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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.).

Competing interests

The authors declare no competing interests.

Author information

Affiliations

  1. Innate Immunity, German Rheumatism Research Center (DRFZ), Leibniz Association, Berlin, Germany

    • Quirin Hammer
    • , Timo Rückert
    • , André Haubner
    • , Marina Babic
    •  & Chiara Romagnani
  2. Institute for Virology, Hannover Medical School, Hannover, Germany

    • Eva Maria Borst
    • , Adriana Tomic
    •  & Martin Messerle
  3. Inflammation Biology, German Rheumatism Research Center (DRFZ), Leibniz Association, Berlin, Germany

    • Josefine Dunst
  4. Cell Biology, German Rheumatism Research Center (DRFZ), Leibniz Association, Berlin, Germany

    • Pawel Durek
  5. Microbiota and Inflammation, German Rheumatism Research Center (DRFZ), Leibniz Association, Berlin, Germany

    • Pawel Durek
  6. Therapeutic Gene Regulation German Rheumatism Research Center (DRFZ), Leibniz Association, Berlin, Germany

    • Frederik Heinrich
    •  & Mir-Farzin Mashreghi
  7. Department of Genetics, University of Saarland, Saarbrücken, Germany

    • Gilles Gasparoni
    •  & Jörn Walter
  8. Department of Experimental Medicine, University of Genoa, Genoa, Italy

    • Gabriella Pietra
  9. Immunologia, IRCCS Ospedale Policlinico San Martino, Genoa, Italy

    • Gabriella Pietra
  10. Medical Clinic I, Marien Hospital Herne, Ruhr University Bochum, Herne, Germany

    • Mikalai Nienen
    •  & Nina Babel
  11. Department of Hematology, Oncology and Tumor Immunology, Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Igor Wolfgang Blau
    • , Il-Kang Na
    • , Philipp Hemmati
    •  & Renate Arnold
  12. Institute of Virology Charité, Universitätsmedizin Berlin, Berlin, Germany

    • Jörg Hofmann
  13. Virology, Labor Berlin - Charité Vivantes GmbH, Berlin, Germany

    • Jörg Hofmann
  14. Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Il-Kang Na
  15. Berlin Institute of Health (BIH), Berlin, Germany

    • Il-Kang Na
  16. Institute of Immunology, Hannover Medical School, Hannover, Germany

    • Immo Prinz
    •  & Christian Koenecke
  17. Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany

    • Christian Koenecke
  18. Berlin-Brandenburg Center for Regenerative Therapies, Institute of Medical Immunology, Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Nina Babel
  19. Systems Biology of Inflammation, German Rheumatism Research Center (DRFZ), Leibniz Association, Berlin, Germany

    • Kevin Thurley
  20. Medical Department I, Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Chiara Romagnani

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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.

Corresponding author

Correspondence to Chiara Romagnani.

Integrated supplementary information

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  1. Supplementary Text and Figures

    Supplementary Figures 1-7 and Supplementary Tables 3-5

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    List of 165 published and 52 newly determined HCMV UL40-encoded peptide sequences

  4. Supplementary Table 2

    Individual Patient Characteristics

  5. Supplementary Tables

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DOI

https://doi.org/10.1038/s41590-018-0082-6