419 Utilization of memory NKG2CHi NK cells to target viral antigen peptides demonstrates a novel cellular immunotherapy strategy

Background Natural Killer (NK) cells play a crucial role in innate immune responses against virally infected and transformed cells. The management of human cytomegalovirus (HCMV) infections relies on a subset of NKG2C+ NK cells. The NKG2C/CD94 receptor complex recognize and respond to HCMV-infected cells expressing HLA-E loaded with viral gpUL40 antigen peptide. Upon activation, NKG2C+ NK cells mount a response akin to adaptive immunity and establish a reservoir of memory cells. As a result, individuals possess significantly elevated levels of long-lived memory NKG2C+ NK cells that persist within the host. The long-term locality of memory NK cells and their developmental and functional outcomes following incubation with viral neoantigens remains largely unknown. In our current study, we find that NK cell effector functions can be induced by viral antigens and we identify a unique subset of Memory NKG2CHi NK cells within the human spleen. Methods Eight healthy adult human spleens were obtained from eight donors. Donor median age was 59 [IQR 48.5–56.5], 50% (n=4) were identified as female, 50% (n=4) were identified as males. Spleens were provided by the Versiti Organ Donor Center of Wisconsin and were processed to a single cell suspension. CD7+ CD3e- CD14- CD19- CD20- NK cells were isolated, using the BD FACSAria sorter. NK cells were then incubated with the following viral antigen peptides VMAPRTLFL, VMAPRTLIL, and VMAPQSLLL. Peptides were utilized and loaded on HLA-E*01:03 BV421-conjugated tetramers that were made by the National Institutes of Health Tetramer Core Facility, Emory University. Results Our studies demonstrate Memory NKG2CHi NK cells isolated from the human spleen elicit a strong effector function response to the viral antigen peptides loaded on HLA-E tetramers. We find that Memory NKG2CHi NK cells have significantly elevated levels of IL-7R and cyto-CD3e compared to immature NK cells. In addition, we find that immature NK cells can develop a memory-like phenotype and function following incubation with the viral antigen peptides. These findings could implicate a potential methodology to generate Memory NKG2CHi NK cells. Conclusions Here, we demonstrate that viral antigen peptides can induce the formation of Memory NKG2CHi NK cells that display a distinct phenotypical profile. These findings can influence the future isolation and application of Memory NK cells in cellular immunotherapies. Acknowledgements We express sincere gratitude to the organ donors and their families for their generous gift of life and knowledge through organ donation. We also extend our appreciation to Lauren Patzman, and the remainder of the Versiti Organ Donor Center team for their role in subject recruitment and sample processing. We thank the Terhune and Hudson labs for their feedback. This work was supported by the National Center for Advancing Translational Sciences, National Institutes of Health award number TL1TR001437 (M.K), the National Institute of Allergy and Infectious Diseases division of the National Institutes of Health under award number R01AI083281 (S.S.T.), and R01 AI102893 and NCI R01 CA179363 (S.M). Additional support obtained from HRHM Program of MACC Fund (S.M.), Nicholas Family Foundation (S.M.), Gardetto Family’s Everyday Good Foundation Inc. (S.M.), and Nan Gardetto Endowed Chair (S.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.