| Popis: |
The propagation of human B cells in the laboratory holds potential as basis for novel therapies for the treatment of currently uncurable diseases. In the first part of this work, we applied B cell expansion protocols via pokeweed mitogen (PWM) stimulation. Furthermore, we improved the state-of-the-art suspension culturing approaches via soluble CD40L protein with defined cytokine cocktails and used this knowledge to establish a novel tissue engineering-like culturing platform for B cells in sodium cellulose sulfate (SCS) poly-diallyl-dimethyl-ammonium chloride (PDADMAC) capsules. We used human primary tonsillar B cells and blood derived B cells to demonstrate the mitogenic potential of PWM for cheap and easy expansion of B cells of both sources. We were able to show, for the first time, that the proliferative response to PWM was lower in younger donors. PWM stimulated the proliferation of specific subpopulations and induced Antibody secreting cell (ASC) development, indicated by CD20 reduction. This showed the functionality of our B cell isolation protocols and describes a simple essay to evaluate B cell responsiveness utilizing PWM. We developed two culturing media with different properties to stimulate specific B cell proliferation and differentiation via CD40L. Using a memory expansion medium without IL-21 maintained high memory cell content, led to comparably low expansion, showed upregulation of Activation-induced deaminase (AID) and caused immunoglobulin (Ig) class switch recombination (CSR) in B cells. ASC expansion medium with IL-21 caused plasmablast (PB) and plasma cell (PC) development and led to higher B cell expansion. A pre-culture with memory expansion medium and later transition to ASC expansion medium led to boosted B cell expansion exceeding pure ASC culture after 11 days. AID upregulation additionally indicated CSR and somatic hypermutation (SHM) in memory pre-culture. Contrary to PWM stimulation, the proliferative response to a memory pre-culture was higher in younger donors. PC development was more pronounced in adult-derived B cells, indicating a still developing immune system in children and confirming age-dependent differences. For a closer mimicking of in vivo B cell development, we applied the newly generated knowledge regarding B cell suspension cultures, stimulated with soluble CD40L, and developed a novel culturing platform. Microcapsules made of SCS-PDADMAC were 2 shown to possess features similar to the tissue environment inside the human body. We showed that this system can facilitate human primary tonsillar B cell cultures, as previously demonstrated for primary human T cells. Proliferation and subclass development were tracked during encapsulated culture in comparison to suspension culture. The differentiation of initially mainly memory B cells into various subtypes, particularly PC, was altered significantly compared to suspension cultures. The development of germinal center (GC)-like phenotype and PC survival are major features of this novel culturing platform. We additionally describe possible adjustments to guide B cells in encapsulated culture toward PC or GC phenotype. Variation of these parameters during encapsulation offers a novel tool for finetuning the B cell response. Hence, this platform may pave the way for developing ex vivo human immune organoids. |
