Mesenchymal Stem Cells Engineered to Inhibit Complement-Mediated Damage

Abstract

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.

Painting factor H onto mesenchymal stem cells protects the cells from complement- and neutrophil-mediated damage

“…Although MSCs have been considered to be hypoimmunogenic or immunoprivileged and can escape from host immune surveillance [38], we [14] and others [15,16] have reported that MSCs activate complement in the blood, leading to cell damage and impairment of function. In our previous study [14], we focused on the direct effect of complement activation on MSC viability and function, i.e., direct MAC-mediated MSC injury, using serum as the source of complement in the absence of blood cells.…”

“…Because of this belief and other advantages, such as cost and convenience, many MSCs in clinical development are allogeneic MSCs. We and others previously reported that, although MSCs seem to be able to escape from adaptive immune surveillance, infused MSCs can be recognized by complement from the innate immune system [14][15][16]. Using serum as a source of complement, we further demonstrated that MACs are assembled on MSCs and directly damage the cells, leading to cellular injury and impaired function 14 .…”

“…In brief, following previously published protocols [29,30], WT B6 mice were immunized subcutaneously (s.c.) with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] in complete Freund's adjuvant (200 μg/mouse), then 2 weeks later, spleen cells were collected and re-stimulated with 10 μg/ml of IRBP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] peptide in vitro, together with 10 ng/ml of IL-23 (Biolegend, CA). After 72 h of culture, blasting T cells were enriched by Ficoll centrifugation and adoptively transferred into naïve B6 mice by tail vein injection (5 × 10 6 /mouse), which were then randomly divided into 2 groups.…”

Do Cryopreserved Mesenchymal Stromal Cells Display Impaired Immunomodulatory and Therapeutic Properties?

“…The IBMIR elicits deleterious effects on cellular therapeutics through a cascade of events; first initiated by triggering of the innate immune cascades and then followed by subsequent effector cell infiltration and graft destruction. Within this process both MSCs procoagulant and complement activating properties have been implied to hamper graft performance . Known risk factors attributed to recognition of MSCs by IBMIR are induction of tissue factor expression during culture , long‐term expansion ex vivo , and potentially allogeneic mismatch .…”

Painting factor H onto mesenchymal stem cells protects the cells from complement- and neutrophil-mediated damage

“…Because of this belief and other advantages, such as cost and convenience, many MSCs in clinical development are allogeneic MSCs. We and others previously reported that, although MSCs seem to be able to escape from adaptive immune surveillance, infused MSCs can be recognized by complement from the innate immune system[14–16]. Using serum as a source of complement, we further demonstrated that MACs are assembled on MSCs and directly damage the cells, leading to cellular injury and impaired function 14 .…”