Phenotype and function of IL-10 producing NK cells in individuals with malaria experience.

Plasmodium falciparum infection can trigger high levels of inflammation that lead to fever and sometimes severe disease. People living in malaria-endemic areas gradually develop resistance to symptomatic malaria and control both parasite numbers and the inflammatory response. We previously found that adaptive natural killer (NK) cells correlate with reduced parasite load and protection from symptoms. We also previously found that murine NK cell production of IL-10 can protect mice from experimental cerebral malaria. Human NK cells can also secrete IL-10, but it was unknown what NK cell subsets produce IL-10 and if this is affected by malaria experience. We hypothesize that NK cell immunoregulation may lower inflammation and reduce fever induction. Here, we show that NK cells from subjects with malaria experience make significantly more IL-10 than subjects with no malaria experience. We then determined the proportions of NK cells that are cytotoxic and produce interferon gamma and/or IL-10 and identified a signature of adaptive and checkpoint molecules on IL-10-producing NK cells. Lastly, we find that co-culture with primary monocytes, Plasmodium -infected RBCs, and antibody induces IL-10 production by NK cells. These data suggest that NK cells may contribute to protection from malaria symptoms via IL-10 production.

Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C).

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease.

Ablation of SYK Kinase from Expanded Primary Human NK Cells via CRISPR/Cas9 Enhances Cytotoxicity and Cytokine Production.

CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses.

The magnitude of SARS-CoV-2-specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.