Characterizing the Functional Profile of NK Cells Engaged by Antibodies

Abstract

The immune system is a complex network of cells working together to protect us against infections and malignancies. Among its components, natural killer (NK) cells are innate lymphocytes capable of identifying and eliminating virally infected or malignant cells without prior sensitization. NK cells also utilize antibodies to recognize additional targets through a process known as antibody-dependent cellular cytotoxicity (ADCC).Despite the versatility of NK cells, key gaps remain in our understanding of NK cell heterogeneity and function during active immune responses. First, most studies focus on NK cells in steady-state conditions, which may not reflect their transcriptomic profiles under activation. Furthermore, compared to human NK cells, the heterogeneity and functional dynamics of non-human primate (NHP) NK cells are poorly understood. This represents a significant knowledge gap, given that NHPs, such as rhesus macaques, serve as crucial preclinical animal models for studying vaccines and immunotherapies, given their immunological similarity to humans. However, despite the similarity in physiology, there are several cases where vaccine efficacy observed in preclinical trials involving NHPs did not translate well into the efficacies in clinical trials, suggesting the underlying differences in immune responses that remain unexplored. My work aims to address the potential difference that exists between rhesus and human NK cells. To achieve this goal, I first characterized rhesus macaque NK cells using single-cell RNA sequencing during ADCC. Using CD107a as a surrogate marker for NK cells actively engaged in ADCC, I identified distinct transcriptomic profiles associated with functional heterogeneity. Clustering analyses revealed that in addition to inducing ADCC, antibody engagement also resulted in the potent production of both CC chemokines CCL3 and CCL4L1 as well as C chemokine XCL1 by a subset of degranulating cells. Of note, we also observed a small subset of non-degranulating NK cells that are poorly cytotoxic yet upregulated chemokine production. Flow cytometry and intracellular staining confirmed these results, demonstrating that degranulating NK cells initiated chemokine production within three hours of activation, with secretion increasing over time. Moreover, engagement of CD16 with non-ADCC mediating antibodies induced strong degranulation but minimal chemokine production, underscoring the importance of receptor-ligand interactions in shaping functional outcomes. Last, to explore potential species-specific differences, I conducted pilot studies on human NK cells. Similar chemokine-producing subsets were observed following antibody engagement, suggesting conserved functional mechanisms. However, I identified notable differences between rhesus and human NK cells. For instance, human NK cells exhibited variable CCL5 expression levels across clusters, while rhesus NK cells displayed uniformly high CCL5 expression. Additionally, the expression patterns of CD16 following antibody engagement differed markedly between the two species. These differences likely contribute to observed discrepancies in vaccine efficacy and immune responses between preclinical models and humans. In summary, this study provides a comprehensive characterization of rhesus macaque NK cells during ADCC, identifying novel chemokine-producing subsets with potential roles in antiviral immunity. Comparative analyses with human NK cells highlight critical species-specific differences, offering insights into NK cell-mediated protective responses and their implications for vaccine development and immunoprophylaxis.