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. 2019 Jun 18;129(9):3770-3785.
doi: 10.1172/JCI125916.

Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming

Aimee Merino  1 , Bin Zhang  1 , Philip Dougherty  1 , Xianghua Luo  2 , Jinhua Wang  3 , Bruce R Blazar  4 , Jeffrey S Miller  1 , Frank Cichocki  1
Affiliations

Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming

Aimee Merino et al. J Clin Invest. .

Abstract

A population of Natural Killer (NK) cells expressing the activating receptor NKG2C and the maturation marker CD57 expands in response to human cytomegalovirus (HCMV) infection. CD3-CD56dimCD57+NKG2C+ NK cells are similar to CD8+ memory T cells with rapid and robust effector function upon re-stimulation, persistence, and epigenetic remodeling of the IFNG locus. Chronic antigen stimulation drives CD8+ memory T cell proliferation while also inducing genome-wide epigenetic reprograming and dysfunction. We hypothesized that chronic stimulation could similarly induce epigenetic reprograming and dysfunction in NK cells. Here we show that chronic stimulation of adaptive NK cells through NKG2C using plate-bound agonistic antibodies in combination with IL-15 drove robust proliferation and activation of CD3-CD56dimCD57+NKG2C+ NK cells while simultaneously inducing high expression of the checkpoint inhibitory receptors LAG-3 and PD-1. Marked induction of checkpoint inhibitory receptors was also observed on the surface of adaptive NK cells co-cultured with HCMV-infected endothelial cells. Chronically stimulated adaptive NK cells were dysfunctional when challenged with tumor targets. These cells exhibited a pattern of epigenetic reprograming, with genome-wide alterations in DNA methylation. Our study has important implications for cancer immunotherapy and suggest that exhausted NK cells could be targeted with inhibitory checkpoint receptor blockade.

Keywords: Immunology; NK cells.

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Conflict of interest statement

Conflict of interest: FC consults for Fate Therapeutics and has received research funds from this relationship. JSM serves on the Scientific Advisory Board (SAB) and consults for GT BioPharma and Fate Therapeutics. He has received research funds from these relationships. JSM also serves on the SAB for CytoSen and Onkimmune. BRB declares a financial conflict with Tmunity and Kadmon Corporation. He also serves on the SAB for GT Biopharma, Magenta Therapeutics, and Five Prime Therapeutics. He consults for Regeneron and Equillium Inc. None of these companies had a role in funding this research. All conflicts are managed according to institutional policies.

Figures

Figure 1
Figure 1. Chronic stimulation through NKG2C expands adaptive NK cells.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and PBS, IgG2b isotype Ab, anti-NKG2A/C Ab, or anti-NKG2C Ab. (A) Representative FACS plots and summary data showing the percentages of NK cell subsets defined by expression of CD57 and NKG2C before and after a 7-day culture (n = 7). Results are from 3 independent experiments. (B) NK cells were labeled with CellTrace dye prior to culturing. Shown are FACS plots of representative donor cells stratified by CD57 and NKG2C expression and summary data (n = 6). Results are from 3 independent experiments. *P ≤ 0.05 by paired t test. P values for multiple group comparisons (A, each group vs. PBS; B, each group vs. IgG2b isotype Ab) were adjusted using the Hommel method.
Figure 2
Figure 2. NKG2A is upregulated on the surface of NKG2C– and NKG2C+ NK cells during culture with IL-15.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15. FACS plots of cells from a representative donor are shown. Summary data (n = 5) show the frequencies of CD3–CD56+ NK cells gated by CD57 and NKG2C that expressed NKG2A before and after culturing. Results are from 2 independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.001, by paired t test. P values of multiple comparisons (each culture day vs. day 0) were adjusted using the Hommel method. SSC, side scatter.
Figure 3
Figure 3. Chronic stimulation of adaptive NK cells through NKG2C leads to a CD45 isoform switch.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and IgG2b isotype Ab, anti-NKG2A/C Ab, or anti-NKG2C Ab. (A) Representative FACS plots and summary data of the percentages of CD45RA+RO– and CD45RA–CD45RO+ NK cells within the CD3–CD56dimNKG2C– and CD3–CD56dimNKG2C+ subsets prior to culturing (n = 5). (B) Representative FACS plots and summary data on the percentages and MFIs of CD45RA and CD45RO on CD3–CD56+NKG2C– NK cells and CD3–CD56+NKG2C+ NK cells after a 7-day culture (n = 7). Results are from 3 independent experiments. (C) Summary data for CD45RA+CD45RO– and CD45RA–CD45RO+ NK cell proliferation after culturing (n = 5), as measured by CellTrace dye dilution. Results are from 2 independent experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by paired t test. P values for multiple group comparisons (within the NKG2C– NK cell groups, within the NKG2C+ NK cell groups, and between the NKG2C– and NKG2C+ NK cell groups) were adjusted using the Hommel method.
Figure 4
Figure 4. Adaptive NK cells chronically stimulated through NKG2C upregulate LAG-3 and PD-1.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and IgG2b isotype Ab, anti-NKG2A/C Ab, or anti-NKG2C Ab. (A) Representative FACS plots and summary data of the percentages of LAG-3 and PD-1 expression on CD3–CD56dimNKG2C– and CD3–CD56dimNKG2C+ NK cell subsets prior to culturing (n = 7). (B) Representative FACS plots and summary data of the percentages and MFIs of LAG-3 and PD-1 on CD3–CD56+NKG2C– NK cells and CD3–CD56+NKG2C+ NK cells after culturing (n = 7). Results are from 3 independent experiments. (C) tSNE images of FACS data. tSNE plots represent a composite of NK cells from 3 donors stimulated with either IgG2b isotype or anti-NKG2A/C Ab. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by paired t test. P values for multiple group comparisons in B (within the NKG2C– NK cells groups, within the NKG2C+ NK cells groups, and between the NKG2C- and NKG2C+ NK cell groups) were adjusted using the Hommel method.
Figure 5
Figure 5. Chronically stimulated adaptive NK cells expressing LAG-3 exhibit impaired IFN-γ production.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and IgG2b isotype Ab, anti-NKG2A/C Ab, or anti-NKG2C Ab. (A) Representative FACS plots and summary data of intracellular IFN-γ levels in preculture CD3–CD56dimNKG2C– and CD3–CD56dimNKG2C+ NK cells cocultured with K562 targets at an E/T ratio of 2:1 (n = 4). Results are from 2 independent experiments. (B) NK cells after culturing were used as effectors in functional assays with K562 targets at an E/T ratio of 2:1. Shown are representative FACS plots and summary data of IFN-γ production by NK cell subsets stratified by NKG2C and LAG-3 expression (n = 4). Results are from 2 independent experiments. *P ≤ 0.05, by paired t test to compare LAG-3– and LAG-3+ NK cells in each condition.
Figure 6
Figure 6. Chronic stimulation through NKp30 or NKG2D preferentially induces inhibitory checkpoint receptor expression on adaptive NK cells.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either IgG2b isotype Ab, anti-NKG2A/C Ab, anti-NKG2C Ab, anti-NKp30 Ab or anti-NKG2D Ab. (A) Summary data of the frequencies of CD3–CD56+NKG2C+ NK cells in each condition. (B) Summary data of the percentages and MFIs of CD45RO, LAG-3, and PD-1 on CD3–CD56+NKG2C– and CD3–CD56+NKG2C+ NK cells in each condition (n = 5). Results are from 2 independent experiments. *P ≤ 0.05 and **P ≤ 0.01, by t test. P values of multiple group comparisons (A, each group vs. IgG isotype Ab; B, within the NKG2C– NK cell groups and within the NKG2C+ NK cell groups) were adjusted using the Hommel method.
Figure 7
Figure 7. Adaptive NK cells cocultured with HCMV-infected HUVECs upregulate LAG-3 and PD-1 and exhibit impaired IFN-γ production.
HUVECs were infected with a GFP-expressing TB40/e clinical strain of HCMV at a MOI of 0.5 or spun down in parallel without virus (mock-infected) and used for 7-day coculture experiments with CD3/CD19-depleted PBMCs from HCMV-seropositive donors (n = 4). Cultures contained 10 ng/ml IL-15. (A) Representative FACS plots and summary data of the percentages of CD3–CD56+NKG2C+ cells from mock-infected and TB40/e-infected HUVEC cocultures. (B) Representative FACS plots and summary data of the percentages of CD3–CD56+NKG2C– and CD3–CD56+NKG2C+ NK cells expressing CD45RO and CD45RO MFI on each cell subset (n = 4). (C) Representative FACS plots and summary data of LAG-3 and PD-1 expression on CD3–CD56+NKG2C– and CD3–CD56+NKG2C+ NK cells from mock- and TB40/e-infected HUVEC cocultures (n = 4). (D) Summary data of intracellular IFN-γ in CD3–CD56+ NK cells gated by NKG2C and LAG-3 expression from mock-infected and TB40/e-infected HUVEC cocultures stimulated with K562 cells at an E/T ratio of 2:1 (n = 4). Results are from 2 independent experiments. *P ≤ 0.05 and **P ≤ 0.01, by paired t test. P values of multiple group comparisons (A, 4 pairwise comparisons as shown; B, same comparisons as in A) were adjusted using the Hommel method.
Figure 8
Figure 8. Chronically stimulated adaptive NK cells exhibit a whole-genome DNA methylation profile indicative of exhaustion.
CD3/CD19-depleted PBMCs from HCMV-seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either IgG2b isotype Ab, anti-NKG2A/C Ab, or anti-NKG2C Ab. The whole-genome DNA methylation profiles of NK cells from these cultures were analyzed using Illumina Infinium MethylationEPIC BeadChips in 2 independent experiments. (A) Heatmap of annotated genomic sites that exhibited consistent differential methylation across NK cells from 7 donors from IgG2b isotype Ab and anti-NKG2A/C Ab cultures and a list of genes within this data set that match genes shown to be differentially expressed or epigenetically remodeled as a result of CD8+ T cell exhaustion. Genes in blue text were shown to be remodeled in exhausted CD8+ T cells as determined by assay for transposase-accessible chromatin with sequencing (ATAC-Seq) (37). Genes in red text were found to be induced in tumor-specific CD8+ T cells as determined by RNA-Seq (34). Genes in green text were identified in both studies. (B) Heatmap of gene-associated promoter regions that exhibited differential methylation between NK cells cultured with IgG2b isotype Ab, anti-NKG2A/C Ab, or anti-NKG2C Ab and were also reported to be differentially regulated in exhausted CD8+ T cells. (C) Heatmap of gene-associated promoter regions matching to the PD-1 signaling pathway as determined by GSEA.
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