Antibodies are routinely used as therapeutic agents to fight a wide range of disorders and cancers. Humoral immunity depends on the germinal center (GC) differentiation process in the B cell follicles of spleen and lymph nodes. In GCs, somatically mutated high-affinity antibody secreting cells (memory B cells and plasma cells) are generated from naïve B cells in response to T cell-dependent antigen. Recent in vivo studies have uncovered crucial signals (such as CD40 ligand (CD40L) and B cell activation factor (BAFF)) that are required for the formation and maintenance of GCs and for the selection of high-affinity antibodies, in vivo. Yet, substantial number of GC B cells cannot be derived ex vivo using 2D cultures and to date, there are no reports to modulate the kinetics of GC reaction. Here we describe an <i>ex vivo</i> engineered 3D immune organ, i.e. a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymph node that provides the basis to induce an accelerated GC reaction by continuously providing extracellular matrix (ECM) and cell–cell signals to naïve B cells. To provide T cell-mediated CD40L and follicular dendritic cell-mediated BAFF signaling, BALB/c 3T3 fibroblasts were stably transfected with both CD40L and BAFF (called hereafter 40LB). We determined the role of 40LBs in 3D organoids and noted that the presence of 40,000 40LBs in hydrogels resulted in maximum proliferation of CD19+ B cells over 4 days compared with other seeding densities. We observed a partial but significant decrease in CD19+ B cells when blocked with a small molecule inhibitor of αvβ3 integrin binding to RGD. Importantly, of these CD19+ cells, the organoid culture with B+40LB at 40,000 and 60,000 40LBs induced ~100-fold increase in B cells expressing the GC-specific marker GL7 within 4 days (and a 55-fold increase in CD19+GL7+Fas+ GC-like B cells) compared to a 10-fold increase in classic 2D B+40LB co-culture . We observed robust immunoglobulin class switching from IgM to IgG1, a hallmark of GC in vivo, where the number of CD19+IgG1+ B cells increased significantly from 185 cells on day 0 to 7120 cells on day 6 in 3D B+40LB organoids with 40,000 40LBs. In conclusion, our immune organoid provides a simple strategy for recapitulating the functional aspects of a lymphoid tissue to induce accelerated GC-like reaction <i>ex vivo</i> with maintained long-term cell survival by simply adjusting the extracellular signal.

Ex vivo engineered immune organoids for controlled differentiation of germinal center-like B cells

253rd National Meeting (2017)

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