Poster O6 - Bioconjugated Injectable Granular Hydrogels for Regulatory T cell Induction

Introduction: Autoimmune diseases arise due to loss of self-tolerance and chronic inflammation via the overabundance of autoreactive and pro-inflammatory T cells coupled with decreased regulatory T cells (Tregs) populations. Inflammatory environments further compromise Treg function, hindering their proliferation and causing a decline in FOXP3 expression, transitioning into effector T cells. Currently, 5% of the general population is estimated to be afflicted by autoimmune diseases. Conventional treatments focus on systemic pharmacological immunosuppression, but this approach risks fostering opportunistic infections and cancer due to broad immune suppression. Due to the pivotal role of Tregs in immune regulation, therapies targeting them have emerged to curb chronic inflammation and promote self-tolerance. Treg therapies typically involve in vivo Treg induction (e.g., by delivery of soluble IL-2, TGF-β, and rapamycin) or adoptive cell transfer. However, such methods are limited by 1) impure T cell populations, 2) Treg phenotype instability, 3) a lack of precise delivery, and 4) systemic off-target effects. To address these challenges, we have developed an injectable granular hydrogel for the local presentation of a covalently-tethered immune checkpoint (B7x) that is associated with potent Treg-inducing function, facilitated by copper-free azide/alkyne click chemistry.

Materials and

Methods: Hyaluronic acid was modified by methacrylates and DBCO to enable photopolymerization and subsequent click-coupling of azide-terminated B7x, respectively (Fig. 1A). Bulk hydrogels were formed (10 mW/cm2, 15 min, LAP photoinitiator) and processed into granules by extrusion fragmentation (18-22G needles) to yield a shear-thinning and self-healing hydrogel, as assessed by rheology. The viability of Jurkat cells was assessed (CCK-8 assay) over 5 days of hydrogel exposure. Splenic CD4+ T cells were cultured with B7x-azide (10-300µg/mL) to validate the bioactivity of the modified ligand in vitro. T cell populations in the hydrogel and secondary lymphoid organs were profiled by flow cytometry 7 days after subcutaneous injection of the B7x-conjugated granular hydrogel (MeHA-B7x) or controls (unmodified or TGF-β loaded) in C57BL/6J mice.

Results, Conclusions, and Discussions: Extrusion fragmentation yielded microgels with an average diameter of 155±53µm, as shown in fluorescent images and their quantification (Fig. 1B). Granular hydrogel formed demonstrated shear-yielding behavior. As a result of granular packing, the hydrogels are solid under low strain (0.5%), liquify under high strain (500%) to enable injection, and rapidly re-solid for local retention in the tissue (Fig. 1C). The viability of Jurkat cells was unaffected by culture with the granular hydrogel, relative to untreated controls (Fig. 1D). The azide-modified B7x ligand increased Treg populations in a dose-dependent fashion in vitro (Fig. 1E), comparable to unmodified B7x. In the local hydrogel compartment (MeHA-B7x), there was a two-fold increase in Treg populations in the B7x-treated groups relative to the hydrogel alone and a significant increase in Treg populations in the B7x-treated groups compared to soluble delivery of TGF-β (Fig. 1F). In the systemic compartment (spleen), there was also a significant increase in Treg populations compared to the hydrogel alone (Fig. 1G). Overall, the Treg populations accumulated at the local site and were distributed to the systemic compartments, highlighting the potential of the ligand bioconjugation system for Treg induction. Functional activity of the persistent locally-anchored signal is a promising strategy to overcome the limitation of the Treg induction by soluble factors, with applications in the treatment of autoimmune dysfunction, organ rejection, and tissue repair.

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