Abstract
The major objective of this study is to develop a novel and broadly applicable immunotherapy platform against cancer and infectious diseases. We hypothesized that human embryonic stem cells (hESC) could serve as a source for generating dendritic cells (DC) with potent immunostimulatory function. One advantage of using hESC-derived DC in clinical settings is the ability to generate virtually unlimited amounts of antigen presenting cells for vaccination. Although hESC-derived DC are not genetically identical to the recipient patient, antigen processing and presentation can be facilitated by matching hESC to recipients that share HLA class I alleles. Another advantage of this technology is that hESC express highly polymorphic HLA class II molecules that serve as major rejection antigens, thereby augmenting the antigen-specific T-cell response in the cancer patient.
In the current study, we have established a novel three-step method to differentiate hESC (H9 cell line) into mature DC sequentially through hematopoietic stem cell and myeloid precursor stages. During the first step, 10–15% CD34+ hematopoietic stem cells were generated by co-culturing hESCs with the bone marrow stromal cell line OP9. During the second step, these CD34+ hematopoietic stem cells were differentiated into CD45+CD33+ myeloid precursors and further expanded in the presence of GM-CSF. In the final step, all myeloid precursors were differentiated into mature DC using a cytokine cocktail including GM-CSF, Flt-3L and TNF-α. Using this method, we were able to generate approximately 1 × 108 mature hESC-derived DCs (hESDC) with ≥ 80% purity. These hESDC exhibited a similar phenotype than monocyte-derived DC with high expression of MHC class I, MHC class II, CD11c, CD54, CD40 and the co-stimulatory molecule CD86. Upon activation with proinflammatory cytokines, the hESDC secreted IL-12p70 and migrated in response to MIP-3β. In mixed lymphocyte reaction assays, hESDC exhibited strong allo-stimulatory capacity. Moreover, peptide-loaded mature hESDCs were able to stimulate antigen-specific CD8+ T-cell responses against the EBV peptide BMLF1280-288 and the MART-1 peptide (ELAGIGILTV) in a HLA-A2-restricted manner. Most importantly, hESDC stimulated HLA-A2+ MART-1 peptide-specific CD8+ T cells in vitro that were capable of recognizing and killing the HLA-A2+ melanoma cell line Malena-3M. These data suggest the development of a scalable DC platform that could be applied in clinical immunotherapy protocols.
Human Embryonic Stem Cells Suppress T Cell Responses via Arginase I-Dependent Mechanism
