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Human cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells.

Luetke-Eversloh M, Hammer Q, Durek P, Nordström K, Gasparoni G, Pink M, Hamann A, Walter J, Chang HD, Dong J, Romagnani C - PLoS Pathog. (2014)

Bottom Line: However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined.The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells.Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

View Article: PubMed Central - PubMed

Affiliation: Innate Immunity, Deutsches Rheuma-Forschungszentrum - A Leibniz Institute, Berlin, Germany.

ABSTRACT
Memory type 1 T helper (T(H)1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8(+) T cells or T(H)1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

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CNS1 accessibility regulates IFNG transcriptional activity induced by NKG2C engagement.(A) Surface expression of NKG2C and 2B4 on NKL (black line) or isotype control (solid grey histogram) was determined by FC. (B) Intracellular expression of IFN-γ by NKL was detected by FC after crosslinking of NKG2C and/or 2B4 for 16 hours. One representative experiment out of four is depicted. (C and D) Luciferase reporter assay of IFNG sequences transfected in NKL. (C) Construct containing the IFNG promoter (IFNGp) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (Luc). pGL3 reporter vectors were transfected into NKL cells along with Renilla reporter vector pRL-TK as internal control and luciferase activity was measured after stimulation of NKL, as indicated. Relative luciferase units (RLU) were calculated in relation to the activity of the IFNGp (−571 to +71) stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pGL3 activity. Mean RLU ± SEM (n = 3) are depicted. (D) Constructs containing the IFNGp (−49 to +71) region with or without the CNS1 were cloned into the CpG-free vector pCpGL. Luciferase activity of untreated (unmethylated, open circles) and of CpG-methyltransferase (M.SssI)-treated vectors (methylated, black circles) was measured after transfection into NKL cells. RLU were calculated relative to the activity displayed by the unmethylated CNS1+IFNGp (−49 to +71) vector stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pCpGL activity. One representative experiment out of three is depicted.
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ppat-1004441-g006: CNS1 accessibility regulates IFNG transcriptional activity induced by NKG2C engagement.(A) Surface expression of NKG2C and 2B4 on NKL (black line) or isotype control (solid grey histogram) was determined by FC. (B) Intracellular expression of IFN-γ by NKL was detected by FC after crosslinking of NKG2C and/or 2B4 for 16 hours. One representative experiment out of four is depicted. (C and D) Luciferase reporter assay of IFNG sequences transfected in NKL. (C) Construct containing the IFNG promoter (IFNGp) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (Luc). pGL3 reporter vectors were transfected into NKL cells along with Renilla reporter vector pRL-TK as internal control and luciferase activity was measured after stimulation of NKL, as indicated. Relative luciferase units (RLU) were calculated in relation to the activity of the IFNGp (−571 to +71) stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pGL3 activity. Mean RLU ± SEM (n = 3) are depicted. (D) Constructs containing the IFNGp (−49 to +71) region with or without the CNS1 were cloned into the CpG-free vector pCpGL. Luciferase activity of untreated (unmethylated, open circles) and of CpG-methyltransferase (M.SssI)-treated vectors (methylated, black circles) was measured after transfection into NKL cells. RLU were calculated relative to the activity displayed by the unmethylated CNS1+IFNGp (−49 to +71) vector stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pCpGL activity. One representative experiment out of three is depicted.

Mentions: Next, we aimed to understand whether CNS1, which was selectively demethylated in NKG2Chi NK cells, would be able to enhance IFNG transcriptional activity induced by engagement of NKG2C alone or in combination with 2B4. To perform luciferase reporter assays, we took advantage of the NK cell line NKL. NKL expressed 2B4 but only intermediate levels of NKG2C (Figure 6A), and only co-engagement of NKG2C and 2B4, but not of NKG2C alone, efficiently induced IFN-γ expression in NKL (Figure 6B). In line with these data, IFNG transcriptional activity in NKL, measured after transfection with the luciferase reporter vector pGL3 containing the IFNG promoter (IFNGp) (−571 to +71), could be induced only after co-engagement of 2B4 and NKG2C (Figure 6C). Next, we tested whether CNS1 was able to enhance IFNG transcriptional activity induced by the engagement of NKG2C and 2B4 and whether selective methylation would suppress it. To this aim, we cloned the minimal IFNGp (−49 to +71), not containing any CpG site, into the CpG-free luciferase reporter vector pCpGL [48], in combination or without the CNS1 sequence containing the CpG sites of interest. The construct was in vitro treated or not with CpG-methyltransferase (M.SssI), which selectively methylated the CNS1 CpG sites. CNS1 induced a dramatic enhancement of IFNG transcriptional activity in response to co-engagement of 2B4 and NKG2C, which was completely abolished by in vitro methylation of the CNS1 construct (Figure 6D). In further support of the concept that DNA methylation plays an important role in IFN-γ expression in NK cells as in TH1 cells, we treated NKL with the DNA methyltransferase inhibitor 5-azacytidine (AZA). Addition of AZA resulted in partial CNS1 demethylation and consistently enhanced expression of IFN-γ induced by NKG2C alone or by NKG2C and 2B4 co-stimulation (Figure S4A and S4B). Altogether, this data show that accessibility of CNS1 is crucial to enhance IFNG transcriptional activity in response to NKG2C engagement.


Human cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells.

Luetke-Eversloh M, Hammer Q, Durek P, Nordström K, Gasparoni G, Pink M, Hamann A, Walter J, Chang HD, Dong J, Romagnani C - PLoS Pathog. (2014)

CNS1 accessibility regulates IFNG transcriptional activity induced by NKG2C engagement.(A) Surface expression of NKG2C and 2B4 on NKL (black line) or isotype control (solid grey histogram) was determined by FC. (B) Intracellular expression of IFN-γ by NKL was detected by FC after crosslinking of NKG2C and/or 2B4 for 16 hours. One representative experiment out of four is depicted. (C and D) Luciferase reporter assay of IFNG sequences transfected in NKL. (C) Construct containing the IFNG promoter (IFNGp) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (Luc). pGL3 reporter vectors were transfected into NKL cells along with Renilla reporter vector pRL-TK as internal control and luciferase activity was measured after stimulation of NKL, as indicated. Relative luciferase units (RLU) were calculated in relation to the activity of the IFNGp (−571 to +71) stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pGL3 activity. Mean RLU ± SEM (n = 3) are depicted. (D) Constructs containing the IFNGp (−49 to +71) region with or without the CNS1 were cloned into the CpG-free vector pCpGL. Luciferase activity of untreated (unmethylated, open circles) and of CpG-methyltransferase (M.SssI)-treated vectors (methylated, black circles) was measured after transfection into NKL cells. RLU were calculated relative to the activity displayed by the unmethylated CNS1+IFNGp (−49 to +71) vector stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pCpGL activity. One representative experiment out of three is depicted.
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ppat-1004441-g006: CNS1 accessibility regulates IFNG transcriptional activity induced by NKG2C engagement.(A) Surface expression of NKG2C and 2B4 on NKL (black line) or isotype control (solid grey histogram) was determined by FC. (B) Intracellular expression of IFN-γ by NKL was detected by FC after crosslinking of NKG2C and/or 2B4 for 16 hours. One representative experiment out of four is depicted. (C and D) Luciferase reporter assay of IFNG sequences transfected in NKL. (C) Construct containing the IFNG promoter (IFNGp) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (Luc). pGL3 reporter vectors were transfected into NKL cells along with Renilla reporter vector pRL-TK as internal control and luciferase activity was measured after stimulation of NKL, as indicated. Relative luciferase units (RLU) were calculated in relation to the activity of the IFNGp (−571 to +71) stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pGL3 activity. Mean RLU ± SEM (n = 3) are depicted. (D) Constructs containing the IFNGp (−49 to +71) region with or without the CNS1 were cloned into the CpG-free vector pCpGL. Luciferase activity of untreated (unmethylated, open circles) and of CpG-methyltransferase (M.SssI)-treated vectors (methylated, black circles) was measured after transfection into NKL cells. RLU were calculated relative to the activity displayed by the unmethylated CNS1+IFNGp (−49 to +71) vector stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pCpGL activity. One representative experiment out of three is depicted.
Mentions: Next, we aimed to understand whether CNS1, which was selectively demethylated in NKG2Chi NK cells, would be able to enhance IFNG transcriptional activity induced by engagement of NKG2C alone or in combination with 2B4. To perform luciferase reporter assays, we took advantage of the NK cell line NKL. NKL expressed 2B4 but only intermediate levels of NKG2C (Figure 6A), and only co-engagement of NKG2C and 2B4, but not of NKG2C alone, efficiently induced IFN-γ expression in NKL (Figure 6B). In line with these data, IFNG transcriptional activity in NKL, measured after transfection with the luciferase reporter vector pGL3 containing the IFNG promoter (IFNGp) (−571 to +71), could be induced only after co-engagement of 2B4 and NKG2C (Figure 6C). Next, we tested whether CNS1 was able to enhance IFNG transcriptional activity induced by the engagement of NKG2C and 2B4 and whether selective methylation would suppress it. To this aim, we cloned the minimal IFNGp (−49 to +71), not containing any CpG site, into the CpG-free luciferase reporter vector pCpGL [48], in combination or without the CNS1 sequence containing the CpG sites of interest. The construct was in vitro treated or not with CpG-methyltransferase (M.SssI), which selectively methylated the CNS1 CpG sites. CNS1 induced a dramatic enhancement of IFNG transcriptional activity in response to co-engagement of 2B4 and NKG2C, which was completely abolished by in vitro methylation of the CNS1 construct (Figure 6D). In further support of the concept that DNA methylation plays an important role in IFN-γ expression in NK cells as in TH1 cells, we treated NKL with the DNA methyltransferase inhibitor 5-azacytidine (AZA). Addition of AZA resulted in partial CNS1 demethylation and consistently enhanced expression of IFN-γ induced by NKG2C alone or by NKG2C and 2B4 co-stimulation (Figure S4A and S4B). Altogether, this data show that accessibility of CNS1 is crucial to enhance IFNG transcriptional activity in response to NKG2C engagement.

Bottom Line: However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined.The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells.Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

View Article: PubMed Central - PubMed

Affiliation: Innate Immunity, Deutsches Rheuma-Forschungszentrum - A Leibniz Institute, Berlin, Germany.

ABSTRACT
Memory type 1 T helper (T(H)1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8(+) T cells or T(H)1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

Show MeSH
Related in: MedlinePlus