Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Abstract

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

Designer Self‐Assembling Peptide Hydrogels to Engineer 3D Cell Microenvironments for Cell Constructs Formation and Precise Oncology Remodeling in Ovarian Cancer

“…[ 96 ] To regenerate the unique 3D cell microenvironment of the thymic stroma, EAK16‐II peptide hydrogel promotes the thymic epithelial cells (TECs) to form 3D cell aggregates. [ 97 ] Similar to PuraMatrix hydrogel, the functionalized characteristics are reported in EAK16‐II peptide hydrogel as well. EAK16‐II peptide with six histidine residues (His‐tags) can self‐sort or coassemble into stable β‐sheet structures to achieve in situ self‐gelling nanomaterials.…”

Chemically‐Induced Cross‐Linking of Peptidic Fibrils for Scaffolding Polymeric Particles and Macrophages

“…Previously, we have functionalized EAK fibrillar gels and coacervates using a co‐assembly approach. [ 10,11,27–30 ] We showed that peptidic affinity domains appended with the EAK sequence can be incorporated into the fibrillar matrices, resulting in composites by which antibody molecules can be captured. Using this general approach, we have engineered His‐tags, protein G ( Streptococcus ), and subunit protein A ( Staphylococcus ) for displaying immunoglobulin G (IgG) indirectly or directly via interactions with the Fc regions of antibodies.…”

“…Using this general approach, we have engineered His‐tags, protein G ( Streptococcus ), and subunit protein A ( Staphylococcus ) for displaying immunoglobulin G (IgG) indirectly or directly via interactions with the Fc regions of antibodies. [ 27,28,30 ] From these platforms we have constructed gels and coacervates for localizing antibodies in tumors and in skin allografts, and as scaffolds for constructing thymic organoids in vitro and in vivo. [ 10,11,28 ] These systems of proteins and/or cells can be administered with conventional syringes; they do not elicit local inflammation or acute toxicities in mice after repeated subcutaneous injections.…”

“…[ 27,28,30 ] From these platforms we have constructed gels and coacervates for localizing antibodies in tumors and in skin allografts, and as scaffolds for constructing thymic organoids in vitro and in vivo. [ 10,11,28 ] These systems of proteins and/or cells can be administered with conventional syringes; they do not elicit local inflammation or acute toxicities in mice after repeated subcutaneous injections. [ 10,11,27,28,30 ] The fibrillization is presumed to occur in vivo upon the contact of EAK with physiological fluids.…”

Toward reducing biomaterial antigenic potential: a miniaturized Fc-binding domain for local deposition of antibodies

“…The unique pattern of alternating negative and positive charges interspersed with a small hydrophobic residue in AEAEAKAKAEAEAKAK (referred to as EAK hereafter) drives the peptide to undergo sol-gel phase transition, forming cross-linking β-fibrils in the presence of salt. We have used a coassembly strategy to engineer Fc-binding function into the fibrillar materials by mixing EAK with another EAK-containing peptide appended with a His-tag (EAKH6) or the single-chain variable fragment dL5 (dL5_EAK) [4][5][6][7][8][9][10][11] . These Fc-binding composites can concentrate IgG antibodies locally in vivo.…”