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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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Related in: MedlinePlus

Cytokine-primed NK cells undergo epigenetic remodeling of the IFNG CNS1.(A) Alignment of the human IFNG and mouse Ifng locus as presented by VISTA browser, DNA sequence identity >50% over at least 100 bp. Conserved regions with >70% sequence identity are marked in red and are depicted relative to the IFNG/Ifng transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the IFNG promoter and/or CNS1 was analyzed by determining CpG methylation of isolated DNA by bisulfite pyrosequencing. Five CpGs from −53 to +171 bp located in the IFNG promoter and six CpGs from −4399 to −4278 bp in the CNS1 region were analyzed and mean percentage of methylation at each individual CpG is depicted. (B and C) CpG methylation of naïve CD4+ T cells, TH1 cells and NK cells, FACS sorted ex vivo as described in Materials and Methods. One representative experiment out of two (T cells) or out of three (NK cells) is shown. (D) FACS sorted total NK cells were labeled with 500 nM CFSE and cultured in the presence of the indicated cytokines. After five days, viable CFSElo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the IFNG CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).
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ppat-1004441-g001: Cytokine-primed NK cells undergo epigenetic remodeling of the IFNG CNS1.(A) Alignment of the human IFNG and mouse Ifng locus as presented by VISTA browser, DNA sequence identity >50% over at least 100 bp. Conserved regions with >70% sequence identity are marked in red and are depicted relative to the IFNG/Ifng transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the IFNG promoter and/or CNS1 was analyzed by determining CpG methylation of isolated DNA by bisulfite pyrosequencing. Five CpGs from −53 to +171 bp located in the IFNG promoter and six CpGs from −4399 to −4278 bp in the CNS1 region were analyzed and mean percentage of methylation at each individual CpG is depicted. (B and C) CpG methylation of naïve CD4+ T cells, TH1 cells and NK cells, FACS sorted ex vivo as described in Materials and Methods. One representative experiment out of two (T cells) or out of three (NK cells) is shown. (D) FACS sorted total NK cells were labeled with 500 nM CFSE and cultured in the presence of the indicated cytokines. After five days, viable CFSElo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the IFNG CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).

Mentions: Among the described regulatory regions conserved between the human and mouse IFNG/Ifng locus (Figure 1A), we analyzed the methylation status of selected CpG residues present in the frame of the promoter and the CNS1, which are important enhancers of IFNG transcriptional activity and represent epigenetic hallmarks of TH1 differentiation [3], [4]. While the promoter was largely demethylated in NK cells and TH1 cells (Figure 1B), CNS1 was completely methylated in NK cells, resembling the configuration of naïve CD4+ T cells, rather than of TH1 cells (Figure 1C). Previous reports have described that after HCMV infection or priming with IL-15+IL-12+IL-18, NK cells can display memory-like properties and the ability to stably produce high amounts of IFN-γ after rechallenge [19], [30], [31]. Considering the parallels to memory TH1 cell formation, we analyzed the methylation status of the CNS1 in cytokine-primed NK cells and in HCMV-specific NK cell expansions. Cytokine-primed NK cells generated in vitro in the presence of IL-15+IL-12+IL-18 displayed complete demethylation of the CNS1. Conversely, only partial and progressive opening of both regions could be observed in the presence of IL-15+IL-12, IL-15+IL-18 or IL-15 alone. Substituting IL-18 by IL-1 almost led to similar results (Figure 1D). Next, we analyzed the methylation status of the IFNG CNS1 in NKG2C+ and NKG2C− NK cells isolated from HCMV− (Figure 2A) or HCMV+ (Figure 2B) individuals as well as in NKG2C+ NK cell expansions, selectively occurring in some HCMV+ individuals (Figure 2C). These NKG2C+ expansions are characterized by preferential expression of CD57 and strong enrichment in at least one sKIR (Figure 2C). As CD57+ NKG2C+ expansions display higher surface expression of NKG2C (Figure S1), they are further referred to as NKG2Chi NK cells [31], [44]. For a fair comparison of all NK cell subsets, CpG methylation analysis was performed after sorting CD56dim CD57+ NK cells. CD56dim CD57+ NK cells displayed an open configuration of the IFNG promoter independent of NKG2C expression or HCMV status (Figures 2A, 2B and 2C). NKG2C− NK cells derived from HCMV+ or HCMV− individuals as well as NKG2C+ NK cells from HCMV− individuals consistently displayed a closed configuration of the CNS1 (Figures 2A, 2B and 2C). Importantly, among HCMV+ individuals, an open configuration of the CNS1 occurred exclusively in donors displaying a distinctive expansion of NKG2Chi sKIR+ NK cells (Figure 2C). Conversely, if no expansion was present, NKG2C+ NK cells from HCMV+ individuals displayed a closed configuration of the CNS1, similar to the NKG2C− subsets (Figure 2B). Altogether, these data show that among NK cells only HCMV-specific NKG2Chi expansions and cytokine-primed NK cells, which were described as memory-like NK cells [15], [19], [30], [31], share the ability to undergo remodeling of the CNS1 with TH1 cells.


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)

Cytokine-primed NK cells undergo epigenetic remodeling of the IFNG CNS1.(A) Alignment of the human IFNG and mouse Ifng locus as presented by VISTA browser, DNA sequence identity >50% over at least 100 bp. Conserved regions with >70% sequence identity are marked in red and are depicted relative to the IFNG/Ifng transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the IFNG promoter and/or CNS1 was analyzed by determining CpG methylation of isolated DNA by bisulfite pyrosequencing. Five CpGs from −53 to +171 bp located in the IFNG promoter and six CpGs from −4399 to −4278 bp in the CNS1 region were analyzed and mean percentage of methylation at each individual CpG is depicted. (B and C) CpG methylation of naïve CD4+ T cells, TH1 cells and NK cells, FACS sorted ex vivo as described in Materials and Methods. One representative experiment out of two (T cells) or out of three (NK cells) is shown. (D) FACS sorted total NK cells were labeled with 500 nM CFSE and cultured in the presence of the indicated cytokines. After five days, viable CFSElo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the IFNG CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).
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Related In: Results  -  Collection

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ppat-1004441-g001: Cytokine-primed NK cells undergo epigenetic remodeling of the IFNG CNS1.(A) Alignment of the human IFNG and mouse Ifng locus as presented by VISTA browser, DNA sequence identity >50% over at least 100 bp. Conserved regions with >70% sequence identity are marked in red and are depicted relative to the IFNG/Ifng transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the IFNG promoter and/or CNS1 was analyzed by determining CpG methylation of isolated DNA by bisulfite pyrosequencing. Five CpGs from −53 to +171 bp located in the IFNG promoter and six CpGs from −4399 to −4278 bp in the CNS1 region were analyzed and mean percentage of methylation at each individual CpG is depicted. (B and C) CpG methylation of naïve CD4+ T cells, TH1 cells and NK cells, FACS sorted ex vivo as described in Materials and Methods. One representative experiment out of two (T cells) or out of three (NK cells) is shown. (D) FACS sorted total NK cells were labeled with 500 nM CFSE and cultured in the presence of the indicated cytokines. After five days, viable CFSElo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the IFNG CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).
Mentions: Among the described regulatory regions conserved between the human and mouse IFNG/Ifng locus (Figure 1A), we analyzed the methylation status of selected CpG residues present in the frame of the promoter and the CNS1, which are important enhancers of IFNG transcriptional activity and represent epigenetic hallmarks of TH1 differentiation [3], [4]. While the promoter was largely demethylated in NK cells and TH1 cells (Figure 1B), CNS1 was completely methylated in NK cells, resembling the configuration of naïve CD4+ T cells, rather than of TH1 cells (Figure 1C). Previous reports have described that after HCMV infection or priming with IL-15+IL-12+IL-18, NK cells can display memory-like properties and the ability to stably produce high amounts of IFN-γ after rechallenge [19], [30], [31]. Considering the parallels to memory TH1 cell formation, we analyzed the methylation status of the CNS1 in cytokine-primed NK cells and in HCMV-specific NK cell expansions. Cytokine-primed NK cells generated in vitro in the presence of IL-15+IL-12+IL-18 displayed complete demethylation of the CNS1. Conversely, only partial and progressive opening of both regions could be observed in the presence of IL-15+IL-12, IL-15+IL-18 or IL-15 alone. Substituting IL-18 by IL-1 almost led to similar results (Figure 1D). Next, we analyzed the methylation status of the IFNG CNS1 in NKG2C+ and NKG2C− NK cells isolated from HCMV− (Figure 2A) or HCMV+ (Figure 2B) individuals as well as in NKG2C+ NK cell expansions, selectively occurring in some HCMV+ individuals (Figure 2C). These NKG2C+ expansions are characterized by preferential expression of CD57 and strong enrichment in at least one sKIR (Figure 2C). As CD57+ NKG2C+ expansions display higher surface expression of NKG2C (Figure S1), they are further referred to as NKG2Chi NK cells [31], [44]. For a fair comparison of all NK cell subsets, CpG methylation analysis was performed after sorting CD56dim CD57+ NK cells. CD56dim CD57+ NK cells displayed an open configuration of the IFNG promoter independent of NKG2C expression or HCMV status (Figures 2A, 2B and 2C). NKG2C− NK cells derived from HCMV+ or HCMV− individuals as well as NKG2C+ NK cells from HCMV− individuals consistently displayed a closed configuration of the CNS1 (Figures 2A, 2B and 2C). Importantly, among HCMV+ individuals, an open configuration of the CNS1 occurred exclusively in donors displaying a distinctive expansion of NKG2Chi sKIR+ NK cells (Figure 2C). Conversely, if no expansion was present, NKG2C+ NK cells from HCMV+ individuals displayed a closed configuration of the CNS1, similar to the NKG2C− subsets (Figure 2B). Altogether, these data show that among NK cells only HCMV-specific NKG2Chi expansions and cytokine-primed NK cells, which were described as memory-like NK cells [15], [19], [30], [31], share the ability to undergo remodeling of the CNS1 with TH1 cells.

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