Brain, Vol. 126, No. 1, 176-185,
January 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg017
The non-classical MHC molecule HLA-G protects human muscle cells from immune-mediated lysis: implications for myoblast transplantation and gene therapy
1 Department of Neurology, University of Tübingen, Medical School, Tübingen, 2 Department of Anthropology and Human Genetics, 3 Genzentrum and Friedrich-Baur Institut and Department of Neurology and 4 Institute for Clinical Neuroimmunology, Ludwig Maximilians University, Munich, Germany
Correspondence to: Dr Heinz Wiendl, Department of Neurology, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany E-mail: heinz.wiendl{at}uni-tuebingen.de*These two authors contributed equally to this work
Received June 30, 2002. Revised July 19, 2002. Accepted July 29, 2002.
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HLA-G is a non-classical MHC class I molecule with highly limited tissue distribution which has been attributed chiefly immune-regulatory functions. We previously have reported that HLA-G is expressed in inflamed muscle in vivo and by cultured myoblasts in vitro. Here, we used the in vitro models of human myoblasts or TE671 muscle rhabdomyosarcoma cells to characterize the functional role of HLA-G for muscle immune cell interactions. Gene transfer of the two major isoforms of HLA-G (transmembranous HLA-G1 and soluble HLA-G5) into TE671 rendered these cells resistant to alloreactive lysis by direct inhibition of natural killer (NK) cells, and CD4 and CD8 T cells. Further, HLA-G reduced alloproliferation, interfered with effective priming of antigen-specific cytotoxic T cells and reduced antigen-specific alloreactive lysis. HLA-G pre-induced on cultured myoblasts inhibited lysis by alloreactive peripheral blood mononuclear cells. This protection was reversed by a neutralizing HLA-G antibody. Interestingly, a few HLA-G-positive cells within a population of HLA-G-negative muscle target cells conveyed significant inhibitory effects on alloreactive lysis. Our results reveal further insights into the immunobiology of muscle and suggest that ectopic expression of HLA-G may promote the survival of transplanted myoblasts in the future treatment of hereditary muscle diseases. Further, HLA-G could represent a novel self-derived anti-inflammatory principle applicable in strategies against inflammatory aggression.
Keywords: HLA-G; muscle immunobiology; non-classical MHC; immune regulation; myopathy
Abbreviations: IFN-
= interferon-; NK natural killer; PBMC peripheral blood mononuclear cell; TNF-
= tumour necrosis factor-
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Introduction |
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Muscle can be a site of desirable and undesirable immune reactions (Hohlfeld and Engel, 1994
). These develop spontaneously during the course of autoimmune and infectious muscle diseases (Dalakas, 1991; Engel et al., 1994) or are induced deliberately by protein- or DNA-based vaccination (Pardoll and Beckerleg, 1995; Kumar and Sercarz, 1996). Local immune reactions pose a serious problem after intramuscular injection of vectors for gene therapy (Blau and Springer, 1995). They also represent the major obstacle for the success of myoblast transfer therapy, a cell-mediated gene transfer method aimed at restoring normal dystrophin expression in Duchenne muscular dystrophy (Mendell et al., 1995; Smythe et al., 2000). It has been demonstrated that muscle cells can participate actively in local immune reactions (reviewed in Hohlfeld and Engel, 1994). Human myoblasts express co-stimulatory molecules (Behrens et al., 1998; Murata and Dalakas, 1999) and can present antigens to CD4 T cells (Goebels et al., 1992). Myoblasts thus qualify as putative antigen-presenting cells. Cultured muscle cells induce primary immune responses in allogeneic situations (Curnow et al., 1998) and are killed efficiently by cytotoxic T cells and natural killer (NK) cells (Hohlfeld and Engel, 1990). Multiple factors such as humoral immunity, complement activation, cytokine secretion or tissue culture processing are likely to contribute to the failure of survival of donor myoblasts in vivo (reviewed in Smythe et al., 2000). However, the unique immunological properties of myoblasts themselves seem to represent a major challenge to resolving the problem of rapid donor myoblast cell death after injection into host muscle in myoblast transfer. A better understanding of myoblast immunobiology could therefore result in the development of successful strategies circumventing this problem.
We have reported recently that muscle fibres in inflammatory myopathies and cultured myoblasts express the histocompatibility antigen HLA-G (Wiendl et al., 2000). HLA-G is a ‘non-classical’ MHC class I molecule (class Ib) structurally related to classical MHC class Ia (HLA-A, -B and -C) which, in contrast to class Ia molecules, exhibits a restricted tissue distribution (reviewed in Carosella et al., 2000, 2001). In contrast to MHC class Ia molecules, HLA-G is characterized by a limited polymorphism and the alternative transcription of spliced mRNAs that encode at least seven different isoforms, including membrane-bound HLA-G1, -G2, -G3 and -G4, and soluble HLA-G5 (formerly HLA-G1s), -G6 (formerly HLA-G2s) and -G7 proteins (Ishitani and Geraghty, 1992; Fujii et al., 1994; Paul et al., 2000; reviewed in Carosella et al., 2000). The function of HLA-G remains elusive. It is thought that by unknown mechanisms, HLA-G prevents maternal lymphocytes from attacking foetal tissue. Like classical HLA class I molecules, HLA-G binds CD8 and antigenic peptides, therefore acting as a possible antigen-presenting molecule (Sanders et al., 1991; Diehl et al., 1996; Münz et al., 1999). However, HLA-G has been identified chiefly as a key mediator of immune tolerance (reviewed in Carosella et al., 2001). HLA-G protects target cells from the cytotoxic activity of T lymphocytes and NK cells through direct or indirect interaction with several inhibitory receptors (Rouas-Freiss et al., 1997b; Riteau et al., 2001a).
In order to elucidate the functional role of HLA-G expression in human muscle, immune-regulatory properties of this molecule were investigated using alloreactive, HLA-A2-mismatched co-culture experiments with peripheral blood mononuclear cells (PBMCs), different lymphocyte subsets and antigen-specific T cells. In TE671 rhabdomyosarcoma cells, HLA-G1 as well as HLA-G5 significantly inhibited primary and secondary alloreactive immune responses. HLA-G inhibited NK cell lysis and prevented direct alloreactive killing by CD8 as well as CD4 cells. Furthermore, HLA-G inhibited secondary alloreactive immune responses, as it efficiently blocked priming of antigen-specific cytotoxic lymphocytes. HLA-G inducibly expressed on myoblasts inhibited lysis by HLA-A2-mismatched alloreactive PBMCs. Interestingly, a few HLA-G-positive muscle cells within a population of HLA-G-negative cells conveyed lysis inhibition under primary alloreactive culture conditions.
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Material and methods |
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Antibodies and reagents
The following antibodies were used: 87G [IgG2b, anti-HLA-G1 and -G5; kindly provided by D. Geraghty (Fred Hutchinson Cancer Research Center, Seattle, WA, USA)]; 4H84 [mIgG1, anti-HLA-G, all denatured isoforms; kindly provided by M. McMaster (University of California, San Francisco, CA, USA)]; MEM-G/9 (mIgG1, anti-HLA-G1 and -G5; Exbio, Prague, Czech Republic); W6/32 [mIgG2a, anti-HLA-A, -B, -C, -G, -E and ß2-microglobulin (ß2m); Biozol, Eching, Germany]; L243 (mIgG2a, anti-HLA-DR), BB7.2 (mIgG2b, anti-HLA-A2; ATCC, Rockville, MD, USA); RPA-T4 [mIgG1
, anti-CD4 fluorescein isothiocyanate (FITC) and phycoerythrin (PE) labelled], HIT8A (mIgG1, anti-CD8 PE labelled); B9.11 (mIgG1, anti-CD8 FITC labelled); N901 (NKH-1; mIgG1, anti-CD56 PE labelled) (Immunotech); and goat anti-mouse F(ab)2 fragment (Dianova, Hamburg, Germany). The antibodies were titrated for flow cytometry and used at the concentrations indicated (usually 20 µg/ml) in the functional assays. AnnexinV–FITC was from Pharmingen (Heidelberg, Germany). Propidium iodide was purchased from Sigma (St Louis, MO, USA). CD95L-containing supernatant was obtained from murine CD95L-transfected Neuro2A mouse neuroblastoma cells (Weller et al., 1997).
Cell culture
The TE671 rhabdomyosarcoma cell line, the JEG-3 human choriocarcinoma cell line and the K-562 human erythroleukaemia cell line (ATCC) were maintained in RPMI 1640 supplemented with 1 mM sodium pyruvate (Gibco Life Technologies, Paisley, UK), penicillin (100 IU/ml)/streptomycin (100 µg/ml) (Gibco) and 10% foetal calf serum (FCS) (Biochrom KG, Berlin, Germany). The cells were routinely tested for contamination with mycoplasma. Where indicated, the cells were irradiated using a Gammacell 1000 Elite (Nordion, Ontario, Canada).
Myoblasts were isolated from normal subjects and from patients with inflammatory myopathy, purified by magnetic bead separation and cultured as described (Wiendl et al., 2000). The myoblasts exhibited >95% positive staining for the neural cell adhesion molecule (NCAM) by flow cytometry. For cytokine induction, myoblasts were cultured in the presence of 500 U/ml interferon- (IFN-; Roche, Basel, Switzerland) and/or 500 U/ml tumour necrosis factor- (TNF-; Roche, Basel, Switzerland). Surface expression of HLA molecules and other molecules was assessed by flow cytometry on a FACSCalibur (Becton Dickinson, Heidelberg, Germany).
HLA-G transfectants
HLA-G transfectants were obtained as described (Le Gal et al., 1999). In brief, HLA-G1 (encoding the full-length HLA-G transcript) and HLA-G5 (formerly HLA-G1s; coding for the secreted HLA-G heavy chain) plasmids were generated by cloning HLA-G1 and HLA-G5 cDNAs into a GFP (green fluorescent protein) construct (pIRES2-pEGFP; Clontech, Palo Alto, CA, USA). GFP vector transfectants (pIRES2-pEGFP) were used as controls. Transfection was done using Effectene® transfection reagent (Qiagen, Hilden, Germany). Cells were selected in media containing 0.5 mg/ml neomycin (G418; Calbiochem, Darmstadt, Germany). The following transfectants were used in the study: K-562-HLA-G1, TE671-pEGFP, TE671-HLA-G1 and TE671-HLA-G5.
PBMCs and purified lymphocyte populations
PBMCs were isolated from the peripheral blood of normal healthy volunteers by density gradient centrifugation using Biocoll Separating Solution (Biochrom KG). Cells were analysed for their HLA-A2 isotype by flow cytometry. CD4, CD8 T cells and CD56 NK cells were purified by positive selection with CD4, CD8 or CD56 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). To rule out alterations of the activation threshold by positive selection, CD4, CD8 and NK cells were also isolated by a negative procedure in some experiments (Dynal Biotech, Hamburg, Germany). The purity of the isolated populations used in the experiments was >95%.
Flow cytometry
Adherent cell lines were collected after treatment with non-enzymatic cell dissociation buffer (Gibco) at 37°C for 5 min. JEG-3, K-562-HLA-G1 or K-562 cells were used as positive and negative control cell lines for HLA-G or MHC expression as indicated. Cells were washed with PBS (phosphate-buffered saline) containing 0.1% bovine serum albumin (BSA) and 0.1% sodium acide, and blocked with human immunoglobulins (Alphaglobin, Grifols, Langen, Germany) for 10 min at 4°C. After one washing step, the unlabelled first antibody was added at the final concentration. Isotype control monoclonal antibodies (mAbs) were used at the same concentrations as the primary antibody. Incubation was done on ice for 30 min, followed by two washes. Goat anti-mouse IgG F(ab)2–PE (5 µg/ml, Sigma) or IgG F(ab)2– di-chlorotriazinyl-fluorescein (DTAF) (10 µg/ml, Dianova) were used as secondary antibodies. After washing, propidium iodide was added to the cells at a final concentration of 0.02 µM. Propidium iodide-positive (nonviable) cells were excluded from analysis. Specific fluorescence indexes (SFIs) were calculated by dividing the mean fluorescence obtained with a specific antibody by the mean fluorescence obtained with isotype control antibody. Analysis of PBMC and lymphocyte subsets was performed similarly. Cells were blocked with human immunoglobulins and stained with either directly labelled antibodies or the respective combinations of an unlabelled first antibody and a fluorescent secondary antibody. Flow cytometry was performed using a FACSCalibur.
Detection of apoptosis by AnnexinV binding
PBMC or purified lymphocyte subsets undergoing apoptotic cell death were analysed by staining with FITC-labelled AnnexinV (Pharmingen). After various incubation times with the TE671 cells, lymphocytes were collected, washed with PBS and resuspended in a buffer containing 10 mM HEPES/NaOH pH 7.4, 140 mM NaCl and 2.5 mM CaCl2. Then AnnexinV–FITC and propidium iodide were added. Following incubation for 30 min, the cells were analysed by flow cytometry. When analysis of lymphocyte subsets was desired, PBMCs were first incubated with the respective PE-labelled cell surface antibodies (CD4–PE, CD8–PE; Pharmingen; CD56–PE; Immunotech) before the AnnexinV staining was performed.
Primary alloreactive proliferation
To suppress proliferation of stimulator cells, TE671 cells (HLA-A2 negative) were incubated in serum-free medium for 24 h, irradiated at 50 Gy and maintained in serum-free medium for another 24 h. Cells were detached using cell dissociation buffer, counted and seeded in RPMI. HLA-A2-positive responder PBMCs (105) were co-incubated with 104 irradiated TE671 cells in a volume of 100 µl. The assays were performed in 96-well plates in triplicate. PBMCs without muscle cells, muscle cells without PBMCs, and phytohaem agglutinin (PHA)-stimulated PBMCs (5 µg/ml) were used as controls. After 4 days, the cells were pulsed for 24 h with 0.5 µCi of [3H]methyl-thymidine (Amersham-Pharmacia Biotech, Freiburg, Germany). The cells were harvested automatically (Inotech, Dottikon, Switzerland) and the incorporated radioactivity was bound to a glass fibre filtermat (Wallac, Turku, Finland). The filtermat was wetted with Ultima Gold Scintillation Cocktail (Packard, Dreieich, Germany) and radioactivity was determined in a Wallac 1450 Microbeta Plus Liquid Scintillation Counter.
Secondary alloreactive immune response (51Cr release assay)
To test for lytic activity of primed cytotoxic T cells after co-culturing with HLA-A2-mismatched muscle cells for 5 days, a 51Cr release assay was performed. A total of 106 growth-suppressed TE671 cells or myoblasts were seeded into 25 cm2 flasks. HLA-A2-mismatched PBMCs (1.5 x 107) were added in 5 ml of RPMI containing 10% FCS. Cells were co-cultured for 5 days. Target muscle cells (106) were labelled by addition of 100 µCi of 51Cr (1 Ci = 37 GBq) in 1 ml of RPMI medium (NEZ147, NEN, Boston, MA, USA), and 104 cells per well were seeded as targets in a U-shaped 96-well plate. Primed alloreactive cytotoxic lymphocytes obtained after 5 days of co-culturing were removed and added to the labelled target cells at different effector : target ratios (ranging from 10 to 80) in a total volume of 100 µl. After co-incubation for 4 h, 50 µl of the supernatant was transferred to a Luma-PlateTM-96 (Packard), dried overnight and measured. To correct for spontaneous 51Cr release, a control of labelled target cells in medium only was included (0% specific lysis). The maximum 51Cr release was determined by addition of Nonidet P-40 (NP-40; 100% lysis). Lysis was calculated as [c.p.m. (effector cells) – c.p.m. (spontaneous)]/[c.p.m. (NP-40) – c.p.m. (spontaneous)] x 100%.
Primary alloreactive lysis (51Cr release assay)
Direct lysis of muscle target cells (TE671 or myoblasts) by alloreactive HLA-A2-mismatched lymphocytes and subpopulations was measured in a 51Cr release assay. Target cells were radioactively labelled and seeded into 96-well U-bottom plates as described above. For blocking experiments, myoblasts or HLA-G transfectants were pre-incubated with specific antibodies or the corresponding isotype control antibodies. After incubation for 1 h at 37°C, cross-linking by a secondary antibody [goat anti-mouse F(ab)2 fragment, 10 µg/ml, Dianova] was performed to immobilize the antibody on the target cells. Purified effector cells (PBMCs, CD4, CD8 and NK cells) were incubated with human immunoglobulin to saturate possible Fc receptor engagement in the effector population. Afterwards, effector cells were added at different effector : target ratios, ranging from 10 to 100, in a total volume of 100 µl. After co-incubation for 8 h, 50 µl of the supernatant were transferred to a Luma-PlateTM-96 (Packard), dried overnight and measured. In experiments using purified NK cells, incubation was for 4 h. To correct for spontaneous 51Cr release, a control of labelled target cells in medium only was included (0% specific lysis). The maximum 51Cr release was determined by addition of NP-40 (100% lysis).
Immunoblot analysis
Cells were lysed for 30 min at 4°C in lysis buffer [20 mM Tris–HCl pH7.5, 1% Triton X-100, 2% trasylol (Bayer, Leverkusen, Germany), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma]. After removal of cellular debris by centrifugation, the protein content of the lysates was quantified using a Micro-Bradford assay (Bradford, 1976). Lysates equivalent to 100 µg of protein were separated by 10% SDS–PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) using standard immunoblotting techniques. HLA-G heavy chains were detected with the mAb 4H84 and the enhanced chemiluminescence (ECL) immunoblotting analysis system (Amersham-Pharmacia).
Statistical analysis
Data are representative of experiments performed at least three times with similar results. Significance was assessed by two-sided t test (*P < 0.05, **P < 0.01).
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Gene transfer of HLA-G1 or -G5 into TE671 muscle cells
We first studied the immune-regulatory properties of HLA-G in a cell line with stable growth characteristics and with phenotypic features of myoblasts, the rhabdomyosarcoma-derived muscle cell line TE671. Parental TE671 cells do not express significant amounts of HLA-G protein, as determined by immunoblot analysis or flow cytometry. In order to dissect the immune-regulatory effects of HLA-G in muscle, we transfected expression plasmids encoding the two major HLA-G isoforms, HLA-G1 and HLA-G5, into TE671 cells. The HLA-G-transfected cell lines showed high levels of HLA-G expression, as assessed by immunoblot for HLA-G1 and HLA-G5 (Fig. 1A) and flow cytometry for HLA-G1 (Fig. 1B). The ectopic HLA-G expression did not alter the expression levels or inducibility of classical MHC I or MHC II molecules by IFN-
or IFN-/TNF-, as determined by flow cytometry (data not shown).
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Gene transfer of HLA-G1 or -G5 into TE671 muscle cells inhibits alloproliferation and renders muscle cells resistant to alloreactive lysis
HLA-A2-mismatched PBMCs were co-cultured with TE671-pEGFP, TE671-HLA-G1 or TE671-HLA-G5 for 5 days, and primary proliferation of responder cells was assessed by [3H]thymidine incorporation. Both HLA-G isoforms significantly suppressed the proliferative immune response towards muscle cells (Fig. 2A).
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Gene transfer of HLA-G1 also rendered TE671 cells less susceptible to the lytic activity of HLA-A2-mismatched PBMCs as assessed in an 8 h 51Cr release assay (Fig. 2B). HLA-G1 also inhibited lysis by CD56 NK cells in the MHC class I-negative K-562-HLA-G1 transfectants which were used as positive controls for NK cell lysis experiments (Pazmany et al., 1996
; and data not shown). HLA-G co-expressed with MHC class I on TE671-HLA-G1 also inhibited lysis by freshly isolated CD56 NK cells (Fig. 2B). Interestingly, experiments with purified effector T-cell subpopulations (CD8 and CD4) demonstrated that HLA-G also inhibited primary alloreactive lysis mediated by CD4 and CD8 T cells (Fig. 2B). To rule out alterations of the activation threshold by positive selection, CD4, CD8 and NK cells were also isolated by a negative procedure in some experiments (Dynal Biotech, Hamburg, Germany). The obtained immune-inhibitory effects of HLA-G were not different between the isolation techniques. The effects seen in the TE671-HLA-G1 cells could be attributed to HLA-G rather than to HLA-G-dependent upregulation of other inhibitory molecules such as HLA-E since lysis inhibition conveyed by the HLA-G transfectants was reversed by a neutralizing HLA-G antibody (87G) (Fig. 2C). HLA-G5 transfectants were also less susceptible to alloreactive lysis by PBMCs (data not shown). HLA-G1 and HLA-G5 display similar immune-inhibitory effects in our experiments. One possible explanation would be binding to common receptor(s) expressed on different lymphocyte subsets. Stages of muscle cell lysis after co-incubation with alloreactive PBMCs were followed under phase optics. The protective effect of HLA-G mirrored the results of the 51Cr release assay. Photographs taken at 72 h after incubation of TE671 cells with PBMCs demonstrated significantly higher survival rates in TE671 cells engineered to express HLA-G1 than in control transfectants (Fig. 2D).
Gene transfer of HLA-G1 or -G5 into TE671 cells inhibits antigen-specific killing: effects on priming of alloreactive antigen-specific cells
The effect of HLA-G on the development of secondary alloreactive immune responses was assessed by generating antigen-specific cytotoxic lymphocytes during co-culture of TE671-pEGFP, TE671-HLA-G1 or TE671-HLA-G5 with HLA-A2-mismatched PBMCs. Lysis of TE671 wild type target cells by primed effector cells was quantitated by measuring 51Cr release at different effector : target ratios at day 5. The presence of HLA-G1 and HLA-G5 in the priming phase significantly diminished the lytic activity of alloreactive cytotoxic T lymphocytes [45% versus 12% (HLA-G1) and 17% (HLA-G5)] (Fig. 2E). Thus HLA-G1 as well as HLA-G5 inhibited efficient priming of antigen-specific cytotoxic T cells.
HLA-G does not induce apoptosis in the effector population
To assess whether induction of apoptosis on effector cells (Fournel et al., 2000) contributed to the observed effects of HLA-G1 and HLA-G5 in primary and secondary alloreactive immune responses, cellular viability within the effector cell population was determined after 24 and 48 h of co-culture of freshly isolated lymphocyte subpopulations (CD8 and CD4) with control-transfected TE671, TE671-HLA-G1 or TE671-HLA-G5 transfectants. HLA-G1 and HLA-G5 did not induce apoptosis in the effector cell populations as detected by Annexin V staining (Fig. 2F).
Small numbers of HLA-G-positive cells within a cell population are sufficient to inhibit direct alloreactive lysis
The immune-inhibitory effects shown above make HLA-G gene transfer a suitable strategy to enhance the survival of grafted myoblasts in the setting of gene therapy for muscle disorders. Therefore, we asked whether transmembraneous HLA-G would protect only those cells which express HLA-G from immune-mediated lysis. This issue was addressed by mixing experiments where HLA-G-expressing TE671 cells (TE671-HLA-G1) were titrated into co-cultures with control transfected TE671 cells. Lysis of the muscle cell target population was quantified by 51Cr release after addition of HLA-A2-mismatched PBMCs as effector cells. Surprisingly, a significant inhibition of lysis by PBMCs was observed when only 3.2% of the target cells expressed HLA-G (Fig. 3). Since direct interaction of HLA-G with certain receptors on immune effector cells is assumed to be the predominant pathway of HLA-G action, a possible explanation for these observations would be shedding of HLA-G into the co-culture suspension. However, using an ELISA (enzyme-linked immunosorbent assay) for soluble HLA molecules, we did not detect a release of soluble HLA-G by the HLA-G1 transfectants during co-incubation with alloreactive PBMCs, thus making that possibility rather unlikely (data not shown).
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HLA-G expressed on cultured myoblasts inhibits alloreactive lysis by HLA-A2-mismatched PBMCs
To demonstrate the functional relevance of HLA-G expression also on primary muscle cells, we performed different alloreactivity assays with purified human myoblasts. Effector populations and target myoblasts were typed for HLA-A2, and co-incubation experiments were performed using HLA-A2-mismatched effector target populations. HLA-G protein surface expression on myoblasts is upregulated by inflammatory cytokines (Wiendl et al., 2000
). Myoblasts were therefore cultured in the presence of IFN- or IFN-/TNF- before the co-culture experiments. Upregulation of the classical MHC (HLA-class I and II) and non-classical MHC (HLA-G) molecules was verified by flow cytometry analysis performed in parallel to the functional experiments (Fig. 4A). The relevance of HLA-G molecules for the protection of myoblasts from direct alloreactive lysis by cytotoxic effector cells was shown indirectly by pre-incubating muscle cells with HLA-G-specific antibody 87G (Fig. 4B). HLA-G conveyed protection of myoblasts against alloreactive lytic activity of PBMCs as recorded in an 8 h 51Cr release assay (Fig. 4B). Since myoblasts are susceptible to NK cell lysis (Hohlfeld and Engel, 1990) and since MHC class I expression downregulates NK cell lysis via interaction with killer inhibitory receptors, blocking MHC class I with W6/32 facilitates lysis by NK cells present within the PBMC population (Fig. 4B). Although alloreactive lysis by MHC class I-restricted CD8 T cells is blocked by W6/32, the observed net effect of W6/32 was an increase in specific lysis of myoblasts by PBMCs. However, since W6/32 cross-reacts with HLA-G and the negative immune-regulatory molecule HLA-E, part of the observed effects of this antibody on lysis may have been mediated by an interaction with HLA-G and HLA-E.
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The present report provides insights into the functional significance of HLA-G expression that has been found recently in human muscle tissue and in cultured myoblasts (Wiendl et al., 2000
). We demonstrate that HLA-G either expressed on human myoblasts or transfected into TE671 muscle rhabdomyosarcoma cells conveys potent inhibition of primary and secondary alloreactive cellular immune responses. Gene transfer of HLA-G1 and -G5 rendered TE671 muscle cells resistant to alloreactive immune effector mechanisms, as shown under different experimental conditions: (i) HLA-G inhibited the primary alloproliferative response to muscle cells (Fig. 2A); (ii) HLA-G directly inhibited primary alloreactive lysis by CD4 and CD8 T cells as well as NK cells (Fig. 2B); and (iii) HLA-G prevented efficient priming of cytotoxic effector cells and protected muscle target cells from antigen-specific cytotoxic T-cell lysis (Fig. 2E). HLA-G inducibly expressed on primary human myoblasts down-modulates lysis by alloreactive PBMCs and NK cells. These observations have several implications for the assumed immune-regulatory role of this MHC class Ib molecule in many immune reactions that occur in muscle.
HLA-G exhibits a limited tissue distribution: first detected on extravillous cytotrophoblast cells (Kovats et al., 1990), HLA-G protein has been found since then in thymic epithelial cells (Crisa et al., 1997), cytokine-activated monocytes (Yang et al., 1996), some tumours (e.g. Paul et al., 1998; Davies et al., 2001; Wiendl et al., 2002) and in inflamed muscle and cultured myoblasts (Wiendl et al., 2000). Although HLA-G possibly could act as a local antigen-presenting molecule (reviewed in Le Bouteiller and Solier, 2001), its interaction with different receptors expressed on certain types of lymphocytes, macrophages/monocytes and dendritic cells is more suggestive of a relevance in immune regulation (reviewed in Carosella et al., 2000, 2001). Expression on extravillous cytotrophoblast cells (Kovats et al., 1990) suggested an immune-inhibitory function in foetal semi-allograft reaction (Rouas-Freiss et al., 1997a). HLA-G functional studies were therefore carried out mainly with HLA class I-negative cell lines such as LCL 721.221 or K-562 that became protected from NK cell-mediated cytolysis when transfected with HLA-G (Rouas-Freiss et al., 1997b). HLA-G has been shown to interact with several receptors on NK cells, namely KIR2DL4, ILT-2, ILT-4 and, putatively, others (Mandelboim et al., 1997; Münz et al., 1997; Rouas-Freiss et al., 1997b). Cultured myoblasts constitutively express classical MHC class I and, while MHC class II has never been found consistently in vivo, it can be induced in vitro by stimulation with the pro-inflammatory cytokine IFN- (Goebels et al., 1992; Michaelis et al., 1993). Our experiments demonstrate that HLA-G expressed on muscle cells conveys immunoprotection not only by inhibition of NK cells, but also by direct interaction with CD8 and CD4 alloreactive T cells (Fig. 2). Furthermore, HLA-G expressed on muscle cells prevents efficient priming of antigen-specific cytotoxic T cells and reduces antigen-specific lysis (Fig. 2). Our study therefore provides further evidence for an important immune-regulatory role of HLA-G co-expressed in the presence of MHC class I and class II (Riteau et al., 2001b). Taken together, our observations therefore advocate the hypothesis that HLA-G is capable of modulating antigen-specific as well as non-antigen-specific cytotoxic T-cell responses during primary and secondary immune responses.
HLA-G1 as well as HLA-G5 inhibited the primary alloproliferative response in the responder population but did not render effector cells apoptotic (Fig. 2). Previous studies have suggested that soluble HLA-G molecules induce apoptosis in CD8 T cells (Fournel et al., 2000), extending observations of such an effect of soluble HLA molecules in general (Zavazava and Kronke, 1996). However, PBMCs in those studies (Zavazava and Kronke, 1996; Fournel et al., 2000; Puppo et al., 2000) had already been pre-stimulated before encountering the soluble HLA molecules. Further, the apoptotic effect of soluble HLA molecules strongly correlated with the duration of the previous stimulation (Puppo et al., 2000). By co-incubating unstimulated responder cells with HLA-G, our results favour the mechanism of an HLA-G-induced cell cycle arrest as a possible explanation for its action on immune effector cells.
The expression of HLA-G can be modulated by different cytokines, namely interferons (Yang et al., 1995, 1996; Lefebvre et al., 2001) and interleukin-10 (Moreau et al., 1999). Further, proliferative allogeneic responses in a mixed lymphocyte reaction induce soluble HLA-G production by CD4 T cells, an observation that supports the immune-regulatory capabilities of HLA-G being broader than previously assumed (Lila et al., 2001). Myoblasts and myotubes themselves are potent inducers of primary and secondary allogeneic immune responses (Hohlfeld and Engel, 1990; Curnow et al., 1998), thus partly explaining the rapid graft destruction after myoblast transfer. Our data provide strong functional evidence for a protective role of muscle-related HLA-G expression in allograft rejection, as it occurs after therapeutic transfer of myoblasts or during heart muscle transplantation. Recently, it has been shown that expression of HLA-G in heart muscle is associated with improved heart graft acceptance (Lila et al., 2000, 2002). This observation corresponds nicely to our data of enhanced survival of HLA-G-expressing muscle cells co-cultured with alloreactive immune effector cells, and supports the immune-regulatory relevance of HLA-G in transplantation. We therefore surmise that gene transfer of HLA-G into myoblasts could constitute a possible future strategy for circumventing the problem of myoblast death after therapeutic injection in vivo.
A major drawback of most approaches in gene therapy is the efficiency of gene delivery (reviewed in Smythe et al., 2000). Mixing experiments carried out in vitro indicate that a surprisingly low number of HLA-G-bearing muscle cells within a population of HLA-G-negative myoblasts could already sufficiently suppress primary alloresponses in vivo (Fig. 3). A direct interaction of HLA-G with certain receptors on immune effector cells is assumed to be the predominant pathway of HLA-G action. Shedding of transmembranous HLA-G into the co-culture suspension would be a possible explanation for some of our observations. However, we could not measure any release of soluble HLA-G by the HLA-G1 transfectants during co-culture with alloreactive PBMCs, thus making that possibility rather unlikely (data not shown). Our surprising observation could be interpreted as a ‘negative bystander’ effect: few HLA-G-positive cells render neighbouring HLA-G-negative cells ‘resistant’ to immune-mediated killing or induce non-reactivity in cytotoxic T cells. However, the molecular explanation for this phenomenon remains to be clarified.
Our data provide novel insights into the functional significance of HLA-G in human muscle immunobiology. Muscle-related HLA-G has broad implications as it could play an important role in many other immune reactions that occur in this tissue (Hohlfeld and Engel, 1994). For example, HLA-G could protect muscle fibres from cell-mediated injury in autoimmune muscle disorders (polymyositis, dermatomyositis or inclusion body myositis) or in various muscle infections. Further, HLA-G could modulate the immune responses after protein- or DNA-based vaccinations. Since HLA-G downregulates primary as well as secondary allogeneic immune responses by interacting with virtually all cellular cytotoxic immune effectors, and since only low numbers of HLA-G-expressing cells are required to convey significant immune inhibitory effects, this molecule represents an appealing self-derived anti-inflammatory principle possibly applicable for different approaches as gene therapy or strategies against inflammatory aggression.
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We wish to thank Drs M. McMaster (University of California, San Francisco) and D. Geraghty (Fred Hutchinson Cancer Research Center, Seattle) for kindly providing anti-HLA-G mAbs, and E. Schmidtmeyer and S. Galuschka for expert technical assistance. Human myoblast cultures were obtained from the Muscle Tissue Culture Collection at the Friedrich-Baur-Institute (Department of Neurology, Ludwig-Maximilians-University, Munich, Germany). The Muscle Tissue Culture Collection is supported by generous grants of the ‘Deutsche Gesellschaft für Muskelkranke’ (Freiburg, Germany) and the ‘Association Francaise contre les Myopathies’ (Paris, France). This work was supported by grants from the Deutsche Forschungsgemeinschaft (Wi 1722/2-1, to H.W.; SFB571 A-1, to R.H.; Lo 549/2-3 to H.L.). M.M. received a scholarship from the IZKF Tübingen. H.L. is supported by grants from the Duchenne Parents Project of Germany (Aktion Benni & Co). The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling foundation.
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