Patent Application
20090081226
The classical MHC class I (Ia) molecules (HLA-A, HLA-B and HLA-C) are highly polymorphic and are ubiquitously expressed on most somatic cells. In contrast, non classical MHC class I (Ib) molecules (HLA-E, HLA-F and HLA-G) are broadly defined by a limited polymorphism and a restricted pattern of cellular expression.
Among class Ib molecules, HLA-E is characterized by a low polymorphism and a broad mRNA expression on different cell types (Lee et al. (1998) J Immunol 160, 4951-60). Cell surface expression of HLA-E requires the availability of β2-microglobulin (Ulbrecht et al. (1999) Eur J Immunol 29, 537-47) and of a set of highly conserved nonameric peptides derived from the leader sequence of various HLA class I molecules including HLA-A, -B, -C, and -G (Braud et al. (1997) Eur J Immunol 27, 1164-9; Ulbrecht et al. (1998) J Immunol 160, 4375-85). Efficient loading of HLA-E with class Ia leader sequence peptide requires the transporter associated with antigen processing (TAP) protein which translocates short peptides from the cytoplasm to the endoplasmic reticulum (Braud et al. (1998) Curr Biol 8, 1-10). HLA-E also associates with peptides which derive either from viruses, including cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus, or from stress proteins (i.e. hsp60) (Ulbrecht et al. (1998) J Immunol 160, 4375-85; Tomasec et al. (2000) Science 287, 1031; Michaelsson et al. (2002) J Exp Med 196, 1403-14).
HLA-E tetramers binding to natural killer (NK) cells and a subset of T cells (αβ and γδ CD8 T cells), allowed the identification of CD94/NKG2A and CD94/NKG2C molecules as receptors for HLA-E (Braud et al. (1998) Nature 391, 795-9; Borrego et al. (1998) J Exp Med 187, 813-8; Lee et al. (1998) Proc Natl Acad Sci USA 95, 5199-204). The interaction of membrane-bound HLA-E with NK cells results in inhibition of NK cell-dependent lysis, mediated by the inhibitory CD94/NKG2A receptors (Braud et al. (1998) Nature 391, 795-9). CD94/NKG2A has also been implicated in down-regulation T-cell function in various pathological situations in humans such as melanoma (Speiser et al. (1999) J Exp Med 190, 775-82), ovarian carcinoma (Malmberg et al. (2002) J Clin Invest 110, 1515-23), arthritis (Dulphy et al. (2002) Int Immunol 14, 471-9) or astrocytoma (Perrin et al. (2002) Immunol Lett 81, 125-32). The function of the non-classical HLA-E molecules is not restricted to the modulation of NK cell responses as it also plays a role in the regulation of T cell function and represents a restriction element for the TCRαβ-mediated recognition (Pietra et al. (2001) Eur J Immunol 31, 3687-93; Li et al. (2001) J Immunol 167, 3800-8; Garcia et al. (2002) Eur J Immunol 32, 936-44; Heinzel et al. (2002) J Exp Med 196, 1473-81). HLA-E complexed with peptides can interact with αβTCRs expressed on CD8+T cells to trigger conventional CTL function Li et al. (2001) J Immunol 167, 3800-8; Lo et al. (1999) J Immunol 162, 5398-406; Speiser et al. (1998) Transplantation 66, 646-50; Leibson et al. (1998) Immunity 9, 289-94). Recent in vitro studies in human (Li et al. (2001) J Immunol 167, 3800-8) and the demonstration that Qa-1 (homologous to HLA-E in mice)-deficient mice (Hu et al. (2004) Nat Immunol 5, 516-23) have defects in immunoregulation mediated by CD8+T cells provide evidence of the involvement of HLA-E-restricted CD8 suppressor cells in controlling the adaptative immune response to both foreign and self antigens.
It is to be noted that the above-mentioned binding experiments between HLA-E and CD94/NKG2A have been mostly conducted with soluble HLA-E tetramers, mainly because the recombinant soluble HLA-E monomers used to date have been demonstrated to be inactive on CD94/NKG2A-expressing cells (Braud et al. (1998) Nature 391, 795-9). However, HLA-E tetramers are cumbersome to synthesize, since they result from the in vitro association of four bacterially-produced individual HLA-E molecules. Furthermore, their heavy molecular weight renders them unfit for in vivo use, and they lead to the inactivation of CD94/NKG2A-expressing cells.
Thus, one of the objects of the present invention is to provide a new soluble HLA-E molecule which is devoid of the defaults and inhibiting properties of the previously known soluble HLA-E molecules.
Another object of the present invention is to provide diagnostic and therapeutic methods using the new soluble HLA-E molecule.
The present invention relates to a purified soluble HLA-E molecule, characterized in that:
it is a monomeric protein,
it presents a sequence identity of at least 80% with membrane-bound HLA-E (SEQ ID NO: 1),
it has a molecular weight from about 36 kDa to about 42 kDa,
it binds to the CD94/NKG2A receptor.
HLA-E belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (β2-microglobulin). The heavy chain is anchored in the membrane. HLA-E binds a restricted subset of peptides derived from the leader peptides of other class I molecules. The heavy chain is approximately 45 kDa and its gene contains 8 exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode the α1 and α2 domains, which both bind the peptide, exon 4 encodes the α3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail.
As intended herein “soluble” notably relate to molecules which are not bound to lipidic membranes, in particular to cell membranes. In particular, “soluble HLA-E molecule” relates to a protein found as a cell-free protein released in the extracellular medium such as cell culture supernatant or biological fluids (i.e. serum, plasma, urine).
As intended herein “purified” relates to a protein which is substantially free of contaminants, such as other soluble HLA molecules such as classical Ia soluble HLA-A, B, C or non classical including MICA and MICB. The purification grade which can be obtained is of at least 95%.
Purity and specificity can be determined by methods well known to the man skilled in the art, such as SDS-PAGE and BB Coomassie staining and also by western blotting assays using HLA-E specific antibodies after protein transfer onto nitrocellulose membranes.
As intended herein the expression “monomeric protein” relates to a single protein or a protein complex which is not constituted of a plurality of identical protein chains covalently linked together; however the monomeric protein can be constituted of one or more different subunits. In particular, as intended herein, the monomeric soluble HLA-E molecule is constituted of a HLA-E heavy chain, or fragments thereof, optionally non-covalently associated to a β2-microglobulin subunit, or fragments thereof, this binary complex being itself optionally associated to a peptide; this whole association thus corresponds to a single MHC:peptide complex. It is, in particular different from HLA-E tetramers, which are constituted four specific MHC:peptide complexes bound to a single molecule of streptavidin.
As will be apparent to the man skilled in the art, the sequence identity percentage relates to the comparison of the sequence of the heavy chain of membrane-bound HLA-E with the corresponding chain of the soluble HLA-E molecule.
As intended herein the molecular weight of the proteins are determined by electrophoretically migrating sodium-dodecyl-sulfate-denatured protein samples on a polyacrylamide gel (SDS-PAGE), in particular as described in the following examples.
In vitro, the binding of the soluble HLA-E molecule to the CD94/NKG2A receptor can be assessed, for instance, by competition experiments involving the soluble HLA-E of the invention and known ligands of the CD94/NK2A receptor, such as antibodies or HLA-E tetramers.
In vivo, the binding of the soluble HLA-E molecule can be assessed according to the examples hereafter described.
The CD94/NKG2A receptor notably results from the association of CD94 (SEQ ID NO: 2) and NKG2A (SEQ ID NO: 3).
The present invention also relates to a process for obtaining a soluble HLA-E molecule, comprising a step of recovering soluble HLA-E molecules from a culture medium in which tumor cells, in particular melanoma cells, melanocytes, Natural Killer cells and/or endothelial cells have been grown.
The cells which produce soluble HLA-E can derive from immortalized cells lines or from primary cultures.
In a preferred embodiment, the invention relates to a process as defined above, wherein the culture medium contains at least one cytokine.
In a further preferred embodiment of the above defined process, the cytokine is selected from the list comprising IFNγ, IL1β and TNFα.
The present invention also relates to a soluble HLA-E molecule such as obtainable according to the above-defined process.
The present invention also relates to ligands of soluble HLA-E molecules as defined above which neither bind to membrane bound HLA-E molecules nor to bacterially produced soluble HLA-E molecules.
As intended herein, the above-defined ligands of soluble HLA-E molecules according to the invention are specific of the soluble HLA-E molecule according to the invention and do not bind to other known soluble HLA-E molecules, i.e. those recombinantly produced in bacteria, under a monomeric form, or resulting from the association from monomeric HLA-E molecules, which are recombinantly produced in bacteria, such as the previously known HLA-E tetramers for example.
According to a preferred embodiment, the above-defined ligands are selected from the list comprising antibodies or paratope-containing fragments thereof, and aptamers.
Preferably the antibodies are monoclonal antibodies.
As intended herein the expression “paratope-containing fragments” of antibodies notably relates to Fab, F(ab)′2 or scFv fragments.
The expression “aptamers” relates to RNA molecules having specific binding capabilities vis-à-vis soluble HLA-E molecules.
The present invention also relates to an in vitro method for diagnosing cancers or inflammatory diseases in a patient, characterized in that HLA-E presence is detected in a biological sample, in particular a sample of solid tissues, such as skin, or liquid tissues, such as serum or plasma, taken from the patient.
In a particular embodiment of the above defined in vitro method for diagnosing cancers or inflammatory diseases in a patient, the biological sample is substantially depleted of cells which normally carry membrane-bound HLA-E, such as endothelial cells, B lymphocytes, T lymphocytes, macrophages, urothelial cells, secretory endometrial cells, or megakaryoblasts.
In a particular embodiment of the above defined in vitro method for diagnosing inflammatory diseases in a patient, the method is used for determining the disease activity or progression, particularly during inactive or active phase, relapse or remission of the disease.
In a preferred embodiment of the above defined in vitro method, HLA-E concentration is measured in a biological sample to test and compared to the HLA-E concentration in a normal corresponding biological sample, a higher HLA-E concentration in the biological sample to test as compared to the normal biological sample being indicative of a pathology.
By <<normal corresponding biological sample>> is meant a biological sample taken from a substantially healthy tissue, wherein the tissue is of the same histological type than the tissue from which the biological sample to test was taken.
HLA-E concentration can be measured according to methods well known to man skilled in the art, such as immunostaining or ELISA.
According to a preferred embodiment of the above defined in vitro method, the presence of soluble HLA-E is detected in a biological sample, in particular a sample selected from a sample of blood, serum, or plasma, taken from the patient
According to the present invention, soluble HLA-E is specifically present in biological samples of patients suffering from cancers or inflammatory diseases. It is essentially absent from samples of healthy patients.
In a preferred embodiment of the above defined in vitro method, soluble HLA-E concentration is measured in a biological sample to test and compared to the soluble HLA-E concentration in a normal corresponding biological sample, a higher soluble HLA-E concentration in the biological sample to test as compared to the normal biological sample being indicative of a pathology.
According to a further preferred embodiment of the above defined in vitro method, the presence of HLA-E is detected by contacting the sample taken from the patient with a HLA-E ligand.
According to another preferred embodiment of the above-defined in vitro method, the HLA-E ligand is selected from a list comprising anti-HLA-E antibodies or paratope-containing fragments thereof, anti-HLA-E aptamers, and CD94/NKG2A molecules or binding site-containing fragments thereof.
The expression “binding site-containing fragments” of CD94/NKG2A molecules relates to fragments of CD94/NKG2A which have retained its HLA-E binding capability.
According to a particularly preferred embodiment of the above-defined in vitro method, the cancers are melanomas, and the inflammatory diseases are vasculitides, in particular anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitides.
In particular the ANCA-associated systemic vasculitides comprise Wegener's granulomatosis and microscopic polyangitiis. Anti-neutrophil cytoplasmic antibody (ANCA) associated systemic vasculitis (AASV) is a well-defined primary vasculitis subgroup, invariably associated with a systemic inflammatory response, which usually normalizes in remission. Thus AASV provides a useful clinical model to investigate the relation between clinical inflammation and endothelial dysfunction. Small vessel vasculitides, such as Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA), are strongly associated with anti-neutrophil cytoplasmic antibodies (ANCA), which are either directed to myeloperoxidase (MPO) or proteinase 3 (PR3) (Cohen Tervaert J W et al, (1990) Kidney Int. 37: 799-806; Jennette J C and Falk R J (1997) N Engl J Med 337: 1512-1523; Velosa J A et al., (1993) Mayo Clinic Proc 68: 561-565). These diseases can occur in any organ system but the respiratory tract and the kidneys are most frequently involved. Untreated, WG results in death within weeks to months.
The present invention also relates to a kit for diagnosing cancers or inflammatory diseases in a patient, characterized in that it comprises:
at least one HLA-E ligand,
a mean for detecting the binding of the HLA-E ligand to a soluble HLA-E,
optionally a soluble HLA-E, in particular a soluble HLA-E as defined above, as a standard.
In a particular embodiment of the above defined kit, the HLA-E ligand is selected from a list comprising anti-HLA-E antibodies or paratope-containing fragments thereof, anti-HLA-E aptamers, and CD94/NKG2A molecules or binding site-containing fragments thereof.
In a particular embodiment of the above defined kit, the HLA-E ligand is an anti-HLA-E monoclonal antibody. Such an antibody can be used, for instance, for Western blotting experiments or for ELISA experiments.
The present invention also relates to a pharmaceutical composition, characterized in that it comprises at least one soluble HLA-E molecule as defined above in association with a pharmaceutically acceptable vehicle.
The present invention also relates to the use of at least one soluble HLA-E molecule as defined above for the manufacture of a medicament intended for the treatment of cancers, in particular melanomas.
In vivo, cancerous cell membranes present high quantities of membrane-bound HLA-E, which inactivates CD94/NKG2A-expressing cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL), which normally destroy cancerous cells. As demonstrated herein, the addition of soluble HLA-E according to the invention prevents CD94/NKG2A inactivation, which favours tumor destruction.
The present invention also relates to a pharmaceutical composition, characterized in that it comprises at least one HLA-E ligand, in particular at lest one soluble HLA-E ligand, more particularly at least one soluble HLA-E ligand as defined above, in association with a pharmaceutically acceptable vehicle.
In a preferred embodiment of the above-defined pharmaceutical composition, the HLA-E ligand is selected from a list comprising anti-HLA-E antibodies or paratope-containing fragments thereof, anti-HLA-E aptamers, and CD94/NKG2A molecules or binding site-containing fragments thereof.
The present invention also relates to the use of at least one HLA-E ligand for the manufacture of a medicament intended for the treatment of inflammatory diseases, in particular vasculitides, such as anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitides.
Inflammatory diseases, such as vasculature inflammation (vasculitis), results, in particular, from an erroneous or exaggerated activation of NK cells or CTL. As demonstrated herein, erroneous or exaggerated activation of NK cells or CTL can be notably induced by soluble HLA-E molecules according to the invention produced by activated endothelial cells. Thus the use of ligands of soluble HLA-E molecules helps reduce the activation of NK cells or CTL.
In a preferred embodiment of the above-defined use, the HLA-E ligand is selected from a list comprising anti-HLA-E antibodies or paratope-containing fragments thereof, anti-HLA-E aptamers, and CD94/NKG2A molecules or binding site-containing fragments thereof.
In particular the HLA-E ligand is a soluble HLA-E ligand according to the invention.
The present invention also relates to the use of cytokines, in particular selected from the list comprising IFNγ, IL1β and TNFα, for producing a soluble HLA-E molecule from cells in vitro.
The present invention also relates to the use of a soluble HLA-E molecule as defined above or of a HLA-E ligand, for modulating the activity of CD94/NKG2A cells, in particular in vitro.
According to a preferred embodiment the invention relates to the above defined use of a soluble HLA-E molecule as defined above for activating CD94/NKG2A cells, in particular in vitro.
According to another preferred embodiment the invention relates to the above defined use of a HLA-E ligand, for inhibiting CD94/NKG2A cells, in particular in vitro.
According to a preferred embodiment of the above defined use, the HLA-E ligand is selected from a list comprising anti-HLA-E antibodies or paratope-containing fragments thereof, anti-HLA-E aptamers, and CD94/NKG2A molecules or binding site-containing fragments thereof.
The following antibodies were used in the examples below:
Antibodies specific for CD94 (HP-3B1) and CD94/NKG2A (Z199) were purchased from Immunotech (Marseille, France). The CD94-specific antibody Y9 was kindly provided by A. Moretta (Genova, Italy). All flow cytometry stainings for CD94 were done with the mAb HP-3B1, while Y9 was used in the cytotoxic assays, as well as irrelevant mouse IgM Ab purchased from MedacGmbH (Hamburg, Germany).
For HLA-E staining we used antibodies MEM-E/2 for immunohistology and western blotting, MEM-E/6 for blocking experiments and MEM-E/7 or MEM-E/8 for flow cytometry, which were provided by V. Horejsi (Prague, Czech Republic) (Menier et al. (2003) Hum Immunol 64, 315-26). MEM-E/02 mAbs bind the denatured HLA-E protein whereas -E/06 (IgG1), -E/07 (IgG1) and -E/08 (IgG1) mAbs bind native cell surface HLA-E molecules. MEM-G/01 recognizes (similar to the 4H84 mAb) the denatured HLA-G heavy chain of all isoforms.
Anti-HLA class I antibodies (W6/32) were purchased from Immunotech (Marseillle, France).
The frequency of HLA-E cell surface expression in melanoma-derived cell lines was determined by flow cytometry.
The melanoma cell lines <<M>> used were mainly established from metastatic tumor fragments as previously described (Gervois et al. (1990) Eur J Immunol 20, 825-31). Other melanoma cell lines were obtained from different laboratories (IPC277/5, C. Aubert (Unite INSERM U119, Marseille, France); DAGI, J. F. Dore (Lyon, France); FM25 and FM29, J. Zeuthen (Copenhagen, Denmark); G-mel, A; Houghton (New York, USA), MW75, D. Schadendorf, Mannheim, Germany and Mel17, S. Perez (Athens, Greece). Normal melanocytes (00M10, 00M33, 011M03, 01M10, 01M11, 01M20 and 97M10) were obtained from M. Regnier (Clichy, France). All cell lines were cultured in complete medium (RPMI 1640 supplemented with 10% fetal calf serum (FCS)).
For flow cytometry, 105 cells (melanoma cell lines or freshly isolated melanocytes) were stained with the anti-HLAE (MEM-E/07) mAb, anti-HLA class I (W6/32) mAb or the isotype control mAb for 30 min at 4° C. After two washes, cells were incubated for 30 min with the secondary PE-labeled antibody. Labeling was analyzed on a FACScan flow cytometer using Cellquest software (Becton Dickinson, Grenoble, France). 10 000 cells were gated with FSC/SSC parameters and analyzed. Ratio Fluorescence Intensity (RFI) has been calculated for HLA-E expression as follows: mean fluorescence intensity obtained with the test/mean fluorescence intensity obtained with the negative control.
It was observed that IFNγ up-regulates the HLA-E expression on the majority of melanoma cell lines (
All short term cultured melanocytes tested (
Therefore, all melanoma cell lines express low levels of HLA-E on their membrane and this expression is up regulated by IFNγ. In contrast, short cultured melanocytes significantly express HLA-E, but this expression is poorly increased by IFN-γ.
In the present study, using anti-HLA-E specific (MEM-E/02, MEM-E/07, MEM-E/08) mAbs, the Inventors showed that HLA-E expression in human non lymphoid organs is mainly restricted to endothelial cells (EC).
First, HLA-E expression in human tissues was examined by immunohistochemistry using monoclonal antibodies specific for HLA-E (MEM-E/02) or HLA-G (MEM-G/01), as a control.
Comparative expression of HLA-E and HLA-G proteins were studied in human first-trimester placenta tissue and kidneys. HLA-E expression was also studied in various normal tissues included epithelial tissues (salivary gland, urinary bladder, thyroid, endometrium, skin, liver), kidney biopsies (obtained from patients suffering of lupus nephritis and vasculitis), lymphoid organs (lymph node and spleen), mesenchymal tissues and hematopoietic cells. Tissues were fixed in 10% formalin and routinely processed for paraffin embedding. Four micrometers-thick paraffin sections were then mounted on pre-treated slides, deparaffinized using toluene, rehydrated through a graded series of ethanol, and rinsed in distilled water. Tissue sections were then subjected to epitope retrieval in microwave oven using citrate buffer (pH 6.0). Tissue sections were stained using a two-step visualization system based on a peroxidase-conjugated dextran backbone, which avoid endogenous biotin detection (Dako Envision+™ System, Dako, Trappes, France). The following antibodies were used: mouse monoclonal anti-human HLA-E (MEM-E/02) and anti human HLA-G (MEM-G/1) mouse monoclonal Abs. Working dilutions were 1:100 for both. Tissue sections were rinsed in buffer, then endogenous peroxidase activity was blocked with the peroxidase-blocking solution for 5 min. Sections were then incubated with primary antibody at room temperature for 30 min. This was followed by incubation with secondary antibody coupled to the peroxidase-conjugated polymer for 30 min at room temperature. Immunostaining was visualized using the substrate system provided in the kit (DAB/H202 substrate) and tissues were counterstained with haematoxylin. Immunopositive cells showed cytoplasmic staining with both antibodies.
In placenta, comparative immunostaining indicated that HLA-E, as well as HLA-G (
To determine whether this staining observed in tissues indicated the presence of HLA-E on the extracellular side of the plasma membrane, immunofluorescence studies on non-permeabilized cultured ECs were performed.
For immunofluorescence, ECs were grown to confluence on glass coverslips. Cultures were washed with PBS and fixed for 20 min in PBS containing 4% paraformaldehyde. Cells were washed again with PBS and incubated over-night at 4° C. with blocking buffer (5% BSA in PBS) and then incubated with an anti-HLA-E mAb (MEM/E-7: 10 μg·ml−1) in blocking buffer with 0.1% Triton X-100 (permeabilized) or without Triton X-100 (non-permeabilized) for 1 h. Slides were rewashed and incubated with FITC-conjugated goat anti-mouse antibodies (5 μg·ml−1, Jackson Lab., West Grove, Pa.) for 1 h. ER and Golgi staining were preformed using rhodamine-B-hexyl ester (2.5 μg·ml−1, Molecular Probes, Eugene, Oreg.) and anti-golgi mAbs (5 μg·ml−1; anti-golgin-97, clone CDF4, Molecular Probes), respectively. Anti-golgi mAbs were revealed using TRITC-conjugated goat anti-mouse antibodies (5 μg·ml−1, Jackson Lab., West Grove, Pa.). Nuclear staining was performed using To-Pro-3 (1:1000 dilution, Molecular Probes). Slides were washed in PBS, dried and mounted with ProLong®& antifade reagent (Molecular Probes). Fluorescence microscopy was performed with a Leica DM-IRBE® laser scanning confocal microscope (Leica AG, Heerbrugg, Switzerland) using a 63×1.4 oil p-Aplo lens and analyzed using Leica TCS NT® software.
The intracellular location of HLA-E was studied by immunofluorescence and confocal microscopy on cultured ECs. Although weaker in intensity than staining for HLA-A, -B and -C, HLA-E staining was found on non-permeabilized cells (
A quantification of HLA-A, -B and -E mRNA levels was performed in cultured ECs by competitive RT-PCR using locus-specific primers and a competitor template with an internal deletion. RNA was isolated using Trizol reagent (Invitrogen Corp.) according to the instructions of the manufacturer. RNA was analyzed by competitive PCR as described previously (Vincent et al (1996) J Immunol 156, 603-610). Briefly, total RNA (2 μg) was reverse transcribed with oligo (dT), treated with RNase H, and made up to 50 μl. cDNAs were diluted 1/2 for competitions. Competitor templates were initially diluted 1/106, followed by four serial dilutions of 1/3. The primer sequences were:
PCR products were sequenced by Genosys (Sigma). Internal standards were obtained by mutagene PCR amplications to generate mutated fragment by the deletion of 5 nucleotides as described (Vincent et al (1996) J Immunol 156, 603-610). PCR products were run on a 4% acrylamide gel and analyzed by capillary electrophoresis on an ABI PRISM 310 DNA Sequencer (PE Applied Biosystem, Foster City, Calif.) using GeneScan® Analysis software.
Semi-quantitative PCR for HLA-E and Pactin was carried out for 20 and 18 cycles, respectively, as follows: 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds, with a final extension at 72° C. for 3 minutes. PCR primers were:
PCR products were separated on a 1.5% agarose gel in the presence of ethidium bromide.
Transcripts encoded by all three HLA loci were detected in resting ECs, demonstrating that ECs express HLA-E constitutively. Whereas there are less HLA-B transcripts than HLA-A transcripts, ECs express HLA-E at levels nearly equal to those of HLA-A (
Cell surface expression for HLA-E was further investigated by flow cytometry on cultured ECs (isolated from artery: HAEC and from umbilical vein: HUVEC) using MEM-E/7 or MEM-E/8 mAbs. Since inflammation deeply affects endothelial cell phenotype and functions, the effects of pro-inflammatory cytokines TNFα, IFNγ and IL1β on HLA-E expression and regulation were studied.
Briefly, human umbilical vein ECs (HUVEC) and arterial endothelial cells (HAEC), isolated from renal artery patches taken from cadaveric transplant donors before kidney transplantation, were isolated as previously described28. ECs were HLA-typed and selected to avoid non-HLA-E-specific cross-reactivity with MEM/E-7 reported for HLA-B7. ECs were cultured in Endothelial Cell Growth Medium (ECGM) supplemented with 10% fetal calf serum (FCS), 0.004 ml·ml−1 ECGS/Heparin, 0.1 ng·ml−1 hEGF, 1 ng·ml−1 hbFGF, 1 μg·ml−1 hydrocortisone, 50 μg·ml−1 gentamicin and 50 ng·ml−1 amphotericin B (C-22010, PromoCell, Heidelberg, Germany). For activation, confluent EC monolayers were incubated with recombinant human TNFα(100 U·ml−1), IFNγ(100 U·ml−1), IL1β (5 ng·ml−1) for the indicated period of time in Endothelial Cell Basal Medium supplemented with 2% FCS. For experiments, ECs were used between the second and fifth passage. Culture supernatants were collected at the indicated times post-activation and kept frozen. When needed, culture supernatants were concentrated (10×) using Microcon YM-3 (Millipore, Bedford, Mass.).
For flow cytometry, cells (1-2×105 cells/sample) were suspended with Trypsin-EDTA (Gibco BRL), washed twice with PBS containing 1% BSA and 0.1% NaN3, and then incubated on ice for 30 min with a saturating concentration of first antibody. After three washes in 1% BSA/0.1% NaN3/PBS, cells were incubated with a FITC-labeled goat anti-mouse F(ab′)2 IgG (Jackson Lab., West Grove, Pa.) at 4° C. for 30 min. This step was followed by three washes in cold 1% BSA/0.1% NaN3/PBS, and cells were suspended in 1% paraformaldehyde in PBS. Negative controls were performed by incubating the cells with isotype-matched control antibody. Mouse monoclonal antibodies (mAbs) used for this study were anti-pan HLA class I (clone W6/32) (ATCC), monomorphic anti-HLA-A2/A28 (clone), anti-HLA-E (MEM/E7 or MEM-E/08), PE or FITC-labeled anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD56, anti-CD14 (all purchased from BD Biosciences, Mountain View, Calif.). Fluorescence was measured on 10,000 cells/sample using a fluorescence activated cell sorter (FACScalibur®: Becton Dickinson, Mountain View, Calif.) and analyzed using CellQuestPro® software (Becton Dickinson). Data are depicted in histograms plotting mean fluorescence intensity (MFI) on a four-decade logarithmic scale (x-axis) versus cell number (y-axis).
Western blot analysis on cultured ECs revealed a single band at 42 kDa, consistent with the molecular weight for HLA-E protein and distinct from classical HLA class I protein (data not shown). Basal levels of cell surface HLA-E protein were low but significant and consistently observed on HAECs and HUVEC (
Treatment of ECs with TNFα, IL1β and IFNγ resulted in a significant increase in membrane-bound HLA-E detected by flow cytometry (
In the present study, using anti-HLA-E specific (MEM-E/02, MEM-E/07, MEM-E/08) mAbs, the Inventors showed HLA-E expression in human melanoma in vitro and in vivo.
First, HLA-E expression in melanoma was examined by immunohistochemistry using monoclonal antibodies specific for HLA-E (MEM-E/02).
Comparative expression of HLA-E protein was studied on sections of 5 normal skin samples and of 11 melanoma tumor samples.
In all the normal skin sections, HLA-E was consistently expressed by melanocytes while a faint expression was observed in the epidermis with level variations according to samples and individuals (
The Inventors then questioned whether HLA-E density on melanoma cells could modulate the susceptibility of these cells to lysis by melanoma Ag specific CTL clones expressing the CD94/NKG2-A receptor. To this end, the lysis of different melanoma cell lines treated or not by IFNγ by melanoma specific CTL clones, expressing or not the inhibitory receptor, was compared.
Briefly, melanoma-reactive CD8 αβ T cell clones specific for Melan-A/MART-1 (M77.84, M199.7.5, M17.29ELA.1, M17.29ELA.3, MEL.C8, and MEL2.46), tyrosinase (M117.14) and NA17-A (M17.221, D126, H2, MEL.F5, CDM39.91A and CDM39.91H) antigens were obtained from TIL (Tumor Infiltrating Lymphocytes) or from PBL (Peripheral Blood Lymphocytes) stimulated ex vivo by peptide pulsed antigen presenting cells (APC). The γδ T cell clone C4.112 was obtained from colon tumor-infiltrating lymphocytes. T cell clones were expanded using a polyclonal T cell stimulation protocol, as described previously (Jotereau et al. (1991) J Immunother 10, 405-11). Briefly, 2000 T cell clones were distributed/well in 96 wells-plates with 200 μl of culture medium (RPMI with 8% human serum and IL-2 150 U/ml) and irradiated feeder cells: LAZ EBV-B cells (2·104/well), allogeneic PBL (105/well) and 15 μg/ml of PHA-L (Difco, Detroit, USA). Peripheral blood polyclonal NK cells were sorted by negative isolation kit from Miltenyi.
Representative examples are shown on
It was then investigated if soluble HLA-E (sHLA-E) proteins might be secreted by melanoma cells and melanocytes, either spontaneously or after INFγ treatment. HLA-E proteins were studied by Western blot analysis in supernatants of a panel of melanoma cell lines cultured 2 days in the presence or not of IFNγ (500 U/ml), using the MEM-E/02 mAb that reacts specifically with the denaturated heavy chain of human HLA-E.
For Western blot analysis, aliquots of total cells supernatants, lysates or sera were separated in 12% SDS-PAGE. The gels were blotted onto nitrocellulose membranes (Hybond; Amersham, Buckinghamshire, U.K.), and the membranes were blocked by incubation with TBS containing 0.1% Tween 20 and 5% nonfat dry milk. The membranes were then probed with the anti-HLA-E Ab (MEM-E2) overnight at 4° C. and washed in TBS containing 0.1% Tween 20. After washing, the membranes were incubated for 2 hours at room temperature with peroxidase-conjugated sheep anti-mouse IgG Ab (Ozyme), washed thoroughly during 2 hours, stained with enhanced chemiluminescence reagent (Roche), and exposed to x-ray film. Cells were stimulated by IFNγ (500 U/ml) for various times. In some experiments, cells were stimulated by IFNγ for 24 h and cultured for a further 4 h with chloroquine (100 μM), leupeptin (100 μM), PMSF (1 mM), EDTA (0.5 mM), Galardin (1 mg/ml).
The results revealed a band of 37 kDa, corresponding to the detection of a soluble HLA-E form (see for examples
Western blotting also revealed the presence of soluble HLA-E (sHLA-E) in the supernatants of activated ECs.
Briefly, cells were maintained for 12-18 h in culture medium without growth supplements and containing 2% serum before treatment. When applicable, cells were treated for 12-72 h with 100 U·ml−1 TNFα, 100 U·ml−1 IFNγ, and/or 2.5 ng·ml−1 IL1β. When applicable, cells were incubated with brefeldin A, galardin or protease inhibitors (Sigma-Aldrich, Saint Quentin Fallavier, France) for the last 6 h. Alternatively, cells were treated for 2 h with cyclohexamide, actinomycin D, monensin or tunicamycin before stimulation. Supernatants were collected and centrifuged for 10 min at 1,500 g to remove cell debris and then used immediately or stored at −70° C. Cells (3×106) were washed in PBS and incubated in 300 μl lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.05% Triton X-100 and complete protease inhibitor cocktail (Sigma-Aldrich)) for 15 min on ice. Cell lysates were centrifuged at 12,000 g for 10 min at 4° C. the supernatants were then recovered and the protein extracts were used immediately or stored at −70° C. Equal amounts of protein (15 μg) were loaded under reducing conditions and resolved by 12% SDS-PAGE gels. Western immunoblot analysis were performed on nitrocellulose membranes (Amersham-Pharmacia, Orsay, France) using anti-HLA-E monoclonal antibodies (MEM/E-2). Anti-mouse IgG, HRP-linked Abs (Cell Signaling Technology, Beverly, Mass.) were used as secondary Abs in cheluminescent western blot assays using ECL® system (Amersham-Pharmacia). When applicable, blots were reprobed with mouse monoclonal anti-GAPDH Ab (Chemicon, Temecula, Calif.) to verify the amount of loaded proteins.
HLA-E release is driven by TNFα or IL1β and the combination of IFNγ and TNFα have an additive effect on its surface expression and release (
HLA-E expression by leucocytes was confirmed in vitro by flow cytometry performed on fresh PBMC.
It was next examined whether they could also produce soluble HLA-E. To this aim, culture supernatants were collected from either non-stimulated or 48 h-activated fresh human PBMC (Peripheral Blood Mononuclear Cells) or purified subsets and subjected to Western blotting.
Briefly, PBMC from random healthy volunteers were purified by Ficoll/Hypaque density centrifugation. NK cells were purified (>95% of CD3-CD56+ and/or CD16+) by negative selection using the NK Cell Isolation kit according to manufacturer's recommendations (Miltenyi Biotec, Paris, France). The human NK cell line, NKL, was grown in RPMI 1640 media (Gibco BRL, Life technologies, Cergy-Pontoise, France) supplemented with 10% FCS, 4 mM glutamine, 1 mM sodium pyruvate, and 200 U/ml of rIL-2 (R&D Systems, Lille, France). U937, Raji, and Jurkat cell lines were from American Tissue Culture Collection (Rockville, Md.). NKL cell line was kindly provided by Dr. Eric Vivier (CIML, Marseille-Luminy, France).
The presence of the soluble HLA-E protein was not detected in most conditions excepted for purified NK cells activated with IL-2 (
Soluble HLA-E (sHLA-E) was purified from culture supernatants according to the following procedure.
Solid ammonium sulfate was be added to the culture supernatant at 4° C. to 40% saturation, the resulting solution was centrifuged and the pellet discarded, additional ammonium sulfate was then added to resulting supernatant to achieve 70% saturation. After centrifugation, the soft pellet was be placed in dialysis bags and dialyzed overnight against 12 volumes of 20 mM Tris-HCl, 0.02% sodium azide, pH 7.4. The dialysate was applied to an anti-HLA-E mAb AffiGel-10 immunoaffinity column (6 ml resin; 5 mg IgG/ml resin) equilibrated in 20 mM Tris-HCl, 0.1 M NaCl, 0.02% sodium azide, pH 7.4. The column was washed with at least 12 volumes of the same buffer, and then eluted with 50% (vol/vol) ethylene glycol in 20 mM Tris-HCl, pH 7.4. The peak fractions from the elution were pooled, concentrated (Centriprep 30; Amicon Inc., Beverly, Mass.), and the buffer changed to 20 mM Tris-HCl, 0.1 M NaCl, 3 mM CaCl2, 0.6 mM MgCl2, 0.02% sodium azide, pH 7.4. Fractions were monitored for absorbance at 280 nm, and for sHLA-E antigen using an ELISA assay. Purity of sHLA-E was assessed on SDS-PAGE gels with Coomassie BB staining, and on Western blots with anti-sHLA-E specific antibody.
To assess the functional role of sHLA-E, melanoma-specific T cell clones were pre-incubated with sHLA-E-positive melanoma supernatant before they were tested against IFNγ-treated melanoma cell target.
Standard 4-h 15Cr-release assays were used to assess antigen-specific target cell lysis. Target cells (human melanoma cell lines or B-EBV cell lines) were labeled with 100 μCi Na251CrO4 (Oris Industrie, Gif-sur-Yvette, France) for 1 h at 37° C. 1000 or 5000 labeled target cells were incubated in the presence of T cell clones at different lymphocyte:target cell ratios (from 0.6 to 20). For some experiments, the melanoma target cells were previously stimulated with IFNγ (500 U/ml) for 48 h to increase MHC expression. In blocking experiments, the effector cells were preincubated with Y9 Ab (pure hybridoma supernatant) or irrelevant isotype control (1 μg/ml), sHLA-E containing supernatants or irrelevant supernatant (pure) at 4° C. for 20 min. prior to addition to the labeled target cells.
Analysis by cytotoxicity assays showed that IFNγ-protection of DAGI from CD94+ T cell clones was suppressed by pre-incubation of T cells with sHLA-E-positive supernatant but not with sHLA-E-negative supernatant (
The increase of cytolytic responses may be caused by the presence of soluble HLA-E in supernatants of IFNγ-treated melanoma cells. This hypothesis was confirmed by using the blocking anti-HLA-E mAb MEM-E6 which abrogated the effect of sHLA-E-positive supernatant on M88 (as well as IFNγ-treated M88) lysis by NK cells (
Thus, these data indicate that soluble HLA-E are physiologically active in modulating the responsiveness of different tumor effectors by increasing CD94/NKG2A+ cell activation.
To investigate the role of shedding of HLA-E as a potential immune control mechanism of inflammatory processes in vivo, sHLA-E levels in sera of patients with ANCA-associated vasculitis were analyzed (
Serum samples (n=22) from 13 patients with ANCA-associated vasculitis (Wegener's granulomatosis) or other autoimmune diseases (systemic lupus erythematosus) were analyzed. All patients with kidney involvement had renal biopsy-proven vasculitis. Serum samples were obtained in the active phase (at diagnosis or relapse) and in remission (3 to 9 months later). The study was performed according to the guidelines of the local ethics committee (CHU de Nantes, France). Sera from healthy blood donors were provided by EFS (Nantes, France) and used as controls.
The presence of sHLA-E in patient sera, examined by immunoblot analysis of sera, revealed a band comparable to that of sHLA-E in culture supernatants (37 kDa). Presence sHLA-E was not detected in all investigated sera of healthy volunteers. Sera from 9 out of 13 patients with vasculitis showed presence of sHLA-E. This strong correlation clearly suggests that HLA-E is released at significant amounts from cells in vivo.
Further results (Table 2 and Table 3) indicated that presence of sHLA-E in patient's sera was positively correlated with disease activity in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (Wegener's granulomatosis and microscopic polyangitiis).
Sera from patients with vasculitis (n=22) or healthy controls (n=9) were assayed for the IL-2 soluble receptor a, IL-8 and VEGF by ELISA and the means were compared by Student's t-test with receptor levels found in normal sera. In both patient groups, we found significant elevations in soluble IL-2Rα and VEGF levels relative to normal volunteers (**p<0.01).
In the frame of the present invention, the presence of tumor-associated HLA-E was looked for in the circulation of healthy donors and melanoma patients. By a Western blot analysis, the presence of soluble HLA-E was detected in 18 of 29 blood serum samples obtained from patients bearing primary (n=14) or metastatic (n=15) melanoma tumors (see for examples
The respective functions of membranous and soluble HLA-E molecules, expressed and released by ECs, were evaluated in cell-mediated cytotoxicity assays. To this aim, NK cells were purified from PBMC and used as effector cells in cytotoxicity assays where target cells were the class I-deficient cell lines C1R and K562, primary cultures of smooth muscle cells (SMC), which do not expressed HLA-E, or ECs (resting or activated for 48 h with IFNγ). Experiments were performed in the presence or absence of a blocking anti-HLA-E mAb.
As depicted on
In an attempt to determine the biological activity of sHLA-E, cytotoxic activity of NK cells toward cells with no or low HLA-E expression (untreated EC and SMC) was measured in the presence of conditioned medium containing sHLA-E. These experiments indicated that cell lysis was significantly decreased when sHLA-E was present (
| Number | Date | Country | Kind |
|---|---|---|---|
| 04293023.0 | Dec 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/013565 | 12/16/2005 | WO | 00 | 7/16/2007 |
