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Keywords:

  • Antigen-presenting cells;
  • Antigen-processing machinery;
  • CTL;
  • Immunogenicity;
  • Mesenchymal stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen-presenting cells (APC) for CD4+ T-cell responses; here we have investigated their ability to serve as APC for in vitro CD8+ T-cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)-γ and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN-γ. To investigate the mechanisms involved in MSC resistance to CTL-mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I-related antigen-processing machinery (APM) components and of immunosuppressive HLA-G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB-1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN-γ. Surface HLA-G was constitutively expressed on MSC but was not involved in their protection from CTL-mediated lysis. MSC supernatants containing soluble HLA-G (sHLA-G) inhibited CTL-mediated lysis, whereas those lacking sHLA-G did not. The role of sHLA-G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA-G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I-restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL-mediated lysis through a mechanism that is partly sHLA-G-dependent.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Human mesenchymal stem cells (MSC) are a rare subset of nonhematopoietic stem cells localized around the vasculature and trabeculae in the bone marrow (BM), representing 0.01%–0.001% of total BM cells. MSC have been also isolated from many other human tissues [1]. Because of the limited stemness of MSC, a recent consensus conference has proposed to denominate them “mesenchymal stromal cells” [2].

In the BM, MSC contribute to the formation of the hemopoietic stem cell (HSC) niche, supporting growth, maturation, differentiation, and survival of HSC [3]. On these grounds, human MSC have been successfully administered to breast cancer patients to improve engraftment after allogeneic HSC transplantation [4, 5]. MSC can differentiate into cells of mesodermal origin in vivo (osteoblasts, adipocytes, and chondrocytes), thus representing a promising tool for tissue repair [6, 7], and into cells of other lineages in vitro (muscle cells, hepatocytes, endothelial cells, and neurons), through a process called transdifferentiation [8].

MSC mediate immunoregulatory activities by inhibiting the functions of different cell types [9, 10]. As far as the effects on T lymphocytes are concerned, MSC (a) inhibit proliferation in response to mitogens [11, 12], anti-CD3 and anti-CD28-specific antibodies [13], or alloantigens [14, 15]; (b) induce anergy in naïve T cells [11, 15, 16]; (c) induce expansion of regulatory T cells [14, 17]; and (d) inhibit cytotoxic T lymphocytes (CTL)-mediated cytotoxicity against allogeneic cells [18, 19]. As far as the effects on natural killer (NK) cells are concerned, MSC (a) inhibit cytotoxicity against virus-infected cells [20], (b) inhibit interleukin (IL)-2-driven NK cell interferon (IFN)-γ secretion and proliferation [12, 21, 22], and (c) exert “veto” function for allogeneic cells [18]. In dendritic cells (DC), MSC (a) downregulate expression of costimulatory molecules [17, 23, 24], (b) inhibit in vitro differentiation of DC from monocytes and CD34+ progenitors [25, 26], and (c) reduce proinflammatory cytokine secretion (IL-12, IFN-γ, and tumor necrosis factor-α) and increase IL-10 secretion [14, 23, 25]. Furthermore, human MSC are poorly immunogenic, in spite of constitutive human leukocyte antigen (HLA)-class I expression and IFN-γ-inducible HLA-class II expression [27].

The immunoregulatory functions of human MSC, coupled with their low immunogenicity, provide a rationale for the use of allogeneic MSC to treat severe graft-versus-host disease (GvHD) [28] and, possibly, autoimmune disorders [29, 30]. Encouraging results have been obtained in patients with GvHD [31], whereas in two murine HSC transplantation models, MSC neither were immunoprivileged [32, 33] nor prevented GvHD [34].

It has been reported that, in a narrow window of IFN-γ concentration, human MSC can exert antigen-presenting cell (APC) functions for HLA-class II-restricted recall antigens, such as Candida albicans and Tetanus toxoid. MSC upregulate their HLA-class II antigen expression by autocrine secretion of low IFN-γ levels; however, when IFN-γ concentration in culture increases, HLA-class II antigen expression is downregulated and the APC function is inhibited [35]. Furthermore, IFN-γ-induced upregulation of class II major histocompatibility complex molecules on both murine and human MSC was found to be modulated by transforming growth factor-β, serum factors, and cell density in vitro [36]. Recently, it has been reported that MSC do not trigger effector functions in activated CTL, inducing an abortive activation program in the latter cells [37]. Here, we have investigated for the first time (a) the ability of MSC to process and present viral or tumor-associated antigens to HLA-class I-restricted CD8+ T cells, (b) the expression of HLA-class I-related antigen-processing machinery (APM) components in human MSC, and (c) the expression of immunoregulatory molecules, such as HLA-G, HLA-E, and the Granzyme B inhibitor protease inhibitor 9 (PI-9), in human MSC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Monoclonal and Polyclonal Antibodies

MSC were characterized using the following monoclonal antibodies (mAbs): anti-CD105, anti-CD73, anti-CD14 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), anti-CD34, anti-CD45, and anti-CD44 (Caltag Laboratories, Burlingame, CA, http://www.caltag.com). The mAb HC-10, which recognizes a determinant of β2 microglobulin (β2m)-free HLA-B heavy chain (HC) and HLA-A10, -A28, -A29, -A30, -A31, -A32, and -A33 HC [38, 39]; the anti-β2m mAb L368 [40]; the mAb TP25.99, which recognizes a conformational determinant of β2m-associated HLA-A, -B, and -C HC, and a linear determinant expressed by β2m-free HLA-B (except HLA-B73), HLA-A1, -A3, -A9, -A11, and -A30 HC [41] were developed and characterized as described [44]. The anti-MB1 mAb SJJ-3, the anti-delta mAb SY-4, the anti-zeta mAb NB1, the anti-LMP-2 mAb SY-1, the anti-LMP-7 mAb SY-3, the anti-LMP10 mAb TO-7, the anti-TAP-2 mAb SY-2, the anti-calnexin mAb TO-5, the anti-ERp-57 mAb TO-2, the anti-calreticulin mAb TO-11, and the anti-tapasin mAb TO-3 were developed and characterized as described [42, 43, 44]. MEM-G/9 and 87G [46] mAbs (anti-HLA-G) were purchased from Exbio (Vestec, CZ, http://www.exbio.cz). 7G3 and 3D12 [47] mAbs (anti-HLA-E) were kindly provided by Dr. Daniel E. Geraghty. 7D8 mAb (anti-PI-9) was purchased from Serotec Ltd. (Oxford, U.K., http://www.serotec.com).

All mAbs are of the IgG1 isotype, except HC-10 mAb, which is an IgG2a, and 7G3 mAb, which is a IgG2b. Irrelevant isotype-matched mouse immunoglobulins (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com) were used as controls. Fluorescein isothiocyanate (FITC)-conjugated F(ab′)2 fragments of rabbit anti-mouse IgG antibodies (Dako, Glostrup, Denmark, http://www.dako.com) or PE-conjugated F(ab′)2 fragments of goat anti-mouse IgG1 antibody (SouthernBiotech) were used as secondary reagents.

Cell Separation and Culture

Human MSC were expanded in vitro from healthy donors' BM obtained after informed consent. Mononuclear cells were isolated by Ficoll-Hystopaque (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com; 1,077 g/ml density) gradient centrifugation at 2,500 rpm for 30 minutes, washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich), counted, and plated at 20–30 × 106 cells per 75-cm2 flask in MesenCult basal medium supplemented with mesenchymal Stem Cell Stimulatory Supplement (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). After 1 week of culture at 37°C and 5% CO2, nonadherent cells were removed, and medium was replaced every other day. MSC were trypsinized (trypsin-EDTA solution; Cambrex, Verviers, Belgium, http://www.cambrex.com) when cultures reached 80%–100% confluence. The purity of MSC suspensions was assessed by flow cytometry on the basis of the expression of CD105, CD73, and CD44 and the absence of CD34, CD45, and CD14 (Fig. 4A). MSC were cultured in vitro for 1–2 passages. MSC supernatants were collected after 24–48 hours of culture. Depletion of soluble HLA-G was performed using Dynabeads Pan Mouse IgG (Dynal Biotech, Oslo, Norway http://www.invitrogen.com/dynal), coated with anti-HLA-G1/-G5 mAb MEM-G/9 (Exbio) for 1 hour at 4°C, following the manufacturer's protocol. The TAP-deficient HLA-A2+ lymphoma T2 cell line, the Epstein-Barr virus (EBV)-positive human B-cell lymphoma Jy cell line (purchased from American Type Culture Collection, Rockville, MD, http://www.atcc.org), the EBV-infected Raji Burkitt's lymphoma cell line, and the lymphoblastoid cell line 721.221.G1 (kindly provided by Dr. Francesco Puppo, University of Genoa, Genoa, Italy) were cultured in RPMI 1640 medium (Euroclone, Wetherby, U.K., http://www.euroclone.net) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, http://www.invitrogen.com), HEPES buffer, nonessential amino acids, and antibiotics (Cambrex).

Flow Cytometry

The intracellular staining [45] and the surface staining [48] of MSC were performed as previously described. Cells were subsequently subjected to flow cytometry using a FACSCalibur instrument (BD Biosciences).

CellQuest software (BD Biosciences) was used for data analysis. Results are expressed as percentage of positive cells or as mean relative fluorescence intensity (MRFI), obtained as a ratio between mean fluorescence intensity (MFI) of cells stained with specific mAb and MFI obtained with isotype control.

MSC Transfection and Infection

mRNA was extracted from four human neuroblastoma (NB) cell lines (GI-ME-N, SKNBE, SHSY5Y, and IMR-32) or from normal donor peripheral blood mononuclear cells (PBMNC) using the mRNA Isolation Kit (Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com) according to the manufacturer's protocol, pooled in equal ratio at 200 μg/ml, and stored at −80°C until use. MSC transfection was performed using Transmessenger Transfection Reagent (Qiagen, Chatworth, CA, http://www1.qiagen.com) following the manufacturer's protocol. After transfection, MSC were cultured for an additional 24 hours in fresh medium before being used, as reported [49]. The Jy cell line and MSC were infected with NS3Ag-expressing vaccinia virus (VV) (5 plaque-forming units per cell) for 1 hour at 37°C and then washed twice and cultured overnight in fresh medium before being used, as reported [50, 51].

Peptides

The peptides GILGFVFTL [52] (Flu matrix 58–66 peptide from Influenza A virus) and CINGVCWTV [53] (NS3 1,073–1,081 peptide from Hepatitis C virus [HCV]) were manually synthesized using the standard method of solid phase peptide synthesis, which follows the 9-fluorenylmethoxycarbonyl strategy with minor modifications [54]. The HLA-A2 binding of the selected peptides was tested by in vitro cellular binding assay using the T2 cell line, as described [55]. Cells were pulsed with peptides (10 μM) for 2 hours at 37°C, washed twice, and then used as APC for in vitro assays.

CTL Generation

NB-specific CTL were generated by weekly restimulation of freshly isolated CD8+ T cells from normal donors with autologous monocyte-derived DC transfected with NB mRNA, as previously described [49]. Flu-specific CTL were generated by weekly restimulation of freshly isolated CD8+ T cells with autologous DC pulsed with Flu peptide. A CTL clone specific for NS3 peptide 1,073–1,081 was generated by weekly restimulation of freshly isolated CD8+ T cells with autologous DC pulsed with NS31,073–1,081 peptide, followed by limiting dilution cloning.

Soluble HLA-G Enzyme-Linked Immunosorbent Assay

Soluble HLA-G (sHLA-G) enzyme-linked immunosorbent assay (ELISA) was performed using MaxiSorp Nunc-Immuno 96-microwell plates (Nunc A/S, Roskilde, Denmark, http://www.nuncbrand.com) coated overnight at 4°C with mAb MEM-G/9 (Exbio; 10 μg/ml) in 0.001 M PBS, pH 7.4. After three washes with PBS 0.05% Tween 20 (washing buffer), plates were saturated with 200 μl/well of PBS with 2% bovine serum albumin for 30 minutes at room temperature (RT).

One hundred microliters of samples (supernatants from MSC) or standards (serial dilutions of calibrated 721.221.G1 cell line supernatant) was added to each well and incubated at RT for 1 hour. Plates were washed three times with washing buffer and then incubated with 100 μl/well of horseradish peroxidase-conjugated anti-β2m mAb NAMB-1 (1 μg/ml) at RT for 1 hour. After three washes, plates were incubated with the substrate (3′,3′,5′,5′-tetramethylbenzidine; Sigma-Aldrich) for 30 minutes at RT. H2SO4 5 M (100 μl/well) was added, and optical densities were measured at 450 nm. The assay's lowest threshold was 1.95 ng/ml sHLA-G. Each sample was tested in duplicate.

ELISpot Assays

IFN-γ and Granzyme B ELISpot assays were carried out using Multiscreen-IP Millipore plates (Millipore, Billerica, MA, http://www.millipore.com) coated overnight at 4°C with anti-IFN-γ (clone 1-DK-1; 1 μg/ml; Mabtech, Nacka, Sweden, http://www.mabtech.com) or anti-Granzyme B (clone GB10; 15 μg/ml; Mabtech) mAbs, respectively. Plates were then washed and blocked with PBS with 2% human albumin (Kedrion SpA, Lucca, Italy, http://www.kedrion.com). Specific CTL (3 × 104) were cultured together with 6 × 104 target cells (1:2 cell ratio) in 200 μl of RPMI 1640 5% human AB serum. The T2 cell line, pulsed with Flu peptide (10 μM), or MSC (unmanipulated, pulsed with Flu peptide, or transfected with NB mRNA) were used as targets. All targets were γ-irradiated (45 Gy) before being plated. Blocking experiments were performed by adding 10 μg/ml anti-HLA class I mAb TP25.99 to target cells 30 minutes before culture with lymphocytes. After 20 hours of incubation at 37°C and 5% CO2, ELISpot was developed according to the manufacturer's protocol. Spots were counted using Bioreader 2000 (Biosys, Karben, Germany, http://www.biosys.de).

NS3 Antigen Presentation

Cells of a CTL clone specific for the HLA-A2-restricted peptide NS31,073–1,081 (CINGVCWTV) were stimulated for 4 hours at 37°C and 5% CO2 with peptide-pulsed APC (Jy or MSC) or NS3Ag-VV-infected APC in U-bottomed microculture wells at 6 × 104 APC per 3 × 104 T cells per well in 0.2 ml of RPMI 1640 10% fetal bovine serum. Brefeldin-A (10 μg/ml; Sigma-Aldrich) was added after 2 hours of culture. Cells were washed and stained with anti-CD8 FITC (Caltag Laboratories) for 15 minutes at 4°C, fixed, permeabilized using Cytofix/Cytoperm solution (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) at 4°C for 20 minutes, rewashed with Perm Wash Buffer (BD Pharmingen), intracellularly stained with PE-labeled anti-IFN-γ antibody (BD Pharmingen) for 15 minutes at 4°C, and finally subjected to flow cytometry.

Cytotoxicity Assays

CTL-mediated cytotoxicity was evaluated by standard 4-hour 51Cr release assay. The effector-to-target (E:T) cell ratio ranged from 100:1 to 1:1. A 10-fold excess of unlabeled K562 cells was added to minimize NK-like activity. Blocking experiments were performed by adding anti-HLA class I TP25.99, anti-HLA-G 87G, or anti-HLA-E 7G3 and 3D12 mAbs (10 μg/ml) to target cells, 30 minutes before culture with lymphocytes. Cold target inhibition was performed only for Flu-specific CTL by adding a 10-fold excess of unlabeled Flu-pulsed T2 cell line. Specific lysis was determined by the following formula: % specific lysis = cpm (sample-spontaneous)/cpm (total-spontaneous) × 100.

Assay for PI-9 Expression

MSC (unmanipulated, transfected with NB mRNA, or pulsed with Flu peptide) were tryspinized, washed, fixed, and permeabilized using Cytofix/Cytoperm solution (BD Pharmingen) at 4°C for 20 minutes, rewashed with Perm Wash Buffer (BD Pharmingen), and intracellularly stained with anti-PI-9 mAb (1 μg per 106 cells) in permeabilization buffer. Cells were then washed twice in permeabilization buffer and intracellularly stained with anti-mouse IgG1 PE mAb (Serotec) and then subjected to flow cytometry. Human tonsil mononuclear cells were used as a positive control. Results were expressed as MRFI.

Statistical Analysis

Data were analyzed by Prism 3.0 (GraphPad Software, Inc., San Diego, http://www.graphpad.com), using the two-tailed Wilcoxon rank test and the Kolmogorov-Smirnov test. p values <.01 or <.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

MSC Act as APC for HLA-Class I-Restricted Viral Peptides

To investigate whether HLA-class I molecules on MSC can present exogenously loaded HLA-class I-restricted peptides derived from viral antigens to specific CTL, HLA-A2+ MSC from four different donors were pulsed with Flu peptide or with an irrelevant peptide and used as APC for Flu-specific HLA-A2+ CTL. T2 cells pulsed with the same peptides were tested as positive control.

CTL efficiently recognized MSC pulsed with Flu peptide (137.3 spots per 30,000 blasts) in the IFN-γ ELISpot assay (Fig. 1A). The recognition was specific, since the number of IFN-γ producing CTL detected was significantly lower against MSC pulsed with an irrelevant peptide (31.5 spots per 30,000 blasts; p = .0135). Furthermore, the recognition is HLA class I-restricted, since addition of the HLA class I-specific mAb TP25.99 significantly reduced the number of IFN-γ producing CTL (14.33 spots per 30,000 blasts; p = .0095).

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Figure Figure 1.. Processing and presentation of viral antigens by MSC to CTL. (A): HLA-A2+ Flu-specific CTL were tested in IFN-γ ELISpot assay against an HLA-matched MSC or T2 cell line pulsed with Flu peptide (black bars) or with an irrelevant peptide (white bars). Gray bars indicate the inhibition of IFN-γ secretion obtained by adding anti-HLA class I monoclonal antibody (mAb) TP25.99 to target cells before coculture with CTL. Results are means ± SD from three different experiments. (B): HLA-A2+ Flu-specific CTL were tested in cytotoxicity assays against HLA-matched Flu-pulsed MSC (black bars) at three different effector-to-target ratios. Blocking experiments were performed by the addition of anti-HLA-class I mAb (white bars) or by cold target inhibition with an unlabeled Flu-pulsed T2 cell line (gray bars). Results are means ± SD from three different experiments. (C): IFN-γ secretion by NS3-specific HLA-A2+ CTL clone was investigated by flow cytometry after culture with an HLA-matched Jy cell line or MSC, both pulsed with NS3 peptide (black bars) or infected with NS3Ag vv (gray bars). White bars indicate the percentage of IFN-γ+ cells obtained incubating the CTL clone with unmanipulated Jy or MSC cells. Results are means ± SD from three different experiments. (D): HLA-A2+ NS3-specific CTL clone was tested in cytotoxicity assays against an HLA-matched MSC or Jy cell line, pulsed with NS3 peptide (black bars) or infected with NS3Ag vv (gray bars). Results are means ± SD from three different experiments. Abbreviations: IFN, interferon; MSC, mesenchymal stem cells; vv, vaccinia virus.

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In cytotoxicity assays, Flu-pulsed MSC were lysed with very low efficiency by specific CTL (specific lysis, 11.43% at a 50:1 E:T ratio) in an HLA-class I-restricted manner, as demonstrated by blocking experiments (specific lysis, 5% with anti-HLA-class I mAb [p = .0022] and 5.67% with cold target inhibition [p = .0022]) (Fig. 1B). CTL-mediated lysis of peptide-pulsed MSC was lower than that obtained with T2 cell line (specific lysis, 98.4% at a 50:1 E:T ratio). Next, to compare the efficiency of MSC to present an endogenously processed antigen versus an exogenously loaded peptide derived from the same antigen to specific CTL, MSC and Jy cells (tested as a positive control) were pulsed with NS31,073–1,080 peptide or infected with NS3Ag-VV and then used as APC for the NS3-specific CTL clone.

The CTL clone specifically secreted IFN-γ in response to infected or peptide-pulsed Jy cells (86.7% and 92.4% IFN-γ+ cells, respectively). Peptide-pulsed MSC were recognized more efficiently than infected MSC (41.2% IFN-γ+ cells vs. 6.8% IFN-γ+ cells, respectively; p = .047) (Fig. 1C). Peptide-pulsed MSC were lysed by the CTL clone, whereas NS3-VV infected MSC were not (specific lysis, 14.2% and 0.75%, respectively, at an E:T ratio of 10:1; p = .0008). The Jy cell line, either peptide-pulsed or infected, was recognized and lysed at a 10:1 E:T ratio (specific lysis, 46.5% and 40.03%, respectively) (Fig. 1D).

Tumor-Associated Antigen Processing and Presentation by MSC

To test the ability of MSC to process and present tumor-associated antigens in an HLA-class I-restricted manner, HLA-A2+ MSC were transfected with NB mRNA. Unmanipulated or transfected MSC were used as APC for HLA-matched NB-specific CTL.

CTL efficiently recognized transfected (262.7 spots per 30,000 blasts) but not unmanipulated MSC (90.3 spots per 30,000 blasts; p < .0001) in the IFN-γ ELISpot assay. Such recognition occurred in an HLA-class I-restricted manner, as demonstrated by inhibition with anti-HLA class I mAb (123.3 spots per 30,000 blasts; p = .0001) (Fig. 2A). When tested in cytotoxicity, CTL did not lyse either transfected or unmanipulated MSC at any E:T ratio (Fig. 2B).

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Figure Figure 2.. Processing and presentation of tumor-associated antigens by MSC to CTL. (A): HLA-A2+ NB-specific CTL were tested in IFN-γ ELISpot assay against HLA-matched MSC, unmanipulated or transfected with NB mRNA (black bars). Gray bars indicate the inhibition of IFN-γ secretion obtained by adding anti-HLA class I mAb to target cells before they were cultured with lymphocytes. Results are means ± SD from five different experiments. (B): HLA-A2+ NB-specific CTL were tested in cytotoxicity assay against HLA-matched MSC, either unmanipulated (gray bars) or transfected with NB mRNA (black bars). Results are means ± SD from five different experiments. Abbreviations: ctr, control; CTR, control; IFN, interferon; mAb, monoclonal antibody; MSC, mesenchymal stem cells; NB, neuroblastoma.

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Immunogenicity of MSC Is Not Affected by IFN-γ

MSC from three different HLA-A2+ donors, either unmanipulated or transfected with NB mRNA, were cultured for 72 hours with medium alone or recombinant human IFN-γ (1,000 U/ml), which upregulated surface expression of HLA class I on MSC (data not shown; [23]). These cells were used as APC for HLA-matched NB-specific CTL in the IFN-γ ELISpot assay.

CTL efficiently recognized transfected but not unmanipulated MSC (237 spots per 30,000 blasts and 99 spots per 30,000 blasts, respectively; p = .0008) in an HLA-restricted manner, as demonstrated by inhibition with anti-HLA class I mAb (155 spots per 30,000 blasts; p = .01). IFN-γ treatment did not significantly increase specific recognition of transfected MSC (274.5 spots per 30,000 blasts; p = .4957) (Fig. 3A). In the cytotoxicity assay, no lysis of unmanipulated or transfected MSC was observed at an E:T ratio of up to 80:1, either untreated or treated with IFN-γ (Fig. 3B).

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Figure Figure 3.. Effects of IFN-γ on antigen-presenting cell function of MSC. (A): HLA-A2+ NB-specific CTL were tested in IFN-γ ELISpot assays against HLA-matched MSC, either unmanipulated or transfected with NB mRNA, following treatment with IFN-γ or medium alone (black bars). Gray bars indicate the inhibition of IFN-γ secretion obtained by adding anti-HLA class I monoclonal antibody to target cells before they were cultured with lymphocytes. Results are means ± SD from three different experiments. (B): HLA-A2+ NB-specific CTL were tested in cytotoxicity assays at three different effector-to-target ratios against HLA-matched MSC, unmanipulated (white bars), transfected with NB mRNA (gray bars), or transfected with NB mRNA and pretreated with IFN-γ (black bars). Results are means ± SD from three different experiments. Abbreviations: ctr, control; IFN-g, interferon γ; MSC, mesenchymal stem cells; NB, neuroblastoma.

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Taken together, the above results demonstrated that MSC are able to present exogenously loaded peptides to specific CTL and to process endogenous antigens and present them, although with lower efficiency, in an HLA-class I-restricted manner. Antigen-loaded MSC evoked IFN-γ secretion in specific CTL but were resistant to CTL-mediated lysis in all experimental conditions.

Expression of HLA Class I-Related APM Components in MSC

To analyze the completeness of APM in MSC, intracellular expression of APM components was investigated by flow cytometry in MSC isolated from the BM of normal individuals and expanded in culture, following 48 hours of incubation with 1,000 U/ml IFN-γ or medium alone. The Raji cell line was tested as a positive control. As shown in Figure 4B, the chaperone ERp57 and the immunoproteasomal components LMP7 and LMP10 were virtually undetectable in MSC cultured with medium alone. β2-Microglobulin (β2m), β2m free HC, and the proteasomal components MB-1 and zeta were downregulated compared with control. The chaperones calnexin, calreticulin, and tapasin, the proteasomal component delta, the immunoproteasomal component LMP2, and the transporter TAP2 were consistently expressed in untreated MSC. IFN-γ treatment strongly enhanced expression of β2m free HC, β2m, and, to a lesser extent, TAP2 but had no effect on the expression of the remaining APM components (Fig. 4C).

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Figure Figure 4.. Immunophenotypic characterization of MSC and expression of APM components. (A): Flow cytometric analysis of MSC immunophenotype. (B): Intracellular expression of antigen-processing machinery (APM) components was evaluated by flow cytometric analysis of MSC and Raji Burkitt's lymphoma cell line, used as positive control. Empty profiles represent staining with specific antibodies, whereas filled profiles represent staining with isotype-matched controls. One representative experiment of five performed is shown. (C): The intracellular expression of APM components in MSC was evaluated by flow cytometry, under basal conditions (white bars) and after 48 hours of treatment with IFN-γ (gray bars). Results are expressed as MRFI. Results are means ± SD from five different experiments. Abbreviations: IFN, interferon; MRFI, mean relative fluorescence intensity; MSC, mesenchymal stem cells.

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Expression of the Immunosuppressive Molecules HLA-G and HLA-E in MSC

To further analyze the mechanism(s) involved in the inhibition of CTL-mediated killing of antigen-loaded MSC, the expression of two immunosuppressive HLA-class Ib molecules, HLA-G and -E, was investigated by flow cytometry on MSC expanded in culture from five different donors, in the following experimental conditions: unmanipulated (control), transfected with NB mRNA, transfected with normal PBMNC mRNA, and infected with NS3Ag-VV. As shown in Figure 5A, HLA-G was expressed on the surface of unmanipulated MSC (MRFI, 3.13 ± 1.05), and its expression was not increased after transfection or infection (MRFI, 2.69 ± 0.37).

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Figure Figure 5.. Expression of HLA-G and HLA-E in mesenchymal stem cells (MSC). (A): HLA-G and HLA-E surface expression was evaluated by flow cytometry on MSC from five different donors in basal conditions, after NB mRNA transfection or after infection with NS3Ag-vaccinia virus. Empty profiles represent staining with specific antibodies, whereas filled profiles represent staining with isotype-matched controls. One representative experiment of five performed is shown. (B): HLA-G (black bars) and HLA-E (gray bars) expression was evaluated by flow cytometry on MSC under the following experimental conditions: unmanipulated or transfected, both cultured with medium alone or with IFN-γ. One representative experiment of five performed is shown. Abbreviations: IFN, interferon; MRFI, mean relative fluorescence intensity; NB, neuroblastoma; PBMNC, peripheral blood mononuclear cells.

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HLA-E was virtually undetectable in unmanipulated MSC or infected MSC (MRFI, 1.19 ± 0.18 and 0.66 ± 0.01). Its expression was significantly induced after transfection, either with NB or PBMNC mRNA (MRFI, 3.15 ± 1.68; p = .022). This latter control excluded the possibility that HLA-E was encoded by tumor mRNA.

Next, HLA-G and HLA-E expression was evaluated by flow cytometry on MSC in the same experimental conditions described above, following culture with IFN-γ. IFN-γ treatment strongly upregulated HLA-E but not HLA-G expression (Fig. 5B).

The presence of HLA-E only in transfected MSC argued against its role in CTL inhibition. Blocking the interaction between HLA-G and its receptors using anti-HLA-G mAb 87G did not restore CTL-mediated cytotoxicity, ruling out a role of surface HLA-G in CTL inhibition (data not shown).

sHLA-G Secreted by MSC Is Involved in the Resistance of MSC to CTL-Mediated Cytotoxicity

Additional experiments tested whether sHLA-G released by MSC played a role in their resistance to CTL-mediated cytotoxicity. sHLA-G concentration in MSC supernatants, tested by ELISA, ranged from 0.17 to 24.3 ng/ml, with significantly higher levels in earlier than in later passages of culture (passages 0–2, 6.81 ± 2.12 ng/ml; passages 3–4, 1.18 ± 0.38 ng/ml; p = .0237) (Fig. 7A). To analyze the role of soluble factors secreted by MSC in CTL-mediated cytotoxicity inhibition, we selected 20 supernatants, half of them containing sHLA-G (range, 3.28–24 ng/ml) and the others lacking sHLA-G. Flu-specific or NB-specific HLA-A2+ CTL were incubated with MSC supernatants or fresh medium for 2 hours at 37°C and then cultured with target cells (Flu-pulsed T2 cell line at a 50:1 E:T ratio or autologous DC transfected with NB mRNA at an 80:1 E:T ratio, respectively) in cytotoxicity assay.

As shown in Figure 7B, Flu-specific CTL treated with fresh medium caused 51Cr release by 96.2%, which was significantly reduced when CTL were incubated with MSC supernatants containing sHLA-G (specific lysis, 49.66%; p < .0001) but not with supernatants lacking sHLA-G (specific lysis, 94.9%). Depletion of sHLA-G from MSC supernatants by immunomagnetic beads coated with anti-HLA-G1/-G5 mAb partially restored CTL-mediated lysis (specific lysis, 62.3%; p = .019).

Similar results were obtained with NB-specific CTL (Fig. 7C). Specific lysis of target cells was 25.5% when CTL were treated with fresh medium, and it decreased at 16.6% when CTL were treated with MSC supernatants containing sHLA-G (p = .0079). sHLA-G depleted supernatants did not inhibit lysis of target cells (specific lysis, 25.24%; p = .0143). In this experiment, lysis of target cells was also significantly reduced when MSC were added in the assay, at a 1:5 or 1:10 effector/MSC ratio (specific lysis, 19.2% and 17.7% respectively; p = .004). These data proved unambiguously that soluble factors secreted by MSC inhibited CTL-mediated lysis, and sHLA-G played a role in this inhibition.

Expression of Granzyme B Inhibitor PI-9 in MSC

Antigen-loaded MSC induced Granzyme B secretion by antigen-specific CTL in ELISpot assay. Figure 6A shows experiments performed with NB-mRNA-transfected MSC cultured with HLA-matched NB-specific CTL.

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Figure Figure 6.. GrB secretion in CTL upon incubation with MSC and PI-9 expression in antigen-loaded MSC. (A): HLA-A2+ NB-specific CTL were tested in GrB ELISpot assay against HLA-matched MSC, either unmanipulated or transfected with NB mRNA (black bars). Gray bar indicates the inhibition of GrB secretion obtained by adding anti-HLA class I mAb to target cells before they were cultured with lymphocytes. Results are means ± SD from three different experiments. (B): The expression of the GrB inhibitor PI-9 was evaluated by flow cytometry in MSC transfected with NB mRNA or pulsed with Flu peptide, using unmanipulated MSC cultured in the same experimental conditions as negative control. Empty profiles represent staining with specific antibodies, whereas filled profiles represent staining with isotype-matched controls. One representative experiment of three performed is shown. Abbreviations: GrB, Granzyme B; mAb, monoclonal antibody; MSC, mesenchymal stem cells; NB, neuroblastoma; PI-9, protease inhibitor 9.

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Figure Figure 7.. Soluble HLA-G (sHLA-G) secretion by MSC and its role in CTL-mediated lysis inhibition. (A): sHLA-G levels in sup from 50 MSC cultures were evaluated by enzyme-linked immunosorbent assay. sHLA-G concentration was measured in all MSC sup tested (black bar), in sup from MSC cultures at passages 0–2 (gray bar) and from MSC cultures at passages 3–4 (light gray bar). Results are means ± SD. (B): HLA-A2+ Flu-specific CTL were cultured with fresh medium (white bar), with MSC sup lacking sHLA-G (light gray bar), with MSC sup containing sHLA-G (black bar) or with MSC sup containing sHLA-G after antibody-mediated depletion of the latter molecule (gray bar). Effector cells were then tested in cytotoxicity assay against T2 cell line pulsed with Flu peptide, at a 50:1 effector-to-target (E:T) ratio. Means ± SD from four different experiments are shown. (C): HLA-A2+ neuroblastoma (NB)-specific CTL were cultured with fresh medium (white bar), with MSC sup containing sHLA-G (black bar), with MSC sup containing sHLA-G after depletion of the latter molecule (gray bar), or in the presence of MSC at a 1:5 or 1:10 ratio with effector cells (light gray bars). Effector cells were then tested in cytotoxicity assay against autologous dendritic cells transfected with NB mRNA, at an 80:1 E:T ratio. Means ± SD from four different experiments are shown. Abbreviations: MSC, mesenchymal stem cells; sup, supernatants.

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PI-9 is the only known physiological antagonist of Granzyme B [56]. We tested by flow cytometry the intracellular expression of PI-9 in MSC transfected with NB mRNA or pulsed with Flu peptide, using unmanipulated MSC (cultured in the same experimental conditions) as control. As shown in Figure 6B, PI-9 was not expressed in the cytoplasm of unmanipulated MSC (MRFI, 1.14 and 1, respectively) but was significantly induced in NB mRNA-transfected or Flu-pulsed MSC (MRFI, 2.78 and 3.76, respectively; p < .01), suggesting a potential role of PI-9 in MSC protection from CTL-mediated lysis.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

MSC are immunosuppressive and poorly immunogenic, and these features make them attractive candidates as therapeutic agents for diseases characterized by abnormal activation of the immune system, such as graft-versus-host disease (GvHD) and autoimmune disorders [14, [15]16, 57, [58]59]. Ringdén et al. [31] and Le Blanc et al. [61] paved the way to the clinical use of MSC in allogeneic HSC transplanted patients undergoing severe GVHD. The successful control of experimental autoimmune encephalomyelitis, a model of multiple sclerosis, and collagen arthritis, a model of rheumatoid arthritis, achieved following MSC infusion holds promise for the treatment of different human autoimmune disorders [29, 62].

In spite of the encouraging results obtained in preclinical models and, to a limited extent, in the human setting, some caveats related to the issue of MSC immunogenicity have emerged from recent mouse studies [32, 33]. In addition, it has been demonstrated that human MSC can act as APC for HLA-class II-restricted antigens depending on the IFN-γ concentration in their microenvironment [35, 36]. Because of the limited information available as to the APC function of human MSC in HLA-class I-restricted T-cell responses [63], we have here addressed this issue using different in vitro models. The result obtained cannot be easily extrapolated to the in vivo situation due to the complexity of the microenvironments where MSC can interact with CTL. Rasmusson et al. recently demonstrated that MSC pulsed with an HLA-class I-restricted viral peptide did not trigger effector functions in specific CTL, downregulating T-cell receptor and CD25 expression and damping protein phosphorylation [37].

However, the mechanism(s) used by MSC to inhibit CTL function have not been clarified. Moreover, the expression of HLA-class I-related APM components in human MSC and their ability to process HLA-class I-restricted antigens and to present them to specific CTL have not yet been analyzed. Here, we addressed the APC function of MSC, using two different experimental systems to dissect exogenous peptide presentation from endogenous antigen processing: (a) pulsing of MSC with HLA-A2-restricted peptides from influenza A virus (Flu) or HCV (NS3), and (b) transfection of MSC with the NS3 gene.

MSC pulsed with viral peptides induced IFN-γ release by specific CTL in an HLA-A2-restricted and antigen-specific manner. When different aliquots of the same MSC suspensions were either transfected with the HCV NS3 gene or pulsed with the NS3 peptide and subsequently incubated with the same specific CTL suspension, the latter displayed a significantly reduced IFN-γ production following challenge with transfected versus peptide-pulsed MSC. These experiments demonstrated that MSC are partly defective in their ability to process a viral antigen, whereas they are competent at presenting an exogenously loaded peptide from the same antigen to CTL. This latter observation is in contrast with the report by Rasmusson et al. [37], who showed that MSC loaded with an EBV peptide did not induce IFN-γ release in specific CTL. The reasons for this discrepancy are not easily apparent and may be related to the different experimental conditions. Notably, in the present study, two different viral peptides were tested using two different assays with similar results.

Cytotoxicity experiments against NS3 peptide- or Flu peptide-pulsed MSC showed that lysis of the latter cells by specific CTL was detectable at E:T ratios ranging from 10:1 to 50:1. This finding is in agreement with the results of Rasmusson et al., who showed in their study that antigen-specific CTL did not lyse EBV peptide-pulsed MSC when tested at a 3:1 ratio [37]. Whereas NS3 peptide-pulsed MSC were killed, although at very low efficiency, by specific CTL, NS3 gene-transfected MSC were not.

MSC injected systemically in tumor-bearing mice are attracted to the tumor site, where they can inhibit or stimulate malignant cell growth, depending on the model and the experimental conditions tested [64, [65], [66]67]. The ability of human MSC to present tumor-associated antigens to specific CTL has not been investigated so far. To address this issue, we transfected MSC with pooled mRNA from four NB cell lines. This protocol has been used successfully with professional APC (i.e., myeloid dendritic cells and B cells) to generate NB-specific CTL [49, 68]. NB mRNA-transfected MSC stimulated IFN-γ and Granzyme B production by specific CTL but were completely protected from lysis, similar to what was observed with NS3 gene-transfected MSC, even at very high E:T ratios.

In this study, CTL-mediated lysis of MSC was low to absent in all experimental systems tested, and it was not enhanced by IFN-γ treatment, an observation that is at variance with that reported for HLA-class II-restricted-antigen presentation by MSC [35]. The protection of MSC from CTL-mediated lysis can be due to (a) defects in the HLA-class I-related APM, which transforms native proteins into small peptides, which are loaded onto nascent HLA class I molecules and then exported to the cell surface and presented to specific CTL [69]; or (b) the expression of immunosuppressive molecules by MSC.

Here we demonstrate for the first time that human MSC display several defects in the expression of some APM components. MSC lacked expression of the chaperone ERp57, which assists in intracellular trafficking of newly generated peptides and in their loading onto HLA class I molecules, and showed downregulated MB1 and zeta, which form the cylinder backbone of the 20S proteasome, where protein degradation takes place [70]. Furthermore, the immunoproteasomal components LMP7 and LMP10, which are induced in professional APC by IFN-γ treatment [71], were not expressed. However, MSC incubation with IFN-γ did not upregulate MB1 or zeta or induce ERp57, LMP7, or LMP10 expression. Taken together, these findings suggested that protein antigen processing and peptide generation may be impaired in MSC, irrespective of IFN-γ stimulation.

The nonpolymorphic HLA class Ib molecule HLA-G exerts many immunosuppressive activities, including inhibition of cell-mediated cytotoxicity [72]. Surface HLA-G was expressed constitutively by MSC and not upregulated by IFN-γ stimulation. Blocking of surface HLA-G did not reinstate lysis of NB mRNA-transfected MSC by specific CTL. In contrast, MSC in early culture passages were found to release sHLA-G in their supernatants, which significantly inhibited the lysis of target cells by specific CTL. sHLA-G depletion from the latter supernatants significantly reduced such inhibition, thus demonstrating that HLA-G is one of the factors involved in protection of MSC from CTL-mediated killing. Other investigators have shown that MSC constitutively release sHLA-G in culture supernatants and that the latter molecule inhibits T-cell proliferation in response to alloantigens [73].

Another potential mechanism involved in MSC resistance to CTL-mediated lysis is related to de novo induction of the expression of the Granzyme B inhibitor PI-9 in MSC pulsed with peptide or transfected with NB mRNA. PI-9 is an intracellular serpin expressed predominantly in lymphocytes and monocyte-derived cells that protects them from lysis [56].

MSC express HLA-class I but not costimulatory molecules, such as CD80 and CD86, which are essential for antigen stimulation of naïve T cells [15]. Accordingly, it has previously been reported that in the absence of costimulation, MSC induce anergy in naïve T cells [15, 16, 23]. However, MSC could act as APC for antigen-specific memory CD8+ T cells, as the expression of costimulatory molecules by APC is not a crucial requirement for these cells [74, 75].

Human BM contains a microenvironment that allows interactions between APC and circulating naïve antigen-specific T cells, leading to the induction of primary memory CD8+ T-cell responses [57]. Zhang et al. have demonstrated the presence of “effector memory” CTL specific for viral antigens with potent recall function in the BM, which could be restimulated and clonally expanded [76]. Moreover, the APC function of infected nonhematopoietic cells and their role in amplifying clonal expansion of effector CD8+ T cells have been emphasized by Thomas et al. [77] We speculate that MSC or their progeny in the BM act as APC for CD8+ effector memory T cells in response to antigenic stimulation, for example in the course of viral infections. In vivo interactions between MSC and CTL may result in cytokine production, leading to recruitment and/or activation of other cell types to the BM. Since MSC appear to be rather resistant to CTL-mediated lysis, they can perform different cycles of antigen presentation to CTL. In this respect, mice immunized with ovalbumin-pulsed, IFN-γ-treated MSC have previously been shown to develop antigen-specific CTL and acquire protection against ovalbumin-expressing tumors [78].

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Human MSC can process and present HLA class I-restricted viral or tumor antigens to specific CTL. The limited efficiency of antigen processing and presentation is likely due to defects in the expression of some APM components, whereas MSC resistance to CTL-mediated lysis appears to be partly sHLA-G-dependent.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

We thank Chiara Bernardini for excellent secretarial assistance. This study was supported by grants from Fondazione Carige, Genova, and Ministero della Salute, Progetto di Ricerca Finalizzata 2006 “Cellule staminali e terapie cellulari rigenerative” (to V.P.) and Public Health Service grants R01-CA67108, R01-CA110249, and P01-CA109688 awarded by the National Cancer Institute DHHS (to S.F.). F.M. was the recipient of a fellowship from Fondazione Italiana Ricerca sul Cancro. L.R. was the recipient of a fellowship from Fondazione Italiana per la Lotta al Neuroblastoma.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References