Genome Association Scanning Research Articles

Optimized Whole Genome Association Scanning for Discovery of HLA Class I-Restricted Minor Histocompatibility Antigens

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A∗01:01, A∗02:01, A∗03:01, B∗07:02, B∗08:01, C∗07:01, and C∗07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A∗24:02 and HLA-A∗23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation.

Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase.

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML.

S890 NEW WHOLE GENOME ASSOCIATION SCANNING APPROACH FOR THE DISCOVERY OF HLA CLASS I‐RESTRICTED MINOR HISTOCOMPATIBILITY ANTIGENS

Background:Patients that undergo allogeneic stem cell transplantation (SCT) as treatment for hematological diseases face the risk of relapse as well as Graft‐versus‐Host Disease (GvHD). GvHD as well as the favorable Graft‐versus‐Leukemia effect are mediated by donor T cells. These T cells recognize minor histocompatibility antigens: polymorphic peptides, which are presented on the cell surface by HLA molecules and are caused by genetic differences in single nucleotide polymorphisms (SNPs) between patient and donor. Knowledge about mismatched minor histocompatibility antigens may enable a more directed donor search as well as better prediction and measurement of the Graft‐versus‐Leukemia effect and GvHD for personalized treatment after transplantation, thereby contributing to a better outcome for patients treated with allogeneic SCT.Aims:Identification of minor histocompatibility antigens has been a laborious process in the past. Therefore, whole genome association scanning was developed in our laboratory. However, this method was restricted to two HLAs and screening of one million SNPs. The aim of this study is to develop and use an optimized method to identify the dominant repertoire of minor histocompatibility antigens in common HLA class I alleles.Methods:Donor T‐cells isolated from patients after allogeneic SCT are tested for recognition of a new panel of 191 selected B‐cell lines, which are sequenced in the 1000 Genome Project. This panel enables the inclusion of seven common HLAs and increases SNP coverage to 12 million (MAF > 0.01). SNPs that strongly associate with T‐cell recognition are subsequently validated to encode minor histocompatibility antigens.Results:A total of 19 novel minor histocompatibility antigens have been identified that are presented in seven common HLA class I alleles, showing the potential of this optimized approach. Besides conventional antigens, cryptic peptides were found as well as peptides restricted to HLAs that were not primarily targeted.Summary/Conclusion:This improved approach for whole genome association scanning has been successfully applied in rapidly discovering new minor histocompatibility antigens. The new method surpasses previous methods in its rapidness and coverage of HLAs and SNPs across the human genome, which additionally allows for identification of cryptic peptides. With the increasing number of patients that are analyzed in this study, T‐cells recognizing known minor histocompatibility antigens are more often isolated, indicating that the repertoire of minor histocompatibility antigens is limited. Our optimized method is expected to discover the majority of unknown antigens in this repertoire, thereby contributing essential knowledge for new strategies to improve clinical outcome after allogeneic SCT.

Integrated Whole Genome and Transcriptome Analysis Identified a Therapeutic Minor Histocompatibility Antigen in a Splice Variant of ITGB2.

In HLA-matched allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells recognizing minor histocompatibility antigens (MiHAs) can mediate desired antitumor immunity as well as undesired side effects. MiHAs with hematopoiesis-restricted expression are relevant targets to augment antitumor immunity after alloSCT without side effects. To identify therapeutic MiHAs, we analyzed the in vivo immune response in a patient with strong antitumor immunity after alloSCT. T-cell clones recognizing patient, but not donor, hematopoietic cells were selected for MiHA discovery by whole genome association scanning. RNA-sequence data from the GEUVADIS project were analyzed to investigate alternative transcripts, and expression patterns were determined by microarray analysis and qPCR. T-cell reactivity was measured by cytokine release and cytotoxicity. T-cell clones were isolated for two HLA-B*15:01-restricted MiHA. LB-GLE1-1V is encoded by a nonsynonymous SNP in exon 6 of GLE1 For the other MiHAs, an associating SNP in intron 3 of ITGB2 was found, but no SNP disparity was present in the normal gene transcript between patient and donor. RNA-sequence analysis identified an alternative ITGB2 transcript containing part of intron 3. qPCR demonstrated that this transcript is restricted to hematopoietic cells and SNP-positive individuals. In silico translation revealed LB-ITGB2-1 as HLA-B*15:01-binding peptide, which was validated as hematopoietic MiHA by T-cell experiments. Whole genome and transcriptome analysis identified LB-ITGB2-1 as MiHAs encoded by an alternative transcript. Our data support the therapeutic relevance of LB-ITGB2-1 and illustrate the value of RNA-sequence analysis for discovery of immune targets encoded by alternative transcripts. Clin Cancer Res; 22(16); 4185-96. ©2016 AACR.

Integrated whole genome and transcriptome analysis identified a therapeutic minor histocompatibility antigen encoded by an alternative ITGB2 transcript

Patients with hematological malignancies can be successfully treated with allogeneic stem cell transplantation (alloSCT). In HLA-matched alloSCT, donor T cells can mediate desired anti-tumor immunity as well as undesired side effects by recognizing minor histocompatibility antigens (MiHA) on patient cells. MiHA are polymorphic peptides with amino acid changes that are created by genetic variants and recognized by specific T cells. MiHA with hematopoiesis restricted expression are relevant targets for immunotherapy to augment anti-tumor immunity after alloSCT without side effects. For high-throughput discovery of MiHA, we previously developed whole genome association scanning (WGAs) in which T cell recognition of a panel of EBV-B cell lines is investigated for association with single nucleotide polymorphisms (SNPs) to identify the genomic region that encodes the antigen. Although WGAs is an efficient strategy for MiHA discovery, strong association with SNPs in introns or other regions outside coding exons can be found for approximately one third of the T cell clones, whereas no SNP disparity in the primary transcript is present between patient and donor, suggesting that the antigen may be encoded by an alternative transcript. To identify MiHA encoded by alternative transcripts, we developed an integrated approach in which WGAs is combined with whole transcriptome data from the GEUVADIS project. By performing WGAs, we identified associating SNPs for two HLA-B*15:01-restricted MiHA that were targeted by donor CD8 T cells in a patient with strong anti-tumor immunity after HLA-matched alloSCT. One antigen (LB-GLE1-1V) was encoded by an associating SNP in exon 6 of the GLE1 gene. For the other antigen, an associating SNP in intron 3 of the ITGB2 gene was found, but no SNP disparity was present in the normal ITGB2 transcript between patient and donor. Therefore, we investigated whether the antigen may be encoded by an alternative transcript. Using RNA-sequence data, we identified an alternative ITGB2 transcript in which part of intron 3 was retained. Q-PCR analysis showed that expression of this transcript was restricted to hematopoietic cells from SNP-positive individuals. In silico translation of the transcript revealed a peptide with strong predicted binding to HLA-B*15:01 (LB-ITGB2-1), which was recognized by specific T cells. T cells for LB-ITGB2-1 also recognized and lysed leukemic cells of different origins, while no reactivity against patient fibroblasts could be detected. In summary, RNA-sequence analysis enabled discovery of LB-ITGB2-1 as immune epitope encoded by an alternative gene transcript. Our data support the therapeutic relevance of LB-ITGB2-1 as target for T cell therapy to stimulate anti-tumor immunity after alloSCT.

Linking Genetics of Brain Changes to Alzheimer's Disease: Sparse Whole Genome Association Scan of Regional MRI Volumes in the ADNI and AddNeuroMed Cohorts.

Alzheimer's disease (AD) is a highly heritable disease, but until recently few replicated genetic markers have been identified. Markers identified so far are likely to account for only a tiny fraction of the heritability of AD and many more genetic risk alleles are thought to be undiscovered. Identifying genetic markers for AD using combined analysis of genetics and brain imaging data. Imaging quantitative trait loci (iQTLs) has recently emerged as an interesting research area for linking genetics of brain changes to AD. We consider a genome-wide association scan of 109 brain-wide regional imaging phenotypes to identify genetic susceptibility loci for AD from a combined set of 1,045 subjects from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and the AddNeuroMed studies. We use one-SNP-at-a-time as well as multi-SNP Hyperlasso based iQTL methods for the analysis. We identified several novel markers associated with AD, namely HOMER2 (rs1256429; intronic, p = 8.7 × 10⁻¹⁰), EOMES (rs2724509; flanking), JAM2 (rs2829841; intronic), and WEE1 (rs10770042; coding). The SNP rs1256429 (HOMER2) was one of the top hits in Hyperlasso as well as in the single-SNP analysis showing an association with the volume of the right thalamus and AD, a brain region reported to be linked with AD in several studies. We believe that the markers identified in this study are novel additions to the existing list of genetic variants associated with AD which can be validated in future replicated studies.

Graft Versus Leukemia Separates From Graft Versus Host Disease By Magnitude and Avidity Of The Allo-Reactive T Cell Response After Allogeneic Stem Cell Transplantation and Donor Lymphocyte Infusion

Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can be a curative treatment for patients with hematological malignancies due to the capacity of allo-reactive donor derived T cells to mediate a curative potent graft versus leukemia (GVL) effect. However, associated acute graft versus host disease (GVHD) remains a major risk. To study the role of CD8+ T cells in GVL reactivity and GVHD, we selected patients who responded to DLI (without preceding cytoreductive treatment) for recurrent disease or incomplete donor chimerism after alloSCT. The patients were grouped according to absence (7 patients) or presence (6 patients) of GVHD. To quantify the number of circulating activated CD8+ T cells before DLI and at the time of disease regression or conversion to full donor chimerism we measured the frequencies of CD8+ HLA-DR+ T cells in peripheral blood samples by flowcytometry. Before DLI, highly variable numbers of CD8+ HLA-DR+ T cells were found (37.8 ± 42.9 x106/L), that significantly increased after DLI (309±473 x106/L, p<0.005), demonstrating involvement of CD8+ HLA-DR+ T cells in immune responses after DLI. To determine the specificity and functional avidity of the CD8+ HLA-DR+ T cells, these cells were isolated using flowcytometric cell sorting and clonally expanded. From a total of 30 samples, on average 225 T cell clones per sample were obtained and tested for recognition of patient and donor derived EBV-LCL, CD40L stimulated B cells (CD40L-B cells) and monocyte derived dendritic cells (monoDC). Surprisingly, in many samples from both patient cohorts high percentages of clones recognizing EBV-LCL derived from both patient and donor but not recognizing CD40L-B cells and monoDC were found. These T cells may be involved in anti-EBV responses irrespective of the presence of a GVL effect or GVHD. To investigate whether the magnitude of the allo-immune response was different in patients with or without GVHD coinciding the GVL effect, we compared the frequencies of allo-reactive T cell clones in samples from both patient groups. Significantly lower percentages of allo-reactive T cell clones were found in patients without GVHD as compared to patients with GVHD (5.1 ± 7.0% versus 32.5 ± 20.0% respectively, p<0.01), showing that coinciding GVHD is associated with an increased magnitude of the allo-reactive T cell response. Per patient, we determined the number of unique antigens targeted by the isolated T cell clones by characterizing the targeted MiHA using whole genome association scanning. In line with the lower total number of allo-reactive T cells, a lower number of unique MiHA was targeted in patients without GVHD (2.7±3.5) as compared to patients with GVHD (10.2±5.8, p=0.015). To determine whether occurrence of GVHD could be explained by the tissue specificity and functional avidity of the allo-reactive T cell response after DLI, we tested the T cell clones obtained from both patient cohorts for recognition of fibroblasts (FB) derived from skin biopsies of the patient. To mimic pro-inflammatory conditions, FB were pretreated for 4 days with 100 IU/ml IFN-γ. Recognition of untreated FB was exclusively mediated by T cell clones obtained from patients with GVHD, whereas recognition of IFN-γ pretreated FB was found for clones isolated from patients with or without coinciding GVHD. In addition, several T cell clones isolated from patients without GVHD were found to be directed against MiHA encoded by genes with a broad expression profile in non-hematopoietic cells comprising FB, despite absence of FB recognition under non-inflammatory conditions. This suggests that in addition to the tissue expression profile of the MiHA other factors, comprising the local inflammatory milieu, play a role in the risk of developing GVHD. In conclusion, our data show a strong correlation between the magnitude and the functional avidity of the allo-reactive CD8+ T cell response and the occurrence of GVHD after DLI. We hypothesize that the limited production of pro-inflammatory cytokines due to the moderate magnitude of the immune response in patients mounting a GVL response without coinciding GVHD reactivity may have prevented the induction of GVHD by the lower avidity allo-reactive T cells, that under pro inflammatory conditions can mediate GVHD by recognition of normal non-hematopoietic cells of the patient. Disclosures:No relevant conflicts of interest to declare.

Emerging paradigms in genomics-based crop improvement.

Next generation sequencing platforms and high-throughput genotyping assays have remarkably expedited the pace of development of genomic tools and resources for several crops. Complementing the technological developments, conceptual shifts have also been witnessed in designing experimental populations. Availability of second generation mapping populations encompassing multiple alleles, multiple traits, and extensive recombination events is radically changing the phenomenon of classical QTL mapping. Additionally, the rising molecular breeding approaches like marker assisted recurrent selection (MARS) that are able to harness several QTLs are of particular importance in obtaining a “designed” genotype carrying the most desirable combinations of favourable alleles. Furthermore, rapid generation of genome-wide marker data coupled with easy access to precise and accurate phenotypic screens enable large-scale exploitation of LD not only to discover novel QTLs via whole genome association scans but also to practise genomic estimated breeding value (GEBV)-based selection of genotypes. Given refinements being experienced in analytical methods and software tools, the multiparent populations will be the resource of choice to undertake genome wide association studies (GWAS), multiparent MARS, and genomic selection (GS). With this, it is envisioned that these high-throughput and high-power molecular breeding methods would greatly assist in exploiting the enormous potential underlying breeding by design approach to facilitate accelerated crop improvement.

Purified CD4 T Lymphocyte Infusion Can Result in Graft-Versus-Leukemia Reactivity without Gvhd by Recognition of Broadly Expressed Minor Histocompatibility Antigens in HLA Class-II

AbstractAbstract 4116Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can mediate curative Graft-versus-Leukemia (GVL) reactivity although frequently at the cost of Graft-versus-Host Disease (GVHD). We previously illustrated that donor CD8 T lymphocytes recognizing HLA class-I restricted minor histocompatibility antigens (MiHAs) that are broadly expressed on tissues of the recipient cause GVL associated with GVHD, whereas T lymphocytes recognizing MiHAs selectively expressed on hematopoietic cells, including the malignant cells, can selectively mediate GVL without GVHD. Since in contrast to HLA class-I, expression of HLA class-II molecules is predominantly restricted to hematopoietic cells, we hypothesized that infused purified donor CD4 T lymphocytes may selectively recognize and eliminate hematopoietic cells from the recipient resulting in GVL without GVHD.We treated a patient with CML in blastic phase in remission after intensive chemotherapy with T cell depleted alloSCT from his HLA-identical sibling donor after myelo-ablative conditioning. After donor engraftment, recipient hematopoiesis reoccurred within 3 months to 90% of CD8 T lymphocytes, 13% of CD4 T lymphocytes and 5% of myelopoiesis. As part of a clinical trial, the patient was treated with 106/kg positively selected purified donor derived CD4 T lymphocytes resulting within 19 weeks in conversion into full donor chimerism in all hematopoietic cell lineages in the total absence of GVHD.To characterize the nature of this hematopoiesis restricted immune response, in vivo activated HLA-DR positive CD4 and CD8 T lymphocytes were clonally isolated by flowcytometric cell sorting at the time of the clinical response, expanded and tested for alloreactivity on patient and donor derived hematopoietic target cells using IFNγ ELISA. From the 204 expanding CD4 T lymphocyte clones 31 clones were alloreactive, whereas none of the 66 expanding CD8 T lymphocyte clones showed alloreactivity.To further identify the fine specificity of this hematopoiesis directed HLA class-II restricted immune response, target molecules of several T lymphocyte clones were molecularly characterized using whole genome association scanning. We first performed blocking studies with HLA class-II restricted monoclonal antibodies and identified HLA-DR to be the restriction molecule. Next, a large panel of third party EBV-LCLs was retrovirally transduced with each of the possible restriction molecules being HLA-DRB1*11:01, HLA-DRB1*15:01, HLA-DRB3*02:02 and HLA-DRB5*01:01. By comparing the recognition pattern of the transduced EBV-LCLs with the 1.1 million single nucleotide polymorphisms in each EBV-LCL, we identified 3 novel MiHAs. Synthesis and analysis of the patient and donor derived allelic peptide variants further confirmed the specificity of the MiHAs as LB-KHNYN-1K in the context of HLA-DRB5*01:01, LB-CTSB-1G in HLA-DRB1*11:01 and LB-ZDHHC13-1K in HLA-DRB1*15:01. Gene expression profiles of KHNYN (located on chromosome 14), CTSB (chromosome 8) and ZDHHC13 (chromosome 11) illustrated that the genes encoding these MiHAs were not only transcribed in hematopoietic cells, but also in other tissues including GVHD target tissues. These results further illustrated that the hematopoietic specificity of the CD4 T lymphocyte response was mainly defined by the restricted expression of the HLA-DR molecules on hematopoietic cells.We conclude that purified CD4 DLI can lead to GVL without GVHD by a selective HLA class-II restricted immune response against patient hematopoiesis. By molecular characterization of 3 novel HLA-DR restricted MiHAs we illustrated that the relative specificity of HLA class-II molecules on hematopoietic cells under non inflammatory conditions was probably responsible for this effect. Since HLA class-II is predominantly expressed on hematopoietic cells only, infusion of donor CD4 T lymphocytes under non inflammatory conditions after HLA identical alloSCT can result in efficient induction of GVL without the toxicity of GVHD. Disclosures:No relevant conflicts of interest to declare.

Durable Remission of Renal Cell Carcinoma After Donor Lymphocyte Infusion Is Unavoidably Linked with Graft Versus Host Disease As Illustrated by the Detection of Allo Reactive T Cells Recognizing a Novel Minor Histocompatibility Antigen Encoded by the FUCA2 Gene

AbstractAbstract 4467Allogeneic stem cell transplantation (allo SCT) followed by donor lymphocyte infusion (DLI) can be applied as immunotherapeutic intervention to treat malignant diseases. In hematological malignancies, remissions can be observed both in the presence and absence of graft versus host disease (GvHD), illustrating that graft versus leukemia and GvHD can be clinically separated. For efficient induction of an allo immune response, professional (hematopoietic) antigen presenting cells (APC) are required, which may result in skewing towards recognition of minor histocompatibility antigens (MiHA) selectively expressed by hematopoietic cells. However, for treatment of solid tumors including renal cell carcinoma (RCC), recognition of MiHA on non hematopoietic cells is essential. Therefore in these malignancies APC have to present broadly expressed MiHA to be targeted on non hematopoietic cells, making it inevitable that the graft versus tumor (GvT) effect is accompanied by GvHD.Here we describe the treatment of a patient who presented with progressive metastatic clear cell RCC with T cell depleted non myeloablative allo SCT. At 7 months after allo SCT the patient received DLI at a single dose of 0.5*107 T cells/kg. Acute skin GvHD occurred within 30 days after DLI and developed soon thereafter into persistent extensive chronic GvHD for several years that eventually resolved after prolonged topical and systemic immune suppression. Whereas the patient was mixed chimeric after allo SCT (84%, 80% and 95% donor after 2, 3 and 5 months, respectively), treatment with DLI resulted in conversion to full donor chimerism, which remained complete during the following years. The strong immune response was accompanied by an approximately 50% reduction in size of the measurable lung metastasis (according to RECIST criteria) and slight regression of the smaller lung lesions. To characterize the specificity of the immune response, we measured known MiHA, and detected a disparity for the previously identified MiHA LRH-1. T cells specific for this MiHA were detectable in peripheral blood at the onset of GvHD (0.14% of CD8+ T cells). Isolated LRH-1 specific T cells strongly recognized patient derived EBV-LCL, but were irresponsive to monocyte derived dendritic cells (monoDC), fibroblasts, keratinocytes, and a panel of LRH-1+ RCC cell lines and are therefore considered irrelevant for the GvT effect. In addition, we directly cloned activated CD8+ T cells and isolated an HLA-B*07 restricted T cell clone with unknown specificity. Whole genome association scanning unraveled a patient specific single nucleotide polymorphism in the FUCA2 gene encoding a novel MiHA which we designated LB-FUCA2-1V. High frequencies of circulating LB-FUCA2-1V specific T cells were detected after DLI (1.9% of CD8+ cells). Moreover, in contrast to LRH-1 specific T cells, LB-FUCA2-1V specific T cells recognized monoDC, and skin derived keratinocytes and fibroblasts after IFN-γ pretreatment. Our data also show recognition of 4 RCC cell lines, and therefore support a dominant role for LB-FUCA2-1V specific T cells in mediating tumor regression. Unfortunately, coinciding with the gradual resolvement of chronic GvHD, the GvT effect declined and eventually the metastases became progressive almost 3 years after allo SCT. In an attempt to re-initiate the GvT effect, escalating DLI doses of 0.2*107, 0.5*107, 1*107 and 5*107 T cells/kg were given. No GvHD developed but also no GvT effect could be achieved and the patient died of progressive disease 8.5 years after allo SCT.In conclusion, we observed a strong donor derived immune response targeting the broadly expressed LB-FUCA2-1V MiHA that durably controlled metastatic clear cell RCC. Initiation of this response likely depended on the presence of patient hematopoiesis, including APC, since all attempts to re-induce the MiHA specific response to treat disease progression with escalating doses of DLI under full donor chimerism were ineffective. Our data suggest that successful treatment of solid tumors may require induction of an effective immune response against broadly expressed MiHA, inevitably inducing damage to normal tissue cells resulting in GvHD. Disclosures:No relevant conflicts of interest to declare.

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease. We analyzed in detail CD8(+) T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8(+) T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens. Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones. Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.

Copy Number Variations at the Prader‐Willi Syndrome Region on Chromosome 15 and associations with Obesity in Whites

Obesity is a serious health problem with strong genetic determination. Copy number variation (CNV) is a common type of genomic variant associated with some complex human diseases. However, it is not clear how CNVs contribute to the etiology of obesity. In this study, we examined 1,000 unrelated US whites to search for CNVs that may predispose to obesity. We focused our analyses on the Prader-Willi syndrome (PWS) critical region (chromosome 15q11-q13), because the PWS region is a hotspot for CNV generation and obesity is one of the major clinical manifestations for chromosome abnormalities at this region. We constructed a map containing 39 CNVs at the PWS critical region with CNV occurrence rates higher than 1%. Among them, three CNVs were significantly associated with body fat mass (P < 0.05), with a higher copy number (CN) associated with an increase of 5.08-9.77kg in body fat mass. These three CNVs are close to two known PWS genes, NDN (necdin homolog) and C15orf2 (chromosome 15 open reading frame 2), and partially overlap with another obesity gene PWRN1 (Prader-Willi region nonprotein-coding RNA 1). Interestingly, our recently published whole genome association scan study using the same sample by examining single-nucleotide polymorphisms (SNPs) did not find any significant associations at these CNV regions, suggesting the importance of examining both CNVs and SNPs for better understanding of genetic basis of obesity. Further studies are warranted to validate these CNVs and their importance to obesity.

New Perspectives On Gene-Environment Interactions In Schizophrenia

We review the salient recent findings on gene-environment interaction in schizophrenia. The issue of cognitive impairment and metabolic risks in modulating pharmacological treatment responses remain a challenge. The intriguing nature-nurture diathesis is discussed in light of recent breakthroughs in genetic studies from genome association scan methodology and epigenomics. Stem-cell driven neurogenesis offers fresh paradigm to unravel nature-narture interaction. We highlight the relevance of genetics towards resolving Krapaelian dichotomy of classification in schizoaffective disorder at the cross-roads of psychosis and affective disorders. We envisage that future advances in Pharmaco-genomics, Bioinformatics will catalyse the applicaton of personalized medicine in practice of clinical psychiatry.

High-Throughput Characterization of 10 New Minor Histocompatibility Antigens by Whole Genome Association Scanning

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

Utility of the pooling approach as applied to whole genome association scans with high-density Affymetrix microarrays

BackgroundWe report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism) microarrays and DNA pooling (SNP-MaP) employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations). The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes.FindingsIn the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods.ConclusionsDespite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.

High Throughput Minor Histocompatibility Antigen Discovery by Whole Genome Association Scanning.

Abstract 685Allogeneic stem cell transplantation (allo-SCT) followed by donor lymphocyte infusion (DLI) is used as a curative treatment for patients with malignant diseases. Donor derived T cells mediate graft versus tumor responses by targeting minor histocompatibility antigens (mHags) that are encoded by patient specific single nucleotide polymorphisms (SNPs). Various approaches have been applied for mHag discovery resulting in the characterization of more than 20 mHags. However, restriction to specific HLA types and unfavourable gene expression strongly limits the number of clinically relevant mHags. Recently, it has been demonstrated that whole genome association scanning (WGAs) can be a tool for mHag identification. Here, we present WGAs as a powerful method for high throughput identification of new mHags. From 2 patients that entered complete remission with limited graft versus host disease after allo-SCT and DLI, activated T cells were cloned by flowcytometric cell sorting. After expansion, 232 stably growing T cell clones were obtained. Patient specific recognition in the absence of donor recognition was demonstrated for 78 clones. By using blocking antibodies and a test panel consisting of partially HLA matched EBV-transformed B cell lines (EBV-LCL), we demonstrated that these 78 T cell clones comprised 20 unique mHag reactivities which could be identified to be restricted to HLA-A*02 or B*07. Since WGAs is based on a balanced segregation of test cells in a positive and a negative group, 15 T cell clones were selected recognizing mHags with population frequencies between 20% to 80% for further analysis. To perform WGAs, a test panel was generated containing 80 HLA-A*02 and B*07 positive EBV-LCL for testing of recognition by all selected T cell clones using Interferon-γ Elisa. In parallel, all EBV-LCL were genotyped for 1 million SNPs using bead arrays. All SNP genotype patterns were combined with each individual T cell recognition pattern. The level of matching between both patterns was statistically analyzed using Fisher's exact test, resulting in p-values indicating the significance of association. Significant association (p-value<10-12) between SNP genotypes and a T cell recognition pattern identified a single genomic region for 12 out of 15 T cell clones. In 2 cases no clear discrimination between positive and negative EBV-LCL could be made, suggesting that these T cell clones may not recognize mHags. Incomplete coverage of a genomic region by SNPs on the bead array may explain the lack of association for 1 T cell clone. For 7 T cell clones, significant association was found with array SNPs located in exons of the genes WNK1, SSR1, PRCP, ARHGDIB, PDCD11, EBI3 and APOBEC3B. For 3 other T cell clones, the genes ERAP1, BCAT2 and GEMIN4 were identified based on significant association with SNPs located in non coding regions. Sequence analysis of the coding regions of these genes from patient and donor revealed additional patient specific SNPs that were not included in the bead array. For the remaining associating TTK and ERGIC1 genes, the coding regions were identical between patient and donor, showing that these mHags are not encoded by exon SNPs in the identified TTK and ERGIC1 genes. Differential mHag expression may be induced in these cases by SNPs in adjacent genes that were not identified by SNPs on the bead array or may be the result of SNPs in non coding regulatory regions or in mRNA splice variants. According to the BioGPS gene expression database, a number of genes as identified by WGAs were predominantly expressed in hematopoietic cells, and may therefore encode relevant targets for T cell therapy. Next, we investigated the amino acid polymorphisms encoded by all identified coding SNPs. Peptides spanning the patient type amino acid polymorphism were submitted to HLA binding prediction algorithms. Candidate peptides were synthesized and T cell recognition was demonstrated at concentrations varying from 0.5 to 5000 nM. Recognition of donor type peptides was absent in all cases, validating the identification of 10 novel mHags. In conclusion, these data demonstrate that activation marker based T cell selection and cloning combined with WGAs results in high throughput discovery of multiple mHags. This strategy therefore allows broad characterization of mHags in donor derived T cell responses and selection of clinically relevant mHags for development of T cell based immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Genome‐wide SNP genotyping study using pooled DNA to identify candidate markers mediating susceptibility to end‐stage renal disease attributed to Type 1 diabetes

Genetic factors play a major role in the progression of kidney disease in diabetes. To identify candidate single nucleotide polymorphisms (SNPs) with potential effects on susceptibility to end-stage renal disease (ESRD), we performed a whole genome association scan using pooled DNA from Caucasian individuals with Type 1 diabetes. We utilized the Illumina Infinium II HumanHap 550 beadchip platform to genotype 555 352 SNPs in DNA pools comprised of 547 cases with ESRD and 549 control subjects with Type 1 diabetes duration > 20 years and no ESRD. Pooled probe intensity was used to predict mean allele frequency (MAF) for each locus. Individual genotyping was performed using the iPLEX assay in conjunction with the MassARRAY platform (Sequenom). We identified 2870 markers showing substantial differences in MAF (5.0-10.7%) between pools. To initiate validation of these findings, we genotyped 22 high-ranking markers in 462 individuals with ESRD and 470 unaffected control subjects selected from the genome-wide SNP genotyping study sample. We observed the strongest evidence for association between ESRD and rs1749824, located in the ZMIZ1 gene [OR = 1.47 (1.21-1.78) per copy of T allele; P = 8.1 x 10(-5)] and rs9298190, located in the musculin gene [OR = 1.56 (1.28-1.91) per copy of C allele; P = 1.6 x 10(-5)]. Evidence for nominal association with markers in or near the IRS2, TMPO, BID, KLRA1, ELMO1 and CNDP1 genes was also observed (P < or = 0.0006). These findings identify several novel loci which may contribute to ESRD susceptibility in individuals with Type 1 diabetes.

HapMap scanning of novel human minor histocompatibility antigens

AbstractMinor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Patterns of genetic diversity and linkage disequilibrium in a highly structured Hordeum vulgare association-mapping population for the Mediterranean basin

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT((R)) markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r (2) of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT((R)) markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.