Hamilton, S. E. | bioRxiv Science

bioRxiv2023

IL-15 complex-induced IL-10 enhances Plasmodium-specific CD4+ Tfh differentiation and antibody production

Malaria, which results from infection with Plasmodium parasites, remains a major public health problem. While humans do not develop long-lived, sterilizing immunity, protection against symptomatic disease develops after repeated exposure to Plasmodium parasites and correlates with the acquisition of humoral immunity. Despite the established role antibodies play in protection from malaria disease, dysregulated inflammation is thought to contribute to the sub-optimal immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously demonstrated that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15R-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death. Using a novel MHC class II tetramer to identify PbA-specific CD4+ T cells, herein we demonstrate that IL-15C treatment enhances Tfh differentiation. Moreover, genetic deletion of NK cell-derived IL-10 or IL-10R expression on T cells prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment results in increased anti-PbA IgG antibody levels and improves survival following reinfection. Overall, these data demonstrate that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the dysregulated inflammation during Plasmodium infection to promote Tfh differentiation and antibody generation, correlating with improved survival from reinfection. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.

immunology↗

bioRxiv2022

Ablation of SYK kinase from primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production

Cytomegalovirus (CMV) infection alters natural killer (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 Fc receptor {gamma} chain (Gene: FCER1G, FcR{gamma}), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (Fc{gamma}RIIIA) signaling apparatus, which includes FcR{gamma}, CD3{zeta}, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcR{gamma} chain caused a modest increase in TNF production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating 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 FcR{gamma} or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3{zeta} chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.

immunology↗