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Title: Human Cytomegalovirus Drives Epigenetic Imprinting of the IFNG Locus in NKG2Chi Natural Killer Cells}
Article:
Abstract:
Memory type 1 T helper( TH1) 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 TH1 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 NKG2Chi 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.
Introduction:
In order to successfully fight infections caused by intracellular pathogens, interferon( IFN)-γ is expressed during an immune response primarily by T cell lineages and natural killer( NK) cells. While NK cells display constitutive IFNG promoter activity and express IFN-γ at early maturation stages[1], the expression by CD8+ and CD4+ T cells is restricted to differentiated effector/memory cells. In particular, naïve CD4+ T cells must undergo a differentiation process towards type 1 T helper cells( TH1), in order to acquire the ability to stably express IFN-γ[2],[3]. A key mechanism stabilizing TH1-lineage commitment is epigenetic imprinting of the IFNG locus, which leads to heritable DNA and histone modifications of cis-elements, such as the promoter and several conserved non-coding sequences( CNS). The IFNG cis-elements that show the most stringent TH1-specific hypersensitivity include the promoter and the activation-induced proximal upstream element CNS1, which is located 6 kb or 4 kb upstream of the mouse Ifng and human IFNG promoter, respectively. These regulatory regions display binding sites for T-bet, STAT4, NF-κB, and NFAT. Once in an open configuration, both regions function as crucial enhancers of IFNG transcriptional activity in TH1 cells, especially in response to TCR stimulation, due to the presence of binding sites for NFAT, which is activated after engagement of the TCR but not of the cytokine receptors in T cells[3]–[8]. Moreover, effector/memory but not naïve CD8+ T cells were shown to display an open configuration in these two IFNG regions[9],[10]. Although epigenetic analysis of the IFNG locus in total NK cells revealed an open configuration in some regions analyzed[1],[9],[11]–[13], the IFNG promoter undergoes partial demethylation during NK cell differentiation[14]. Thus, a more detailed analysis of additional IFNG regulatory regions in NK cell subsets is of great importance. Though being part of the innate immune system, defined subsets of NK cells display adaptive features[15]. NK cells with memory-like properties have been described during mouse cytomegalovirus( MCMV) infection[16], after hapten-induced contact hypersensitivity[17], as well as after cytokine( IL-15+IL-12+IL-18) priming[18]–[20]. Primary MCMV infection of C57BL/6 mice induces the clonal expansion of Ly49H+ NK cells, which can directly interact with the m157 protein of MCMV[21]. Ly49H+ memory-like NK cells persist for several months and, upon rechallenge, undergo secondary expansion and display enhanced effector functions[16]. The mechanisms underlying the generation of memory-like features in NK cells are a field of intense research[22]–[27]. For instance, it has been shown that proliferation of MCMV-specific Ly49H+ NK cells relies on IL-12R and STAT4 signaling and PLZP-mediated antagonism of Blimp-1[22],[24]. However, it is not clear whether this phenomenon might be associated with imprinting of the IFNG locus, similar to memory CD8+ T cells and CD4+ TH1 cells. Human CMV( HCMV) infection also plays an important role in shaping the human NK cell repertoire. In HCMV seropositive( HCMV+) individuals, CD94/NKG2C+, further referred to as NKG2C+, NK cells are present at higher frequency compared to HCMV seronegative( HCMV−) ones[28] and can be expanded in vitro in the presence of HCMV-infected fibroblasts[29]. In the course of HCMV reactivation after solid organ or hematopoietic cell transplantation, NKG2C+ NK cells from certain donors undergo extensive proliferation in vivo[30]–[33]. These NKG2C+ expansions are characterized by bright expression of NKG2C( NKG2Chi) and by a terminally differentiated phenotype, as suggested by the preferential expression of CD57[31],[34]. The specific mechanisms how HCMV might mediate “priming” and expansion of NKG2Chi NK cells is still not completely clear. Both NKG2C and its inhibitory counterpart CD94/NKG2A, further referred to as NKG2A, recognize the non-classical HLA class I molecule HLA-E[35]–[37], whose surface expression is stabilized by peptides derived from the leader sequence of other HLA class I molecules in a transporter associated with antigen processing( TAP)-dependent fashion[38],[39]. Interestingly, a peptide derived from the gpUL40 protein of HCMV strains and clinical isolates is able to stabilize HLA-E in a TAP-independent manner and enables recognition by both NKG2A and NKG2C[40],[41]. HCMV exploits several strategies to induce down-regulation of MHC class I molecules in infected cells, for instance by blocking TAP[42]. Thus, while classical MHC class I molecules are down-regulated, gpUL40 induces surface expression of HLA-E on infected cells[40]. As the inhibitory receptor NKG2A generally displays a higher affinity for HLA-E compared to NKG2C[41],[43], gpUL40-induced up-regulation of HLA-E may represent an escape route for HCMV against NK cell mediated elimination of infected cells. However, while NKG2C is often co-expressed with NKG2A in HCMV− individuals, NKG2Chi expansions in HCMV+ individuals typically lack NKG2A, while expressing killer cell immunoglobulin-like receptor( KIR) specific for self-MHC( sKIR), especially KIR2DL1 or KIR2DL2/3, and LIR-1[28],[33],[34],[44]. Importantly, NKG2Chi expansions persist over time, are highly competent IFN-γ producers and exhibit even higher IFN-γ expression upon HCMV reactivation[30]–[32]. For all these reasons, it has been suggested that NKG2Chi NK cell expansions found in HCMV infected individuals might also represent NK cells with memory-like properties[15],[31],[32]. With the aim to analyze whether epigenetic regulation of the IFNG locus could contribute to determine high IFN-γ competence and stability in NK cells described to display memory-like properties, we here showed that only HCMV-specific NKG2Chi expansions and cytokine-primed NK cells displayed an open configuration at the IFNG CNS1, which acted as important enhancer of IFNG transcriptional activity in response to NKG2C and activating receptors( actR) engagement.
Discussion:
Our present study showed that epigenetic remodeling of the CNS1 region in the IFNG locus is a hallmark of NKG2Chi NK cells expanded in HCMV+ individuals as well as cytokine-primed NK cells, which have been previously described as memory-like NK cells[15],[19],[31],[32] and share an open configuration of this region with TH1 cells. CNS1 is a crucial enhancer of IFNG transcriptional activity in TH1 cells, especially in response to TCR triggering[4],[8]. In line with its important role as TCR-dependent enhancer in TH1 cells, we showed that accessibility of CNS1 enhanced IFNG transcriptional activity of NK cells upon triggering of actR, especially NKG2C. Whereas resting NK cells generally need simultaneous engagement of multiple actR[49], the sole engagement of NKG2C was sufficient to induce IFN-γ production in expanded NKG2Chi cells, while 2B4 mainly functions as coreceptor[50]. We propose that CNS1 accessibility contributes to the robust IFN-γ expression in response to NKG2C engagement as one important mechanism. Epigenetic remodeling of the IFNG locus, together with high expression of NKG2C, might help NK cells to compensate for other actR deficiencies. Several characteristics displayed by NKG2Chi NK cells, such as their in vivo expansion, persistence in time and high effector functions after HCMV reactivation support the concept that these cells display adaptive properties[15]. Indeed, our genome wide analysis of DNA methylation showed that NKG2Chi NK cell expansions show remarkably lower epigenetic distance to TH1 and CD8+ memory T cells as compared to the other NK cell subsets analyzed. This observation further strengthens the idea that common epigenomic features might be functional hallmarks of memory-like NK cells and memory T cells. It is still not clear to which extent some of the adaptive features of NKG2Chi expansions depend on low-level viral reactivation events occurring in healthy HCMV+ individuals[51]. In line with this interpretation, it was shown that during allogeneic hematopoietic stem cell transplantation from HCMV+ donor to HCMV+ recipient, NKG2C+ NK cells also expand in the absence of clinically detectable HCMV viremia[32]. Similarly, clinically silent reactivation of HCMV or of other herpesviruses also stimulates HCMV-specific T cells and dramatically shapes the T cell repertoire[52],[53]. Which signals are driving CNS1 demethylation exclusively in expanded NKG2Chi NK cells? Co-culture with 221-AEH cells, which express the NKG2C ligand HLA-E, or with HCMV-infected fibroblasts leads to expansion of NKG2C+ NK cells[29],[44]. Although engagement of NKG2C might contribute to induce CNS1 opening in these cells, we do not have evidences for that. On the other hand, we show that IL-12+IL-15+IL-18 stimulation is sufficient to drive CNS1 demethylation in NK cells. Priming by pro-inflammatory cytokines, which largely contribute to anti-viral defense[54], might not only induce CNS1 opening but also contribute to select NKG2C+ cells with proliferative and/or survival advantage[34],[55], thus enabling the persistence of a progeny, which has undergone epigenetic remodeling of the CNS1, similar to memory TH1 cells. Further supporting a central role of pro-inflammatory cytokines in this process, it was shown that generation of MCMV-specific Ly49H+ memory-like NK cells is strongly dependent on IL-12R/STAT4 signaling[22] and that induction of PLZP, required for the proliferative burst of Ly49H+ NK cells, is accomplished by IL-12 and IL-18 but not by Ly49H engagement[24]. A preferential expansion/survival in response to inflammatory cytokines might also partially explain why NKG2C+ NK cells from HCMV+ individuals could expand in the course of other viral infections, such as with Hantavirus, Chikungunya or Hepatitis C virus[34],[55],[56]. This unique ability of NKG2Chi NK cells to expand and become activated in different infections closely resembles what has been observed for HCMV-specific T cell responses. It was shown that the broad HCMV-specific T cell compartment not only controls viral reactivation, but can also alter the outcome of unrelated infections[57], a phenomenon known as heterologous immunity[58]. Herpesvirus-specific CD8+ T cells, especially HCMV- and EBV-specific ones, are frequently activated in the setting of other infections, among which Hantavirus and Hepatitis virus have been reported[57],[59]. This phenomenon does not rely exclusively on T cell receptor cross-reactivity but also acts through antigen-independent bystander mechanisms, such as cytokines, in particular IL-15[51],[57]. Similarly, during Hantavirus and Hepatitis virus infections proinflammatory cytokines might promote the expansion of NKG2Chi NK cells, which were previously primed during HCMV infection. Interestingly, we also showed that, among CD56dim NKG2Chi NK cells, CNS1 demethylation is not confined to the CD57+, but also present in the CD57− subset, which is highly responsive and proliferates in response to cytokines[44],[60]. Thus, our and other data[44] suggest that CD57− NKG2Chi cells, rather than their CD57+ counterpart, might represent memory-like precursors replenishing and stably maintaining the peripheral pool of NKG2Chi NK cells. In summary our data identified epigenetic imprinting in the CNS1 region of the IFNG locus as a hallmark and mechanism underlying adaptive features and IFN-γ memory in NK cells. We also showed that a broader epigenetic remodeling accompanies these adaptive features demonstrating that both inflammatory and pathogen cues induce epigenetic remodeling of NK cells. These two observations extend our view of the molecular control caused by inflammation in innate immune cells[61],[62] and furthermore highlight adaptive epigenetic similarities among the two branches of the immune system.
Upon viral infection , the innate interferon ( IFN ) -γ producing Natural Killer ( NK ) cells provide fast , but short-term protection , while adaptive T cells confer delayed , but long-lasting immunity . Once acquired , effector properties remain stably imprinted in the T cell memory progeny . Recently , it was shown that human cytomegalovirus ( HCMV ) infection can shape the human NK cell repertoire and drive the generation and maintenance of NK cell expansions , which express the activating receptor CD94/NKG2C and have been described as memory-like NK cells . However , the molecular mechanisms underlying NK cell adaptive properties driven by HCMV infection have not been completely defined . In this study , we 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 , thus providing a molecular basis for the regulation of adaptive features in innate cells .