Generation of functional mini thymus-like units with self-assembling EAK16-II/EAKIIH6 hydrogel

Abstract A functional thymus is crucial to produce mature, self-tolerant T cells. Crosstalk between the residing thymic stromal cells, especially the predominant population of thymic epithelial cells (TECs), and the developing thymocytes is essential for thymopoiesis. The survival and proliferation of TECs depend on a unique 3 dimensional (3-D) configuration of the thymus microenvironment. Disorganization of TECs results in a loss of gene expressions essential for TEC growth and survival, as well as inability to produce functional T cells. Recapitulating the property of thymus has been proven to be challenging. In our study, we have incorporated EAK16-II, a low molecular weight peptide that self-assembles into beta-sheet fibrils, and its histidinylated analogue, to induce 3-D aggregation of TECs. When the adaptor complex comprised of anti-EpCAM IgG, anti-His and recombinant protein A/G molecules were mixed with TECs in the hydrogel, TECs were captured into small clusters. TECs cultured in this condition maintained their molecular properties and were viable up to 2 weeks. Furthermore, when the TEC clusters in the hydrogel were transplanted into athymic nude mice, T cell development was observed, and these newly generated T cells proliferated upon stimulation with allogenic cells. These results demonstrate that self-assembling EAK16-II/EAKIIH6 system with addition of tri-component adaptor complex may be a useful tool to organize TECs in a 3-D-like structure to support T cell development, suggesting a possibility to generate injectable mini thymus-like units to restore adaptive immune system.