PROJECT SUMMARY Aged individuals, who are often at higher risk of fatality in life-threatening infectious diseases, do not form high- quality antibodies against new infections. Humoral immunity against infections depends on the germinal center (GC) differentiation process in the B cell follicles of lymph nodes. In GCs, naïve B cells rapidly proliferate in response to T cell-dependent antigens and somatically mutate into high-affinity antibody-secreting cells, i.e., plasma cells. A significant concern is that with aging, B cells exhibit a decreased expansion of B cells and GC reaction in response to antigen. However, it is unclear whether this is solely due to immune senescence and a defect in B cells or the follicular T helper cell (TFH), and whether the lymphoid microenvironment of B cell follicles plays a role. B cells in young mice assume heterogeneous cell fates upon stimulation, with only a fraction differentiating into antibody-secreting cells (ASC), however, this phenomenon remains unknown in B cells from old mice. Notably, plasma cell differentiation in young B cells is controlled by multiple cell division-coupled epigenetic programs, which also remains understudied in old B cells. Because aged mice cannot generate sufficient GCs to provide insight into ASC fate and epigenomic remodeling of GC B cells, necessitating the development of a tissue-engineered model of aged lymphoid tissues. The long-term goals of this R01 are 1) to develop an ex vivo “aged B cell follicle” organoid technology capable of providing lymphoid microenvironment signals in a plug-and-play manner to induce early GC programming of aged B cells from mice and human B cells, therefore enabling the study of plasma cell fate and regulation of the epigenome of B cells; and 2) to identify checkpoint targets that can be suppressed to boost GC response in aged B cells. PI has demonstrated that GCs phenotype could be generated in organoids using B cells from >2-yr old mice when differentiated under young lymphoid microenvironment conditions, however to a lesser extent than young B cells. However, true differentiation fate of aged B cells ex vivo can only be realized in a microenvironment representative of old lymph nodes.The R01 is highly significant and innovative because it will 1) establish lymphoid microenvironment of aged lymph nodes and engineer a hydrogel-based “aged” immune organoid to study vaccine and infection responses, 2) enable control over the ex vivo B cell differentiation of aged B cells in ex vivo cultures and generate antigen-specific antibodies against infections, 3) define the cell division kinetics of B cell differentiation and identify the molecular and epigenetic trajectories of aged B cell fate and ASC formation, and 4) identify checkpoint targets to accelerate GCs in aging.

Research Narrative Elderly people have a reduced ability towards brisk and sustained responses to new antigens, causes of which are poorly understood but can be attributed to a defective T cell and B cell interaction. Consequently, animal models are limited in their ability to generate antibody responses, necessitating the development of a tissue- engineered model of an aged lymph node. The proposed work will generate aged immune organoids that could serve as a rapid testing platform to identify non- or poor-responder aged individuals to infectious disease vaccines.