Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch

Modulation of mesenchymal stem cell immunogenicity through stable expression of human cytomegalovirus proteins

Mesenchymal stem cells (MSC) have received considerable attention for their promising potential in cellular replacement therapies as a result of several characteristics they exhibit: 1) migration to sites of tissue injury; 2) engraftment into those damaged areas; 3) restoration of injured tissue through both differentiation into required cell types and trophic support of endogenous cellular pools; and 4) immunosuppressive capacity via modulation of both cellular and innate immune responses. Despite their documented immunomodulatory properties, however, MSC are not completely immune-inert and may elicit an immune response after in vivo transplantation in an allogeneic setting, leading to poor MSC allograft outcome and potential failure of cell-based regenerative therapies. In the present dissertation, we explored one strategy to potentially improve the immune evading abilities of MSC by exploiting the inherent immunomodulatory mechanisms of human cytomegalovirus (HCMV). To accomplish this, we genetically engineered MSC to express proteins coded for by the unique short (US) regions of HCMV: US2, US3, US6, and US11, which have been shown to be involved in down-regulation of HLA-I molecules and consequent inhibition of CTL proliferation. Study of immune responses to US-transduced MSC revealed a reduction in HLA-I surface expression, particularly for US6 and US11, and a resultant decrease in PBMNC stimulation and proliferation. Despite the decrease of HLA-I surface levels resulting from expression of US proteins on MSC, NK cell responses were also reduced due to surface upregulation of HLA-G1 molecule on MSC-US11 cells and CD155 protein on MSC-US6 cell line. Furthermore, study of US-transduced MSC ability to stimulate and activate the complement system cascade revealed that MSC expressing US2, US3 and US6 exhibited substantially elevated levels of the complement inhibitor, CD59. As a conclusion of the in vitro analysis of US-transduced MSC cell lines, cells expressing US6 or US11 proteins possessed significantly increased protection from CTL and NK killing, and, in the case of US6, an enhanced defense against complement attack. We thus concluded that these US-MSC lines should be less immunogenic and therefore exhibit increased engraftment potential in vivo. To test this experimentally, we transplanted MSC-US6 and MSC-US11 cell lines, together with an MSC-E control cell line into fetal sheep recipients and subsequently analyzed the levels of MSC engraftment within the liver and heart of the recipients. Analysis of fetal liver revealed that expression of either US6 or US11 significantly increased MSC engraftment capabilities compared to injection of the control cell line, but also decreased the ability of the engrafted US-MSC to differentiate into mature hepatocytes and hepatic stem/progenitor cells. In contrast, analysis of cardiac tissue indicated that expression of US6 or US11 proteins not only allowed engraftment within the heart, but also broadened the differentiative potential of these cells, enabling them to form mature cardiomyocytes and cardiac stem cells.In conclusion, expression of HCMV US6 or US11 proteins on MSC increased their ability to evade the immune system, both in vitro and in vivo. This increased immuno-invisibility led to enhanced engraftment capabilities within the liver and heart following transplantation in utero, and also broadened the differentiative capacity of these cells following engraftment within the heart. The observation that US-transduced MSC gave rise to not only phenotypically mature cells within the liver and heart, but also to stem/progenitor cells within these tissues suggests that MSC transduced to express HCMV US proteins could represent a clinically valuable source of cells for replenishing/repairing damaged/diseased cells within these organs

Mesenchymal stem cells engineered to inhibit complement-mediated damage.

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool.

To identify human bone marrow stromal cell(BMSC) subsets with enhanced ability to engraft/contribute to the resident intestinal cellular pool, we transplanted clonally derived BMSCs into fetalsheep. Analysis at 75 d posttransplantation showed 2 of the 6 clones engrafting the intestine at 4­ to 5­fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor­derived Musashi­1 (12.01–14.17 vs. 1.2–3.8%; P<0.01) or leucine­rich repeat­containing G­protein coupled receptor 5 (Lgr5) cells within the intestinalstem cell(ISC) region. Phenotypic and transcriptome analysis determined that the clones with enhanced intestinal contribution expressed high levels of Ephrin type B receptor 2 (EphB2). Intestinal explants demonstrated proliferation of the engrafted cells and ability to generate crypt­like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post­transplant, the EphB2 BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2 BMSCs engrafted at 0.3 ± 0.1% (P<0.01). Therefore we identified a marker for isolating and culturing an expandable subpopulation of BMSCs with enhanced intestinal homing and contribution to the ISC region.— Colletti, E., El Shabrawy, D., Soland, M., Yamagami, T., Mokhtari, S., Osborne, C., Schlauch, K., Zanjani, E. D., Porada, C. D., Almeida­Porada, G. EphB2 isolates a human marrow stromal cellsubpopulation with enhanced ability to contribute to the resident intestinal cellular pool