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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

RRBS-based global methylation analysis of NK cell and T cell subsets.Genomic DNA of ex vivo FACS sorted NK and T cell subsets was analyzed for CpG methylation by RRBS. (A–C) Beta-values for each methylation site identified by RRBS were averaged from two donors for each T or NK cell subset as indicated. Sites with coverages below 5 were removed from further analysis. (A) Clustering analysis of differentially methylated CpG sites found by RRBS. Clustering was performed on 1,000 most variant methylation sites out of 30,000 randomly chosen sites, localized within gene bodies and promoter regions. The indicated cell subsets cluster according to the similarities of their methylation profile. Beta-values depicted from dark-red to yellow represent level of methylation of the individual CpG sites. (B) Similarities between the cell subsets as judged by the Euclidian distance. (C) Principle component analysis (PCA) of mean methylation levels from two donors for indicated cell subsets. The PCA revealed a clear separation of CD4+ TH1 and CD8+ memory cells, naïve CD4+ and CD8+ cells, and NK cells by the first two components. (D) CpG methylation sites of T and NK cell subsets of one representative donor identified by RRBS in gene bodies of TXB21, EOMES, PRF1, TNF, PRDM1 and ZBTB32. Line diagram represents sequence conservation within vertebrates. Blue and violet bars indicate the methylation level (0–100%) and coverage for each identification, respectively. Coverages of 5 and more reads are represented as a full violet bar.
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ppat-1004441-g004: RRBS-based global methylation analysis of NK cell and T cell subsets.Genomic DNA of ex vivo FACS sorted NK and T cell subsets was analyzed for CpG methylation by RRBS. (A–C) Beta-values for each methylation site identified by RRBS were averaged from two donors for each T or NK cell subset as indicated. Sites with coverages below 5 were removed from further analysis. (A) Clustering analysis of differentially methylated CpG sites found by RRBS. Clustering was performed on 1,000 most variant methylation sites out of 30,000 randomly chosen sites, localized within gene bodies and promoter regions. The indicated cell subsets cluster according to the similarities of their methylation profile. Beta-values depicted from dark-red to yellow represent level of methylation of the individual CpG sites. (B) Similarities between the cell subsets as judged by the Euclidian distance. (C) Principle component analysis (PCA) of mean methylation levels from two donors for indicated cell subsets. The PCA revealed a clear separation of CD4+ TH1 and CD8+ memory cells, naïve CD4+ and CD8+ cells, and NK cells by the first two components. (D) CpG methylation sites of T and NK cell subsets of one representative donor identified by RRBS in gene bodies of TXB21, EOMES, PRF1, TNF, PRDM1 and ZBTB32. Line diagram represents sequence conservation within vertebrates. Blue and violet bars indicate the methylation level (0–100%) and coverage for each identification, respectively. Coverages of 5 and more reads are represented as a full violet bar.

Mentions: In order to understand whether IFNG CNS1 demethylation was part of a broader epigenetic remodeling occurring in NKG2Chi expansions from HCMV+ individuals, we performed RRBS-based global methylation analysis of CD57+ NKG2Chi, CD57+ NKG2C− and CD57− NKG2C− NK cells in comparison with CD8+ memory and CD4+ TH1 cells as well as with CD8+ or CD4+ naïve T cells isolated from the same HCMV+ individuals. CD8+ memory and CD4+ TH1 cells, CD8+ or CD4+ naïve T cells, and NK cells constitute three different clusters. Interestingly, NKG2Chi NK cells distinctively shared the methylation profile of a large set of CpG sites with CD8+ memory or CD4+ TH1 cells. Conversely, CD57− NKG2C− NK cells were more similar to naïve CD8+ or CD4+ T cells, with CD57+ NKG2C− NK cells displaying a methylation profile intermediate to the two other NK cell subsets (Figure 4A). The NK cell subsets show a decreasing gradient of similarities to naïve CD8+ and CD4+ T cells from CD57− NKG2C− via CD57+ NKG2C− to CD57+ NKG2Chi, as judged by the Euclidian distance, and a complementary, increasing gradient of similarities to CD8+ memory and CD4+ TH1 cells (Figure 4B). This observation was further confirmed by employing an unsupervised strategy and performing principal component analysis (PCA) (Figure 4C). Most of the variation among the different lymphocyte subsets was captured by the first principal component (PC1). CD8+ and CD4+ naïve T cells were at one extreme, while CD8+ memory together with TH1 cells were at the other extreme of the PC1-axis, suggesting that PC1 defined the direction of lymphocyte differentiation, which was characterized by a global epigenetic remodeling (Figure 4B). NK cell subsets distributed along the PC1, with CD57− NKG2C− cells displaying the highest PC1 score, similar to naïve T cells, while CD57+ NKG2Chi NK cells displayed the lowest PC1 score. T cell and NK cell lineage-defining epigenetic modifications were likely described by the second principal component (PC2). Analyzing the methylation pattern of other genes of interest, which are shared between NK cells and TH1 or CD8+ memory T cells, such as TBX21 (T-bet), EOMES (Eomesodermin), and PRF1 (perforin), we observed that all NK cell subsets displayed similar or even more pronounced demethylation pattern at several CpG regions compared to TH1 or CD8+ memory T cells (Figure 4D). TNF was mainly demethylated in both CD57+ NKG2Chi and CD57+ NKG2C− NK cell subsets, similar to TH1 or CD8+ memory T cells. Recently, it was shown that PRDM1 (Blimp-1) and ZBTB32 (also known as PLZP, ROG, FAZF and TZFP), which are not expressed in naïve T cells but are up-regulated in differentiated T cells [45]–[47], regulate the proliferative burst of Ly49H+ memory NK cells during MCMV infection [24]. Interestingly, some CpGs of the PRDM1 and ZBTB32 genes were consistently demethylated in NKG2Chi NK cells and TH1 or memory CD8+ T cells, compared to other NK cell subsets or naïve T cells. Altogether, these data suggest that CNS1 demethylation in NKG2Chi expansions from HCMV+ individuals is part of a broader epigenetic remodeling, which is partially shared by TH1 and memory CD8+ T 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)

RRBS-based global methylation analysis of NK cell and T cell subsets.Genomic DNA of ex vivo FACS sorted NK and T cell subsets was analyzed for CpG methylation by RRBS. (A–C) Beta-values for each methylation site identified by RRBS were averaged from two donors for each T or NK cell subset as indicated. Sites with coverages below 5 were removed from further analysis. (A) Clustering analysis of differentially methylated CpG sites found by RRBS. Clustering was performed on 1,000 most variant methylation sites out of 30,000 randomly chosen sites, localized within gene bodies and promoter regions. The indicated cell subsets cluster according to the similarities of their methylation profile. Beta-values depicted from dark-red to yellow represent level of methylation of the individual CpG sites. (B) Similarities between the cell subsets as judged by the Euclidian distance. (C) Principle component analysis (PCA) of mean methylation levels from two donors for indicated cell subsets. The PCA revealed a clear separation of CD4+ TH1 and CD8+ memory cells, naïve CD4+ and CD8+ cells, and NK cells by the first two components. (D) CpG methylation sites of T and NK cell subsets of one representative donor identified by RRBS in gene bodies of TXB21, EOMES, PRF1, TNF, PRDM1 and ZBTB32. Line diagram represents sequence conservation within vertebrates. Blue and violet bars indicate the methylation level (0–100%) and coverage for each identification, respectively. Coverages of 5 and more reads are represented as a full violet bar.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199780&req=5

ppat-1004441-g004: RRBS-based global methylation analysis of NK cell and T cell subsets.Genomic DNA of ex vivo FACS sorted NK and T cell subsets was analyzed for CpG methylation by RRBS. (A–C) Beta-values for each methylation site identified by RRBS were averaged from two donors for each T or NK cell subset as indicated. Sites with coverages below 5 were removed from further analysis. (A) Clustering analysis of differentially methylated CpG sites found by RRBS. Clustering was performed on 1,000 most variant methylation sites out of 30,000 randomly chosen sites, localized within gene bodies and promoter regions. The indicated cell subsets cluster according to the similarities of their methylation profile. Beta-values depicted from dark-red to yellow represent level of methylation of the individual CpG sites. (B) Similarities between the cell subsets as judged by the Euclidian distance. (C) Principle component analysis (PCA) of mean methylation levels from two donors for indicated cell subsets. The PCA revealed a clear separation of CD4+ TH1 and CD8+ memory cells, naïve CD4+ and CD8+ cells, and NK cells by the first two components. (D) CpG methylation sites of T and NK cell subsets of one representative donor identified by RRBS in gene bodies of TXB21, EOMES, PRF1, TNF, PRDM1 and ZBTB32. Line diagram represents sequence conservation within vertebrates. Blue and violet bars indicate the methylation level (0–100%) and coverage for each identification, respectively. Coverages of 5 and more reads are represented as a full violet bar.
Mentions: In order to understand whether IFNG CNS1 demethylation was part of a broader epigenetic remodeling occurring in NKG2Chi expansions from HCMV+ individuals, we performed RRBS-based global methylation analysis of CD57+ NKG2Chi, CD57+ NKG2C− and CD57− NKG2C− NK cells in comparison with CD8+ memory and CD4+ TH1 cells as well as with CD8+ or CD4+ naïve T cells isolated from the same HCMV+ individuals. CD8+ memory and CD4+ TH1 cells, CD8+ or CD4+ naïve T cells, and NK cells constitute three different clusters. Interestingly, NKG2Chi NK cells distinctively shared the methylation profile of a large set of CpG sites with CD8+ memory or CD4+ TH1 cells. Conversely, CD57− NKG2C− NK cells were more similar to naïve CD8+ or CD4+ T cells, with CD57+ NKG2C− NK cells displaying a methylation profile intermediate to the two other NK cell subsets (Figure 4A). The NK cell subsets show a decreasing gradient of similarities to naïve CD8+ and CD4+ T cells from CD57− NKG2C− via CD57+ NKG2C− to CD57+ NKG2Chi, as judged by the Euclidian distance, and a complementary, increasing gradient of similarities to CD8+ memory and CD4+ TH1 cells (Figure 4B). This observation was further confirmed by employing an unsupervised strategy and performing principal component analysis (PCA) (Figure 4C). Most of the variation among the different lymphocyte subsets was captured by the first principal component (PC1). CD8+ and CD4+ naïve T cells were at one extreme, while CD8+ memory together with TH1 cells were at the other extreme of the PC1-axis, suggesting that PC1 defined the direction of lymphocyte differentiation, which was characterized by a global epigenetic remodeling (Figure 4B). NK cell subsets distributed along the PC1, with CD57− NKG2C− cells displaying the highest PC1 score, similar to naïve T cells, while CD57+ NKG2Chi NK cells displayed the lowest PC1 score. T cell and NK cell lineage-defining epigenetic modifications were likely described by the second principal component (PC2). Analyzing the methylation pattern of other genes of interest, which are shared between NK cells and TH1 or CD8+ memory T cells, such as TBX21 (T-bet), EOMES (Eomesodermin), and PRF1 (perforin), we observed that all NK cell subsets displayed similar or even more pronounced demethylation pattern at several CpG regions compared to TH1 or CD8+ memory T cells (Figure 4D). TNF was mainly demethylated in both CD57+ NKG2Chi and CD57+ NKG2C− NK cell subsets, similar to TH1 or CD8+ memory T cells. Recently, it was shown that PRDM1 (Blimp-1) and ZBTB32 (also known as PLZP, ROG, FAZF and TZFP), which are not expressed in naïve T cells but are up-regulated in differentiated T cells [45]–[47], regulate the proliferative burst of Ly49H+ memory NK cells during MCMV infection [24]. Interestingly, some CpGs of the PRDM1 and ZBTB32 genes were consistently demethylated in NKG2Chi NK cells and TH1 or memory CD8+ T cells, compared to other NK cell subsets or naïve T cells. Altogether, these data suggest that CNS1 demethylation in NKG2Chi expansions from HCMV+ individuals is part of a broader epigenetic remodeling, which is partially shared by TH1 and memory CD8+ T 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