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

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

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

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

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

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

IFN-γ modulates expression of MHC class I and class II molecules on US2- and US11-transduced hMSCs.

<p>Flow cytometric analysis of the expression of MHC class I (HLA-ABC; upper panels) or MHC class II (HLA-DR; lower panels) proteins on the surface of untransduced hMSCs (Ctrl) and of cells transduced with RV-US2-eGFP (US2) or with RV-US11-eGFP (US11). The cells were (+ IFN-γ) or were not (- IFN-γ) incubated for 48 hours with 100 ng/ml IFN-γ prior to flow cytometry. The <i>US11</i>- and <i>US2</i>-transduced hMSCs used for this experiment, were sorted on the basis of <i>eGFP</i> expression.</p