Immune responses to SARS-CoV-2: characterization of NK cells following vaccination and virus-specific T cells in the nasal mucosa

The global COVID-19 pandemic has highlighted the urgent need to understand the mechanisms of protective immunity against SARS-CoV-2. While humoral responses have been investigated by various research groups and several clinical trials, important properties of innate and mucosal immunity in shaping protection have remained underexplored. Indeed, emerging evidence has shown the involvement of Natural Killer (NK) cells in both the induction and effector phases of vaccine-induced immunity. Similarly, tissue-resident T cells within the nasal mucosa represent key players in antiviral defense that remained incompletely defined in the context of SARS-CoV-2 infection and vaccination. To investigate these aspects, we conducted two studies focusing on systemic NK cell responses and local nasal T cell immunity. First, we characterized the phenotypic and functional dynamics of NK cells following BNT162b2 mRNA vaccination in SARS-CoV-2 unexposed healthy workers. Peripheral blood samples were collected at baseline and 20 days after the first vaccine dose, as well as at 30 and 240 days following the booster. NK cell responses were assessed using overlapping peptides covering the RBD region of the ancestral SARS-CoV-2 spike protein. Our analyses revealed enhanced NK cell activity upon RBD re-stimulation of PBMC in the presence of IL-12 and IL-18 several months after booster vaccination. Moreover, we observed a decline in the expression frequency of the inhibitory receptor NKG2A, whereas the frequency of the activating receptor NKG2C was increased in cytomegalovirus (CMV)-positive individuals. Notably, RBD peptides did not directly activate purified NK cells, suggesting that other immune cells are required to mediate this activation. Indeed, IFNγ+ NK cells positively correlated with RBD-specific IFNγ+ T cells, supporting a cooperative interaction between innate and adaptive immune cells in response to SARS-CoV-2 vaccination. In the second study, we explored the immune cell composition of the nasal cavity and the role of tissue-resident T cells in local viral control. Paired blood and nasal swab samples were longitudinally collected from healthy donors during six consecutive months. We found that CD8⁺ T cells represent the predominant adaptive immune subset at the level of the upper nasal turbinate. Their transcriptomic profiling revealed that nasal CD8⁺ T cells failed to upregulate STAT1 upon TCR stimulation, a feature that may facilitate clonal expansion despite the antiproliferative effects of IFN signaling that characterize the early phase of an infection. Additionally, these cells exhibited a cytotoxic, Th1-like profile, enriched in tissue residency markers, but lacked TCF7 expression, suggesting a limited self-renewal capacity. In terms of antigen specificity, we observed that only viruses with local respiratory tropism were able to induce the presence of virus-specific CD8⁺ T cells in the nasal mucosa. Indeed, only SARS-CoV-2- and influenza A-specific CD8⁺ T cells were detectable, whereas HCMV-specific CD8⁺ T cells, representative of a systemic, non-respiratory infection, were undetectable. However, their persistence over time in the nasal cavity appeared to be linked to repeated viral encounters. Taken together, our findings provide important insights that may guide the development of novel vaccination strategies, particularly in the context of future emerging infectious diseases. Specifically, they highlight the contribution of NK cells to vaccine-induced immunity against SARS-CoV-2 and emphasize the importance of inducing nasal-resident CD8⁺ T cells in shaping long-lasting immune protection.