Research Summary Humoral immunity against infections requires 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 antigen presentation and somatically mutate into high-affinity antibody-secreting cells (plasma cells). Despite a deep understanding of immunology, numerous challenges exist in understanding disease transmission, pathology, and developing new vaccines against life-threatening infectious diseases. These include a limited understanding of immune correlates of protection, identification of viable vaccine candidates, and off-target effects that must be evaluated in staged clinical trials. Significant pre-clinical research depends on mice, non-human primates, or other animal models. However, to date, no ex vivo technology has shown evidence of hallmark GC functional characteristics and the ability to generate high-affinity, long-lived human plasma and memory B cells. Existing technologies, such as 2D B and T cell co-cultures lead to inefficient GC responses, and human tonsil transwell aggregates are short-lived. The long-term goal of this project is to microenvironment that sustains the survival and differentiation of human peripheral blood mononuclear cells (PBMC)-derived B cells into bona fide human GCs as well as long-lived plasma cells that engraft in vivo. The central hypothesis is that a synthetic hydrogel-based presentation of cellular and extracellular matrix (ECM) microenvironmental cues that mimic lymphoid tissues will induce differentiation of human PBMC-derived naïve B cells into GC B cells and memory cells and prolong the survival of plasma cells ex vivo and in vivo. The central objective of this project is to understand how the lymphoid microenvironment regulates the ex vivo differentiation kinetics of human B cells and generate ex vivo long-lived plasma cells that engraft in vivo. The specific aims are: Aim 1: Bioengineer PEG-4MAL-based lymphoid organoids with human B, T, and stromal cells to recapitulate temporal kinetics of decision-making of human GC B cells against antigens and adjuvants. Aim 2: Integrate engineered GCs with forced affinity maturation to isolate high-affinity B cells. Aim 3: Evaluate the in vivo engraftment of engineered human B cells delivered using injectable gels. This renewal project is highly significant and innovative because it will 1) engineer a hydrogel-based human immune organoid, inspired by lymphoid tissues, for generating antigen-specific GC B cells, 2) enable forced affinity maturation ex vivo, and 3) examine in vivo engraftment of ex vivo engineered B cells.

Research Narrative Despite our understanding of immunology, numerous challenges exist in understanding disease transmission, pathology, and developing new vaccines against life-threatening infectious diseases. These include a limited understanding of immune correlates of protection, identification of viable vaccine candidates, and off- target effects that must be evaluated in staged clinical trials. This project will develop a hydrogel-based human immune organoid, inspired by lymphoid tissues, for generating antigen-specific GC B cells and examine in vivo engraftment of ex vivo engineered B cells.