Exploitation of Herpesvirus Immune Evasion Strategies to Modify the Immunogenicity of Human Mesenchymal Stem Cell Transplants

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS: We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable

Anomer-Equilibrated Streptozotocin Solution for the Induction of Experimental Diabetes in Mice (Mus musculus)

Streptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the solution may change considerably during the first 2 h after dissolution due to equilibration of the 2 anomers (α and β) of streptozotocin. Because of the drug's alleged instability in solution, the typical recommendation is to administer streptozotocin within 10 min after dissolution. We compared the induction of diabetes in NOD/SCID mice by injection of a single high dose of freshly made or anomer-equilibrated streptozotocin solution. Solutions were prepared from dry compound containing 85% of the α anomer, which is the more toxic of the 2. Body weight and nonfasting blood glucose levels were measured weekly for 8 wk. Both solutions induced long-term hyperglycemia, but blood glucose levels and mortality were higher and damage to pancreatic islands more pronounced in the mice receiving freshly prepared solution. A small proportion of mice did not respond in both treatment groups. If stored at 4 °C in the dark, the anomer-equilibrated solution retains its biologic activity for at least 40 d; under those conditions the streptozotocin content decreases by 0.1% daily, as determined by HPLC. Anomer-equilibrated streptozotocin solution has several practical advantages, and we recommend its use as standard for the induction of experimental diabetes because this practice may improve reproducibility and comparison of results between different laboratories

Sphingosine Phosphate Lyase Is Upregulated in Duchenne Muscular Dystrophy, and Its Inhibition Early in Life Attenuates Inflammation and Dystrophy in Mdx Mice

Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy

Different herpesviral immunoevasins inhibit MHC class I expression on hMSCs to a different extent.

<p>(A) Flow cytometric analysis of culture-expanded (Ctrl) hMSCs and of hMSCs transduced with RVs coding for eGFP alone or for eGFP and either the BHV-1 UL49.5, EBV BNLF2A, HCMV US2 or HCMV US11 protein. The data are from cells analyzed 30 days after transduction. Analysis of the hMSCs at 5 and 90 days post-transduction yielded very similar results (data not shown). MFIs of the HLA-ABC signal of eGFP^- (untransduced [UT]) and eGFP⁺ (transduced [T]) cell populations in each sample and their ratio are given below the dot plots (B). (C) Western blot analysis of lysates from untransduced hMSCs (−) and of hMSCs transduced with RV-BLF2A-eGFP, RV-UL49.5-eGFP, RV-US2-eGFP or RV-US11-eGFP (+). The specificity of the antibodies used is indicated at the left. Numbers at the right represent molecular weights in kilodalton (kDa). The 26-kDa and 19.4-kDa protein species recognized by the US2-specific antibodies correspond to N-glycosylated and non-glycosylated forms of the US2 protein, respectively.</p

NK cell levels in C57Bl mice treated with NK1.1-specific MAbs.

<p>Flow cytometric analysis of PBMCs of a representative mouse stained with antibodies directed to murine CD45, CD3 and NK1.1. The upper panels show data of peripheral blood taken before treatment with NK1.1-specific MAbs. The lower panels are derived from a peripheral blood sample taken 7 days after intraperitoneal injection of 100 µg anti-NK1.1 antibodies. Cells stained with isotype-matched control antibodies are depicted in the left column. In the right column only CD45⁺ cells that were gated in R1 (middle column) have been analyzed for surface expression of NK1.1 (gate R2) and CD3 (gate R3). All peripheral blood samples of mice treated with NK1.1-specific MAbs showed similar low levels of NK1.1⁺ CD3⁻ cells.</p

Herpesviral immunoevasins do not compromise the replication rate of hMSCs in culture.

<p>hMSCs were transduced at passage number 8 with bicistronic RVs encoding eGFP and a herpesviral immunoevasin and kept in culture for 7 additional passages. Lines represent untransduced hMSCs (□) and cells transduced with RV-UL49.5-eGFP (♦), RV-BNLF2A-eGFP (▴), RV-US2-eGFP (▪) or RV-US11-eGFP (•) during 5 passages.</p

US2 and US11 inhibit MHC class I surface expression in hMSCs of different donors.

<p>(A) Flow cytometric analysis of HLA-ABC surface expression in hMSCs derived from the BM of three different donors. Gray lines represent untransduced hMSCs. Black lines correspond to hMSCs transduced with RV-US2-eGFP (upper panels) or with RV-US11-eGFP (lower panels). The presented data are from cells analyzed 30 days after transduction. (B) Flow cytometric analysis of CD44 surface expression in hMSCs of donor 4 at 30 days after transduction with RV-US2-eGFP (upper panel) or RV-US11-eGFP (lower panel). Similar results were obtained with cells of all other donors (data not shown). (C) Comparison of down-regulation of HLA-ABC surface expression by RV-US2-eGFP and RV-US11-eGFP. Shown are HLA-ABC MFI values of untransduced (gray bars) and transduced (black bars) cells of single samples of culture-expanded hMSCs from 4 donors (marked D1 to D4; values derived from data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014493#pone-0014493-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014493#pone-0014493-g002" target="_blank">2</a>). The differences in the average MFI ratio (calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014493#pone-0014493-g001" target="_blank">Fig. 1</a>) between the hMSCs exposed to RV-US2-eGFP and those transduced with RV-US11-eGFP is highly significant (*p = 0.0005).</p

Infiltration of recipient's immune cells in the vicinity of the hMSC-implants.

<p>Immunohistological and histochemical analysis of frozen tissue section. (A and B) Pinnea excised 3, 7 and 14 days after implantation of <i>LacZ</i>-transduced hMSCs (A) or <i>LacZ</i>-transduced US11-hMSCs (B). Implanted hMSCs are visualized in the pinnea by X-gal (blue) staining. DAB staining was used to identify granulocytes (endogenous peroxidase) and CD8⁺ cells. The granulocytes are recognized by the intracellular localization of the brown precipitate while the CD8⁺ cells (arrows) are predominantly showing staining of the plasma membrane. The anti-NKp46 antibody was used to identify NK cells (red, cell membrane staining) and nuclei were stained with Hoechst 33342 (blue). (C) Control tissues (pinna and spleen). (D) CD8⁺ and NK cell counts in pinnea transplanted with untreated hMSCs (black bars) or US11-hMSCs (gray bars). The data represent averages ± SD of CD8⁺ and NKp46⁺ cells present in 9 different tissue sections (3 pinnea per experimental group and 3 sections with a mutual distance of 32 µm per pinna). *p<0.05.</p