Evidence of Host Immune Recognition of Allogeneic Mesenchymal Stem Cells.
Authors: Beggs Kirsten J. , Javazon Elisabeth H. , Tebbets Jessica C. , Flake Alan W. 
Published: April 2020

Abstract
The immunologic properties of Mesenchymal Stem Cells (MSCs) are of therapeutic interest. Previous work shows MSCs do not elicit an alloreactive T cell response in vitro due to suppressive mechanisms. These results suggested that allogeneic MSCs could be used in vivo without inducing an immune response. We further explored this hypothesis using a well-characterized population of Adult Murine Mesenchymal Stem Cells (AmMSCs). These AmMSC are free of hematopoietic contamination and have the following immunologic phenotype: Class I +, Class II-, CD40-, CD80+, and CD86−. Upon treatment with interferon gamma, upregulation of Class I, Class II, CD80, and CD86 was observed. T cell proliferation assays were performed using CFDA SE tracking dye and T cell subsets were analyzed using dual color flow cytometry. The AmMSCs were capable of suppressing CD4+ and CD8+ T cell proliferation in response to alloantigen or polyclonal mitogen, independent of MHC matching, when cultured in direct contact with the T cells. Intracellular cytokine staining of CD4+ and CD8+ T cells revealed that interferon gamma and IL-10 production increased in co-cultures with AmMSC. This suggested that AmMSC are recognized by T cells, but are suppressing proliferation due to other mechanisms in vitro.
In order to determine if donor AmMSCs are detected by an immunocompetent host in vivo, we conducted the following study. C57/B6 mice received an intraperitoneal injection of either one million C57/B6 GFP AmMSC (congenic, n=5), one million Balb/c AmMSC (allogeneic, n=5) , or 5 million Balb/c splenocytes (allogeneic control, n=5) at time point zero, and then were given an additional injection of the same cells at 4 weeks. Mice were bled at 0,2,4,5, and 6 weeks after the first injection. Serum was collected and assayed for alloantibody production. Alloantibody results revealed IgG and IgM responses against donor alloantigen at titers of greater than 1:100 in all 5 animals injected with Balb/c MSC. This was significantly increased over titers observed in the 5 mice injected with congenic C57/B6 GFP AmMSC, and similar to the titers seen in mice injected with allogeneic splenocytes. At six weeks post injection animals were sacrificed and a mixed lymphocyte reaction (MLR) was performed using host splenocytes as responders and irradiated donor splenocytes as stimulators. Splenocytes were stained using CFDA SE tracking dye and stained for CD4+ and CD8+ positive T cells. Proliferation of CD4+ and CD8+ T cells was measured using dual color flow cytometry. No increase in proliferation compared to a naïve MLR was noted in the animals injected with the congenic C57/B6 GFP AmMSCs. However all animals injected with allogeneic AmMSCs or allogeneic splenocytes showed increased CD4+ and CD8+ proliferation.
It has been suggested primarily based on their in vitro properties, that MSCs may evade the immune response or induce tolerance after allogeneic transplantation. Our results document immune recognition of AmMSCs in vitro and in vivo. While we observed suppression of T-cell proliferation in vitro, our results after in vivo administration support a specific allogeneic immunization response equivalent to that induced by allogeneic splenocytes. These results argue against the theory that allogeneic MSCs may induce tolerance after transplantation, or that they can escape immune surveillance.