(H-308) A Fully Synthetic Ex-vivo Germinal Center Model Elucidates Differential Responses of B cells to Antigen Presentation Method

Introduction:: High-affinity antigen-specific B cells are generated in vivo within specialized structures called germinal centers (GCs), in which follicular dendritic cells (FDCs) present antigen on their membrane surface to cognate B cells, and follicular helper T cells (T_FHs) present co-stimulatory CD40L, instructing rapid proliferation and differentiation of B cells towards antibody-producing plasma cells^1,2. However, the recapitulation of GC response in vitro is difficult due to a lack of control over key signals that regulate B–FDC and B–T_FH cell interactions. Mimicking the GC response in vitro in a fully synthetic biomaterial-based manner could provide a more quantitative, controllable, and reproducible system for modulating B cell behavior and expanding therapeutic B cells. The FDC's fluid membrane surface allows for the B cell to pull the antigen into clusters and internalize it, a process that frequently involves tearing off and internalizing fragments of the FDC lipid membrane as well. We hypothesized that reproducing the natural FDC antigen presentation using a surface that has membrane fluidity characteristics will have a more profound effect on GC reaction induction than soluble antigen presentation. Our results indicate that fluid membrane-bound antigen induces a strong GC response, resulting in rapid proliferation, induction of the GC reaction, and IgG1 class switching. However, in the in vivo GC, B cells are exposed to both soluble and FDC-bound antigen. Herein, we combined both soluble and liposome-bound antigen in the sGC, revealing how both dose and presentation mode of key GC signaling cues modulate proliferation and differentiation of B cells.

Materials and Methods:: Dynabeads were conjugated to trimeric CD40L as artificial T_FHs while biotinylated liposomes were conjugated to conjugated biotinylated anti-IgM via a streptavidin linker, as previously reported^3,4. Splenic naïve B cells from 6-12-week-old female C57/BL6 mice were cultured in the sGC along with the GC signaling particles and soluble B cell-activating factor (BAFF), IL4, and IL21. GC phenotypes were analyzed at endpoint via flow cytometry.


Results, Conclusions, and Discussions:: Murine B cells were cultured in the sGC with antigen composed of varying ratios of soluble and liposome-bound antigen. As previously published, liposome only conditions had the greatest total number of B220+GL7+ germinal center phenotype B cells compared to soluble only conditions. By day 5, we observed 1.8x more cell expansion than soluble presentation across all doses. EZH2 histone methyltransferase, which is required for B cells to form germinal center and has been reported to facilitate cell proliferation, was significantly upregulated in B cells exposed to antigen-presenting liposomes compared to soluble antigen, suggesting that epigenetic changes are contributing to the proliferative phenotype in the liposome condition. Confocal imaging revealed that liposome-based antigen presentation resulted in significantly greater BCR cluster area after 30 min than both soluble antigen and unstimulated cells, suggesting that liposome-based presentation enhances BCR crosslinking, leading to higher levels of B cell activation.



Interestingly, conditions cultured with both liposome-bound and soluble antigen had intermediary numbers of GC B cells, revealing that the fraction of liposome-bound antigen present in culture is proportional to the number of GC B cells generated. We observed the opposite trend for B220+CD138+ plasmablasts, in which soluble antigen alone had the greatest number of B220+CD138+ cells with less cells generated as the ratio of soluble antigen in the culture decreased to zero. We then incubated the B cells with the same ratios of liposomes and soluble antigen fluorescently labeled with FITC and APC, respectively, to determine the uptake of antigen by the cells using flow cytometry. The B cells became APC+ after 4 hours and FITC+ after 28 hours, revealing that there is a temporal difference in the interactions between B cells and different presentation modes of antigen, potentially resulting in the observed phenotypic differences.


Our results indicate that both concentration and mode of presentation of key GC signaling cues significantly modulate and instruct B cell GC behavior in vitro. This knowledge can be used to tune B cell GC responses for studying GC biology in health and diseases, and for expanding therapeutic B cells ex vivo for potential cell therapy applications.


Acknowledgements (Optional): : Funding provided by the Marcus Center and the Georgia Research Alliance.


References (Optional): : ¹Singh A. Nat Nanotech. 2020; ²Cyster J. Cell. 2010;143:503-505; ³Roh K. Biomaterials, 2018;164:106-120; ⁴Kramer L. Adv NanoBiomed Res. 2022;2: 2100137.