The need for alternative therapies in solid organ transplantation has been obvious for quite some time. Despite standard immunosuppression, rates of acute and chronic rejection, and as a consequence survival, have not been satisfactory (1
). Unfortunately, new therapies for allograft prolongation in humans have been mostly unsuccessful, in part due to the fact that many of these therapies rely on T-cell dependent mechanisms (45
) including regulatory T-cells (Tregs) (46
). A significant obstacle has been the fact that standard immunosuppression with CNI is inhibitory to T-cells - both effector and regulatory (45
). Recently, donor-specific bone marrow transplantation (BMT) (48
) has demonstrated tolerance induction in animal models and humans. However, this therapy is donor-derived and therefore carries the risk of graft versus host disease (GVHD) (52
), as well as other serious immune reactions (49
). Consequently, autologous therapy could form an attractive safe alternative to donor cell therapy. In two studies, autologous
MSC were used to treat fully allogeneic cardiac allografts with no allograft prolongation seen in the first (43
) and robust prolongation only with concomitant Sirolimus therapy in the second (44
). Studies with donor-derived MSC also demonstrate an increase in CD4+
Tregs, which was interpreted as indicating that there may be a mechanistic requirement for Tregs in tolerance induction by MSC (43
). As an alternative approach, we investigated a different autologous
progenitor cell population with the hypothesis that we would attain allograft prolongation through the repair/replacement of donor vascular endothelium. We initially hypothesized that CD117+
PC would differentiate into vascular cells and incorporate into ischemia-reperfusion/allo-immune injured intra-graft vessels, thus partially ‘hiding’ the allograft by limiting the primary target of the acute alloresponse (11
Results demonstrated significant dose-dependent, CD117-dependent cardiac allograft prolongation by CD117+
PC despite a lack of increased recipient vascular chimerism. CD117+
PC differentially engrafted cardiac allografts and peripheral lymphoid tissues in vivo and profoundly inhibited T-cell proliferation in vitro. However, in vitro results demonstrated relatively equal inhibition of T-cell proliferation by autologous CD117+
effluent cells, and unmanipulated BM cells, demonstrating that multiple BM populations can inhibit T-cell proliferation in vitro. Interestingly, neither CD117+
PC nor CD117−
effluent cells significantly effected early T-cell proliferation in vivo
. Despite this, recipient splenocytes, conditioned by CD117+
PC and prolonged exposure to BALB/c allografts, were significantly diminished in their proliferative capacity to ex-vivo re-stimulation with donor-type and third party allo-APCs, suggesting CD117+
PC lead to a dampened or ‘sluggish’ late cellular allo-immune response. At present, we would propose that the benefit to the allograft afforded by CD117+
PC may not be via a clear inhibition of adaptive reactivity per se
, but rather by an unexpected cytoprotective property of the CD117+
PC. We would note that this type of pro-survival benefit of autologous BM-derived cells was demonstrated by Li et al
. who found that severe pancreatic injury in E2f1
deficient mice was rescued by syngeneic, wild-type bone marrow (53
). Clearly, future mechanistic studies will need to include investigation into pro-survival factors that might potentially be involved in the in vivo mechanism of action of CD117+
PC and their ability to prolong cardiac allograft survival.
A potential caveat to these findings is the possibility that a small number of contaminating MSC could be responsible for allograft prolongation (< 0.5% of the CD117+PC prep is CD117−CD45−). However, it is quite unlikely that this is the case. Importantly, treatment with CD117-depleted (effluent) cells which contain approximately 30% CD117−CD45−cells as potential MSCs did not lead to increased cardiac allograft survival, thus arguing against a contribution by MSC and demonstrating a requirement for CD117 expression. Also, CD117+PC therapy does not lead to an increase in CD4+CD25+Foxp3+ Tregs as previously found with MSC therapy. Thus, while MSCs form an important cell-based approach to modifying allograft reactivity, it appears that CD117+PC represent an additional progenitor cell population that has the potential to promote allograft survival.
A surprising finding was in the sometimes large number of GFP+ cells present at day +7 within the spleens of animals receiving GFP+CD117+PC (). In triplicate paired experiments, consistent absolute fold changes between GFP+CD117− control and GFP+CD117+PC-treated recipient spleens were noted (ranging from 13–22 fold increase with CD117+PC-treatment, p < 0.02, paired T-test, not shown). Interestingly, in some cases greater than 40% of total spleen cells were GFP+ after GFP+CD117+PC cell transfer. Given that only a total of 107 GFP+CD117+ cells were injected, it seems likely that expansion of PC-derived cells occurred, although we did not specifically assess for proliferation of these cells. Such potential cell expansion may not be surprising given the enrichment of stem cells and progenitor cells in the donor cell population.
It will be important in future studies to determine the eventual differentiated cell fate(s) responsible for the efficacy of autologous CD117+PC therapy in prolonging allograft survival. As discussed, EPC and CAC are likely candidates. Thus far, immunohistochemical and flow cytometric data did not show any significant difference in the relative allograft content of GFP+CD45+CD31+ (potential CAC) or GFP+CD45−CD31+ (potential EPC) cells in allografts treated with either GFP+CD117+PC or GFP+CD117− effluent cells (not shown). Future studies to selectively remove potential CAC and EPC populations from the injected CD117+PC cell prep should demonstrate any contribution from either of these two candidate progenitors.
In conclusion, results of these studies demonstrate the novel finding that autologous CD117+PC abrogate acute cardiac allograft rejection in a dose-dependent, CD117-dependent fashion and that the allograft promoting effects appear to be unique from those of previously studied MSC. Importantly, as autologous cells, CD117+PC pose presumably limited risk to the host and as such possess the potential to form an adjunct role with other tolerance-promoting agents to promote allograft survival. It will be intriguing to determine if CD117+PC play a general cytoprotective role that promotes survival of tissue vascular and/or parenchymal cells under the duress of transplantation and alloreactivity.