thresholds.Altogether, this defines NR2F6 as an intracellular immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity.

View Article: PubMed Central - PubMed

Affiliation: Translational Cell Genetics, Department for Pharmacology and Genetics, Medical University of Innsbruck, 6020 Innsbruck, Austria. Electronic address: natascha.kleiter@i-med.ac.at.

No MeSH data available.


Related in: MedlinePlus