The EZH2 histone methyltransferase is required for B cells to form germinal centers (GC). Here we show that EZH2 mediates GC formation through repression of cyclin-dependent kinase inhibitor CDKN1A (p21Cip1). Deletion of Cdkn1a rescues the GC reaction in Ezh2 −/− mice. Using a 3D B cell follicular organoid system that mimics the GC reaction, we show that depletion of EZH2 suppresses G1 to S phase transition of GC B cells in a Cdkn1a-dependent manner. GC B cells of Cdkn1a −/− Ezh2 −/− mice have high levels of phospho-Rb, indicating that loss of Cdkn1a enables progression of cell cycle. Moreover, the transcription factor E2F1 induces EZH2 during the GC reaction. E2f1 −/− mice manifest impaired GC responses, which is rescued by restoring EZH2 expression, thus defining a positive feedback loop in which EZH2 controls GC B cell proliferation by suppressing CDKN1A, enabling cell cycle progression with a concomitant phosphorylation of Rb and release of E2F1.
Ex vivo engineered three-dimensional organotypic cultures have enabled the real-time study and control of biological functioning of mammalian tissues. Organs of broad interest where its architectural, cellular, and molecular complexity has prevented progress in ex vivo engineering are the secondary immune organs. Ex vivo immune organs can enable mechanistic understanding of the immune system and more importantly, accelerate the translation of immunotherapies as well as a deeper understanding of the mechanisms that lead to their malignant transformation into a variety of B and T cell malignancies. However, till date, no modular ex vivo immune organ has been developed with an ability to control the rate of immune reaction through tunable design parameter. Here we describe a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymphoid tissue that provides the basis to induce an accelerated germinal center (GC) reaction by continuously providing extracellular matrix (ECM) and cell–cell signals to naïve B cells. Compared to existing co-cultures, immune organoids provide a control over primary B cell proliferation with ~100-fold higher and rapid differentiation to the GC phenotype with robust antibody class switching.
Induction of B-cell immunity against infection depends on the initiation of the germinal center (GC) reaction in secondary lymphoid organs. Ex vivo recapitulation of the GC reaction in 2D cultures results in transient cell growth, with poor yield and short-term survival. Furthermore, no reported 2D ex vivo system can modulate the kinetics of a GC-like phenotype or the rate of antibody class switching. This protocol describes a methodology for developing immune organoids that partially mimic the B-cell zone of a lymphoid tissue, for efficient and rapid generation of B cells with a GC-like phenotype from naive murine B cells. The organoid is composed of a bioadhesive protein, gelatin, that is transformed into an ionically cross-linked hydrated network using biocompatible silicate nanoparticles (SiNPs). We explain how to establish the immune organoid culture to sustain immune cell proliferation and transformation into a GC-like phenotype. Starting with cell encapsulation in digested lymphoid tissues, clusters of proliferating B cells with a GC-like phenotype can be generated in the organoids at controlled rates, within ∼1 week. The culture methodology described here is currently the only one that allows the accelerated induction of a GC-like phenotype in B cells and supports a controllable immunoglobulin class-switching reaction. This method can be easily implemented in a typical tissue culture room by personnel with standard mammalian cell culture expertise.
Most antigen discovery and vaccine development aimed at driving functional B cell responses rely on mouse immunizations studies. To date, there is no 3D ex vivo immune tissues, which are capable of driving antigen-specific B cell responses to rapidly determine the humoral immunogenicity of antigens, understand the role of extracellular matrix in humoral immunity, and generate high affinity antibody responses. This can be attributed to the complexity of B cell differentiation and affinity maturation process in the germinal center (GC) reaction, which makes these highly specialized cells susceptible to rapid apoptosis ex vivo. We have previously reported immune tissues that show ex vivo GC-like response, however in a non-antigen specific manner. Here, we report a maleimide (MAL)-functionalized polyethylene glycol (PEG)-based designer immune tissues that modulate B cell differentiation and enriches antigen-specific GC B cells in the presence of T-cell like signals. With the 3D synthetic immune tissue platform, we assessed various hydrogel design parameters to control ex vivo GC reaction. Using an Ezh2 Cγ1-cre transgenic mouse model, we demonstrated ex vivo IgG1 antibody class switching. Using immune tissues developed from a B1-8 mutant mouse that represents a recombined antibody variable region derived from a 4-hydroxy-3-nitrophenylacetyl (NP) hapten binding antibody (B1-8), we demonstrate antigen specificity and selective enrichment of antigen-specific B cells with high affinity at both cell surface and secreted levels in integrin ligand-dependent manner. The ex vivo antigen-specific platform technology offers use in scientific understanding of immunobiology, matrix immunology, and in biotechnology applications, ranging from the antigen testing, vaccine development, and generation of antibodies against diseases.
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