We and others have previously reported that ES cell-derived hematopoietic progenitor cells (ESC-HPCs) induce transplantation tolerance. However the mechanisms by which this process occurs have remained elusive. Here, we focused on whether poor MHC expression by ESC-HPCs alone is sufficient to explain this unique characteristic.

We established a protocol for the derivation of ESC-HPCs. When compared to bone marrow cells, ESC-HPCs were found to be less immunogenic due to their poor expression of MHC antigens and the costimulatory molecules CD80 and CD86. As target cells to alloreactive T cells, ESC-HPCs are not lysed. We used IFN-r to upregulate MHC expression, which significantly upregulates class I but not class II antigens in HPCs. These ESC-HPCs were also not susceptible to CTL killing. To investigate this characteristic further, C57Bl/6 mice were either sensitized with Balb/c ESC-HPCs or with Balb/c splenocytes. Surprisingly, splenic T cells from mice sensitized with HPCs showed a significantly poor response to both the Balb/c splenocytes and to CD3 stimulation. However, their response to Ionomycin was normal as compared to those of the splenic T cells from mice sensitized with Balb/c splenocytes. IL-2 failed to restore T cell responses to normal levels, suggesting T cell exhaustion rather that T cell anergy. These results seemed to suggest that HPCs downregulated T cells by a process that involved T cell receptor signaling. Suppressor myeloid cells have been shown to express arginase-1, an enzyme that scavenger L-arginine. The metabolites of arginine hydrolysis are ornithine and urea. These products downregulate the CD3 z-chain rendering T cells nonresponsive to TcR stimulation. Here, we hypothesized that ESC-HPCs secrete arginase-1. Indeed western blotting revealed that ES cells and their HPCs secrete arginase-1. Further, expression of the CD3 z-chain was significantly downregulated by HPCs recovering in the presence of the arginase inhibitor. Thus, our data show for the first time that ESC-HPCs use multiple mechanisms to downregulate T cells. First, they poorly express MHC antigens, second, they express low levels of co-stimulatory molecules and thirdly they secrete arginase-1. A better understanding of these mechanisms will enable better exploitation of pluripotent stem cells and their derivatives in future therapies.

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