A synthetic human 3D in vitro lymphoid model enhancing B-cell survival and functional differentiation.

To investigate B-cell differentiation and maturation occurring in the germinal center (GC) using in vitro culture systems, key factors and interactions of the GC reaction need to be accurately simulated. This study aims at improving in vitro GC simulation using 3D culture techniques. Human B-cells were incorporated into PEG-4MAL hydrogels, to create a synthetic extracellular matrix, supported by CD40L cells, human tonsil-derived lymphoid stromal cells, and cytokines. The differentiation and antibody production of CD19+B-cells was best supported in a 5.0%-PEG-4MAL, 2.0 mM-RGD-peptide composition. The 3D culture significantly increased plasmablast and plasma cell numbers as well as antibody production, with less B-cell death compared to 2D cultures. Class switching of naive CD19+IgD+B-cells toward IgG+ and IgA+B-cells was observed. The formation of large B-cell clusters indicates the formation of GC-like structures. In conclusion, a well-characterized and controllable hydrogel-based human 3D lymphoid model is presented that supports enhanced B-cell survival, proliferation, differentiation, and antibody production.

Differences in IgG autoantibody Fab glycosylation across autoimmune diseases.

BACKGROUND: Increased prevalence of autoantibody Fab glycosylation has been demonstrated for several autoimmune diseases. OBJECTIVES: To study whether elevated Fab glycosylation is a common feature of autoimmunity, this study investigated Fab glycosylation levels on serum IgG and its subclasses for autoantibodies associated with a range of different B cell-mediated autoimmune diseases, including rheumatoid arthritis, myasthenia gravis subtypes, pemphigus vulgaris, antineutrophil cytoplasmic antibody-associated vasculitis, systemic lupus erythematosus, anti-glomerular basement membrane glomerulonephritis, thrombotic thrombocytopenic purpura, and Guillain-Barré syndrome. METHODS: The level of Fab glycosylated IgG antibodies was assessed by lectin affinity chromatography and autoantigen-specific immunoassays. RESULTS: In 6 of 10 autoantibody responses, in 5 of 8 diseases, the investigators found increased levels of Fab glycosylation on IgG autoantibodies that varied from 86% in rheumatoid arthritis to 26% in systemic lupus erythematosus. Elevated autoantibody Fab glycosylation was not restricted to IgG4, which is known to be prone to Fab glycosylation, but was also present in IgG1. When autoimmune diseases with a chronic disease course were compared with more acute autoimmune illnesses, increased Fab glycosylation was restricted to the chronic diseases. As a proxy for chronic autoantigen exposure, the investigators determined Fab glycosylation levels on antibodies to common latent herpes viruses, as well as to glycoprotein 120 in individuals who are chronically HIV-1-infected. Immunity to these viral antigens was not associated with increased Fab glycosylation levels, indicating that chronic antigen-stimulation as such does not lead to increased Fab glycosylation levels. CONCLUSIONS: These data indicate that in chronic but not acute B cell-mediated autoimmune diseases, disease-specific autoantibodies are enriched for Fab glycans.

A Human Hematopoietic Niche Model Supporting Hematopoietic Stem and Progenitor Cells In Vitro.

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk, a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability, clonogenic hematopoietic potential, and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells, together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel, the supportive cells self-assemble into a hypoxic stromal network, stimulating CD34+ CD38+ cell formation, while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks, in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly, the primary in vitro niche model supports HSPCs with no cytokine addition. Overall, the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches, in the context of normal hematopoiesis or blood-related diseases.

Cellular immunotherapy on primary multiple myeloma expanded in a 3D bone marrow niche model.

Bone marrow niches support multiple myeloma, providing signals and cell-cell interactions essential for disease progression. A 3D bone marrow niche model was developed, in which supportive multipotent mesenchymal stromal cells and their osteogenic derivatives were co-cultured with endothelial progenitor cells. These co-cultured cells formed networks within the 3D culture, facilitating the survival and proliferation of primary CD138+ myeloma cells for up to 28 days. During this culture, no genetic drift was observed within the genomic profile of the primary myeloma cells, indicating a stable outgrowth of the cultured CD138+ population. The 3D bone marrow niche model enabled testing of a novel class of engineered immune cells, so called TEGs (αßT cells engineered to express a defined γδTCR) on primary myeloma cells. TEGs were engineered and tested from both healthy donors and myeloma patients. The added TEGs were capable of migrating through the 3D culture, exerting a killing response towards the primary myeloma cells in 6 out of 8 donor samples after both 24 and 48 hours. Such a killing response was not observed when adding mock transduced T cells. No differences were observed comparing allogeneic and autologous therapy. The supporting stromal microenvironment was unaffected in all conditions after 48 hours. When adding TEG therapy, the 3D model surpassed 2D models in many aspects by enabling analyses of specific homing, and both on- and off-target effects, preparing the ground for the clinical testing of TEGs. The model allows studying novel immunotherapies, therapy resistance mechanisms and possible side-effects for this incurable disease.

Endosteal and Perivascular Subniches in a 3D Bone Marrow Model for Multiple Myeloma.

The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also, the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology. We used a bioprintable pasty calcium phosphate cement (CPC) scaffold with seeded osteogenic multipotent mesenchymal stromal cells (O-MSCs) to model the endosteal niche, and Matrigel containing both endothelial progenitor cells (EPCs) and MSCs to model the perivascular niche. Within the model containing one or both of the niches, primary CD138+ myeloma cells were cultured and analyzed for both survival and proliferation. The 3D bone marrow model with combined subniches significantly increasing the proliferation of CD138+ myeloma cells compared to both environments separately. The developed model showed an essential role of the perivascular niche over the endosteal niche in supporting myeloma cells. The developed model can be used to study the expansion of primary myeloma cells and their interactions with varying bone marrow subniches.