Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses

Abstract

Introduction: Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive.

Methods: We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses.

Results: Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs.

Discussion: These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Keywords: Adaptive NK cells; HLA-E; dendritic cells; memory; peptides.

Figure 1

Co-culture of mDC loaded with an HCMV-associated pp65-derived peptide pool significantly expands the frequency of aNK cells. NK cells from HCMV-seropositive donors were cultured for 14 days with mDC loaded with the pools of overlapping 15-mer peptides derived from either HCMV-associated pp65 or HIV-1-associated Gag proteins, or left unloaded in the presence of 10 ng/ml IL-15 relative to cocultures with monocytes and imDC. (A) Representative flow cytometry plots and gating strategy of CD56/NKG2C cNK and aNK cell expansion are shown. (B) Cumulative (n = 10) data showing the percentages of cNK and aNK cells within the total CD57+ NK cell population. Results from five independent experiments are presented as mean ± SEM. A One-way ANOVA test was used for statistical analyses. (C) Cumulative (n = 9) data showing the percentages of aNK and cNK cell proliferation (Ki67), degranulation (CD107a), and IFNγ production following co-culture with M, imDCs, mDCs, or mDCs+pp65. The presented results are from three independent experiments. All the cumulative data are shown as mean ± SEM. A two-way ANOVA test was used for statistical analyses. (D) Representative data from three independent experiments showing NK cell phenotype based on CD45RA and CD45RO expression levels when in co-culture with mDC or mDC+pp65. * indicating p-values ≤ 0.05.

Figure 2

aNK cell proliferation is dependent on HLA-E/NKG2C interactions. (A) Representative gating strategy of NK cells (n = 6-10) cultured with mDC+pp65 for 14 days in the presence of either a control isotype-matched antibody IgG, or anti-HLA-E, anti-NKG2C, or anti-NKG2A blocking antibodies (5 μg/ml), thereafter assessed for their (B) (top panel) frequency and (bottom panel) number of cells, (C) viability, and (D, E) proliferation by flow cytometry. All blood donors were HCMV seropositive. (F) Percentage change (blocking – IgG) from (E) is shown. The results from 2-5 independent experiments are presented, and data are shown as representative plots or for each donor, where the donor in (A, C) is the same, and the donor in (D) is included in accumulative data in (E). Student’s T-test and Two-way ANOVA were used for statistical analyses. * indicating p-values ≤ 0.05, ** indicating p-values ≤ 0.001, *** indicating p-values ≤ 0.0001.

Figure 3

Identification of three 15-mer and 9-mer pp65-derived HLA-E-restricted epitopes. (A) NanoDSF studies to assess the thermal stability of HLA-E in complex with the following peptides: SEVENVSVNVHNPTG, DSDEELVTTERKTPR, and TSGSDSDEELVTTER. The F350/F330 was plotted against temperatures varying from 20°C to 95°C. Red dashed lines indicate the calculated melting temperatures ( Table 1 ). (B) K562 cells transfected with HLA-E*0101 were loaded with the indicated peptides overnight, washed, and analyzed for their HLA-E expression. Peptide titration from three independent experiments and one representative overlay histogram and MFI are presented out of 3 independent experiments (10 µM). (C) NanoDSF studies to assess the thermal stability of HLA-E in complex with the following peptides: SGSDSDEEL, DSDEELVTTERKTPR, VTTERKTPR, DSDEELVTT, DMDEELVLL, and DQDEELVLL. The F350/F330 was plotted against temperature varying from 20°C to 95°C. Red dashed lines indicate the calculated melting temperatures ( Table 1 ). (D) K562 cells transfected with HLA-E*0101 were loaded with the indicated peptides overnight, washed, and analyzed for their HLA-E expression. Peptide titration and one representative overlay histogram and MFI are presented out of 3 independent experiments (10 µM). Positive (UL40) and negative (no peptide) peptide controls are included in (B). A Two-way ANOVA was used for statistical analyses of cumulative data in (B, D). *Multiple comparisons were corrected by using the FDR method <0.05.

Figure 4

aNK cells recognize pp65-derived HLA-E-restricted 15-mer epitopes presented on mDC and perform recall responses. NK cells were cocultured with mDC in the absence of peptides or pulsed with the indicated peptides (10 µM) in the presence of 10 ng/ml IL-15 for 14 days. Later, NK cells were restimulated with HCMV-infected or uninfected MRC-5 cells before analysis of aNK cell function. Blood donors were either HCMV-seropositive (red lines) or -seronegative (black lines). Data are shown from three independent experiments and individual donors (n = 9). Representative plots (A) and cumulative data (B) are shown. A Two-way ANOVA test was used for statistical analyses and * indicating p-values ≤ 0.05.

Figure 5

aNK cells antigen priming is dependent on HLA-E and no other MHC molecules. NK cells (n = 3) were cultured with mDC+pp65 for 14 days in the presence of either a control isotype-matched antibody IgG, or anti-HLA-E, anti-MHC I, or anti-MHC II blocking antibodies (5 μg/ml), at (A, B) the priming phase (day 0 and 7) or at (C) the restimulation phase (day 14) and thereafter assessed for their frequency and proliferation by flow cytometry. All blood donors were HCMV seropositive. The results from two independent experiments are presented, and data are shown as representative histograms for each donor. A Two-way ANOVA was used for statistical analyses. * indicating p-values ≤ 0.05, ** indicating p-values ≤ 0.001, *** indicating p-values ≤ 0.0001.

Figure 6

Multidimensional investigations of aNK cells confirm their specific function in response to peptides. (A) Dimensional reduction opt-SNE analyses of NK cells are shown following coculture with mDC loaded with pp65405-419 (DSDEELVTTERKTPR) peptide and assessed phenotypically distinct clusters. One representative opt-SNE, out of three independent experiments is shown. (B–D) Dimensional reduction opt-SNE analyses of NK cells are shown following coculture with mDC unloaded or loaded with different peptides and assessed for the phenotype (CD57 and NKG2C) and function (Ki67 and TNFα) of aNK and cNK cell clusters. One representative out of three independent experiments is shown. (E) NK cells were cultured with mDC peptide unloaded or loaded for 14 days and assessed for the killing capacity of infected or uninfected MRC-5 cells 6 hours prior to staining. Pooled data are shown from two independent experiments (n=11). Data are shown in boxplots and statistical analysis was performed using a Two-way ANOVA test * indicating p-values ≤ 0.05, ** indicating p-values ≤ 0.01.