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

Abstract

IntroductionHuman 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.MethodsWe 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.ResultsHere, 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.DiscussionThese 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.

CXCR2-mediated recruitment of adaptive NK cells with NKG2C/HLA-E dependent antigen-specific memory enhances tumor killing in ovarian cancer

“…Our hypothesis suggests that these molecules play a pivotal role in discerning between self and nonself-peptides. On the other hand, we found that HLA-E and NKG2C are important for the recall responses in aNK cells, which has been earlier reported by us and others in defense against viruses (12,47). Moreover, we found e.g., a greater TNFα secretion by aNK cells following a secondary stimulation with specific tumor cells, which facilitates the activation of the transcription factor AP-1 that is activated both in antigen specific T cells and clonal expanded NK cells (46,48).…”

“…However, the interaction of cDC1 was more restricted to aNK cell (Figure 2E). Exploring the top ligand-receptor underlying cDC1-aNK cell interaction, identified HLA-E-KLRC2 which may play a role in antigen presentation as was demonstrated in our previous study (12). Other significant interactions were revealed including CXCL1-CXCR2, HLA-C-KIR2DL3, CLEC2B-KLRF1, which are involved in chemotaxis, NK cell education, and antigen presentation and cytotoxicity, respectively.…”

“…Recently, we demonstrated that aNK cells, when primed by viral peptide-loaded DCs, exhibit antigen-specific responses with the capability of immune memory generation (12). In this study, we sought to examine the hypothesis that aNK cells develop antigenspecific memory in response to desmoplastic tumors.…”

“…Certainly, these aNK cells exhibited the ability to initiate recall responses upon secondary stimulation with viable, virally infected cells, distinguishing them from conventional (c)NK cells (12). In this study, we sought to investigate whether aNK cells can establish antigen-specific memory responses to tumors and elucidate their role in desmoplastic tumors as a potential immunotherapy agent.…”