These data are consistent with a previous study that found that pDCs induce human blood B cell differentiation through type I IFN41 but contrast with a murine influenza challenge study that showed pDCs were dispensable to the response42, highlighting a difference between murine and human systems. in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation Pifithrin-β and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system. Antigen recognition by lymphocytes has been studied by immunologists since the discovery of antibodies and their specificities over a century ago1C4, followed by the more recent discovery of T cells and their antigen receptors in the 1960sC1980s5C7. The B cells that are responsible for forming a neutralizing antibody response develop within germinal centers (GCs) and extrafollicular regions in lymphoid organs8C11. Upon antigen presentation by antigen-presenting cells (APCs)12C15, T follicular helper (TFH) cells, and a variety of hematopoietic and non-hematopoietic cells interact and deliver signals to GC B cells for survival, proliferation, antibody affinity maturation, class-switch recombination and differentiation16,17. Almost all of these interactions have been elucidated through in vivo studies of inbred mice. While these have produced a wealth of important information8,18C20, the lack of a system that replicates the essential features of human adaptive immunity, such as affinity maturation and class switching, and the effects of adjuvants, leaves many mechanistic aspects inaccessible. This is especially important for vaccine testing since many candidates that worked in animal models ultimately fail in human trials21C24, suggesting that genetic and environmental differences among species are important considerations in vaccine development. Many in vitro systems rely on isolation of small chunks or Pifithrin-β slices of a tissue sample to prepare explant cultures25C31. Explant methods, although useful for studying individual aspects of infection and immunity30,32C34, typically Pifithrin-β do not maintain cell composition for very long (3C4 d), nor do they capture all of the features of an adaptive immune response. Although bioreactor, chip-based and other specialized in vitro differentiation systems show promise, they too have not been able to replicate the complexities of adaptive immunity. We decided to take advantage of the widespread availability of human tonsils, lymphoid organs that are easily procured from tonsillectomy surgeries as discarded tissue, to develop an accessible system that replicates an antigen-specific adaptive immune response to a vaccine and supports key aspects of adaptive immunity. Results Preparation of immune organoids from tonsils and other lymphoid tissues. Over 20 years ago, Owen and Jenkinson demonstrated that dissociated murine thymic cells could reassociate in culture and recapitulate major aspects of T cell selection35,36. We applied a similar approach to develop human tonsil cultures with dissociated cells that reaggregate in culture (Fig. 1a; see Supplementary Table 1 for tissue donor characteristics). For organoid preparation, frozen single-cell suspensions from tonsil tissues were thawed and plated at high density into the wells of permeable membrane plates (commonly known as Transwells) along with the antigen of interest. After several days in culture, reaggregated regions of clustered cells were visible (Fig. 1a). We assessed the cell composition of the reaggregated cultures after 7 d in the presence or absence of antigen and NFE1 our optimized culture conditions (Methods) sustained appropriate tonsil cell composition (Fig. 1b). We used influenza vaccines and viruses as model antigens since much is already known about the features of the human being influenza response in vivo. Upon activation with live attenuated influenza vaccine (LAIV), there were notable raises in B cell differentiation and a more structured tradition morphology developed, suggesting additional activity in response to this immunogen. Open in a separate windowpane Fig. 1 | Adaptive immune reactions in tonsil organoids.a, Workflow for cells disruption and tradition preparation with representative stereoscope images from day time-5 unstimulated or LAIV-stimulated replicate ethnicities. Light areas are areas of high cell denseness. b, Cell composition of immune cell types in freshly isolated, freezing and revived day time-0 tonsil cells, day time-7 unstimulated ethnicities and day time-7 LAIV-stimulated ethnicities. Frequencies were determined by circulation cytometry, and plotted ideals are the mean s.e.m. (= 5.