The variegated expression of the KIR family of class I MHC receptors generates specialized natural killer (NK) cells capable of allele-specific HLA recognition. Understanding the mechanism of KIR gene activation will lead to improved methods for the generation of fully functional NK cells. A central RUNX-binding site in the KIR proximal promoter is required for gene activation. RUNX proteins recruit ten-eleven translocation (TET) proteins that generate 5-hydroxymethylcytosine (5hmC) and drive DNA demethylation. Assessment of 5-methylcytosine (5mC) and 5hmC residues at four stages of NK cell development reveals deposition of 5hmC primarily in a CREB site next to the RUNX site at the CD56Bright stage but not the subsequent CD56Dim stage representing fully mature NK cells. KIR promoter demethylation is delayed relative to other lineage-associated genes, indicating a high threshold for KIR gene demethylation in developing NK cells, and a window of opportunity for RUNX/TET-dependent KIR gene activation in CD56Bright NK cells.

Newly emerging influenza viruses pose a constant threat as they encounter a population lacking neutralizing antibodies against the new strain. However, cross-reactive non-neutralizing antibodies (nnABs) may be present and help mitigate disease symptoms through various effector mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Although nnABs to influenza virus have received more attention lately, little information is available on their age-related prevalence, steady-state levels, functional properties, and changes in these parameters over time. Using longitudinal samples from adolescents, adults, and older adults, collected before and after the 2009 swine flu pandemic, we comprehensively characterized the specificity and functionality of nnAB responses against H1N1 pandemic 2009 (H1N1pdm09) virus. Remarkably, all participants exhibited cross-reactive antibodies to this virus before having encountered it through infection or vaccination, with the highest baseline levels observed in older adults. The levels of these IgG antibodies showed a strong correlation with engagement of fragment crystallizable γ receptor IIIa (FcγRIIIa) and NK cell activation, both of which were notably lower in adolescents than in adults and older adults. Without infection or vaccination, average amounts of H1N1pdm09-reactive antibodies remained relatively stable on the population level over the 5-year study period. However, on an individual level, substantial increases and decreases occurred. H1N1pdm09 infection or vaccination significantly enhanced specific antibody levels and the FcγRIIIa-engaging capacity of these antibodies in all age groups. ADCC-mediating antibodies increased, however, only in adolescents, reaching the same level as observed in the adult groups. Taken together, our results demonstrate the presence of cross-reactive, non-neutralizing, functional, and boostable antibodies against a never-encountered influenza virus strain across all age groups. These antibodies might contribute to protection from severe disease. Accordingly, in case of a newly emerging influenza virus, their further enhancement by vaccination could be beneficial as an immediate protective measure before a strain-specific vaccine becomes available.

Non-Hodgkin lymphoma (NHL), predominantly B cell lymphomas (B-NHL), is currently treated with chemotherapy combined with rituximab (RTX), an anti-CD20 monoclonal antibody. Despite substantial therapeutic advances, treatment resistance and disease relapse continue to affect a significant fraction of patients. The mechanisms by which the tumour microenvironment and other factors influence RTX efficacy are not fully elucidated. Herein, we hypothesized that CD20+ extracellular vesicles (EVs) shed by B cell lymphomas are recognized by RTX forming immune complexes that modulate natural killer (NK) cell activity via Fcγ receptor (FcγR) interactions. EVs isolated from lymph node explants and plasma samples of B-NHL patients contained abundant CD20+ vesicles, which were particularly enriched in advanced disease. RTX specifically bound CD20 on these EVs, generating EV-RTX immune complexes. Functional studies employing EVs from a B-NHL cell line and from patient-derived samples demonstrated that, whereas EVs suppressed NK cell activation and cytotoxicity, EV-RTX immune complexes reversed this inhibitory effect and enhanced NK cell effector functions. Indeed, EV-RTX immune complexes specifically triggered FcγRIIIa-dependent NK cell activation, evidenced by increased Syk phosphorylation, CD69 expression, and enhanced lytic activity against target cells. Our findings uncover a previously unrecognized mechanism by which RTX, through the formation of immune complexes with CD20+ EVs, promotes NK cell activation and may enhance therapeutic efficacy. More broadly, these results demonstrate that antibody binding can endow EVs with novel immunomodulatory properties, revealing a potential mechanism by which therapeutic antibodies reshape EV function and influence anti-tumour immunity.