To investigate the part played by this chemokine in the development of autoantibodies, we gene-engineered MSCs from CCL2/C57BL/6 mice to secrete EPO and transplanted them in C57BL/6 and BALB/c mice (MSC characterization inFigure 3ad). response, experiments were performed using EPO-engineered MSCs derived from CCL2/mice and related results were obtained. In conclusion, MHC-mismatched MSCs can break the tolerance to autoantigens and lead to the development of pathogenic autoantibodies. == Intro == Bone marrow mesenchymal stromal cells (MSCs) are nonhematopoietic progenitor cells possessing immunomodulatory properties and excellentin vitroexpansion potential.1Preclinical studies using genetically altered MSCs have proven them to be ideal delivery vehicles for thein vivoadministration of restorative proteins.2,3,4We have previously reported that murine MSCs genetically modified to secrete erythropoietin (EPO) generate a dose-dependent increase in the hematocrit when subcutaneously implanted in mice5,6,7,8,9and have observed the development of anti-EPO antibodies in some mice without secondary anemia.10In this study, we investigated the potential use of MSCs like a cell therapy platform for the delivery of high levels Rabbit Polyclonal to HEY2 of EPO in immunocompetent mice (where allogeneic MSCs act as universal donor cells). Although both syngeneic and allogeneic MSCs lead to an increase in hematocrit (Hct) levels, we BMS-911543 here describe that only allogeneic MSCs lead to the development of anemia due to the generation of neutralizing anti-EPO antibodies. == Results == The C57BL/6 MSCs used in this study were assessed to have a standard MSC phenotype and maintain mesenchymal plasticity, as explained previously.11C57BL/6 MSCs were transduced using a murine EPO encoding retrovirus, and confirmed to secrete high EPO levels (Number 1a). When injected subcutaneously in mice, a rapid increase in Hct was observed followed by a return to baseline. Interestingly, in the BALB/c allorecipients, the Hct further declined to an average of 30% by week 6 (Number 1b). All mice were then re-treated with BMS-911543 the same MSCs, which led to a transient increase in Hct in both organizations, but eventually to severe anemia again only in the BALB/c allorecipient mice. A high anti-EPO antibody titer was acquired in BALB/c mice, while a progressive increase in antibody levels occurred in C57BL/6 mice (Number 2a). However, the antibodies present in the C57BL/6 plasma at 12 weeks did not neutralize the biological effect of EPO as assessed from the proliferation rate of the EPO-responsive UT7-EPO cells in the presence of murine plasma (Number 2b). This could be related to the specific epitope diversity to which the antibody response is definitely mounted.12Blood platelet and white blood cell counts were normal in an analyzed subset of anemic mice (data not shown). Allogeneic MSCs therefore induced the formation of neutralizing anti-EPO antibodies with secondary anemia. == Number 1. == Transplantation of EPO expressing MSCs.(a) Murine EPO secreted by transduced MSCs (expressed as g/million cells/24 h). (b) Weekly average hematocrit results after the MSC transplantation (N= 10 mice in BALB/c group,N= 5 mice in C57BL/6 group) Mice were in the beginning injected with 10 106MSCs at week 0, then 30 106MSCs at week 6. Error bars symbolize SD. EPO, erythropoietin; MSC, mesenchymal stromal cell. == Number 2. == Development of anti-EPO antibodies.(a) Anti-EPO antibodies detected in murine plasma diluted 20-fold (average of three samples per group). (b) Proliferation of UT7-EPO cells when murine plasma at week 12 is usually added to the EPO made up of medium, as assessed by the formazan formation in the MTS assay (common of three samples per group). Error bars represent standard deviations. *P< 0.05. EPO, erythropoietin. To assess which MSC factor(s) generated by MSCs could modulate the break of tolerance to the EPO autoantigen, we studied the secretome of murine MSCs using a cytokine BMS-911543 array. This qualitative analysis (results not shown) revealed a high secretion of the C-C motif chemokine 2 (CCL2), which was confirmed by enzyme-linked immunosorbent assay (ELISA) (Physique 3a). To investigate the role played by this chemokine in the development of autoantibodies, we gene-engineered MSCs from CCL2/C57BL/6 mice to secrete EPO and transplanted them in C57BL/6 and BALB/c.