In this study, we have shown that allogeneic CPCs are hypoimmunogenic and immunomodulatory. Our in vitro results demonstrate that rCPCs have an allo antigenic phenotype similar to that of rMSCs and that they do not induce T cell proliferation. This is further supported by in vivo studies where, in contrast to xenogeneic hCPCs, allogeneic rCPCs did not induce T cell proliferation, local inflammation, or cellular or humoral immune responses. Furthermore, rCPCs appear to have immunomodulatory effects by reducing T cell proliferation, increasing Tregs, and promoting M2 macrophage polarization. Finally, allogeneic rCPCs induced significant recovery of cardiac function and reduction of scar size in rats subjected to MI. Overall, these results provide the first evidence for the immune tolerance, safety, and efficacy of allogeneic CPC therapy after MI.
We found that the expression of mesenchymal (CD90 and CD105), MHC (both I and II), and costimulatory molecules (CD80 and CD86) is similar among rCPCs and rMSCs. The lack of MHC class II expression prevents recognition by cytotoxic natural killer cells, while the low levels of expression of costimulatory molecules support escape from alloreactive CD4+ T lymphocytes. These observations provide the structural basis for the hypoimmunogenic phenotype of CPCs; the functional consequences were confirmed by mixed lymphocyte reaction assays. In vitro co-culturing of rCPCs in either allogeneic or syngeneic combinations did not induce significant CD3+ T cell proliferation compared with controls but did significantly increase Tregs (FOX-P3+). These in vitro results were further confirmed in vivo by flow cytometry. Allogeneic rCPCs significantly increased the population of Tregs and M2 cells but not CD8+ and CD4+ cells in the rat myocardium.
Allogeneic CDCs and MSCs have been shown to be efficacious in improving cardiac functional recovery32,33. In fact, a recent clinical study (POSEIDON-DCM) showed that allogeneic MSCs are even more efficacious than autologous MSCs in patients with dilated cardiomyopathy33. We evaluated cardiac functional recovery and scar size after allogeneic, syngeneic, and xenogeneic CPC transplantation in a rat MI model. Allogeneic rCPCs improved cardiac function, as measured by LV ejection fraction and fractional shortening. Both allogeneic and syngeneic rCPCs improved cardiac function at day 28 post-transplantation compared with day 1. However, xenogeneic hCPCs failed to improve cardiac function. Histological analysis indicated that the beneficial effects of allogeneic and syngeneic cells were associated with preservation of LV wall thickness and reduction of scar size. In contrast, xenogeneic cells did not produce such effects.
Endogenous CPCs were originally thought to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells during heart development and even in the adult heart34. However, in more recent studies from many groups, including ours, significant differentiation of exogenous CPCs or hCPCs into cardiomyocytes has not been observed. 7,16,35,36. In the present study, fewer than 3% of exogenous CPCs differentiated into endothelial, smooth muscle, or myocardial cells (data not shown). Similar findings have been published for CDCs and MSCs37. Therefore, replacement of damaged cells by differentiation of CPCs cannot explain the observed beneficial effects. It is likely that CPC-derived secretory products (paracrine factors) account for the improvement in cardiac function5. Consistent with this notion, we recently reported that CPC secretory products promote angiogenesis and proliferation of endogenous cardiomyocytes16,35. We observed similar effects in the present study using allogeneic rCPCs although there is no definitive evidence for myocyte proliferation.
Immune cells play a critical role in ischemia-induced adverse cardiac remodeling. This process can be divided into 3 distinct phases: 1) an inflammatory phase, where cardiomyocyte necrosis triggers innate immune responses, promoting infiltration of the infarcted region by neutrophils and monocytes; 2) a proliferative phase, characterized by the appearance of M2 macrophages involved in preliminary tissue stabilization by inducing processes such as angiogenesis; 3) a final phase, characterized by the infarcted area becoming fibrotic, cardiac cells undergoing apoptosis, and the inflammatory response diminishing38. Modulation of immune responses during tissue remodeling is thought to be a therapeutic target for augmentation of tissue healing and repair in MI.
This study was conducted using functional readouts including immune cells in vitro and fully immunocompetent rats for the in vivo studies. We observed that even under fully immunocompetent conditions, rCPCs did not induce significant immune responses in the mixed lymphocyte reaction assays and induced minimal or no tissue infiltration of immune cells at 28 days after cell transplantation in vivo. Our results suggest that rCPCs exert immunomodulatory effects by inhibiting T cell proliferation, promoting Treg proliferation, and enhancing monocyte differentiation into M2 macrophages, all of which are typically associated with immune tolerance. These immunomodulatory effects were similar for allogeneic and syngeneic cells. Our findings are consistent with recent reports suggesting a beneficial role for Tregs in cardiac repair of the infarcted myocardium.18 Tregs inhibit CD4+ and CD8+ T cell proliferation and inhibit the secretion of interferon gamma39. Additionally, Tregs play an important role in polarization of macrophages toward the M2 phenotype40, which, in turn, plays an important role in post-infarct tissue repair41. Our observations support the notion that rCPCs, via regulation of T-reg cells and promotion of M2 macrophage-dependent processes, attenuate ischemia-induced adverse cardiac remodeling, preserving cardiac function. Nevertheless, further research is needed to determine whether there are additional mechanisms by which CPCs exert beneficial effects on the injured myocardium. Overall, our results clearly establish that CPCs are both hypoimmunogenic and immunomodulatory.
The most important question addressed in our study is how CPCs induce cardioprotective, hypoimmunogenic, and immunomodulatory responses. We previously showed that secreted paracrine factors influence cardiac repair/remodeling8. GDF15, also known as macrophage inhibitory cytokine 1, was found to be abundant in CPCs and in their secretome8. It has been shown previously that GDF15 enhances Treg-mediated suppression of T-cell activation by increasing IL-10 activation in Treg 17. In this study, injection of Treg+ (CD4+ & CD25+) cells alone in the myocardium of nude rats (T cell–deficient) did not promote functional recovery, whereas injection of the combination of rCPCs + Treg+ cells promoted significant recovery, which was associated with an increase in cardioprotective M2 cells in the injured myocardium. In vivo experiments with rCPCs-GDF-15KD showed significant decreases in all cardiac parameters in immunocompetent rats, and rCPCs-GDF-15KD were unable to activate Treg and M2 cells. Furthermore, our data suggests that GDF15 secreted by CPCs inactivates NF-κB signaling in Treg cells in the ischemic myocardium, which may decrease apoptosis and increase the polarization of M2 cells. Taken together, these observations support the concept that the NF-κB/GDF15 regulatory axis in transplanted allogeneic CPCs improved cardiac function after MI by attenuating adverse cardiac remodeling and by polarizing cardioprotective M2 cells. Functional improvement in the heart was also associated with histological evidence of increased angiogenesis and cardiomyocyte proliferation. As a previous study has already demonstrated the safety of autologous CPCs in patients with chronic heart failure6, our results support further work toward establishing banks of clinical-grade, readily-available, “off-the-shelf” allogeneic CPCs, as well as clinical trials evaluating the safety and efficacy of this cell-based approach to favorably affect the post-MI cardiac remodeling process.42,43