Three-Dimensional Printing of Regulatory T-Cells: A Novel Immunotherapy to Prevent Transplantation Rejection

Abstract

The work presented in this thesis explores the encapsulation of human natural and induced regulatory T-cells (nTregs and iTregs), and the generation of phenotypically stable iTregs for application in 3D-bioprinting with pancreatic islets. 3D-bioprinting of Tregs with islets could serve as an alternative method of islet transplantation to tackle current challenges associated with intra-hepatic islet transplantation such as side-effects of immunosuppressive drugs and sub-optimal characteristics of liver as the site of islet implantation. Paper one explores in vitro evaluation of nTregs and iTregs encapsulated in alginate-gelatin methacryloyl (GelMA) hydrogel. While encapsulation of islets has been extensively investigated since the 1950s, encapsulation of Tregs has never been done, thus this paper aimed to examine whether Tregs can viably be encapsulated. In this study, the alginate-GelMA hydrogel was supplemented with Treg-specific bioactive factors IL-2 and CCL1, to evaluate the effect of IL-2 on encapsulated Tregs and to investigate the potential of CCL1 to recruit recipient Tregs upon transplantation. This study demonstrated that encapsulated nTregs and iTregs are viable, phenotypically stable and functional. Furthermore, encapsulation prevented migration of Tregs out of the hydrogel structure in the presence of potent chemotactic signals. Supplementation of the hydrogel with IL-2 and CCL1 improved encapsulated Treg viability, phenotype and function, and recruited Tregs to the hydrogel structure, respectively. Moreover, peripheral blood CD4+ T-cells expressing the chemokine receptor for CCL1, CCR8, were highly enriched with Tregs, and selective recruitment of these Tregs from peripheral blood mononuclear cells was demonstrated using CCL1. Paper two investigates the generation of stable human iTregs. Most trials of Treg-therapy have been focusing on nTregs, as iTregs are unstable and can differentiate into proinflammatory Th17 cells. For applications in transplantation, iTregs could be an attractive alternative to nTregs given their TCR repertoire and the ease of generating enough iTregs for clinical dosage. In this study, an iTreg differentiation method was optimized and demonstrated to generate superior iTregs to a commercially available kit in terms of viability, and CD25 and FOXP3 expression. In-house generated iTregs were stable in the absence of IL-2 and in the presence of Th17-polarizing cytokines without upregulation of Th17 signature genes, even though demethylation of the Treg-specific demethylation region (TSDR) was not demonstrated. Moreover, they were stable and highly suppressive when re-stimulated without iTreg differentiation components