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Biomaterials

Biomaterials - Poster Session E

Poster B7 - T follicular helper-like cell differentiation, encapsulation, and culture in degradable microgels

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  T follicular helper cells (Tfh) are a subclass of CD4+ helper T cells that provide functionally crucial signals to B cells in secondary lymphoid tissues like the lymph node during an adaptive immune response. Tfh signals are particularly important for germinal center formation, which are microanatomical sites where B cells undergo rapid proliferation and mutations to generate protective high-affinity against antigens. Activated B cells in germinal centers mature and differentiate due to T cell help, provided mainly through ligands and cytokines presented by Tfh cells, such as CD40L and IL-21. Tfh cells also present CD3, CD4, ICOS, CCR7, PD-1, CXCR5, and BCL-6 ligands, which is important for characterization. This B-T cell interaction in germinal centers is incredibly important, without which the humoral immunity response would not be effective. Hence, understanding how CD4+ T cells differentiate into Tfh-like cells and modeling their role in germinal centers ex vivo is crucial for the fields of infectious disease and immunology. We propose to create this model using high-throughput generation of microgels of controlled size, as it provides a rapid and reproducible methodology for cell encapsulation and has been successfully optimized by our lab for use with other highly sensitive human immune cells.

Materials and

Methods:  Our experimental flow starts with isolation of cells via negative selection kit, differentiation of T cells in 2D culture and finally, encapsulation of T cells into microgels for further culture and analysis (Figure 1a). Dynabeads were added to frozen PBMC isolated naïve CD4+ T cells at a 1:1 ratio in a 96 well plate for activation. Cells were treated with recombinant Activin A and IL-12 or left untreated as a negative control for the duration of culture. On culture day 1, 3, and 5, cells were analyzed using multiparametric flow cytometry to evaluate viability and T cell phenotype. Media from the cultured samples was collected to evaluate the secretion of IL-21 cytokine using an IL-21 Human ELISA Kit. Naive T cells were evaluated for encapsulation and culture within degradable microgels. Microgels composed of four-armed poly(ethylene glycol) (PEG-4MAL) were fabricated using a flow focusing microfluidic device using ethylene glycol bis(mercaptoacetate) (EGBMA) as a hydrolytic crosslinker. After fabrication, microgels were extracted from the oil phase, washed and cultured at 37C with RPMI based lymphocyte media. After culture and degradation over 5 days, T cell microgels were passed through a filter mesh plate and evaluated for viability and phenotype via flow cytometry analysis.

Results, Conclusions, and Discussions: Naive CD4+ T cells treated with combination of Activin A and IL-12 showed an increased expression of ICOS and CCR7 (Figure 1b), which are typical markers for induction of Tfh cell-like phenotype. The critical cell surface ligand, CD40L, also had a significant increase in Activin A and IL-12 treated T cells (Figure 1b), which indicates that the treated T cells could have functional interactions with naïve B cells in future co-culture experiments. In addition, once exposed to treatment, T cells in culture began to stably secrete IL-21 cytokine over the 5 days in culture, which is crucial for germinal center responses in adaptive immunity. We then characterized the diameter, polydispersity and swelling of generated PEG-4MAL microgels without cells under DTT and 0.5 mM, 1 mM and 2 mM EGBMA conditions (Figure 1c). Generated microgels generally began with a diameter of ~80 µm with polydispersity in size that changed with concentration as indicated by recorded microgels in relative frequency. To validate that our T cells could be encapsulated into these PEG-4MAL microgels, we have used live/dead imaging of fluorescently labelled microgels with cells, which show effective encapsulation of the cells as well as high viability maintenance post-encapsulation (Figure 1d). In addition, analysis of confocal live/dead images show that ~50% of microgels generated contain between 10-20 cells per microgel and less than 10% of microgels analyzed had below 10 cells per microgel. This cell density per microgel could be conducive to future co-culture and expansion studies. Looking forward, these findings can be used to create microgel environments with Tfh and B cells co-cultured where their unique cellular interactions can be explored. Our data has shown that Tfh like phenotypes can be obtained using Activin A and IL-12 cytokine to bias cellular differentiation and this treatment can also amplify key signals that are crucial to germinal center responses, like CD40L and IL-21. On a larger scale, we will apply this work to generate a functional model of the human lymph node B and T cell zones for modeling and exploration of adaptive immune responses in vitro.

Acknowledgements (Optional):