Mesenchymal Stromal Cells Engineered to Express ... - Cell Press

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Mesenchymal Stromal Cells Engineered to Express Erythropoietin Induce Anti-erythropoietin Antibodies and Anemia in Allorecipients Philippe M Campeau1,2, Moutih Rafei1, Moïra François1, Elena Birman1, Kathy-Ann Forner1 and Jacques Galipeau1–3 The Montreal Center for Experimental Therapeutics in Cancer, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada; Department of Human Genetics, McGill University, Montreal, Quebec, Canada; 3Division of Hematology/Oncology, Department of Medicine and Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada 1 2

Autologous bone marrow mesenchymal stromal cells (MSCs) have been successfully used for the delivery of erythropoietin (EPO) in murine models of anemia and myocardial infarction. For clinical applications where a transient effect would be adequate, such as myocardial infarction, the use of EPO-engineered universal donor allogeneic MSCs would be a substantial convenience. We thus investigated whether MSCs from C57BL/6 mice would permit robust transient EPO delivery in normal BALB/c allorecipients. Implantation of MSCs overexpressing murine EPO led to increases in hematocrit in syngeneic and allogeneic mice, but the latter eventually developed severe anemia due to acquired neutralizing anti-EPO antibodies. As MSCs constitutively produce the CCL2 chemokine which may behave as an adjuvant to the anti-EPO immune response, experiments were performed using EPO-engineered MSCs derived from CCL2−/− mice and similar results were obtained. In conclusion, MHC-mismatched MSCs can break the tolerance to autoantigens and lead to the development of pathogenic autoantibodies. Received 2 October 2008; accepted 6 November 2008; published online 16 December 2008. doi:10.1038/mt.2008.270

Introduction Bone marrow mesenchymal stromal cells (MSCs) are nonhematopoietic progenitor cells possessing immunomodulatory properties and excellent in vitro expansion potential.1 Preclinical studies using genetically modified MSCs have demonstrated them to be ideal delivery vehicles for the in vivo administration of therapeutic proteins.2–4 We have previously reported that murine MSCs genetically modified to secrete erythropoietin (EPO) generate a dose-dependent increase in the hematocrit when subcutaneously implanted in mice5–9 and have observed the development of anti-EPO antibodies in some mice without secondary anemia.10 In this study, we investigated the potential use of MSCs as a cell therapy platform for the delivery of high levels 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 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 typical MSC phenotype and retain mesenchymal plasticity, as described previously.11 C57BL/6 MSCs were transduced using a murine EPO encoding retrovirus, and confirmed to secrete high EPO levels (Figure 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 (Figure 1b). All mice were then re-treated with the same MSCs, which led to a transient increase in Hct in both groups, but eventually to severe anemia again only in the BALB/c allorecipient mice. A high anti-EPO antibody titer was obtained in BALB/c mice, while a progressive increase in antibody levels occurred in C57BL/6 mice (Figure 2a). However, the antibodies present in the C57BL/6 plasma at 12 weeks did not neutralize the biological effect of EPO as assessed by the proliferation rate of the EPO-responsive UT7-EPO cells in the presence of murine plasma (Figure 2b). This could be related to the specific epitope diversity to which the antibody response is mounted.12 Blood platelet and white blood cell counts were normal in an analyzed subset of anemic mice (data not shown). Allogeneic MSCs thus induced the formation of neutralizing anti-EPO antibodies with secondary anemia. 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 array. This qualitative analysis (results not shown) revealed a high secretion of the C-C motif chemokine 2 (CCL2), which was confirmed by enzymelinked immunosorbent assay (ELISA) (Figure 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 mice (MSC characterization in Figure 3a–d). The Hct increased

Correspondence: Jacques Galipeau, Division of Hematology/Oncology, Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada. E-mail: [email protected] Molecular Therapy vol. 17 no. 2, 369–372 feb. 2009

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Allogeneic MSCs Break Tolerance to Autoantigens

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Figure 1 Transplantation of EPO expressing MSCs. (a) Murine EPO secreted by transduced MSCs (expressed as µg/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 initially injected with 10 × 106 MSCs at week 0, then 30 × 106 MSCs at week 6. Error bars represent SD. EPO, erythropoietin; MSC, mesenchymal stromal cell.

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Figure 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 added to the EPO containing medium, as assessed by the formazan formation in the MTS assay (average of three samples per group). Error bars represent standard deviations. *P < 0.05. EPO, erythropoietin.

rapidly and fell below normal in allogeneic mice within 4 weeks (Figure 4a). A high anti-EPO antibody titer developed in allogeneic BALB/c mice, with very low anti-EPO antibodies in the plasma of syngeneic C57BL/6 mice (Figure 4b). The mice which were not killed normalized their hematocrit within 12 weeks and had normal erythropoiesis on bone marrow examination in the recovery phase (data not shown).

Discussion The potential use of allogeneic MSCs for cell transplantation is generating growing interest.13 To further study the potential of MSCs as universal donor cells, we assayed syngeneic and allogeneic MSCs for the delivery of EPO. Surprisingly, MSCs that secrete very high levels of EPO eventually led to anemia in the allogeneic context. In syngeneic mice, antibodies against EPO could be detected, but these antibodies did not neutralize EPO in a cell culture assay with an EPO-dependent cell line. In our first series of experiments depicted in Figures 1 and 2, MSCs also expressed the prokaryotic β-galactosidase antigen (LacZ), which may play a role in the observed autoimmunization to EPO. However, in a distinct set of experiments performed with CCL2−/− MSCs that were not gene-engineered with β-galactosidase (Figures 3 and 4), we also observed autoimmunization to EPO in the allogeneic implant scenario and not in the syngeneic. This is corroborated by the extent of polycythemia and the anti-EPO antibody levels in our previously published results using C57BL/6 MSCs expressing only murine EPO.12 370

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Figure 3 Characterization of CCL2−/− MSCs. (a) CCL2 secretion from NIH-3T3, WT C57BL/6 MSCs and CCL2−/− MSCs (expressed as pg/million cells/24 h). (b) Immunophenotype of the CCL2−/− MSCs. (c) Adipocytes (lipid droplets stained with Oil Red O) and osteoblasts (mineralization stained with Alizarin red) formed from CCL2−/− MSCs after 4 weeks in respective differentiation medium. (d) Murine EPO secreted by transduced CCL2−/− MSCs (expressed as µg/million cells/24 h). Error bars represent standard deviations. EPO, erythropoietin; MSC, mesenchymal stromal cell.

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Figure 4 Development of anti-EPO antibodies and anemia with CCL2−/− MSCs. (a) Weekly average hematocrit results after the 20 × 106 CCL2−/− MSCs transplantation at week 0 (N = 10 mice in BALB/c group, N = 5 mice in C57BL/6 group). (b) Anti-EPO antibodies detected in murine plasma diluted 20-fold (average of three samples per group). Error bars represent standard deviations. *P < 0.05. EPO, erythropoietin; MSC, mesenchymal stromal cell.

Acquisition of acquired autoantibodies arising from use of recombinant protein pharmaceuticals has been described. Indeed, neutralizing anti-interferon-β antibodies are seen in up to 35% of patients with multiple sclerosis treated with this protein.14 Antibodies to administered human proteins are also seen with insulin,15 interferon-α,16 factor VII,17 and various lysosomal enzymes.18 Concerning the development of antibodies with the use of human MSCs, anti-calf serum antibodies were detected in humans transplanted with MSCs cultured with fetal calf serum.19 More than 200 cases of EPO-related pure red cell aplasia have been reported in humans treated with recombinant human EPO.20 Identified and supposed factors include the adjuvant effect of www.moleculartherapy.org vol. 17 no. 2 feb. 2009


the rubber in contact with the solution, the formation of aggregates modulated by the stabilizers, and the storage and mode of administration of the product. The incidence of EPO-induced pure red cell aplasia was reduced by >90% after the formulation, storage, and administration of EPO was normalized. Furthermore, macaques treated with homologous EPO by gene therapy develop anti-EPO antibodies when the EPO levels are too high.21,22 Despite these results, EPO gene and cell therapy efforts continue, in the hope of developing alternative, sustained solutions for anemia. The absence of pure red cell aplasia in some of the other models23 could be explained by a controlled or more moderate release of EPO. We and others have demonstrated that immune-competent recipients will mount an immune response against allogeneic MSCs and that MSCs can behave as antigen-presenting cells as well.24–27 Interestingly, we observed that syngeneic MSCs were not associated with acquisition of neutralizing anti-EPO antibodies. We can, therefore, speculate that the immune rejection of allogeneic MSCs is accompanied by an immune response that acts as an adjuvant in breaking the tolerance to MSC-associated EPO. The bias of BALB/c mice to develop a Th2 response could also contribute to explain higher anti-Epo levels in an allogeneic context.28–30 Though MSC-driven acquisition of antibodies to xeno-antigens, such as fetal bovine serum,19 is reconcilable with the notion of “immune-suppressive” MSCs, the break of tolerance to a selfantigen such as EPO severely challenges the “immune-privileged” attribute sometimes bestowed upon MSCs. The mechanisms leading to autoimmune responses are still the subject of intensive study. Natural antibodies are ubiquitous in healthy individuals,31 but the events leading to the development of pathogenic, often polyclonal, autoantibodies are still debated32 and have been shown to often implicate dysregulation of Tregs.33 Findings relevant to the development of anti-EPO antibodies following allogeneic MSC transplantation include the association of autoimmune disease following hematopoietic cell transplantation,34 and the fact that EPO can enhance B cell–mediated immune responses.35 Studying the role of Tregs, EPO-enhanced immune response and epitope diversity of anti-EPO antibodies are amongst the interesting investigational avenues to consider in this murine model of acquired anti-EPO antibodies and secondary anemia. In summary, we have observed that allogeneic murine MSCs engineered to overexpress a self-antigen, such as EPO, led to a break of tolerance when administered to mice with a normal immune system. Although the translation of this finding to humans has not been made, caution should be used in clinical trials utilizing allogeneic MSCs for the therapeutic delivery of human proteins. Conversely, these experiments also suggest that allogeneic MSCs may be useful tools for breaking tolerance to cancer-associated autoantigens and may be of use in cancer immunotherapy.

Materials and Methods Animals, cells, and retrovirus. UT7-EPO cells36 are a gift from Anna Rita Migliaccio (Mount Sinai School of Medicine, New York). Female BALB/c, C57BL/6, and CCL2−/− C57BL/6 (B6.129S4-Ccl2tm1Rol/J) mice were from Jackson laboratories (Bar Harbor, ME). Murine Epo-encoding retroviruses were generated as described previously11 and were used to transduce C57BL/6 MSCs (previously transduced with a LacZ-encoding retrovirus, a gift from Bernard Massie, Montreal Biotechnology Research Institute) and CCL2−/− C57BL/6 MSCs. Molecular Therapy vol. 17 no. 2 feb. 2009


MSCs harvest, characterization, and differentiation. Whole bone mar-

row from femurs and tibias of WT female C57BL/6 or CCL2−/− C57BL/6 mice was harvested and placed in culture in complete media. Five days later, nonadherent cells were washed and adherent cells were kept in culture for a period of 5–6 weeks before the appearance of a homogeneous polyclonal population. MSCs were phenotyped by FACS Calibur cytometer (BD Biosciences, San Jose, CA) using R-phycoerythrin (PE)-conjugated anti-CD31, CD44, CD45, CD73, CD90, CD105, MHCI, and MHCII. MSC plasticity was tested by the induction of differentiation into mesenchymal lineages as follows: for osteogenic differentiation, MSCs (70–80% confluent) were cultured in media supplemented with β-glycerol phosphate (10 nmol/l), dexamethasone (10−8 mol/l), and ascorbic acid-2 phosphate (5 µg/ml) for 4 weeks with media changes every 2–3 days. Alizarin Red S was then used to stain calcium in the mineralized extracellular matrix as shown previously.11 To induce adipogenic differentiation, MSCs (50–60% confluent) were cultured in complete media supplemented with indomethacin (46 µmol/l), 3-isobutyl-methylxanthine (0.5 nmol/l), dexamethasone (1 µmol/l), and insulin (10 µg/ml) for 7 days with continual media changes every 2 days. Oil Red O was used for lipid droplet staining. MSC implantation and blood analyses. MSCs were mixed with 250 µl of Matrigel (BD Biosciences) and injected subcutaneously in the flanks of the mice. Animal studies were approved by the McGill University Animal Care Committee. The Hct was measured by blood centrifugation, and other parameters were intermittently analyzed using the Vet ABC animal blood counter (ABX Hematology, Garden Grove, CA) and the VetScan VS2 biochemical analyzer (Abaxis, Union City, CA). ELISAs and cytokine arrays. Murine EPO ELISA, Murine CCL2 ELISAs,

and recombinant murine EPO, were purchased from R&D System (Minneapolis, MN) and used according to manufacturer’s instructions. A rabbit polyclonal antibody against EPO (H-162) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For conducting an ELISA test, 0.5 µg/ml of recombinant murine EPO was used to coat Nunc Maxisorb 96-well plates (Thermo Fisher Scientific, Rochester, NY), using coating buffer, and then using ELISA wash solution and Assay diluent solution from eBioscience (San Diego, CA). After washing three times, murine plasma was applied starting with 1/20 dilutions. After a 2-hour incubation, the wells were washed thrice, and a 1/10,000 dilution of 1 mg/ml rabbit anti-mouse HRP-coupled antibody was used. In the case of the rabbit polyclonal antibody against EPO used to create a standard curve, with a 1/10,000 dilution of 1 mg/ml goat anti-rabbit HRP-coupled antibody (both from Bethyl Laboratories, Montgomery, TX). After a 1-hour incubation and further washing, the ELISA was revealed using ABTS substrate (Invitrogen, Carlsbad, CA). The assay gives values in anti-EPO equivalents, as a commercial mouse anti-mouse antibody is not available for precise quantification. For the cytokine array, we used RayBio Mouse Cytokine Antibody Array 3 from Raybiotech (Norcross, GA). We used C57BL/6 murine MSCs cell culture supernatant and applied it to the membranes, which were then processed according to the manufacturer’s instruction and the blots were analyzed with Image J.37

Antibody neutralization assay. UT7-EPO cells were expanded in Dulbecco’s modified Eagle’s media containing 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10 ng/ml recombinant murine EPO. Furthermore, 20,000 cells were seeded per well of a 96-well plate, in 100 µl of media, and 20 µl of murine plasma were added. After 48 hours of incubation, cell proliferation was assessed using a MTS-based assay (CellTiter, Promega, Madison, WI) according to the manufacturer’s protocol. Statistical analysis. P values were calculated by paired Student’s t-test.

ACKNOWLEDGMENTS This work was supported by a grant from the Canadian Gene Cure Foundation. The authors have no conflict of interest to declare.

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