In this study, we demonstrated that ex vivo expanded HSCs more efficiently overcome MHC barriers and repopulated allogeneic recipient mice than freshly isolated HSCs. As measured by limiting dilution analysis, there was a 40-fold increase in the allograft ability of HSCs cultured for only 8 days compared to that of the freshly isolated HSCs. To identify the underlying mechanisms, we found that both increased numbers of HSCs and cultured-induced elevation of expression of the immune inhibitor CD274 on the surface of HSCs contributed to the enhanced allograft efficiency. As a proof-of-principle that ex vivo expanded HSCs can be used to cure genetic diseases in allogeneic recipients, we used ex vivo expanded allogeneic HSCs for transplantation and successfully rescued the lethal phenotype of DNA-PK knock-in mice.
We used three models of allogeneic transplantation: non-competitive transplantation into lethally irradiated recipients, competitive transplantation into lethally irradiated recipients, and non-competitive transplantation into sublethally irradiated recipients. While the first model was well-established and allows fewer numbers of donor cells for engraftment, it may result in the mouse death if donor HSCs cannot repopulate recipients. The second and third models ensure the survival of all recipients and better mimic the human transplantation scenario in which reduced intensity conditioning is often applied. Nevertheless, due to the enhanced host immune rejection, more than 10-fold of freshly isolated allogeneic donor HSCs are needed for successful engraftment in these models. This also underscores the importance of the increased number and MHC matching of donor HSCs in the clinical setting.
Our findings may shed new light on allogeneic transplantation of human HSCs into patients, which cannot be appropriately modeled by xenograft into immune-deficient mouse recipients. Two major problems, failure of engraftment and GVHD, have limited the progress in allogeneic transplantation. A strategy that significantly improves donor engraftment and reduces the risk of GVHD compared to current practice is needed. Transplantation of freshly isolated allogeneic HSCs indeed decreases the risk of GVHD, but results in much lower engraftment (Shizuru et al., 1996
; Wang et al., 1997
). Here we show that ex vivo
expanded mouse HSCs possess two advantages: increased HSC numbers and the enhanced immune feature to evade host rejection, therefore having dramatically enhanced allogeneic engraftment. Importantly, similar to freshly isolated HSCs (Shizuru et al., 1996
; Wang et al., 1997
), no sign of GVHD was observed after allogeneic transplantation of ex vivo
expanded HSCs. This is expected because the condition of our (or other) HSC culture supports expansion of HSCs, along with production of differentiated myeloid but not much lymphoid cells. The culture thus does not seem to produce the source cells including T cells that may cause GVHD. Therefore, ex vivo
expanded mouse HSCs appear to be an appropriate cell source to solve the problems of allogeneic transplantation in the mouse model. Based on these results of mouse HSCs and the elevation of CD274 on cultured human HSCs, we propose that ex vivo
expansion of human HSCs may benefit the practice of allogeneic transplantation for patients. This would apply to non-matched or low-matched donor human cord blood, BM, or mobilized peripheral blood HSCs. If donor human HSCs can be expanded in culture and engraft non-matched or low-matched patients without GVHD, this strategy will possibly lead to an ultimate solution to problems in allogeneic transplantation.
It is known that some adult stem cells, such as mesenchymal and amnion stem cells, but not embryonic stem cells, are capable of avoiding rejection through production of immunosuppressive molecules and can be used in intra- and even interspecies transplantation (Salem and Thiemermann; Swijnenburg et al., 2008
). Here we demonstrated that the immune inhibitor CD274 is expressed on freshly isolated HSCs and its expression dramatically increased upon culture. Interestingly, CD274 does not appear to significantly affect the repopulation of long-term HSCs before and after culture as determined by syngeneic transplantation, suggesting its main role is not regulation of the regular activity of HSCs, but modulation of immunological properties of these cells. This was confirmed by the result that the deletion of CD274 or treatment with a CD274 neutralizing antibody abrogated the ability of cultured but unexpanded HSCs to cross the MHC barrier. CD274 was shown in previous studies to be expressed on activated immune cells and parenchymal cells and in immune-privileged sites such as eyes and placenta (Francisco et al.; Zou and Chen, 2008
). CD274 is also selectively expressed by various cellular components in the tumor microenvironment, where it inhibits tumor-specific T-cell immunity by inducing T cell apoptosis and delay rejection (Zou and Chen, 2008
). Here we provided an example suggesting that HSCs possess the ability to evade the rejection of the acquired immune system by regulating the expression of their own surface immune inhibitor such as CD274. Besides HSCs, the elevation of CD274 on hematopoietic progenitors produced during culture also might have contributed to the enhanced allograft. However, it is interesting to note that more differentiated Lin+
cells elicit no effect, although they also express CD274. This may be contributed by the different cellular locations of HSCs/progenitors and more differentiated hematopoietic cells home after transplantation. It therefore will be interesting to study where the T cell-mediated immune response occurs for allogeneic transplanted HSCs in the future. In addition, it is noteworthy that CD274 may not be the only immune suppressor acts on the HSC allograft. This is because that, although CD274-null HSCs behave much worse in allogeneic transplantation than their WT counterparts, they still possess a certain ability for allogeneic engraftment. Consistent with the elevation of the expression of immune inhibitor CD274 upon culture, co-stimulatory molecules such as CD80 and CD86 lost their expression on some cells after culture. All these observations clearly indicate that ex vivo
culture significantly modulates the immunogenicity of stem cells. The identification of additional immune molecules whose alterations can regulate allograft will enable the complete resolution of the issue of immune rejection in allogeneic transplantation.
While our study suggests that the upregulation of CD274 on cultured cells including HSCs inhibited allogeneic T cell response, a related example is surface expression of CD47, which enables HSCs and leukemia cells to evade innate macrophage phagocytosis (Jaiswal et al., 2009
). Based on these results, we hypothesize that all homeostatic HSCs express low levels of surface immune suppressors, and the levels of these suppressors can be induced by stress or immune signals. These immune suppressors may thus modulate HSC immunogenicity and, therefore, contribute to the “immune privilege” of HSCs. This regulatable “immune privilege” should be advantageous to HSCs, as it may allow these important stem cells to rapidly adjust to altered environment or to protect them from the excessive immune activation and even potential autoimmune disorder. Whether the expression of CD274 on HSCs or cancer cells can be regulated in vivo
and its biological significance warrants further investigation.
Furthermore, we speculate that a common mechanism exists for regulation of expression of immune inhibitory signals in some other types of stem cells – similar to that in tumor cells. The expression and regulation of immune inhibitors on stem cells per se may allow these cells to survive an unexpected immune attack. It will be interesting to study the immunology of stem cells by investigating the roles of surface immune molecules on embryonic stem cells, induced pluripotent stem cells, other adult stem cells, and cancer stem cells.
In summary, our study demonstrated the great benefits of ex vivo expansion of HSCs for overcoming problems in allogeneic transplantation, and revealed the importance of an immune inhibitor on the surface of HSCs. This work should shed new light on understanding the immunology of HSCs and other stem cells and may lead to development of novel strategies for successful allogeneic transplantation of human patients.