Patent Application
20130338035
The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. The inventors identified rat and human MHC and NKC genes but also non-MHC and non-NKC genes that are regulated during graft versus host reaction (GVHR) in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.
Haematopoietic stem cell transplantation (HSCT) is currently the only potentially curative treatment for many malignant and non-malignant haematological diseases. However, the overall survival rate after transplantation is still only 40% to 60% due to severe posttransplant complications, which include graft versus host disease (GVHD), relapse, and infection. Human leukocyte antigen (HLA) matching is essential to reduce the risk of graft rejection and GVHD. However, non-HLA genes also impact on transplant outcome and acute GVHD can be fatal even in patients receiving transplants from HLA-identical matched sibling donors (MSD). The cumulative incidence of grade 2 to 4 GVHD was 35% in a recent study evaluating 1960 MSD transplants. MSDs are currently available for about one third of the patients and, therefore, alternative donors are needed. HLA-matched unrelated donors (MUD) are more widely accepted than cord blood or mismatched related donors.
The level of HLA matching used for selection of MUDs has changed over time and usually includes now HLA-A, B, C, and DRB1 loci (8/8 match). In some studies matching has been extended to the HLA-DQB1 locus (10/10 match). A large recent study has compared MSD and 8/8 matched MUD transplants in a homogenous cohort of patients with chronic myeloid leukemia and found a 2.44 times higher risk of grade 2 to 4 acute GVHD in 8/8 matched MUD compared to MSD transplants (Arora M, et al. (2009) J Clin Oncol 27: 1644-1652). In another study, the incidence of grade 2 to 4 acute GVHD was still higher in 10/10 matched MUD compared to MSD transplants (Yakoub-Agha I, et al. (2006) J Clin Oncol 24: 5695-5702). The higher risk of GVHD after MUD compared to MSD transplants could be due to a higher degree of similarity in non-HLA genes for siblings, who share 50% of their genome with the respective recipient. However, also the HLA region itself could contribute to this difference since it harbors, in addition to the classical HLA class I and II genes, more than 200 other genes (Consortium T M S (1999) Nature 401: 921-923), many with immunological functions. In accordance with this hypothesis, mismatching of microsatellite markers in HLA class I, class II, and class III regions was associated with an increased risk of death in 10/10 matched MUD transplants. The HLA complex, as is the whole human genome, is organized into segments of closely linked genetic variants that are inherited as haplotypes on the same DNA strand. HLA haplotypes can be defined by HLA class I and II alleles and they are in strong linkage disequilibrium with defined genetic variants of non-class I/non-class II genes within the haplotype blocks within this region. Interestingly, HLA haplotype mismatching in 10/10 matched MUD transplants was associated with an increased risk of severe acute GVHD (Petersdorf E W, et al. (2007) PLoS Med 4: e8). This finding demonstrates that the HLA complex encodes in addition to HLA-A, B, C, DRB1, and DQB1 further unidentified genes that contribute significantly to the risk of developing acute GVHD. In case of disparity between donor and recipient alleles these genes may function as minor histocompatibility antigens. Alternatively, specific allelic variants may also increase the risk of GVHD, e.g., TNFA, a gene located within the class III region of the MHC encoding the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Several TNFA polymorphisms have been associated with an increased risk of GVHD and some of them are associated with increased TNF-alpha levels (Dickinson A M, et al. (2007) Expert Rev Mol Med 9: 1-19). The strong linkage disequilibrium within the HLA complex makes it very difficult to identify further non-class I/non-class II HLA genes involved in the pathophysiology of GVHD by genetic association studies alone.
HLA gene expression profiling may be an alternative strategy to identify HLA genes that are involved in the pathophysiology of GVHD. The inventors assumed that at least some non-class I/non-class II HLA genes that contribute to the risk of GVHD change their expression levels during disease progression. However, the genetic variation between clinical samples complicates the situation because allelic variation of gene expression could interfere with expression change in the pathophysiological process.
Accordingly, there is still a need for the identification of genes that contribute significantly to the risk of developing acute GVHD. These genes or gene markers may be used in the assessment of the risk to develop GVHD or GVHR, for the diagnosis of GVHD or GVHR, for monitoring treatment of GVHD or GVHR, and for screening for immunomodulating substances which may be useful in the treatment of GVHD or GVHR.
In a first aspect, the invention relates to a method of predicting the risk of a subject to develop graft versus host reaction (GvHR) or graft versus host disease (GvHD), comprising
In a second aspect, the invention relates to a method of diagnosing graft versus host reaction (GvHR) or graft versus host disease (GvHD) in a subject following transplantation, comprising:
In a third aspect, the invention relates to a method of monitoring the efficacy of treatment of graft versus host reaction (GvHR) or graft versus host disease (GvHD) in a subject following transplantation, comprising:
In a fourth aspect, the invention relates to a method of screening for a candidate substance for treatment of graft versus host reaction (GvHR) or graft versus host disease (GvHD), comprising:
In a final aspect, the invention pertains to a use of a kit in a method of predicting the risk of developing graft versus host reaction (GvHR) or graft versus host disease (GvHD) according to the first aspect, or in a method of diagnosing GvHR or GvHD according to the second aspect, or in a method of monitoring the efficacy of treatment of GvHR or GvHD according to the third aspect, wherein the kit comprises at least one isolated polynucleotide, wherein each isolated polynucleotide independently comprises
In an exploratory experiment, the inventors analyzed the expression of 169 genes with human homologues, including the respective MHC and NKC region genes, identified in the rat in human skin explant samples (c.f. example 2, and Table 9). These human skin explants were cultured for 1, 2, or 3 days resulting in GVHR of grades I, II, and III, respectively. Notably, 69% of all tested human genes were found to be regulated in at least one of these human samples as predicted by the results of the rat expression profiling experiments. 21%, i.e. 36 of the tested genes, were regulated in all 3 human skin explant samples in accordance with the rat model, but this regulation varied depending on the GVHR grade and the time course of the skin explant assay. Although the inventors only validated these genes firstly on 3 samples, the unexpectedly high concordance rate between the results of rat and human skin explant assays strongly suggests that the rat skin explant assay is an informative model for human GVHR and possibly GVHD.
Interestingly, for some of the genes that were found to be regulated in GVHR and GVHD in the rat, the human homologues are polymorphic and disease associations of gene polymorphisms have been described. These include HLA-DMB, C2, AIF1, SPR1, and possibly UBD. Therefore, these genes are especially interesting candidates of further non-class I/class II HLA genes that might confer an increased genetic risk of GVHD after HSCT depending on the genotype. In addition, the OLR1 gene in the NKC is polymorphic and polymorphisms of this gene have been associated with atherosclerosis, myocardial infarction, and Alzheimer's disease.
Several laboratory tests have been assessed for their ability to predict the risk of GVHD in patients. The skin explant assay has a predictive value of about 80% when cyclosporine alone is used for GVHD prophylaxis. A gene expression analysis of selected genes may help to further improve the predictive value of the assay. Pretransplant gene expression profiling of donor peripheral blood mononuclear cells (PBMC) has recently been shown to be a useful tool to predict the risk of GVHD. Post transplant differences in the gene expression profile of PBMC of patients with acute and chronic GVHD compared to non-GVHD samples have been described.
The inventors identified rat and human MHC and NKC genes but also non-MHC and non-NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.
The inventors observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays.
The inventors aimed to identify genes that are regulated during GVHR in the skin explant assay because these genes could be involved in the pathophysiology of GVHR and contribute to the genetic risk of GVHD. Special attention was given to genes encoded within the MHC region for the following reasons: Firstly, evidence has been presented that further risk genes for GVHD in addition to MHC class I and class II genes are present in this region. Secondly, those genes cannot easily be identified by genetic linkage analysis alone due to the strong linkage disequilibrium with MHC class I and class II genes so that expression profiling could be a worthwhile alternative approach. Thirdly, the inventors wanted to focus in this initial study on a fully characterized genomic region of special immunological importance rather than to follow a whole genome expression profiling approach. Importantly, 39% of the BN rat MHC genes (RT1n haplotype) annotated by Hurt and colleagues (Hurt P, et al. (2004) Genome Res 14: 631-639) were at the time point of array construction not represented in the Agilent database and therefore not represented on the Agilent whole rat genome array. In addition to the MHC region, genes of the NKC region were included because this region encodes Ly49 genes and their products can function as receptors for the numerous MHC class Ia and Ib gene products encoded in the MHC. A higher percentage of MHC genes and NKC genes than genes in other regions of the genome were found to be regulated in the allogeneic skin explants compared to skin samples co-cultured with syngeneic lymphocytes. Of the 25 MHC genes found to be significantly regulated (p<0.05), 5 are known to be involved in antigen processing and presentation. Besides two of three MHC class Ia genes in the BN strain (RT1-A1 and RT1-A2) that present peptides to cytotoxic T lymphocytes (CTL), the genes Tap1 and Psmb8, encoding a subunit of the antigen transporter and a subunit of the immunoproteasome (also known as LMP7), were found to be up-regulated. RT1-DMb encodes a homologue of HLA-DMB, a chaperone in the MHC class II presentation pathway. Furthermore, non-classical MHC class Ib genes (RT1-CE2, RT1-CE3, RT1-CE5, RT1-CE8, RT1-CE10, RT1-CE16, RT1-T24-4, RT-BM1) were up-regulated during GVHR in the skin explants. The function of the RT1-C/E/M class I genes is not well defined. It is known that they can become targets of CTL and function as ligands for activating or inhibitory NK receptors. RT1-C/E/M incompatibility has been shown to induce skin and pancreas graft rejection and to modulate the fate of MHC class IImismatched heart grafts. The RT1-T24-4 gene belongs to a family of genes that was originally identified as pseudogenes in the haplotype r21. In the RT1n haplotype all four family members are presumably functional. However, their actual function has not been experimentally demonstrated so far. The RT-BM1 (RT1-S3) gene is assumed to be orthologous to the mouse H2-T23gene, which encodes the Qa-1 molecule. This is a functional homologue of HLA-E, which presents leader peptides of MHC class I molecules to the inhibitory NK receptor CD94/NKG2A. Interestingly, its expression can vary substantially depending on the RT1 haplotype. It has to be noticed that no human/rat orthology can be established for the class I genes in the various class I clusters. Therefore, with respect to class I genes, the rat cannot serve as a model for the HLA complex. However, the non-class I genes are clearly orthologous.
In addition to Tap1, Psmb8, and RT1-DMb, 12 further non-class I MHC genes were found to be regulated in the rat skin explant assays, some of them also involved in the immune response, such as the complement component C2, while such a role is strongly assumed for other genes. The allograft inflammatory factor 1 (Aif1), was cloned from chronically rejecting rat cardiac allografts and it was also found in transplanted human hearts. Persistent expression of AIF-1 is associated with the development of a cardiac allograft vasculopathy. The expression of AIF-1 is mostly limited to the monocyte/macrophage lineage, and can be augmented by interferon (IFN)-γ. The specific function of the leukocyte specific transcript 1 (Lst1) gene is not known, although its strong expression in dendritic cells and functional data suggest an immunomodulatory role. The expression of human LST1, specifically of splice variants encoding soluble isoforms, was increased in rheumatoid arthritis-affected blood and synovium and was up-regulated in response to IFN-γ. The immediate early response 3 (Ier3) gene is stress-inducible and is involved in the regulation of cell death and oncogenesis. The protein (also known as IEX-1 or IEX-1L) functions in the protection of cells from Fas or TNF-α-induced apoptosis. However, it increases the rate of apoptosis in ultraviolet B irradiated keratinocytes. Distinct domains of the proteins were described to be responsible for pro and anti-apoptotic activities of the protein. The diubiquitin gene (UbM has been shown to be expressed in rat lymphoblasts, thymus, and testis. In the mouse it is expressed in dendritic cells and B cells, is inducible by IFN-γ, and can cause apoptosis. The protein (also known as FAT10) provides an ubiquitin-independent signal for proteasomal degradation. It has been suggested to participate in antigen processing, but its expression did not affect MHC class I expression or antigen presentation. In view of the reported roles of these genes in the immune response, a direct involvement in GVHD is conceivable.
For the other regulated MHC genes an involvement in immune functions has not been established so far. Spr1 (or Psors1c2) is the psoriasis susceptibility 1 candidate 2 gene and was found to be expressed in the thymus of rats. Its human homologue is expressed in normal and psoriasis skin and has been suggested to confer susceptibility to psoriasis. The function of the gene product is not known so far. G18 (Gpsm3) is an activator of G-protein signaling. Pbx2 encodes an ubiquitously expressed transcriptional activator. The Ly6g6e gene belongs to the lymphocyte antigen 6 (Ly-6) superfamily that encodes proteins attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that is directly involved in signal transduction. Mouse Ly6g6e was found to be highly expressed at the leading edges of cells, on filopodia, which are normally involved in cell adhesion and migration. The mitochondrial ribosomal protein S18B (Mrsps18b) gene encodes a 28S subunit protein that belongs to the ribosomal protein S18P family. The functions of the HLA-B associated transcript 5 (Bat5) and Fij13158 (or RGD1303066) genes have not been characterized so far.
Many of the up-regulated MHC genes are inducible by IFN-γ, a type II cytokine that is primarily secreted by activated T and NK cells. Several studies have demonstrated an increased level of IFN-γ in the early phase of GVHD. Therefore, this cytokine might be highly important for the regulation of the expression of MHC genes during GVHR.
The inventors also included the NKC region in the expression profiling which harbors the Ly49 genes that encode NK receptors of the killer cell lectin-like receptor type and some of these have been shown to interact with both MHC class Ia and Ib molecules. In contrast to the MHC region, no reference sequence has been published for the NKC region of the rat. Therefore, 20 genes that were recently assigned to this region in the assembly RGSC v3.4 (Twigger et al. (2008) Nat. Genet. 40: 523-527) were not represented on the array. However, for most of them no function associated with the immune system has been reported. Interestingly, only Ly49 receptor genes which have an ITIM motif in their cytoplasmic region were up-regulated in the allogeneic skin explant assays. This includes also the LOC690045 gene which encodes an immunoreceptor similar to Ly49si1. It is not clear whether one of these gene products interacts with the MHC class Ib molecules that the inventors found to be up-regulated. Ly49 receptors are normally present mainly on NK cells and the skin explants harbored few leukocytes. However, skin resident lymphocytes can become activated in human skin explant assays. Although few NK cells infiltrating a tissue that normally does not contain these cells might cause a drastic relative change in the presence of Ly49 transcripts, the possibility should not be dismissed that other cells may express the receptors under pathological conditions. The role of NK cells for GVHR in skin explants needs to be further explored. In general NK cells are assumed to prevent GVHR, improve engraftment and to exert strong graft-versusleukemia effects without causing GVHD.
In the NKC region the inventors found one non-Ly49 gene to be regulated. The Olr1 gene encodes a receptor protein which belongs to the C-type lectin superfamily. The protein (also known as LOX-1) binds, internalizes and degrades oxidized low-density lipoprotein, which induces vascular endothelial cell activation and dysfunction, resulting in pro-inflammatory responses, pro-oxidative conditions and apoptosis. In addition, it acts as a receptor for extracellular heat shock protein 70 on dendritic cells. Binding and internalization of heat shock protein 70/peptide complexes channels peptides into the MHC class I presentation pathway. Thus, the protein is involved in antigen cross-presentation to naive T cells.
In addition to the MHC and NKC region genes, 168 further genes were significantly regulated in allogeneic skin explants. Many of them also have immunological functions and need to be analyzed in more detail in subsequent studies.
The results obtained in the MHC and NKC gene expression profiling experiment were confirmed in most tested cases by qRT-PCR on the skin explant samples. Some genes, e.g. Aif1 and Ly49i9, appeared to be up-regulated even in grade I GVHR. Olr1, in contrast, was up-regulated predominantly in grade II and III GVHR in all comparisons. Importantly, several of the MHC and NKC genes that were identified to be regulated in the skin explant assays, including Aif1, Lst1, and Olr1, were also regulated in the GVHD affected skin of transplanted animals. Thus, the skin explant assay can model GVHD not only histologically but also with respect to gene regulation. However, the up-regulation of the tested Ly49 genes (Ly49si1 and Ly49i9) that were observed in the skin explant was not clearly confirmed in the GVHD-affected skin of transplanted rats. Skin lesions from transplanted animals are likely to be more heterogeneous with respect to the dynamics of the pathophysiological process than skin explant samples, and this may contribute to the variation in results.
In conclusion, the MHC gene expression profiling approach in the rat skin explant assay identified a number of non-class I/class II genes that might contribute to the MHC-associated risk of GVHD following HSCT. These genes could be directly involved in the pathophysiology of GVHD or serve as molecular markers for GVHD and GVHR. The possibility should not be dismissed, however, that these marker genes could indicate that protective pathways are induced which modulate tissue damage during inflammation. Moreover, their human homologues may be useful for risk assessment, diagnosis, and as potential targets for therapy of GVHD in patients.
Accordingly, in a first aspect, the invention relates to a method of predicting the risk of a subject to develop graft versus host reaction (GvHR) or graft versus host disease (GvHD), comprising
The term “predicting the risk of a subject” is used herein to refer to the prediction of the likelihood of a subject to develop graft versus host reaction (GvHR) or graft versus host disease (GvHD). The method of the invention may be used clinically in order to determine the best treatment modalities and regimen and/or to evaluate whether said patient is likely to respond favourably to a treatment, such as surgical intervention, as for example a transplantation, in particular with regard to dosage and/or drug combinations.
The terms “graft versus host reaction” and “graft versus host disease” may be used synonymously. Usually, 3 criteria must be met in order for GvHD to occur: (1) Administration of an immunocompetent graft, with viable and functional immune cells, (2) the recipient is immunologically disparate—histoincompatible, and (3) the recipient is immunocompromised and therefore cannot destroy or inactivate the transplanted cells. Following transplantation, T cells present in the graft, either as contaminants or intentionally introduced into the host, perceive host tissues as antigenically foreign and attack the tissues of the transplant recipient. GvHD occurs not only when there is a mismatch of a major MHC class I or II antigen but also in the context of disparities between minor histocompatibility antigens. GvHD is a common complication in recipients of bone marrow transplants from, e.g., HLA-identical siblings, who typically differ from each other in many polymorphic proteins encoded by genes unlinked to the MHC.
Clinically, GvHD is divided into acute and chronic forms. Acute and chronic GvHD appear to involve different immune cell subsets, different cytokine profiles, different host targets, and respond differently to treatment. For example, the acute form of GvHD is normally observed within the first 100 days post-transplant, and is a major challenge to transplants owing to associated morbidity and mortality. In contrast thereto, the chronic form of GvHD normally occurs after 100 days. The appearance of moderate to severe cases of chronic GvHD adversely influences long-term survival.
In order to determine the expression level of one or more prognostic RNA transcripts, or their corresponding cDNAs, or their expression products of one or more genes, a sample comprising cells from the subject and, thus, the prognostic RNA transcripts or their expression products is first derived from said subject.
The term “sample”, as used herein, refers to a sample comprising cells of the subject to be tested, which may be the graft or the host in question, which cells may be homogenized and disrupted in order to release and optionally isolate the prognostic RNA transcripts. Preferably, the sample is a biopsy sample, preferably a biopsy sample of the tissue to be transplanted or of the tissue wherein the transplant is grafted, or a sample of Peripheral Blood Mononuclear Cells (PBMC). A peripheral blood mononuclear cell (PBMC) is a blood cell having a round nucleus. In general, these cells are immune cells, such as lymphocytes (e.g., T cells, B cells, and NK cells), monocytes or macrophages. These cells are often extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat containing said PBMCs under a layer of plasma. Alternatively, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. This method results in neutrophils and other polymorphonuclear (PMN) cells which are important in innate immune defence being obtained. However any other suitable method may be used in order to isolate PBMC from the subject.
Said RNA transcripts may subsequently be used directly or processed into another form, such as cRNA, cDNA or PCR amplification products, which still represent the expressed genes in said sample of cells, i.e. the transcripts of these genes. RNA can be isolated according to any of a number of methods well known to those of skill in the art. For example, mRNA is isolated using oligo d(T) column chromatography or glass beads. For example, RNA extraction may be performed by using TRIZOL reagent (Invitrogen, Carlsbad, Calif., USA), as described in more detail in the examples.
Alternatively, a cDNA may be reverse transcribed from said prognostic RNA transcript, RNA transcribed from that cDNA, a DNA amplified from that cDNA, RNA transcribed from the amplified DNA, or the like. Total mRNA can be converted to cDNA and amplified by conventional procedures, for example, by reverse transcription in a per se known manner. A cDNA may be amplified by any of a variety of conventional amplification procedures, including PCR. Suitable PCR primers can be selected using any well-known methods. Further examples of primers are given in the Examples section below.
For example, the level of expression of a prognostic RNA transcript or their corresponding cDNA in a sample is determined by hybridizing said RNA transcript or corresponding cDNA to a detectable probe, e.g. by performing a microarray, such as a DNA microarray. Alternatively, the expression level may be determined by using quantitative PCR. Then, the mRNA copy number may be calculated from the amount of hybridization, which generally reflects the level of expression of the polynucleotide in the cells of the sample, normalized to the amount of total RNA (or cDNA) or to the expression level of one or more housekeeping genes.
Methods for detecting hybridization are well known in the art. For example, the prognostic RNA transcript or corresponding cDNA may be labelled with a fluorescent label and levels and patterns of fluorescence indicative of hybridization are measured, e.g. by fluorescence microscopy, preferably confocal fluorescence microscopy. In this detection method, an argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier and the amount of emitted light detected and quantitated. The detected signals are considered to be proportional to the amount of probe/target hybridization complex at each position of the microarray. Further, the fluorescence microscope may be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensity. The scanned image is examined to determine the abundance/expression level of each hybridized target transcript. Alternatively, a fluorescent imaging device, such as a microarray scanner, may be used.
Typically, array fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one array is used under similar test conditions. This may be achieved by using the intensities derived from internal normalization controls contained on each microarray, e.g. from housekeeping genes. Accordingly, “normalized” refers to the expression level of an RNA transcript relative to the expression level of the total RNA or relative to the expression level of a housekeeping gene. Housekeeping genes are genes that are constitutively transcribed at a relatively constant level across many or all known conditions, since the housekeeping gene's products are typically needed for maintenance of the cell. Examples of housekeeping genes include actin, GAPDH, and ubiquitin.
However, further methods for determining the amount of a polynucleotide are well known in the art and may include any suitable quantitative method. Examples for such further methods are, for example, quantitative PCR, such as real-time PCR, or reverse transcription PCR (RT-PCR), using primers specific for those polynucleotides. Methods for selecting suitable primers for detecting and quantitating the amplified product are known in the art and exemplified in the Examples section below.
Alternatively, the expression level may be determined by the expression product(s), i.e. by the polypeptides encoded by said genes. This may be accomplished using immunological methods involving the use of antibodies directed against said polypeptides, e.g. the expression level of the corresponding expression product(s) is determined by ELISA or immunohistochemistry.
In order to perform an ELISA the sample with an unknown amount of expression is product is immobilized on a solid support either non-specifically via adsorption to the surface of the solid support or specifically by a so called capture-antibody specific to the expression product. After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can itself be covalently linked to an enzyme, or can be detected by a secondary antibody linked to an enzyme. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. Detection occurs by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of expression product in the sample. Immunohistochemistry refers to a method involving localizing the expression product in said cells of the sample using fluorescence labelled antibodies and determining the fluorescence intensity.
However, any suitable method may be used for determining the expression level of said expression product(s), such as by way of Western blotting, protein microarray, flow cytometry or surface plasmon resonance.
Thus, in a preferred embodiment, the expression level is determined by DNA microarray analysis or quantitative PCR and subsequent calculation of the mRNA copy number normalized to the amount of total RNA or to the expression level of one or more housekeeping genes. In another preferred embodiment the expression level of the corresponding expression product(s) is determined by ELISA, Western blotting, protein microarray or immunohistochemistry, flow cytometry or surface plasmon resonance.
The term “every unit of increased expression” and the term “every unit of decreased expression” as used herein refers to an expression level of one or more prognostic RNA transcripts, or their corresponding cDNAs, or their expression product(s) that has been found differentially expressed in subjects suffering or prone to suffer from GvHD or GvHR in comparison to healthy subjects. Thus, in case of “every unit of increased expression”, the higher the expression level of a gene which is predominantly expressed in the cells of a subject who suffers or is prone to suffer from GvHD or GvHR, the higher is the risk that the subject to be tested is expected to develop GvHD or GvHR. Likewise, in case of “every unit of decreased expression”, the lower the expression level of a gene which is predominantly expressed in healthy subjects but not in subjects suffering or prone to suffer from GvHD or GvHR, the higher is the risk that the subject to be tested develops GvHR or GvHD.
The determined expression level may be compared to a corresponding baseline value. As used herein, the term “corresponding baseline value” refers to the level of gene expression in normal cells or PBMCs, e.g. in a sample from a healthy subject or from a “pool” of samples derived from healthy subjects; or from a pool of one or more tissues from healthy subjects. Any of the above types of baseline values may be available in a database compiled from such values. Therefore, in a preferred embodiment, the baseline value may be the expression level of said at least one gene in at least one healthy subject.
An expression level of a gene may be considered as being increased if the log 2-fold change is at least 1, such as at least 1.1, or at least 1.2, preferably at least 1.25, such as at least 1.5 or at least 1.75, more preferably at least 2.0, such as at least 2.25 or at least 2.5, and most preferably at least 2.75 or even at least 3.0. Likewise, an expression level of a gene may be considered as being decreased if the log 2-fold change is at least −1, such as at least −1.1, or at least −1.2, preferably at least −1.25, such as at least −1.5 or at least −1.75, more preferably at least −2.0, such as at least −2.25 or at least −2.5, and most preferably at least −2.75 or even at least −3.0.
Alternatively, the term “increased” amount means herein an amount which is typically at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, preferably at least 200%, at least 225%, at least 250%, at least 275%, more preferably at least 300%, at least 350%, or at least 400%, most preferably at least 500% of the baseline value.
Likewise, the term “decreased”, as meant herein, refers to an amount which is typically less than 90%, less than 85%, less than 80%, less than 75%, more preferably less than 70%, less than 65%, less than 60%, even more preferably less than 50%, less than 40%, or less than 30%, most preferably less than 25%, less than 20%, or even less than 10% of the baseline value.
The term “one or more” as used herein means that either one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen expression level(s) of said genes is/are determined.
The term “corresponding”, as used herein, refers to the baseline value of the same gene as determined in the sample. The genes and their respective reference sequence is given in Table 10 below as well as in SEQ ID NOs 1-25.
The following combinations of biomarkers are contemplated to be particularly useful:
In a preferred embodiment, the subject is a mammal, preferably a mouse, rat, guinea pig, cat, dog, sheep, horse, cow, pig, more preferably the subject is a human.
In another preferred embodiment, the method further comprises determining the prognostic transcript of one or more genes selected from the group of genes consisting of Ubd, C2, Lst1, Aif1, C1QTNF7, CEACAM4, MME, IGFBP5, TAP1, CTGF, ANP32A, HCLS1, HTRA1, LGALS7, PTGER2, PTPN7, TGM2, TREM2 and CARD11; or their corresponding cDNAs, or their expression products, wherein
Accordingly, any combination of genes Ctss, Pbx2, Grem1, Ly6g6e, Olr1, Spr1, Msr1, Spic, Nfe2, Tnfaip8l2, Ier3, Pik3ap1, and Pstpip1 may be combined with any combination of genes Ubd, C2, Lst1, Aif1, C1QTNF7, CEACAM4, MME, IGFBP5, TAP1, CTGF, ANP32A, HCLS1, HTRA1, LGALS7, PTGER2, PTPN7, TGM2, TREM2 and CARD11.
In a second aspect, the invention relates to a method of diagnosing graft versus host reaction (GvHR) or graft versus host disease (GvHD) in a subject following transplantation, comprising:
The preferred embodiments of the first aspect are also preferred embodiments of the second aspect, and the same definitions apply.
However, in one particularly preferred embodiment, the baseline value is the expression level of said at least one gene in said subject prior to said transplantation and/or in at least one healthy subject.
In a preferred embodiment of the second aspect, said method further comprises determining the prognostic transcript of one or more genes selected from the group of genes consisting of Ubd, C2, Lst1, Aif1, C1QTNF7, CEACAM4, MME, IGFBP5, TAP1, CTGF, ANP32A, HCLS1, HTRA1, LGALS7, PTGER2, PTPN7, TGM2, TREM2 and CARD11; or their corresponding cDNAs, or their expression products, wherein
In a third aspect, the invention relates to a method of monitoring the efficacy of treatment of graft versus host reaction (GvHR) or graft versus host disease (GvHD) in a subject following transplantation, comprising:
The preferred embodiments of the first and second aspect are also preferred embodiments of the third aspect, and the same definitions apply.
In another preferred embodiment, the method of the third aspect further comprises determining the prognostic transcript of one or more genes selected from the group of genes consisting of Ubd, C2, Lst1, Aif1, C1QTNF7, CEACAM4, MME, IGFBP5, Tap1, Ctgf, ANP32A, HCLS1, HTRA1, LGALS7, PTGER2, PTPN7, TGM2, TREM2 and CARD11; or their corresponding cDNAs, or their expression products, wherein
In a very important fourth aspect, the invention further relates to a method of screening for a candidate substance for treatment of graft versus host reaction (GvHR) or graft versus host disease (GvHD), comprising:
Preferably, the screening method is carried out in vitro, i.e. in an ex vivo model, with cultured cells or with tissue, and by applying high throughput procedures. One example of such an ex vivo model is the Skin Explant Assay. This unique, non-artificial, (human) in vitro assay technology allows the study of primary and secondary immune responses in the presence of immunomodulatory drugs or allogeneic stem cells, reducing the need for extensive animal testing. Incubation with, for example, human skin, allows skin damage to be assessed by histopathology. The skin is graded for histological damage using criteria similar to that used and observed in the clinical setting. Results correlate with systemic disease and have been shown to predict outcome. The Skin Explant Assay is further exemplified in the Examples section and in the references cited therein.
Candidate substances selected by the screening method according to the invention may be subsequently also tested in vivo.
Alternatively, the screening assay may be directly performed in vivo by using a non-human animal model which suffers from GvHR or GvHD. Suitable non-human animal models include rats, mice, guinea pigs, pigs, dogs, and cats. However, it has to be made sure that the scientific gain outweighs any animal suffering, and that the testings are carried out in accordance with national restrictions for animal testings.
A variety of types of putative candidate substances may be tested and identified as suitable. For example, one can utilize known properties of a target protein to devise agents to stimulate or inhibit its production or activity, as desired. That is, one can devise a means to inhibit the action of, or bind, block, remove or otherwise diminish the presence, activity and/or availability of, a protein whose upregulation is associated with GvHD or GvHR; or one can devise a means to stimulate the action of, or to potentiate or enhance the activity of or availability of, a protein whose down-regulation is associated with GvHD or GvHR.
For example, in the case of a cellular receptor, one could expose the receptor to an antagonist, a soluble form of the receptor or a “decoy” ligand binding site of a receptor (to compete for ligand) to inhibit it. Antibodies may be administered to a cell to bind and inactivate (or compete with), or to enhance the activity of, secreted protein products or expressed cell-surface products of genes of interest.
Another approach is to employ antisense oligonucleotides or nucleic acid constructs that inhibit expression of a gene whose down-regulation is desired, in a highly specific manner. Methods to select, test and optimize putative antisense sequences are routine. Nucleic acid constructs may be used to express an antisense molecule of interest, or antisense oligonucleotides as such may be administered to a cell. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane, hybridization-triggered cleavage agents, or intercalating agents. Multiple antisense constructs or oligonucleotides specific for different genes can be employed together. The sequences of the down-regulated genes described herein can be used to design the antisense molecules. The antisense sequences may range from about 6 to about 50 nucleotides, and may be as large as 100 or 200 nucleotides, or larger. They may correspond to full-length coding sequences and/or may be genomic sequences that comprise non-coding sequences.
Another approach is to use ribozymes that can specifically cleave nucleic acids encoding the overexpressed genes disclosed herein. Such methods are routine in the art and methods of making and using any of a variety of appropriate ribozymes are well known to the skilled worker. A ribozyme having specificity for an mRNA of interest can be designed based upon the nucleotide sequence of, e.g., the corresponding cDNA. Alternatively, the sequence of an overexpressed gene disclosed herein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.
Another approach involves double stranded RNAs called small interfering RNAs. A siRNA is a double-stranded RNA molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary. The siRNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker. The siRNA may be a polynucleotide having a hairpin secondary structure, i.e. having self-complementary sense and antisense regions. The siRNA may be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi. In certain embodiments, the siRNA molecule comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. RNAi molecules may be used to inhibit gene expression, using conventional procedures.
Another approach is to use small molecules, or “compounds”, isolated from natural sources or developed synthetically, e.g., by combinatorial chemistry. In general, such molecules are identified from large libraries of natural products or synthetic (or semisynthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the methods of the invention. Accordingly, virtually any number of chemical extracts or compounds can be used in the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, polypeptide- and nucleic acid-based compounds. Synthetic compound libraries are commercially available, e.g., from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, e.g., Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are generated, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
Methods for introducing candidate substances into cells are conventional. For example, methods of gene transfer may be used, wherein antisense molecules, ribozymes, or siRNAs are introduced into a rectal carcinoma cell of interest, or nucleic acids that encode proteins which modulate (up-regulate or down-regulate) the production or activity of one or more of the genes disclosed herein. Methods of gene transfer are conventional, and include virus-mediated gene transfer, for example, with retroviruses, lentiviruses, and recombinant adenovirus vectors. Adeno-associated virus (AAV) may also be used. Improved efficiency is attained by the use of promoter enhancer elements in the DNA constructs. In addition to virus-mediated gene transfer, physical means well-known in the art can be used for direct gene transfer, including administration of plasmid DNA and particle-bombardment mediated gene transfer. Furthermore, electroporation or calcium phosphate transfection, both well-known means to transfer genes into cell in vitro, may also be used. Gene transfer may also be achieved by using “carrier mediated gene transfer”. Preferred carriers are targeted liposomes such as immunoliposomes, which can incorporate acylated monoclonal antibodies into the lipid bilayer, or polycations such as asialoglycoprotein/polylysine. Liposomes have been used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles. Preformed liposomes that contain synthetic cationic lipids form stable complexes with polyanionic DNA. Cationic liposomes, liposomes comprising some cationic lipid, that contained a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium-bromide (DDAB) have efficiently transferred heterologous genes into eukaryotic cells and can mediate high level cellular expression of transgenes, or mRNA, by delivering them into a variety of cultured cell lines.
In still a final aspect, the invention describes the use of a kit in a method of predicting the risk of developing graft versus host reaction (GvHR) or graft versus host disease (GvHD) according to the first aspect, or in a method of diagnosing GvHR or GvHD according to the second aspect, or in a method of monitoring the efficacy of treatment of GvHR or GvHD according to the third aspect, wherein the kit comprises at least one isolated polynucleotide, wherein each isolated polynucleotide independently comprises
The isolated polynucleotide may have at least 90% identity to a polynucleotide cornprising a nucleotide sequence selected from the group of nucleotide sequences consisting of at least 20 contiguous nucleotides of the nucleotide sequence selected from SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 18, 20, 22, and/or 24; or SEQ ID NO: 26-47; more preferably to the CDS encoded therein. Preferably, said isolated polynucleotide, has a nucleotide sequence having at least 92%, at least 94%, at least 96%, at least 98%, or 99% nucleotide sequence identity to a polynucleotide comprising a nucleotide sequence selected from the group of nucleotide sequences consisting of at least 20 contiguous nucleotides of the nucleotide sequence selected from SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 18, 20, 22, and/or 24; or SEQ ID NO: 26-47; more preferably to the CDS encoded therein.
Generally, a nucleotide sequence has “at least x % identity” with another nucleotide sequence or any of the sequences given above if, when the sequence identity between those to aligned sequences is at least x %. Such an alignment can be performed using for example publicly available computer homology programs such as the “BLAST” program provided at the NCBI homepage at http://www.ncbi.nlm.nih.gov/blast/blast.cgi, using the default settings provided therein. Further methods of calculating sequence identity percentages of sets of nucleic acid sequences are known in the art.
Preferably, the isolated polynucleotides comprise at least 25, preferably at least 30, more preferably at least 35, even more preferably at least 40, most preferably 50, in particular 60 contiguous nucleotides.
In another preferred embodiment, the isolated polynucleotides are arranged in an array, in particular wherein the kit comprises no more than 8000, preferably no more than 7000, more preferably no more than 6000, even more preferably no more than 5000 or even no more than 4000, most preferably no more than 3000 or even no more than 2000, in particular no more than 1000 or even no more than 500 or no more than 100 isolated polynucleotides in total.
The isolated polynucleotides of the kit may be used as probes in a hybridization method, however, in a more preferred embodiment, the isolated polynucleotides are arranged in an array. The term “array”, as used herein, means an ordered arrangement of addressable, accessible, spatially discrete or identifiable, molecules disposed on a surface. Moreover, the array may be a microarray (sometimes referred to as a DNA “chip”). Microarrays allow for massively parallel gene expression analysis. Furthermore, the hybridization signal from each of the array elements is individually distinguishable. Arrays can comprise any number of sites that comprise probes, from about 5 to, in the case of a microarray, tens to hundreds of thousands or more. Microfluidic devises are also contemplated.
Any suitable, compatible surfaces can be used in conjunction with this array. The surface (usually a solid, preferably a suitable rigid or semi-rigid support) may be any organic or inorganic material or a combination thereof, including, merely by way of example, plastics such as polypropylene or polystyrene; ceramic; silicon; (fused) silica, quartz or glass, which can have the thickness of, for example, a glass microscope slide or a glass cover slip; paper, such as filter paper; diazotized cellulose; nitrocellulose filters; nylon membrane; or polyacrylamide gel pad. Substrates that are trans-parent to light are useful when the method of performing an assay involves optical detection. Suitable surfaces include membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles, capillades, or the like. The surface can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the isolated polynucleotides are bound. It can, for example, be a flat surface such as a square, rectangle, or circle; a curved surface; or a three dimensional surface such as a bead, particle, strand, precipitate, tube, sphere, etc.
Methods of making DNA arrays, including microarrays are conventional. For example, the probes may be synthesized directly on the surface; or preformed molecules, such as oligonucleotides or cDNAs, may be introduced onto (e.g., bound to, or otherwise immobilized on) the surface. Among suitable fabrication methods are photolithography, pipetting, drop-touch, piezoelectric printing (ink-jet), or the like.
Furthermore, the probes do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure to the attached nucleic acid probe. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the nucleic acid probe.
The kit may optionally further comprise, isolated polynucleotides that act as internal controls. The controls may be positive controls or negative controls, examples of which will be evident to the skilled worker. The determined amounts obtained by use of the kit should reflect accurately the amounts of control target polynucleotide added to the sample.
The kit may further comprise means for carrying out a method of the invention, means for reading hybridization results and instructions for performing a method, such as a diagnostic method. Hybridization results may be units of fluorescence. Other optional elements of the kit may include suitable buffers, media components, or the like; a computer or computer-readable medium for storing and/or evaluating the assay results; containers; or packaging materials. Reagents for performing suitable controls may also be included. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in single reaction form for diagnostic use. The following examples are meant to further illustrate, but not limit, the invention. The examples comprise technical features, and it will be appreciated that the invention relates also to combinations of the technical features presented in this exemplifying section.
The inventors decided to analyze a rat model of GVHD making use of genetically well-defined inbred stains. Importantly, the non-class I/non-class II genes of human (HLA) and rat (RT1) MHCs are highly conserved. However, the size and organization of MHC class I encoding regions are considerably variable and the rat possesses a significant number of MHC class Ib genes for which no human homologues exist. At least some of these genes have already been proven to encode ligands for inhibitory or activating natural killer (NK) receptors (Naper C, et al. (1999) Eur J Immunol 29: 2046-2053; Naper C, et al. (2005) J Immunol 174: 2702-2711). In the rat, in contrast to human, NK receptors of the Ly49 killer cell lectin-like receptor type predominate over killer cell Ig-like receptor genes. Therefore, the inventors also included the natural killer complex (NKC) in the expression profiling which harbors the Ly49 genes and additional natural cytotoxicity receptor genes.
To reduce the complexity of the experimental approach, the inventors used an invitro-model of the graft versus host reaction (GVHR)—the skin explant assay. This assay has been shown to be a sensitive predictor of GVHD in patients (Sviland L, et al. (2001) Hum Immunol 62: 1277-1281). It was also used to study the pathophysiology of GVHR (Dickinson A M, et al. (2002) Nat Med 8: 410-414). Recently, the inventors developed a rat skin explant assay (Novota P, et al. (2008) Transplantation 85: 1809-1816). This standardized in-vitro-model allows for studying gene expression during GVHR in a setting that is not influenced by undefined genetic differences between tissue samples which is unavoidable in human studies. Presently, the inventors used this model to analyze the MHC and NKC gene expression profiles of GVHR.
For the rat skin explant assays, rats of the inbred strains LEW.1N (RT1n), LEW.1A (RT1a), LEW.1AV1 (RT1av1), LOU/C (RT1u), and BUF (RT1b) were bred in the central animal facility of the Medical Faculty of the University of Gottingen. Rats of the strains PVG/OlaHsd (RT1c) and BN/RijHsd (RT1n) were purchased from Harlan Winkelmann (Borchen, Germany). Animals between 10 and 20 weeks of age were used for the experiments. For transplantation experiments, PVG rats of the RT7.2 allotype (allelic variant RT7b), originally obtained from Harlan OLAC, UK), were bred at the animal facility of the University of Oslo and BN rats were purchased from Harlan.
Rat skin explant assays were performed as previously described in detail (Novota P, Sviland L, Zinöcker S, Stocki P, Balavarca Y, et al. (2008) Correlation of Hsp70-1 and Hsp70-2 gene expression with the degree of graft-versus-host reaction in a rat skin explant model. Transplantation 85: 1809-1816). Briefly, mononuclear cells were obtained from rat spleens. Responder and irradiated (25 Gy) stimulator splenocytes were co-cultured in a MLR and the proliferation of responder lymphocytes was tested by [methyl-3H]-thymidine (Amersham, Braunschweig, Germany) incorporation. The stimulation index was calculated as described (Novota P, et al. (2008), supra). After 7 days 106 responder lymphocytes were added to freshly obtained skin samples from the stimulator strain that were cultured in 200 μl NaHCO3-buffered Dulbecco's modified Eagle's medium (DMEM; Biochrom) supplemented with 3% normal rat serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics in round-bottomed microtitre plates (Sarstedt, Nümbrecht, Germany). The skin samples were excised from the paws of rats after washing with 70% ethanol. The subcutaneous fat tissue was removed and the samples were trimmed to a size of approximately 1.5×1.5 mm. Skin samples cultured in medium only and samples co-cultured with lymphocytes from a “syngeneic MLR” were used as controls. After 3 days, the skin explants were washed with N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)-buffered DMEM and snap frozen in liquid nitrogen and stored at −80° C. for RNA preparation. Parallel samples were fixed in 10% neutral-buffered formalin, sectioned, and stained with hematoxylin and eosin (H&E). The histological evaluation of the skin explants was performed blind by an expert histopathologist (L.S.) based on the grading system described by Lerner (Lerner K G, et al. (1974) Transplant Proc 6: 367-371). To obtain skin explant samples for an expression profiling experiment, the inventors used BN rats (RT1n) as recipients and PVG rats (RT1c) as donors. This combination is mismatched for minor and major histocompatibility antigens, which gives rise to GVHR grades I to IV (Novota P, et al. (2008), supra). PVG splenocytes were stimulated for 7 days in a mixed lymphocyte reaction (MLR) with irradiated BN splenocytes. Syngeneic co-cultures (BN plus irradiated BN splenocytes) were performed as control experiments. The stimulation index indicated a specific proliferation of PVG lymphocytes in response to irradiated BN lymphocytes in contrast to syngeneic cultures of BN lymphocytes (p<0.0001, U test; n=12 responder animals per strain, data not shown). After 7 days the PVG and BN lymphocytes were harvested, added to fresh BN skin samples from 12 individual animals and cultured for 3 further days. For further controls, additional BN skin samples from the same animals were cultured in medium only. On day 3 the skin samples were harvested and snap frozen for RNA preparation. Parallel samples were fixed and assayed for histological evidence of GVHR (
RNA was prepared from the 24 BN skin explants exposed either to syngeneic (BN; n=12) or to allogeneic (PVG, n=12) lymphocytes and used for MHC gene expression profiling.
RNA extraction was carried out using TRIZOL reagent (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's recommendations. Afterwards, the RNA samples were treated with RQ1 RNase free DNase (Promega, Madison, Wis., USA) for 20 min at 37° C. in order to remove genomic DNA contaminations. The RNA was then purified as described previously (Novota P, et al. (2008) Transplantation 85: 1809-1816). Quantity and quality of extracted RNA were controlled by capillary electrophoresis
Microarray Experiment
For the expression profiling, a custom-designed oligo DNA microarray (Agilent) was designed. For this purpose the annotated sequence of the MHC of the BN strain was used (Hurt P, et al. (2004) Genome Res 14: 631-639). The 15K microarray covered 224 MHC genes by 649 oligonucleotide probes and 43 NKC genes by 101 probes. For 88 of these genes, i.e. 39.3%, the inventors had to design custom probes. A list of the MHC genes in the chromosomal order with all results obtained in the expression profiling experiment is given in the Table 5a.
These probes were spotted in triplicates. Further probes representing 6342 genes were added mainly to allow for data normalization. A two-color 12×2 paired swap design (Landgrebe J, et al. (2004) In Silico Biol 4: 461-470) using 24 arrays was applied, comparing RNA samples from 12 independent allogeneic and 12 independent syngeneic skin explant assays. Aliquots of total RNA (200 ng) were used as starting material. The “Low RNA Input linear Amplification Kit Plus, two color” (Agilent, 5188-5340) and the “RNA Spike-In Kit” (Agilent, 5188-5279) were used for cDNA synthesis and in-vitro transcription according to the manufacturer's recommendations. Quantity and dye incorporation rates of the amplified cRNAs were determined using the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 (NanoDrop Technologies, Wilmington, Del., USA). Afterwards, 300 ng aliquots of Cy3 and Cy5-labeled cRNAs from syngeneic and allogeneic skin explant assays, respectively, were mixed and hybridized to the microarrays. The hybridization was performed for 17 hours at 10 rpm and 65° C. After washing, Cy3 and Cy5 intensities were detected by two-color scanning using a DNA microarray scanner (Agilent, G2505B) at 5 micron resolution. Scanned image files were visually inspected for artifacts. The generated raw data were extracted using the Feature Extraction 9.1 software (Agilent).
The normalization of the raw microarray data was done with a non-linear loess regression (Yang Y H, et al. (2002) Nucleic Acids Res 30: e15). Differentially expressed genes were identified by an analysis of variance (ANOVA) mixed effects model (Landgrebe J, et al. (2004) In Silico Biol 4: 461-470) using SAS PROC MIXED. The resulting p-values were adjusted with the Benjamini-Hochberg method to control the false discovery rate (Benjamini Y, Hochberg Y (1995) J Roy Statist Soc Ser B 57: 289-300). The microarray data were generated conforming to the MIAME guidelines and have been deposited in NCBI's Gene Expression Omnibus (accessible through GEO series accession number GSE17928). For a general analysis of the gene expression data the PANTHER (Protein ANalysis THrough Evolutionary Relationships) system (Thomas P D, et al. (2003) Genome Res 13: 2129-2141) was used, which classifies genes by their functions (www.pantherdb.org/tools/genexAnalysis.jsp). The microarray data were mapped to PANTHER molecular function and biological process categories, as well as to biological pathways (Thomas P D, et al. (2006) Nucleic Acids Res 34: W645-650).
For 42 of the 224 MHC genes, a probe on the array indicated a significant regulation (p<0.05) in the allogeneic skin explant assays (n=12) compared to the syngeneic controls (n=12) (Tab. 5b). Eleven of these MHC genes showed on average at least a 2-fold up-regulation (log 2-fold change ≧1) or 50% reduction (log 2-fold change ≦−1) of mRNA levels (
Furthermore, 43 genes of the NKC region, as a second important immune gene cluster, were represented on the array including all Ly49 genes in this region (Tab. 6a). For 8 of the 43 NKC genes represented on the array, a probe indicated a significant regulation (p<0.05) in the allogeneic skin explant assays compared to the syngeneic controls (Tab. 6b, 6c). In addition to the Olr1 gene, 6 Ly49 genes appeared to be up-regulated in the allogeneic skin explant assays (
Probes for 6342 additional genes from all chromosomes were included mainly to allow for data normalization. For 168 of the non-MHC/non-NKC genes, a probe on the array indicated a significant (p<0.05) and strong (log 2-fold change ≧1 or ≦−1) regulation in the allogeneic skin explant assays compared to the syngeneic controls (
Rattus norvegicus endogenous retrovirus mRNA, partial
Rattus norvegicus similar to protein tyrosine phosphatase,
Rattus norvegicus chemokine (C-C motif) ligand 6
The percentage of significantly (p<0.05) and strongly (log 2-fold change ≧1 or ≦−1) up- or down-regulated genes was higher in the NKC region (14.0%) compared to MHC region (4.9%) and the genes encoded in other regions of the genome (2.6%). This difference was even more pronounced for up-regulated genes. 14.0% of the NKC, but only 4.5% of the MHC and 1.5% of the other genes were up-regulated (Tab. 2).
1Only those genes that were both significantly (p < 0.05) and strongly (log2-fold change ≧1 or ≦−1) regulated were taken into account for this comparison.
For a general analysis of the gene expression data the PANTHER system (Thomas P D, et al. (2003) Genome Res 13: 2129-2141) was used. With this tool the inventors found a significant up-regulation of genes taking part in “immunity and defence” (p<0.0001, binominal test). More specifically, genes involved in “T cell-mediated immunity” (p<0.0001), “NK cell-mediated immunity” (p<0.0001), “cytokine and chemokine-mediated signaling” (p=0.0032), and “B cell and antibody-mediated immunity” (p=0.0235) were up-regulated. Genes involved in “complement-mediated immunity” (p=0.0336) and “cell adhesion” (p=0.0003) were down-regulated (data not shown).
Validation of Rat Candidate Genes by Quantitative Real-Time PCR
To determine the reliability of the microarray results, the inventors analyzed the expression of 13 selected genes from the MHC and NKC regions by qRT-PCR experiments in 8 of the sample pairs that had been used for the microarrays (see
For 12 genes the regulation that was observed in the microarray experiment was confirmed by qRT-PCR as indicated by a regulation into the same direction when the allogeneic and syngeneic skin explant assays were compared using the ΔΔ cycle threshold (ct) method for relative quantification of gene expression (
The up-regulation of genes in skin explants could be due to the change of gene expression in cells of the skin or due to infiltration of donor lymphocytes. Non-infiltrating or non-attaching donor lymphocytes were washed off before freezing of the skin explants and therefore would not contribute significantly to the results. Infiltrating lymphocytes were rarely seen in skin explants by histological analysis (data not shown). To further determine T cell infiltration at the RNA level, the inventors analyzed the expression of the CD3 zeta chain in qRT-PCR. Cd3z expression was found to be up-regulated in comparison to syngeneic controls and medium controls (
Next the inventors determined the expression of 10 selected genes in an independent set of skin explant assays. Skin explants derived from BN (RT1n) and LEW.1N (RT1n) rats were co-cultured with pre-stimulated allogeneic lymphocytes from rats with minor (BN lymphocytes and LEW.1N skin), major (LEW.1A (RT1a) or LEW.1AV1 (RT1av1) lymphocytes and LEW.1N skin), or minor and major histoincompatibility (PVG lymphocytes (RT1c) and BN skin or LOU/C (RT1u) lymphocytes and LEW.1N skin). Skin samples cultured with syngeneic lymphocytes (BN or LEW.1N) or cultured in medium only served as controls. The GVHR grading obtained in these experiments is shown in
Regulation of Selected MHC and NKC Genes During GVHR
The expression of 7 MHC and 3 NKC genes was evaluated in the skin explant samples showing grade I, II, III or IV GVHR (
Regulation of Selected MHC and NKC Genes During GVHD
Next, the inventors wanted to know whether the genes found to be differentially expressed in GVHR in skin explant assays were also regulated in vivo in GVHD. For this purpose the inventors analyzed skin samples from BN rats that were transplanted with bone marrow from PVG rats and developed acute GVHD.
Transplantation experiments were approved by the Experimental Animal Board under the Ministry of Agriculture of Norway (ID 09.1514, 09.1515 and VIT 09.1512). Male PVG (RT7b) rats served as bone marrow and lymph node donors. Mononuclear bone marrow cells were purified by density gradient centrifugation in Nycoprep 1.077A (Medinor ASA, Norway). The cells were depleted of T cells by magnetic separation using anti-CD5 (Ox19) and anti-αβ T cell receptor (R73) antibodies conjugated to pan-mouse IgG coated Dynabeads (Dynal Biotech ASA, Norway). This procedure reduced the CD3+ T cell content in the bone marrow from 3% to less than 0.3%. Male BN rats were used as recipients. They were irradiated (9 Gy) and subsequently received an i.v. injection of 30×106 PVG.7b T cell-depleted bone marrow cells. 14 days post transplantation, 1.5×106 lymph node cells were injected i.v. to evoke GVHD. The rats were regularly monitored for GVHD symptoms. Rats suffering from irreversible GVHD were sacrificed and skin samples were processed for RNA preparation and histology in parallel.
The analyzed skin samples showed in histology a grade I or grade II GVHD. The results of qRT-PCR for 7 MHC genes and 3 NKC genes are shown in
Finally, the inventors explored the regulation of the identified genes during GVHR in human skin explant assays.
Validation of the rat candidate genes with human homologues was done by qRT-PCR on clinical samples of GvhD skin and normal skin samples. This was done by relative quantification using custom designed Taqman low density array (TLDA) cards (Applied Biosystems), each card contained 4 replicates of 95 unique genes and a control gene, 185. The qRT-PCR reactions were set up using Taqman x2 gene expression mastermix (Applied Biosystems), 50 ng RNA equivalent of cDNA and the total volume adjusted to 200 μl with nuclease free water (Quiagen). The TLDA cards were run on a 7900 qRT-PCR system (Applied Biosystems) using the TLDA block and analysed using the RQ manager 1.2 software (Applied Biosystems). To normalize variations in the RNA concentration and quality in different samples, the ct values obtained in real-time PCR for the genes were corrected by the ct-value obtained for the housekeeping gene in the same sample (Δct=ct housekeeping gene−ct gene of interest) then the relative changes in RNA expression were calculated using the ΔΔct method (ΔΔct=Δct sample of interest−Δct control sample) using the average Δct values of 5 normal skins as the control sample for each of the 9 GVHD skins.
At 1, 2 and 3 days of co-culture with alloreactive lymphocytes skin samples of one donor were taken and analyzed in comparison to parallel samples cultured in medium only. At day 1a GVHR of grade I was observed that increased to grade II at day 2 and grade III at day 3. The inventors determined the expression of 15 MHC and 1 NKC gene by qRT-PCR (Tab. 3).
↑1
1Explanation of symbols:
Of these 16 genes 12 (75%) were regulated at least in one skin explant sample in the way predicted by the results of the rat expression profiling experiments (Tab. 4). Three genes TAP1, PSMB8, and UBD were up-regulated in all 3 human skin explant samples. The genes C2, FLI13158, and OLR1 were regulated in 2 of the 3 samples as predicted by the rat experiments. In addition, the inventors determined the expression of 153 non-MHC/non-NCR genes that were identified to be regulated in rat skin explant assays. Also of these genes 105 (69%) were regulated in at least one of the human skin explant samples in accordance with the results obtained in the rat model (Tab. 4). These results suggest that the in vitro rat model of the skin explant assay gives evidences of gene expression changes that are very likely to occur also in human skin explant assays during GVHR.
In a follow-up study, 24 genes have been identified in additional validation tests. The results are shown in Table 9. The probes used and the reference sequences are shown in Table 10. The additional validation tests confirmed the significant regulation of gene expression, i.e. up-regulation or down-regulation, preferably down-regulation for Ctss, Pbx2, Spr1, Spic, Nfe2, Tnfaip8l2, Ier3, and Lst1.
Statistical Analyses not Related to Microarray Experiments
Paired comparisons between experimental groups were performed using the non-parametric Mann-Whitney U test. Pearson's and Spearman's correlation coefficients were calculated to determine the correlation between mRNA expression levels of two genes. The statistical analyses were performed using WinSTAT® software.
Further studies were undertaken to evaluate the expression markers also under clinical conditions. Therefore, new tests were performed using skin explant assay as well as mRNA expression profiling studies directly on clinical GVHD biopsies to validate the results from the previous skin explant studies. The clinical GVHD biopsies were taken from hematopoietic stem cell transplantation (HSCT) patients. These data are summarized in Table 11.
Experimental Skin Explants Assays Using Autologous HSCT Patients and Normal Controls
Peripheral blood mononuclear cells (PBMC) and skin samples were obtained from autologous HSCT patients following informed consent and approval from the North Tyneside Research Ethics Committee. Buffy coat from HLA mismatched normal blood donations were obtained from Newcastle National Blood Service with consent. Skin explant assays were performed as previously described [5,6], 1×107 responder PBMC from healthy volunteers was cultured with an equal number of irradiated PBMC from autologous HSCT patients, in 10 ml complete medium (RPMI 1640 supplemented with antibiotics, 2 mM L-glutamine and 10% heat inactivated human AB serum) in a 25 cm2 flask. Standard 4 mm punch skin biopsy specimens were obtained pretransplant from the auto HSCT patients and divided into 12 equal sized pieces. After 7 days of culture, the MLR primed lymphocytes were washed and resuspended in complete medium supplemented with 20% heat inactivated autologous (patient) serum and co-cultured in duplicate with patient skin at a cell concentration of 1×106 cells/well in a volume of 200 μl/well in 96-well round-bottomed microtitre plates. In addition each skin sample was also cultured in duplicate in culture medium alone as a negative or medium only control. A time course experiment was set up to enable RNA expression analysis to be assessed early, (day 1) and late, (days 2 and 3) to monitor the interaction of sensitised T cells with recipient skin. Parallel control skins were incubated in medium only on days 1, 2 and 3 and used as the comparators. The skin samples were removed from the time series, duplicate control and MLR skin explant on days one, two or three, one sample was fixed in 10% buffered formalin, sectioned and stained with H&E and duplicate sample placed in RNAlater (Ambion) and stored at −80° C. prior to RNA extraction.
The histopathological evaluation of the skin explants for graft versus host reaction (GVHR) was performed independently by at least two assessors. Grade I histopathological damage in skin biopsies was regarded as background and was normally observed in the medium control. All biopsies presenting histopathological damage of grade II or above were regarded as GVHR positive.
Clinical Biopsies
Standard 4 mm punch biopsies or scrape biopsies were obtained from 10 patients at various time points post transplant at onset of acute GvHD together with normal skin skin controls (n=10). RNA was extracted from these biopsies as described below.
RNA Extraction and cDNA Production
RNA was extracted from the skin samples stored in RNA later using the Ambion mirVana miRNA Isolation Kit according to the manufacturer's recommendations and quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific). cDNA was generated by random hexamer priming, briefly equal quantities of RNA and 2× strength cDNA mix containing random hexamer primers (Pharmacia), dNTPs (Roche), reverse transcriptase (MMLVRT—Invitrogen) and an RNase inhibitor (Rnasin—Promega) were incubated at 37° C. for 2 hours with a further incubation at 65° C. for 10 minutes to denature the reverse transcriptase.
Validation of Candidate Genes by Quantitative Real-Time PCR
Validation of the candidate genes in the human skin explant assay and clinical biopsies was done by qRT-PCR. For this relative quantification with three custom designed Taqman low density array (TLDA) cards (Applied Biosystems) were used each card contained 4 replicates of 94 unique genes and two control genes, 18S and GAPDH, giving a total of 282 genes. The qRT-PCR reactions were set up using Taqman x2 gene expression mastermix (Applied Biosystems), 50 ng RNA equivalent of cDNA and the total volume adjusted to 200 μl with nuclease free water (Quiagen). The reaction mix was loaded onto the TLDA cards and the cards were run on a 7900 qRT-PCR system (Applied Biosystems) and analysed using the RQ manager 1.2 software (Applied Biosystems). The relative changes in RNA expression were calculated using the ΔΔct method, that is, ΔΔct=Δct sample of interest−Δct control sample, where the Δct is the ct of the control gene−the ct of the gene of interest.
Genes which showed a consistent change in expression between the medium only control skin and the MLR skin or in the clinical aGVHD skin compared to normal skin were investigated further using additional normal (n=10) and clinical aGVHD (n=10) skin samples. Real time PCR was carried out using individual TaqMan assays (Applied Biosystems) for the genes of interest and the control gene GAPDH (Applied Biosystems). The reactions were set up in triplicate using Taqman x2 gene expression mastermix, 10 to 20 ng RNA equivalent of cDNA and the manufacturer's recommended concentration of primer/probe mix. The reactions were run on a 7900 qRT-PCR system (Applied Biosystems) and analysed using the SDS 2.3 software, normalisation of expression was performed using GAPDH gene, expression results and ACT values were calculated as above.
Statistical Analysis
Comparisons between the experimental groups were carried out using the non-parametric Mann-Whitney U test using Graphpad prism 5 software (Graphpad Software inc.).
Table 5. Expression Profiling Results of MHC Genes
In Table 5a, results for all 224 MHC genes are shown in their chromosomal order (Hurt P, et al. (2004) Genome Res 14: 631-639). The expression profiling results of BN skin explant samples exposed to pre-stimulated allogeneic (PVG) lymphocytes in comparison to those exposed to syngeneic (BN) lymphocytes are given. The log 2-fold changes and the fold changes in gene expression are shown for every oligonucleotide probe used. The adjusted p-values are indicated. Significant change is defined by p<0.05 and strong change is defined by log 2-fold change ≧1 or ≦−1; i.e. fold change ≧2 or ≦0.5. In addition, the identification numbers of the probes on the arrays are given (probe ID) together with the information whether these probes were taken from the Agilent database or custom designed. Table 5b contains the same information for all MHC genes for which at least one probe indicated a significant alteration of gene expression. In Table 5c, the data for those genes are summarized that are considered to be regulated significantly because either at least a single probe indicated a significant (p<0.05) and strong (log 2-fold change ≧1 or ≦−1) regulation or at least 50% of the gene probes indicated a significant (p<0.05) regulation of gene expression.
Table 6. Expression Profiling Results of NKC Genes
In Table 6a, results for all 43 NKC genes investigated are indicated in their chromosomal order (Klrg; Pzp to Csda). The expression profiling results of BN skin explant samples exposed to pre-stimulated allogeneic (PVG) lymphocytes in comparison to those exposed to syngeneic (BN) lymphocytes are given. The log 2-fold changes and the fold changes in gene expression are shown for every oligonucleotide probe used. The adjusted p-values are indicated. Significant change is defined by p<0.05 and strong change is defined by log 2-fold change ≧1 or ≦−1; i.e. fold change ≧2 or ≦0.5. In addition, the identification numbers of the probes on the arrays are given (probe ID) together with the information whether these probes were taken from the Agilent database or custom designed. Table 6b contains the information for all NKC genes for which at least one probe indicted a significant alteration of gene expression. In Table 6c, the data for those genes are summarized that are considered to be regulated significantly because either at least a single probe indicated a significant (p<0.05) and strong (log 2-fold change ≧1 or ≦−1) regulation or at least 50% of the probes indicated a significant (p<0.05) regulation of gene expression.
Rattus norvegicus killer cell lectin-like
Rattus norvegicus similar to osteoclast
Rattus norvegicus lysyl oxidase
Rattus norvegicus retinoic acid
Rattus norvegicus glutamate receptor,
LOC684607
Rattus norvegicus cDNA clone IMAGE:
Rattus norvegicus nel-like 2 homolog
Tcfap2b
Nfib
Thbs4
Nfib
LOC684607
Thbs4
Tcfap2b
Il1rn
Il2rb
Rattus norvegicus integrin alpha X
Ccl3
Il1rn
Il2rb
Ccl3
Rattus norvegicus chemokine (C-C
Rattus norvegicus similar to protein
Rattus norvegicus endogenous
1F: forward primer, R: reverse primer
2The real-time PCR efficiency coefficient (E) of one cycle in the exponential phase was calculated according to the equation: E = 10[-1/slope of standard curve]
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
Rattus novegicus
Homo sapiens
| Number | Date | Country | Kind |
|---|---|---|---|
| 1021149.8 | Dec 2010 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP11/72804 | 12/14/2011 | WO | 00 | 8/28/2013 |
