University of Florida
Engineering an Infectious Treg Biomimetic through Surface Bound TGF-β1 Polymers
Ethan Y Yang2, Kerim M Gattas-Asfura1,2, Allison L Bayer2, Cherie Stabler1,2.
1Biomedical Engineering, University of Florida, Gainesville, FL, United States; 2Diabetes Research Institute, University of Miami, Miami, FL, United States
Introduction: Modulation of immunological responses to allografts following transplantation is of pivotal importance to improving graft outcome and duration. Of the many approaches, harnessing the dominant tolerance induced by regulatory T cells (Treg) holds tremendous promise. While soluble TGF-β1 has been shown to stimulate Treg generation, delivery is challenged by the need to elegantly control dosage and its capacity to induce fibrosis. Alternatively, recent studies have highlighted the unique potency of cell surface-bound TGF-β1 on Treg for promoting infectious tolerance, i.e. to confer suppressive capacity from one cell to another. To mimic the infectious characteristic of Treg, encapsulation platforms capable of surface expression of TGF-β1 were engineered. The capacity of these platforms to direct effector T cells towards regulatory pathways was subsequently examined.
Methods: TGF-β1 was chemoselectively linked to surfaces via Staudinger ligation. Naïve CD4+CD62LhighRFP- T cells (from FoxP3RFP reporter mice) were sorted and incubated with TGF-β1 surfaces for up to 5 days, followed by assessment of viable Treg conversion. Subsequent studies coated antigen presenting cells (APC) with TGF-β1 polymer, followed by quantification of Treg generation. For antigen specific Treg, T cells were isolated from OTIxFoxP3-RFP reporter mice and stimulated with OVA peptide. The functional activity of Treg generating using the TGF-β1 polymer was evaluated via T cell suppression assay and compared to native Tregs.
Results and Discussion: To link TGF-β1 to polymers, a functional handle of 1-methyl-2-di-phenylphosphino-terephthalate (MDT) was added, which permits biorthogonal (ie. highly selective, spontaneous, and cell-compatible) linkage to complementary N3 surfaces via Staudinger ligation. MDT modification resulted in no change in protein activity. Following binding of TGF-β1 polymers to an inert spheres, the capacity of this platform to generate Tregs was assessed. TGF-β1 linked beads efficiently converted naïve T cells to Tregs (31.8 ± 5.1% of viable CD4+ T cells) at a level comparable to soluble TGF-β1 (P = 0.15) and significantly different than BSA linked beads (P < 0.001). This platform was subsequently translated to viable cell surfaces, where TGF-β1 polymers were grafted onto APCs. Resulting TGF-β1 expressing APCs efficiently converted naïve T cells to Tregs (45.1 ± 6.9% of viable CD4+ T cells), in stark comparison to BSA expressing control APCs (3.1 ± 0.9%). To evaluate the capacity of these platforms to generate antigen-specific Tregs, naïve T cells with TCR specificity to OVA (OTII) were used. When stimulated with OVA peptide, TGF-β1 expressing APCs generated monoclonal Tregs, demonstrating that that these approach can not only convert naïve T cells into FoxP3-expressing T cells, but generate antigen-specific Treg when co-stimulated with the complementary antigen. The functional capacity of Tregs induced using TGF-β1 expressing APCs was validated via effector T cell suppression studies. As the number of induced Tregs increased, suppression of effector T cells was more pronounced. Trends were comparable to native Tregs, albeit slightly less potent.
Conclusions: These findings demonstrate the unique faculty of a tethered TGF-β1 biomaterial to function as an “infectious” Treg and provide a compelling approach for generating tolerogenic microenvironments for allograft transplantation using bioactive encapsulation platforms.
National Institutes of Health. Diabetes Research Institute Foundation.
17:30 - 18:30
|Joint Poster Session||Bioactive covalent layer-by-layer platforms for cellular encapsulation||Main Foyer 1 & 2|
11:00 - 12:30
|Cellular Encapsulation||Engineering an Infectious Treg Biomimetic through Surface Bound TGF-β1 Polymers||Room 111-112|
12:30 - 13:30
|Islet Encapsulation||Layer-by-layer Encapsulation of Bioactive Polymers for Islet Encapsulation||Room 109|