Memory NKG2C+ nk Cells Undergo Robust Expansion and Exhibit Strong Effector Functions upon Re-Exposure to the HCMV gpUL40 Antigen Peptide

Background: Natural Killer (NK) cells play a crucial role in the immediate immune response against virally infected and transformed cells. Managing human cytomegalovirus (HCMV) infections relies on a subset of NKG2C⁺ NK cells. The NKG2C/CD94 receptor complex identifies and responds to HCMV-infected cells expressing HLA-E loaded with viral gpUL40 antigen peptide. When activated, NKG2C⁺ NK cells exhibit a response similar to adaptive immunity and establish a reservoir of memory cells. As a result, HCMV⁺ individuals have significantly elevated levels of long-lived Memory NKG2C⁺ NK cells that persist within the host. The long-term residency of memory NK cells and their developmental and functional outcomes following re-exposure to the HCMV gpUL40 antigen remains largely unknown. In our current study, we identify a unique subset of Memory NKG2C⁺ NK cells within the human spleen that expand and increase their effector function capabilities upon re-exposure to the HCMV gpUL40 antigen peptide.

Methods & Results: Human spleens were obtained from healthy adult donors, both HCMV⁺ and HCMV^-. These spleens were provided by the Versiti Organ Donor Center of Wisconsin and tissues were processed into single-cell suspensions. NKG2C⁺ NK cells were isolated and then incubated with viral antigen peptides VMAPRTLFL, VMAPRTLIL, VMAPQSLLL and the human self-peptide ALALVRMLI. The peptides VMAPRTLFL, VMAPRTLIL, and VMAPQSLLL haven been previously identified as clinical isolates of the HCMV gpUL40_15-23 peptide. The human self-peptide ALALVRMLI derived from the ATP-binding cassette transporter multidrug resistance-associated protein 7 (MRP7), was used as a control, as previous reports have demonstrated it inhibits NK cell effector functions. These peptides were loaded on HLA-E*01:03 BV421-conjugated tetramers produced by the National Institutes of Health Tetramer Core Facility.

We observed that tetramer staining of splenic NKG2C⁺ NK cells revealed positive binding of all four HLA-E tetramer/peptide complexes, with HLA-E_VMAPRTLFL showing the highest proportion of tetramer-positive NKG2C⁺ cells. To verify specificity to the NKG2C receptor, we performed NKG2C receptor blockage prior to HLA-E tetramer staining and found a significant reduction in tetramer-positive NKG2C⁺ NK cells. We suspected the remaining tetramer-positive cells were NKG2C⁺ NKG2A⁺ NK cells, as this subset constitutes a small percentage of the total NKG2C⁺ NK cell population. Our findings confirmed that nearly all gated NKG2C⁺NKG2A⁺ NK cells were positive for the individual HLA-E tetramer/peptide complexes, suggesting that the NKG2A receptor binds with lower affinity. Notably, the HLA-E_ALALVRMLI tetramer/peptide complex, known to engage the NKG2A receptor with higher affinity, had the highest proportion of tetramer-positive cells. Collectively, these findings indicate our designed HLA-E tetramer/peptide complexes can specifically bind to NKG2C⁺ NK cells with varying affinities, and that a small subset of NKG2C⁺NKG2A⁺ NK cells can also bind to these HLA-E tetramer/peptide complexes with lower affinity via the NKG2A/CD94 receptor complex.

To investigate if Memory NKG2C⁺ NK cells undergo antigen-specific expansion, we performed HLA-E tetramer incubation assays using individual HLA-E tetramer/peptide complexes. Importantly, HLA-E_VMAPRTLFL resulted in the most significant expansion in the total proportion of NKG2C⁺ NK cells from both the HCMV⁺ and HCMV^- donors, with a greater increase in the HCMV⁺ donors. In addition, Memory NKG2C⁺ NK cells displayed a strong recall response to the HCMV gpUL40 antigen, resulting in significantly higher total frequency and production of IFN-g. In contrast, naïve NKG2C⁺ NK cells from HCMV- donors developed a memory-like phenotype following incubation with the HCMV gpUL40 antigen. Together, our studies reveal Memory NKG2C⁺ NK cells isolated from the human spleen of HCMV⁺ donors mount a strong recall response to the HCMV gpUL40 antigen, and that naïve NKG2C⁺ NK cells can develop a memory-like phenotype and functional response following exposure to the HCMV gpUL40 antigen.

Conclusion: Here, we find a distinct subset of long-lived Memory NKG2C⁺ NK cells that persist within the human spleen and demonstrate that the HCMV gpUL40 antigen can induce the formation and expansion of these cells. These findings can contribute to the future isolation and design of Memory NKG2C⁺ NK cell immunotherapies.