Background/Purpose: Germinal centers (GC) are specialized lymphoid structures found within the B cell follicles of secondary lymphoid tissue formed following infection or immunization. They are vital for adaptive immune responses; a stromal cell network within GC governs antigen delivery and cell trafficking, while follicular helper T cells offer cognate interactions and cytokine stimulation to B cells. In chronic inflammatory settings like Sjögren’s syndrome and rheumatoid arthritis (RA), ectopic GC structures in salivary glands and synovial tissue respectively drive antigen-driven B cell autoimmune responses. Targeting B cell differentiation and autoantibody production in GC presents therapeutic avenues for autoimmune diseases. To develop a human 3D culture system that mimics the GC lymphoid structure and functions. This will permit exploration of the mechanisms underlying GC biology and to limit the need for animal experimentation.

Methods: Optimized ratios of human B-cells, irradiated T-cells, T-follicular helper cells, dendritic cells, and human tonsil-derived lymphoid stromal cells were cocultured in a synthetic extracellular matrix composed of matrigel and collagen-infused hydrogels. These 3D cocultures were activated with 0.15 ng/mL Staphylococcal Enterotoxin B (SEB).  At different time-points, the cultures were harvested, and the changing phenotype and function of the cell populations were evaluated. The composition of the cell aggregates was examined using an EVOS microscope, changes in the stage B cell differentiation were analyzed using flow cytometry, and the immunoglobulin levels in the culture supernatants were analyzed by ELISA.  The effects of JAK, BAFF and NIK inhibitors were evaluated.

Results: Upon SEB stimulation proliferation of B and T cells was observed. Clusters of B and T cells with the stromal cells indicate the cells were organizing into structures than resemble GCs.  Over time, we observed the differentiation of naive B-cells (CD19+,IgD-) into plasma cells (CD19⁺,IgD⁺, CD38^hi) which peaked on day 6 making up 6-15% of the CD19+ cells. This was accompanied by class-switch recombination and secretion of IgA and IgG. Additionally, GC specific B-cell populations, including centrocytes (Dark Zone; CXCR4^hiCD83^lowCD86^low), and centroblast (Light Zone; CXCR4^lowCD83^hiCD86^hi ) populations were observed.  The survival of B cells was far superior in culture system in the presence of Matrigel. To varying extents, the JAK, BAFF and NIK inhibitors all exhibited dose-dependent inhibition of aggregate formation, plasma cell differentiation, and Ig production. Interestingly, the NIK inhibitor was more effective at blocking plasma cell differentiation in the 3D model compared to simple B cell only differentiation assays. This may reflect the broader role that NIK plays in the modulating both B cell and T cell biology which is captured within the more complex 3D system.

Conclusion: We present a well-characterized human 3D lymphoid model that resembles the human GC, facilitating enhanced B-cell survival, proliferation, differentiation, and antibody production. This in vitro 3D model system provides the opportunity to explore pathways, targets and drug modalities which may impact GC biology.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/establishment-of-a-human-3d-in-vitro-lymphoid-model-to-evaluate-germinal-center-biology/