Abstract
Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C+ NK cells has remained unclear. Here we found that adaptive NKG2C+ NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C+ NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C+ NK cell populations among HCMV-seropositive people.
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Acknowledgements
We thank P. Wehler, A. Seegebarth and U. Uhlig for technical assistance; the DRFZ FCCF for cell sorting; D. Hernandez for critical reading of the manuscript; A. Moretta (University of Genoa) for antibody clone GL183; E. Weiss (Ludwig Maximilian University) for K562–HLA-E cells; J. Coligan (US National Institutes of Health) for RMA-S–HLA-E cells; G. Smith (Northwestern University) for the pGS403 plasmid; and A. Scheffold, A. Brooks and K.-J. Malmberg for comments during manuscript preparation. Supported by Leibniz Science Campus Chronic Inflammation (http://www.chronische-entzuendung.org), Leibniz Best Minds program (K.T.), the German Research Foundation (SFB 650, RO3565/2-1 and RO3565/4-1 to C.R.; SFB900 to M.M., I.P. and C.K.; and Heisenberg Program RO 3565/1-1 for C.R.), the state of Berlin (for the work of F.H. and M.-F.M.), the Stiftung Charité (for I.-K.N), the European Regional Development Fund (ERDF 2014-2020 and EFRE 1.8/11 for the work of F.H. and M.-F.M.) and the Leibniz Graduate School for Rheumatology (Q.H.).
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The authors declare no competing interests.
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Contributions
Q.H. coordinated the study; Q.H. and C.R. conceived of and designed the study; Q.H., T.R., J.D., A.H. and M.B. performed experiments and analyzed data; E.M.B., A.T., and M.M. designed and generated HCMV variants; P.D., F.H. and M.-F.M. performed transcriptome analysis; G.G. and J.W. performed epigenetic analyses; G.P. provided reagents and expertise; M.N., I.W.B., J.H., I.-K.N., I.P, C.K., P.H., N.B. and R.A. acquired and provided clinical samples and data; K.T. modeled proliferation data; J.H. investigated clinical samples with respect to viral load; Q.H. and C.R. wrote the manuscript; and C.R. supervised the work.
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Supplementary Figure 1 Sequence variations in HCMV UL40-encoded peptides control the activation of adaptive NKG2C+ NK cells but do not differentially affect inhibition of NKG2C– NKG2A+ NK cells.
(a-b) PBMC of healthy HCMV– (n=20) and HCMV+ (n=40) donors were screened by flow cytometry. (a) Frequency of NKG2C+ cells within the CD56dim population and (b) frequency of CD2+ Siglec-7– NKG2A– FcεR1γ– cells within the CD56dim NKG2C+ population. Symbols indicate individual donors and lines median. CV, coefficient of variation. (c) Gating strategy for functional assays using HCMV+ donors with adaptive NKG2C+ NK cells. After culture of purified viable CD3– CD56+ NK cells with peptide-pulsed target cells, adaptive NKG2C+ NK cells were gated as viable single CD56dim NKG2A– CD57+ KIR+ NKG2C+ cells. Depending on the phenotype of the individual donor, KIR were gated as KIR2DL1+, KIR2DL3+, or KIR3DL1+. (d) Purified NK cells from HCMV+ donors were used as effector cells in cytotoxicity assays against labelled peptide-pulsed RMA-S–HLA-E cells and cytotoxicity was calculated as described in the Methods section. Symbols indicate individual data points, lines indicate means, and error bars indicate SEM (n=6 individual donors in 3 independent experiments). Two-way repeated-measures ANOVA with Bonferroni correction between VMAPRTLIL and VMAPRTLFL. (e) RMA-S–HLA-E cells were pulsed with 300 μM of the indicated peptides and geometric mean fluorescence intensity (geoMFI) of HLA-E surface expression was assed. Symbols indicate independent experiments (n=6) and horizontal lines median. Friedman test with Dunn’s post test. (f) Binding affinities were predicted using the NetMHC4.0 algorithm. The HCMV pp65-derived HLA-A2-restricted NLVPMVATV peptide served as a non-HLA-E-binding control. (g) RMA-S–HLA-E cells were pulsed with 300 μM VMAPRTLIL or VMAPRTLFL peptide followed by removal of peptide and chase for 6 h. Decay in HLA-E surface expression was calculated assuming first order kinetics. Symbols indicate individual data points, error bars indicate SEM (n=3 independent experiments), and lines indicate linear regression curves. Slopes of regression curves were compared using ANCOVA. (h) RMA-S–HLA-E cells were pulsed with increasing concentrations of the indicated peptides and geoMFI of HLA-E surface expression upon pulsing was assessed. Symbols indicate individual data points, lines indicate means, and error bars indicate SEM (n=6 independent experiments). Two-way repeated-measures ANOVA with Bonferroni correction between VMAPRTLIL and VMAPRTLFL. (i) Degranulation response of viable CD56dim NKG2C– (triangles) or viable CD56dim NKG2A– CD57+ KIR+ NKG2C+ NK cells (circles) upon culture without or with VMAPRTLFL-pulsed RMA-S–HLA-E cells. Connected symbols represent individual donors (n=12 in 6 experiments). Two-tailed Wilcoxon test. (j) Sorted viable CD56dim NKG2A– NKG2C+ NK cells from HCMV+ donors were treated with IgG1 isotype control or anti-CD94 blocking antibody prior to culture without or with VMAPRTLFL-pulsed RMA-S–HLA-E cells. Summary of degranulation of viable CD56dim NKG2A– CD57+ NKG2C+ NK cells. Connected symbols represent individual donors (n=6 in 3 independent experiments). Two-tailed Wilcoxon test. (k) Purified NK cells from HCMV+ donors were cultured with K562–HLA-E cells pulsed with indicated peptides at indicated concentrations. Summary of CCL3 expression and degranulation gated on viable CD56dim NKG2A– CD57+ KIR+ NKG2C+ NK cells (circles) or CD56dim NKG2C– NKG2A+ cells (triangles). Symbols indicate individual data points, lines indicate means, and error bars indicate SEM (n=6 individual donors in 3 independent experiments). Two-way repeated-measures ANOVA with Bonferroni correction between VMAPRTLIL and VMAPRTLFL. NS not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001.
Supplementary Figure 2 Co-Stimulation via LFA-3 enhances functional responses of adaptive NKG2C+ NK cells.
(a) Purified NK cells from HCMV+ donors were cultured with K562–HLA-E cells pulsed with indicated peptides. Summary of effector functions gated on viable CD56dim NKG2A– CD57+ KIR+ NKG2C+ NK cells. Connected symbols indicate individual donors (n=15 in 8 independent experiments). Friedman test with Dunn’s post test. (b) K562–HLA-E cells were examined for the expression of LFA-3 by flow cytometry. Fluorescence minus one (FMO) control and stained condition gated on viable cells. (c) Purified NK cells from HCMV+ donors were either left untreated or treated with blocking anti-LFA-3 antibody followed by stimulation with VMAPRTLIL-pulsed K562–HLA-E cells. Effector functions gated on viable CD56dim NKG2A– CD57+ KIR+ NKG2C+ NK cells. Connected symbols represent individual donors (n=9 in 5 independent experiments). Two-tailed Wilcoxon test. NS not significant, *P < 0.05, **P < 0.01, ****P < 0.0001.
Supplementary Figure 3 NKG2C– NK cells do not differentially recognize HCMV-encoded peptides during infection.
(a) HUVECs were infected with TB40R and transcript levels of HCMV UL40 relative to human GAPDH were determined at indicated time points by quantitative real-time PCR. Symbols indicate independent infection experiments (n=4) and lines median. ND, not detectable. (b-c) HUVECs were infected with TB40R mutants encoding for distinct UL40 peptides and analyzed by flow cytometry 48 h post infection. (b) Representative FACS staining (left) of uninfected and infected (gated on HCMV–IE+) HUVECs compared to fluorescence minus one (FMO) control and summary (right) of HLA class I expression. Symbols indicate independent experiments (n=10) and lines median. (c) Representative FACS staining (left) of uninfected and infected (gated on HCMV–IE+) HUVECs compared to FMO control and summary (right) of HLA-E expression. Symbols indicate independent experiments (n=9) and lines median. (d) HUVECs (homozygous for both HLA–C1 and HLA–Bw4) were infected with TB40R variants encoding for distinct UL40 peptides. Purified rested NK cells from HCMV+ donors were cultured for 6 h in medium alone or with virus-infected HUVECs. Summary of effector functions gated on viable CD56dim NKG2A– CD57+ KIR2DL1– KIR3DL1– KIR2DL3+ NKG2C+ adaptive NK cells. Connected symbols represent individual donors (n=12 in 3 independent experiments). (e) Purified NK cells from HCMV+ donors were primed with 25 ng/mL IFN–α for 16 h and subsequently cultured as in (d). Summary of effector functions gated on viable CD56dim KIR2DL1– KIR3DL1– KIR2DL3+ NKG2C- NK cells. Connected symbols represent individual donors (n=12 in 3 independent experiments). Friedman test with Dunn’s post test. NS not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001.
Supplementary Figure 4 Co-Stimulation via LFA-3 enhances proliferation of NKG2C+ NK cells from HCMV– donors.
(a-b) Purified CD56dim NK cells from HCMV– donors were cultured for 7 days with peptide-pulsed RMA-S–HLA-E cells in the presence of IL-15. (a) Proliferation indices and (b) replication indices of NKG2C+ NK cells were normalized to NKG2C– NK cells of the same donor. Connected symbols represent individual donors (n=8 in 3 independent experiments). Friedman test with Dunn’s post test. (c) Purified CD56dim NK cells from HCMV– donors were cultured for 7 days with either RMA-S–HLA-E or RMA-S–HLA-E–LFA-3 cells in the presence of IL-15. Proliferation and replication indices were normalized as in (a). Connected symbols represent individual donors (n=8 in 3 independent experiments). Two-tailed Wilcoxon test. (d-f) Purified CD56dim NK cells from HCMV– donors were cultured with peptide-pulsed RMA-S–HLA-E–LFA-3 cells in the presence of IL-15. (d) NKG2C+ NK cell numbers per μl of culture medium and (e) precursor frequency of NKG2C+ NK cells over time. Symbols indicate individual donors (n=8 in 2 independent experiments) and lines median. Two-way repeated-measures ANOVA with Bonferroni correction. (f) Frequency of NKG2C+ NK cells after 14 days of culture. Symbols indicate individual donors (n=18 in 7 independent experiments) and lines median. Friedman test with Dunn’s post test. NS not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001.
Supplementary Figure 5 Analysis of NKG2C+ NK cell proliferation.
(a) Purified CD56dim NK cells from HCMV– donors were cultured with peptide-pulsed RMA-S–HLA-E–LFA-3 cells in the presence of IL-15 combined with treatment with IL-12 plus IL-18 during the initial 20 h of culture. Precursor frequency of NKG2C+ NK cells over time. Symbols indicate individual donors (n=6 in 2 independent experiments) and lines median. Two-way repeated-measures ANOVA with Bonferroni correction. (b-f) Mathematical analysis of NKG2C+ NK cell proliferation dynamics. (b-c) Symbols and error bars indicate mean±SEM of experimentally obtained precursor frequencies of NKG2C+ NK cells (b) with (data from Supplementary Fig. 5a) or (c) without (data from Supplementary Fig. 4e) treatment with IL-12 plus IL-18 during the initial 20 h of culture. Lines indicate best-fit gamma distributions, which are used as input for Fig. 5f and Supplementary Fig. 5d. (d) Modified Gett–Hodgkin model describing NKG2C+ NK cell proliferation and accumulation dynamics in the absence of treatment with IL-12 plus IL-18. Symbols and error bars indicate mean±SEM of experimentally obtained NKG2C+ NK cell numbers after normalization to day 1 values (set as 1); lines indicate best-fit curves of the model. Precursor frequencies were experimentally obtained (Supplementary Fig. 4e, Supplementary Fig. 5c), while division times and death rates (both mean±SEM) were inferred as best-fit parameters by non-linear optimization. (e-f) Modified Gett–Hodgkin models with fixed input parameters in the (e) presence or (f) absence of treatment with IL-12 plus IL-18. Symbols and error bars indicate mean±SEM of experimentally obtained NKG2C+ NK cell numbers after normalization to day 1 values (set as 1); lines indicate curves of the model. Precursor frequencies were experimentally obtained; division time and death rate values were inferred by non-linear optimization for the VMAPQSLLL peptide (as in Fig. 5f and Supplementary Fig. 5d, respectively) and set as fixed parameters for both peptides. NS not significant, ***P < 0.005, ****P < 0.0001.
Supplementary Figure 6 Phenotypic alterations of NKG2C+ NK cells.
(a-b) Purified CD56dim NK cells from HCMV– donors were cultured for 14 days with peptide-pulsed RMA-S–HLA-E–LFA-3 cells in the presence of IL-15 alone or in combination with IL-12 plus IL-18. (a) Summaries of Syk, CD161, FcεR1γ, CD7, NKG2A, and DNAM-1 expression on viable NKG2C+ NK cells. Connected symbols represent individual donors (n=6 for FcεR1γ; n=8 for CD161, CD7, and DNAM-1; n=10 for NKG2A; n=12 for Syk in 2-5 independent experiments). Friedman test with Dunn’s post test. (b) Comparison of NKG2C– and NKG2C+ NK cells after 14 days of culture with VMAPRTLFL-pulsed RMA-S–HLA-E–LFA-3 cells in the presence of IL-15 and IL-12 plus IL-18. Connected symbols represent individual donors (n=6 for FcεR1γ; n=8 for educating-KIR, CD161, CD7, and DNAM-1; n=10 for CD2, Siglec-7, and NKG2A; n=12 for Syk in 2-5 independent experiments). Two-tailed Wilcoxon test. NS not significant, *P < 0.05, **P < 0.01, ***P < 0.005. (c-d) Gene expression analysis of sorted viable CD56+ NKG2C+ NK cells cultured in the presence of VMAPQSLLL-pulsed targets (n=3 donors) or VMAPRTLFL plus IL-12 plus IL-18 (n=5 donors) for 7 days. Heat maps of selected (c) adaptive NK cell signature genes and (d) activation and exhaustion markers based on z-scores of rlog-transformed read counts clustered by Pearson correlation and Ward minimum variance. Asterisk-marked genes indicate adjusted P < 0.05.
Supplementary Figure 7 Analysis of the phenotype of adaptive NKG2C+ NK cells in HCMV-infected patients.
(a) Study design. (b) Expression of CD2, Siglec-7, FcεR1γ, and NKG2A by NKG2C+ and NKG2C– CD56dim NK cells. Symbols represent individual patients (white circles, HCMV– without detectable viremia, n=10; blue circles, HCMV reactivation with VMAPRTLIL peptide, n=10; red circles, HCMV reactivation with VMAPRTLFL peptide, n=2) and lines depict median. (c) Frequency of NKG2C+ cells within the CD3– CD56dim compartment over time of n=4 individual patients analyzed in one experiment. Black arrow heads indicate time points of initial HCMV detection.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1-7 and Supplementary Tables 3-5
Supplementary Table 1
List of 165 published and 52 newly determined HCMV UL40-encoded peptide sequences
Supplementary Table 2
Individual Patient Characteristics
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Hammer, Q., Rückert, T., Borst, E.M. et al. Peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells. Nat Immunol 19, 453–463 (2018). https://doi.org/10.1038/s41590-018-0082-6
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DOI: https://doi.org/10.1038/s41590-018-0082-6
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