Remodeling of secretory lysosomes during education tunes functional potential in NK cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation;however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI (3,5) P-2 synthesis, or genetic silencing of the PI(3,5) P-2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education

Adaptive Natural Killer Cells Integrate Interleukin-18 during Target-Cell Encounter

Human cytomegalovirus (HCMV) infection induces adaptations in the natural killer (NK)-cell compartment. Expanded subsets of adaptive NK cells display potent effector functions against cellular targets, despite their apparent unresponsiveness to stimulation with classical dendritic cell-derived cytokines interleukin (IL)-12 and IL-18. However, it remains unclear whether adaptive NK cells have completely lost their ability to sense inflammation via IL-12 and IL-18 or whether these pro-inflammatory signals can be functionally integrated into defined contexts. Here, we demonstrate that adaptive NKG2C+ NK cells can be costimulated by the presence of pro-inflammatory cytokines during target cell-induced activation. Cytokine costimulation of adaptive NK cells resulted in elevated interferon (IFN)-gamma and tumor necrosis factor (TNF) production, which promoted protein expression of HLA class I and adhesion molecules as well as transcription of genes involved in antigen processing and antiviral states in endothelial bystander cells in vitro. We further show that IL-18 drove costimulation in functional assays and was sufficient for elevated cytokine production in the absence of IL-12. Hence, adaptive NKG2C+ NK cells—although poorly responsive to IL-12 and IL-18 as an isolated stimulus—integrate IL-18 as a costimulatory signal during target-cell encounter

Human Cytomegalovirus Drives Epigenetic Imprinting of the <i>IFNG</i> Locus in NKG2C^hi Natural Killer Cells

<div><p>Memory type 1 T helper (T_H1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the <i>IFNG</i> 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 NKG2C^hi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the <i>IFNG</i> conserved non-coding sequence (CNS) 1, similar to memory CD8⁺ T cells or T_H1 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 <i>IFNG</i> 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.</p></div

CNS1 demethylation occurs independent of CD57/sKIR expression and is stably imprinted in NKG2C^hi NK cells.

<p>(A–C) Phenotype and methylation status of the CNS1 was analyzed in ex vivo NKG2C^hi NK cell expansions from representative HCMV⁺ donors (n = 4), as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#ppat-1004441-g001" target="_blank">Figure 1</a>. (A and B) CD56 and NKG2C expression was analyzed by FC on PBMC, after gating on viable CD3⁻ CD56⁺ NK cells, while CD57 and sKIR (KIR2DL3 in HLA-C1⁺ donor) expression was analyzed after gating on CD56^dim NKG2C^hi NK cells. CpG methylation of the <i>IFNG</i> CNS1 was analyzed in FACS sorted CD56^dim NKG2C⁺ NK cell subsets as indicated and is depicted as mean percentage of methylation at each CpG site. (C) The same HCMV⁺ individual displaying an expanded NKG2C^hi population was analyzed twice with an interval of one year between the two measurements. NKG2C and CD57 expression was analyzed by FC on PBMC, after gating on viable CD3⁻ CD56^dim NK cells, while sKIR expression was analyzed after gating on CD56^dim CD57⁺ NKG2C^+/− NK cells. CpG methylation of the <i>IFNG</i> CNS1 was analyzed in FACS sorted CD56^dim CD57⁺ NKG2C⁻ (gray bars) and in CD56^dim CD57⁺ NKG2C⁺ NK cells (black bars) and is depicted as mean percentage of methylation at each CpG site.</p

Expanded NKG2C^hi NK cells display complete demethylation of the <i>IFNG</i> CNS1.

<p>Analysis of surface marker expression and methylation status of the <i>IFNG</i> promoter and CNS1 was analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#ppat-1004441-g001" target="_blank">Figure 1</a> in ex vivo NK cell subsets derived from representative HCMV⁻ (n = 2) (A) or HCMV⁺ (n = 4) donors (B and C), without (B) or with expansion of NKG2C^hi NK cells (C). NKG2C and CD57 expression was analyzed by FC on PBMC, after gating on viable CD3⁻ CD56^dim NK cells, while sKIR (KIR2DL3 in HLA-C1⁺ donor) expression was analyzed after gating on viable CD3⁻ CD56^dim CD57⁺ NKG2C^+/− NK cells. CpG methylation of the <i>IFNG</i> promoter and CNS1 was analyzed in FACS sorted CD56^dim CD57⁺ NKG2C⁻ (gray bars) and in CD56^dim CD57⁺ NKG2C^+/hi NK cells (black bars) from each donor and is depicted as mean percentage of methylation at each CpG site.</p

CNS1 accessibility regulates <i>IFNG</i> transcriptional activity induced by NKG2C engagement.

<p>(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 <i>IFNG</i> sequences transfected in NKL. (C) Construct containing the <i>IFNG</i> promoter (<i>IFNG</i>p) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (<i>Luc</i>). 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 <i>IFNG</i>p (−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 <i>IFNG</i>p (−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+<i>IFNG</i>p (−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.</p

Cytokine-primed NK cells undergo epigenetic remodeling of the <i>IFNG</i> CNS1.

<p>(A) Alignment of the human <i>IFNG</i> and mouse <i>Ifng</i> 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 <i>IFNG</i>/<i>Ifng</i> transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the <i>IFNG</i> 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 <i>IFNG</i> 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, T_H1 cells and NK cells, FACS sorted ex vivo as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#s4" target="_blank">Materials and Methods</a>. 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 CFSE^lo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the <i>IFNG</i> CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).</p

NKG2C⁺ NK cells express IFN-γ in response to NKG2C engagement.

<p>(A and B) Viable CD3⁻ CD56^dim NK cells were FACS sorted and stimulated by cross-linking of NKG2C and/or 2B4 for 16 hours. Analysis of intracellular IFN-γ expression was performed by FC after gating on CD56^dim CD57⁺ NKG2A⁻ sKIR⁺ cells from HCMV⁺ donors displaying or not an expansion of NKG2C^hi cells. Gating strategy is depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#ppat.1004441.s003" target="_blank">Figure S3D</a>. One representative experiment (A) and mean percentage of IFN-γ producing cells ± SEM (B) are depicted (n≥8). Percentage of IFN-γ producing cells was calculated after subtracting the value observed in unstimulated cells. ***<i>p</i><0.001 calculated with Mann-Whitney test. (C) Surface expression of CD48 and HLA-E was analyzed by FC on 221 (dashed line) or 221-AEH cells (black line), with isotype control or secondary staining only (solid grey histogram). One representative staining out of two is depicted. (D and E) Viable CD3⁻ CD56^dim NK cells were FACS sorted and stimulated by co-culture with 221 or 221-AEH for 6 hours. Analysis of intracellular IFN-γ expression was performed by FC after gating on CD56^dim CD57⁺ NKG2A⁻ sKIR⁺ NKG2C^hi/+/− cells from HCMV^+/− donors. One representative experiment (D) and mean percentage of IFN-γ producing cells ± SEM (E) are depicted (n = 6). *<i>p</i><0.05, **<i>p</i><0.01, calculated with Mann-Whitney test.</p