Tonsil explants as a human in vitro model to study vaccine responses.

Introduction: Vaccination is one of the most effective infection prevention strategies. Viruses with high mutation rates -such as influenza- escape vaccine-induced immunity and represent significant challenges to vaccine design. Influenza vaccine strain selection is based on circulating strains and immunogenicity testing in animal models with limited predictive outcomes for vaccine effectiveness in humans. Methods: We developed a human in vitro vaccination model using human tonsil tissue explants cultured in 3D perfusion bioreactors to be utilized as a platform to test and improve vaccines. Results: Tonsils cultured in bioreactors showed higher viability, metabolic activity, and more robust immune responses than those in static cultures. The in vitro vaccination system responded to various premanufactured vaccines, protein antigens, and antigen combinations. In particular, a multivalent in vitro immunization with three phylogenetically distant H3N2 influenza strains showed evidence for broader B cell activation and induced higher antibody cross-reactivity than combinations with more related strains. Moreover, we demonstrate the capacity of our in vitro model to generate de novo humoral immune responses to a model antigen. Discussion: Perfusion-cultured tonsil tissue may be a valuable human in vitro model for immunology research with potential application in vaccine candidate selection.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.