Immunogenicity of allogeneic mesenchymal stem cells
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Allogeneic mesenchymal stem cells (allo-MSC) are a promising “off-the-shelf” therapy and are often cited as failing to elicit anti-donor immune responses. Intra-muscular (IM) administration may be the optimal route for allo-MSC therapy in heart disease and limb ischemia. In the project described in this thesis, anti-donor cellular and humoral immune responses were compared in fully MHC-mismatched mice following single or repeated IM injections of allo-MSC. As positive and negative controls, additional groups of mice received IM injections of allogeneic splenocytes (allo-splenocyte), of syngeneic MSC (syn-MSC) or of vehicle alone. A panel of assay techniques was developed to assess anti-donor immune responses in secondary lymphoid organs including multi-colour flow cytometry panels, dendritic cell (DC)-stimulated mixed lymphocyte reactions (MLRs), cytotoxicity assays and assays of donor-specific IgG antibody. Analyses of anti-donor T cell responses 1 week following the final IM injections demonstrated donor-specific hypo-responsiveness in recipients of repeated (but not single) injections of allo-MSC. In contrast, repeated allo-splenocyte injection was associated with enhanced anti-donor T cell responses. The donor-specific hypo-responsiveness associated with repeated IM allo-MSC was present in both CD4+ and CD8+ T cell compartments. It was not accompanied by expansion of CD4+/FOXP3+ regulatory T cells but was associated with increased production of interferon gamma and interleukin 10 in donor antigen-stimulated MLRs as well as with increased numbers of CD8+/CD11c+ T cells and alterations to the myeloid and natural killer cell/natural kill T cell compartments in the spleen. Analyses of humoral (B cell) responses 1 week following the final injections revealed that recipients of single and repeated IM injections of allo-MSC consistently developed anti-donor IgG antibodies. The donor-specific IgG antibodies of allo-MSC recipients were of similar titres to those observed in recipients of allo-splencoyte injections but had higher ratio of IgG1 to IgG2a isotype. Nonetheless, anti-donor IgG induced by single or repeated IM injection of allo-MSC supported complement-mediated lysis of donor cells, indicating potential to mediate rejection in vivo. In additional experiments involving mice with induced hind limb ischaemia, it was shown that the development anti-donor IgG following IM injection of allo-MSC could be prevented by a short course of the T cell-specific immunosuppressive drug tacrolimus. Taken together, the results from this project provide new insights into the immunogenicity of allo-MSC delivered by the IM route on a single or multiple occasions. Specifically, it was found that IM injection of allo-MSC consistently induced donor-specific T cell and B cell (antibody) immune responses – likely via indirect alloantigen presentation. Importantly, repeated IM administration of allo-MSC resulted in the emergence of donor-specific T cell hypo-responsiveness which we propose may reflect a multi-faceted anti-inflammatory response to MSC-delivered donor antigen that is dependent on an initial “priming” exposure. Also of importance to future clinical application, IM injection of allo-MSC proved to be a potent inducer of IgG1-predominant anti-donor antibody with potential to mediate donor cell lysis. The findings highlight the fact that allo-MSC are, in fact, inherently immunogenic when delivered intramuscularly and that harnessing their tolerogenic potential may, paradoxically, require an initial sensitising phase. These complex interactions between allo-MSC and the recipient immune system should be considered and studied in more detail in human participants in clinical trials involving Allo-MSC administration.
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