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Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3and KMT2Awere detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.

CD4+T cells play a central role in adaptive immunity. The acknowledgment of their cytolytic effector function and the finding that endogenous antigens can enter the HLA class II processing pathway make CD4+T cells promising tools for immunotherapy. Expression of HLA class II and endogenous antigen, however, does not always correlate with T-cell recognition. We therefore investigated processing and presentation of endogenous HLA class II epitopes that induced CD4+T cells during in vivo immune responses. We demonstrate that the peptide editor HLA-DM allowed antigen presentation of some (DM-resistant antigens) but abolished surface expression of other natural HLA class II epitopes (DM-sensitive antigens). DM sensitivity was shown to be epitope specific, mediated via interaction between HLA-DM and the HLA-DR restriction molecule, and reversible by HLA-DO. Because of the restricted expression of HLA-DO, presentation of DM-sensitive antigens was limited to professional antigen-presenting cells, whereas DM-resistant epitopes were expressed on all HLA class II–expressing cells. In conclusion, our data provide novel insights into the presentation of endogenous HLA class II epitopes and identify intracellular antigen processing and presentation as a critical factor for CD4+T-cell recognition. This opens perspectives to exploit selective processing capacities as a new approach for targeted immunotherapy.

CD4+ T cells play a central role in adaptive immunity. The acknowledgment of their cytolytic effector function and the finding that endogenous antigens can enter the HLA class II processing pathway make CD4+ T cells promising tools for immunotherapy. Expression of HLA class II and endogenous antigen, however, does not always correlate with T-cell recognition. We therefore investigated processing and presentation of endogenous HLA class II epitopes that induced CD4+ T cells during in vivo immune responses. We demonstrate that the peptide editor HLA-DM allowed antigen presentation of some (DM-resistant antigens) but abolished surface expression of other natural HLA class II epitopes (DM-sensitive antigens). DM sensitivity was shown to be epitope specific, mediated via interaction between HLA-DM and the HLA-DR restriction molecule, and reversible by HLA-DO. Because of the restricted expression of HLA-DO, presentation of DM-sensitive antigens was limited to professional antigen-presenting cells, whereas DM-resistant epitopes were expressed on all HLA class II–expressing cells. In conclusion, our data provide novel insights into the presentation of endogenous HLA class II epitopes and identify intracellular antigen processing and presentation as a critical factor for CD4+ T-cell recognition. This opens perspectives to exploit selective processing capacities as a new approach for targeted immunotherapy.

Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8+T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7–restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1)gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1–specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8+ T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7–restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1–specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Introduction

Borras: GenomeScan B.V.: Employment. Janssen:GenomeScan B.V.: Employment.

Janssen: GenomeScan BV: Employment.

Background

No relevant conflicts of interest to declare.

Abstract 4097

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Abstract 1273

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No relevant conflicts of interest to declare.

Abstract 685

Abstract 4084

In unrelated donor hematopoietic stem cell transplantation (URD-SCT) patients are preferably transplanted with stem cells from a fully HLA matched donor, usually defined as identical for HLA-class I, -DR and -DQ. Since HLA-DPB1 is often not taken into consideration in donor selection, 80–90% of URD-SCTs are mismatched for HLA-DPB1. The role of HLA-DPB1 as transplantation antigen has been unclear, since clinical reports on the impact of matching for HLA-DPB1 on transplant outcome showed conflicting results. HLA-DPB1 mismatching has been associated with an increased risk of graft versus host disease (GVHD). However, we recently demonstrated that HLA-DPB1 specific T cells can mediate a potent graft versus leukemia effect without inducing GVHD. It has been suggested that the controversial effects of matching for HLA-DPB1 in URD-SCT could partly be explained by the assumption that not all HLA-DPB1 differences are immunogenic. This theory was based on the cross-reactive recognition of two HLA-DPB1* 09 specific T cell clones that recognized other HLA-DPB1 alleles sharing amino acids (aa) in position 8–11 of HLA-DPB1 (Zino et al, blood 2004). It was hypothesized that there would be no induction of T cell responses between individuals expressing HLA-DPB1 molecules sharing this aa sequence. This was translated into a classification of permissive and non-permissive HLA-DPB1 mismatches in order to allow a broader donor selection. To investigate whether cross-reactive recognition of other HLA-DPB1 molecules by our previously generated HLA-DPB1*02 or *03 specific CD4+ T cell clones depended on the presence of specific aa sequences we tested recognition of a panel of 14 EBV-LCL expressing 9 different HLA-DPB1 molecules. All HLA-DPB1*02 as well as all *03 specific T cell clones showed cross-reactivity with other HLA-DPB1 alleles and each T cell clone exhibited its own pattern of cross-reactivity. Two HLA-DPB1*0201 specific T cell clones with different TCR-Vβ showed also recognition of EBV-LCL expressing HLA-DPB1*1001 and *1701 or HLA-DPB1*1001, *0901 and *1601 respectively. Five HLA-DPB1*03 reactive T cells clones with different TCR-Vβ showed differential cross-recognition of EBV-LCL expressing HLA-DPB1*0101, *0601, *1101, *1301 and *1401. To identify immunogenic differences the aa sequences of the HLA-DPB1 molecules recognized by the various T cell clones were compared. The HLA-DPB1 molecules recognized by the HLA-DPB1*02 specific T cell clones shared an aa substitution at position 69 compared to the responder cell. However, HLA-DPB1*0601,*0901 and *1901 with the same substitution were not recognized by both T cell clones. This phenomenon was also observed for the HLA-DPB1*03 specific T cell clones, indicating that the cross-reactive recognition of HLA-DPB1 could not be predicted by aa sequences. Next, we analyzed the immunogenicity of various HLA-DPB1 alleles in different stimulator/responder combinations to verify the classification of permissive and non-permissive mismatches. We developed a model to generate allo-HLA-DP responses by transducing HLA-class II negative HELA cells with various HLA-DP molecules and used these cells to stimulate purified CD4+ T cells from HLA-DPB1 homozygous donors. HELA cells transduced with HLA-DPB1*0101, *0201, *0301, *0401, *0402, *0501, *0601, *0901, *1101, *1301, *1401 or *1701 were used as stimulator cells. Responder CD4+ T cells were typed HLA-DPB1* 0201, *0301, *0401 or *0402. 14 days after stimulation, CD4+ T cells were tested for recognition of the stimulator cells and of HELA cells transduced with the responder HLA-DPB1 molecule as a negative control. For these 4 responders, stimulation with 12 different HLA-DP transduced HELA cell lines resulted in specific IFN-γ production in response to the stimulator cells in 47 out of 48 stimulations. 28 CD4+ T cell lines also showed cross-reactive recognition of HELA cells transduced with at least one other HLA-DPB1 molecule. In conclusion, we showed that cross-reactive recognition of various HLA-DPB1 molecules by HLA-DPB1 specific T cells is a common observation. However, we demonstrated that cross-reactivity between HLA-DPB1 molecules by allo-HLA-DPB1 specific T cells does not exclude the generation of immune response between individuals expressing these HLA-DPB1 molecules. By generating multiple allo-HLA-DP specific T cell lines, we showed that all HLA-DPB1 mismatch combinations are immunogenic.

Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T cells recognizing minor histocompatibility antigens (mHags) on malignant cells of the recipient. These donor originated T cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues of the patient. In this study, we performed a detailed analysis and characterization of mHags recognized by CD8+ T cells contributing to Graft-versus-Leukemia (GvL) reactivity in a patient treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. The GvL effect in this patient was accompanied with only mild GvHD of the skin.

Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T cells recognizing minor histocompatibility antigens (mHags) on malignant cells of the recipient. These donor originated T cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues of the patient. In this study, we performed a detailed analysis and characterization of mHags recognized by CD8+ T cells contributing to Graft-versus-Leukemia (GvL) reactivity in a patient treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. The GvL effect in this patient was accompanied with only mild GvHD of the skin. To investigate the specificity of the CD8+ T cell response induced in this patient, activated (HLA-DR+) CD8+ T cells were single cell sorted from a bone marrow sample obtained five weeks after DLI by flowcytometry. A number of isolated CD8+ T cell clones were shown to be specific for mHags, as determined by differential recognition of patient and donor EBV-transformed B cells (EBV-LCL) in IFN-g ELISA and 51Crrelease assays. By screening a panel of third party EBV-LCL sharing one or more HLA class I restriction molecules with the patient, CD8+ T cell clones directed against 7 different mHags were identified, including the known hematopoiesis restricted mHags HA-1 and HA-2, which are presented in HLA-A*0201. Of the 5 remaining specificities, 4 mHags were presented in HLA-B*4001 (B60) and one mHag in HLA-B*0801. To determine the tissue distribution patterns in more detail, we tested recognition of selected non-malignant hematopoietic cells (monocytes, B cells, T cells), malignant CD34+ CML precursor cells, and skin-derived fibroblasts. One HLA-B*4001-restricted T cell clone (clone ZRZ16) failed to recognize all primary hematopoietic cells and skin fibroblasts. The four remaining T cell clones were all capable of recognizing and lysing (specific subsets of) non-malignant hematopoietic cells and malignant CD34+ CML precursor cells. Fibroblast recognition could be demonstrated for two of these four T cell clones. Since clone ZRZ16 failed to recognize all hematopoietic and non-hematopoietic cells, except for EBV-LCL, we tested whether this clone selectively recognizes antigen presenting cells (APC), which are known to be required for efficient induction of immune responses in vivo. Clone ZRZ16 showed indeed strong recognition of monocyte-derived dendritic cells as well as in vitro differentiated CD34+ CML cells with APC phenotype. To identify the mHag recognized by the CD8+ T cell clone, we screened a cDNA expression library constructed from EBV-LCL from the patient. One single cDNA was isolated as the target for B*4001 restricted CD8+ T cell clone ZRZ16. The epitope recognized by this clone was derived from the 3’ untranslated region (UTR) of a cDNA encoding thyroid hormone receptor interactor 10 (TRIP10). The peptide epitope was translated in a reading frame different from the TRIP10 protein and comprises three single nucleotide polymorphisms, which were all different between patient and donor. Two of the three SNPs were shown to be important for recognition by clone ZRZ16. Despite ubiquitous tissue expression of the TRIP10 gene as determined by public microarray analysis, CD8+ T cells specific for the newly-identified LB-TRIP10-1EPC mHag selectively recognized APC and failed to recognize CD34+ CML precursor cells, suggesting a predominant role in the initiation, but not effector, phase of the anti-tumor response. In conclusion, our data show a detailed analysis of mHag specific CD8+ T cell immunity induced in a patient successfully treated with DLI for relapsed CML and provide evidence for differential involvement of HLA class I restricted mHags in the onset and execution of GvL reactivity.

In unrelated donor hematopoietic stem cell transplantation (URD-SCT) patients are preferably transplanted with stem cells from a fully HLA matched donor, usually defined as identical for HLA-class I, -DR and -DQ. Since HLA-DPB1 is often not taken into consideration in donor selection, 80–90% of URD-SCTs are mismatched for HLA-DPB1. The role of HLA-DPB1 as transplantation antigen has been unclear, since clinical reports on the impact of matching for HLA-DPB1 on transplant outcome showed conflicting results. HLA-DPB1 mismatching has been associated with an increased risk of graft versus host disease (GVHD). However, we recently demonstrated that HLA-DPB1 specific T cells can mediate a potent graft versus leukemia effect without inducing GVHD. It has been suggested that the controversial effects of matching for HLA-DPB1 in URD-SCT could partly be explained by the assumption that not all HLA-DPB1 differences are immunogenic. This theory was based on the cross-reactive recognition of two HLA-DPB1* 09 specific T cell clones that recognized other HLA-DPB1 alleles sharing amino acids (aa) in position 8–11 of HLA-DPB1 (Zino et al, blood 2004). It was hypothesized that there would be no induction of T cell responses between individuals expressing HLA-DPB1 molecules sharing this aa sequence. This was translated into a classification of permissive and non-permissive HLA-DPB1 mismatches in order to allow a broader donor selection. To investigate whether cross-reactive recognition of other HLA-DPB1 molecules by our previously generated HLA-DPB1*02 or *03 specific CD4+ T cell clones depended on the presence of specific aa sequences we tested recognition of a panel of 14 EBV-LCL expressing 9 different HLA-DPB1 molecules. All HLA-DPB1*02 as well as all *03 specific T cell clones showed cross-reactivity with other HLA-DPB1 alleles and each T cell clone exhibited its own pattern of cross-reactivity. Two HLA-DPB1*0201 specific T cell clones with different TCR-Vβ showed also recognition of EBV-LCL expressing HLA-DPB1*1001 and *1701 or HLA-DPB1*1001, *0901 and *1601 respectively. Five HLA-DPB1*03 reactive T cells clones with different TCR-Vβ showed differential cross-recognition of EBV-LCL expressing HLA-DPB1*0101, *0601, *1101, *1301 and *1401. To identify immunogenic differences the aa sequences of the HLA-DPB1 molecules recognized by the various T cell clones were compared. The HLA-DPB1 molecules recognized by the HLA-DPB1*02 specific T cell clones shared an aa substitution at position 69 compared to the responder cell. However, HLA-DPB1*0601,*0901 and *1901 with the same substitution were not recognized by both T cell clones. This phenomenon was also observed for the HLA-DPB1*03 specific T cell clones, indicating that the cross-reactive recognition of HLA-DPB1 could not be predicted by aa sequences. Next, we analyzed the immunogenicity of various HLA-DPB1 alleles in different stimulator/responder combinations to verify the classification of permissive and non-permissive mismatches. We developed a model to generate allo-HLA-DP responses by transducing HLA-class II negative HELA cells with various HLA-DP molecules and used these cells to stimulate purified CD4+ T cells from HLA-DPB1 homozygous donors. HELA cells transduced with HLA-DPB1*0101, *0201, *0301, *0401, *0402, *0501, *0601, *0901, *1101, *1301, *1401 or *1701 were used as stimulator cells. Responder CD4+ T cells were typed HLA-DPB1* 0201, *0301, *0401 or *0402. 14 days after stimulation, CD4+ T cells were tested for recognition of the stimulator cells and of HELA cells transduced with the responder HLA-DPB1 molecule as a negative control. For these 4 responders, stimulation with 12 different HLA-DP transduced HELA cell lines resulted in specific IFN-γ production in response to the stimulator cells in 47 out of 48 stimulations. 28 CD4+ T cell lines also showed cross-reactive recognition of HELA cells transduced with at least one other HLA-DPB1 molecule. In conclusion, we showed that cross-reactive recognition of various HLA-DPB1 molecules by HLA-DPB1 specific T cells is a common observation. However, we demonstrated that cross-reactivity between HLA-DPB1 molecules by allo-HLA-DPB1 specific T cells does not exclude the generation of immune response between individuals expressing these HLA-DPB1 molecules. By generating multiple allo-HLA-DP specific T cell lines, we showed that all HLA-DPB1 mismatch combinations are immunogenic.

Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+and CD4+T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLI). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, mHag specific CD4+ T cells may selectively mediate Graft-versus-Leukemia (GvL) reactivity without Graft-versus-Host Disease (GvHD). Clinical studies have shown that adoptive transfer of CD4+ donor lymphocytes after HLA-matched alloSCT may lead to clinical remissions with a reduced incidence of GvHD, emphasizing the relevance of CD4+ T cells and HLA class II associated mHags for development of effective anti-tumor T cell therapies after alloSCT with a low risk for GvHD. The aim of this study was to identify the HLA class II associated mHag that is recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLA-matched alloSCT who developed strong GvL reactivity with mild GvHD of the skin after treatment with DLI. We previously developed recombinant bacteria cDNA expression libraries based on delivery of exogenous antigens for identification of HLA class II antigens and used this method for identification of the first autosomal HLA class II (HLA-DQB1*0603) associated mHag LB-PI4K2B-1S. LB-PI4K2B-1S has a population frequency of 40–50% and is encoded by the broadly-expressed phosphatidylinositol 4-kinase type II β gene. In the patient with CML, a polyclonal CD4+ T cell response against LB-PI4K2B-1S and simultaneous mHag specific CD8+ T cells were demonstrated. LB-PI4K2B-1S specific CD4+ T cells were shown to recognize the CD34+ CML cells of the patient as well as other leukemic cells. Recognition and lysis of normal hematopoietic cells by LB-PI4K2B-1S specific CD4+ T cells critically depended on the number of HLA-DQ molecules expressed at the cell surface and was restricted to high HLA-DQ-expressing B cells, mature dendritic cells (DC) and EBV-transformed B cells. HLA-DQ expression on T cells, PHA-stimulated blasts, monocytes and immature DC was absent or low and not sufficient for T cell recognition. We also demonstrated that HLA-DQ expression on normal cells of non-hematopoietic origin after extensive culturing with IFN-γ was moderately upregulated as compared to HLA-DR and -DP and not sufficient for recognition by LB-PI4K2B-1S specific CD4+ T cells. In conclusion, the data suggest that LB-PI4K2B-1S specific CD4+ T cells mediated tumor rejection by directly eliminating the malignant cells of the patient as effector cells and stimulating the induction and maintenance of CD8+ T cell immunity as helper cells, and HLA-DQ associated mHags may be appropriate targets for T cell therapies with the aim to selectively stimulate GvL after HLA-matched alloSCT with a low risk for GvHD.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLI). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, mHag specific CD4+T cells may selectively mediate Graft-versus-Leukemia (GvL) reactivity without Graft-versus-Host Disease (GvHD). Clinical studies have shown that adoptive transfer of CD4+donor lymphocytes after HLA-matched alloSCT may lead to clinical remissions with a reduced incidence of GvHD, emphasizing the relevance of CD4+T cells and HLA class II associated mHags for development of effective anti-tumor T cell therapies after alloSCT with a low risk for GvHD. The aim of this study was to identify the HLA class II associated mHag that is recognized by CD4+T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLA-matched alloSCT who developed strong GvL reactivity with mild GvHD of the skin after treatment with DLI. We previously developed recombinant bacteria cDNA expression libraries based on delivery of exogenous antigens for identification of HLA class II antigens and used this method for identification of the first autosomal HLA class II (HLA-DQB1*0603) associated mHag LB-PI4K2B-1S. LB-PI4K2B-1S has a population frequency of 40–50% and is encoded by the broadly-expressed phosphatidylinositol 4-kinase type II β gene. In the patient with CML, a polyclonal CD4+T cell response against LB-PI4K2B-1S and simultaneous mHag specific CD8+T cells were demonstrated. LB-PI4K2B-1S specific CD4+T cells were shown to recognize the CD34+CML cells of the patient as well as other leukemic cells. Recognition and lysis of normal hematopoietic cells by LB-PI4K2B-1S specific CD4+T cells critically depended on the number of HLA-DQ molecules expressed at the cell surface and was restricted to high HLA-DQ-expressing B cells, mature dendritic cells (DC) and EBV-transformed B cells. HLA-DQ expression on T cells, PHA-stimulated blasts, monocytes and immature DC was absent or low and not sufficient for T cell recognition. We also demonstrated that HLA-DQ expression on normal cells of non-hematopoietic origin after extensive culturing with IFN-γ was moderately upregulated as compared to HLA-DR and -DP and not sufficient for recognition by LB-PI4K2B-1S specific CD4+T cells. In conclusion, the data suggest that

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated cytotoxic CD8+ T cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma (MM) after allogeneic HLA-matched SCT who suffered only from mild graft-versus-host disease (GVHD). The CTL clones were isolated by direct cloning of T cells producing IFN-? upon stimulation with irradiated bone marrow cells harvested from the patient before SCT. The tumor-reactivity of the CTL clones was demonstrated by the recognition of MM cells in the bone marrow using the CFSE-based cytotoxicity assay. In addition to tumor cells, the CTL clones also lysed the patient-derived EBV-transformed lymphoblastoid cell line (EBV-LCL) and PHA-blasts, whereas donor-derived EBV-LCL cells and PHA blasts were not recognized demonstrating that these CTL clones were directed against a minor histocompatibility antigen (mHag). Using cDNA expression cloning, the target molecule of a HLA-B7-restricted CTL clone was identified. The CTL clone recognized a mHag produced by a non-synonymous single nucleotide polymorphism in the angiogenic endothelial cell growth factor-1 (ECGF-1) gene also known as thymidine phosphorylase. Analysis of the expression of the ECGF-1 gene in a micro array study demonstrated high levels of expression in hematopoietic cells, in particular CD14+ monocytes and DC, but ECGF-1 expression could also be detected in lung, liver and heart. We confirmed the expression pattern of ECGF-1 using quantitative real-time RT-PCR. CD14+ cells showed the highest levels of expression, although expression in other hematopoietic cells, like CD4+, CD8+ and CD19+ cells could also be detected. In agreement with the microarray study, expression of ECGF-1 in lung, liver and heart was found. By immunohistochemical analysis, it has been demonstrated that the expression of ECGF-1 in these tissues was mainly due to the presence of macrophages, although weak expression in stroma cells could also be detected. Although the patient from whom the CTL clone was isolated had more than 1% circulating ECGF-1-specific CD8+ T cells as determined with tetramer staining, she only suffered from mild GVHD indicating that the low level of ECGF-1 expression in some normal tissues has no major detrimental side effects. ECGF-1 is not only expressed in hematological tumors like multiple myeloma, CML and AML, but is also expressed in various other tumors, like melanoma, breast carcinoma and renal cell carcinoma. In these solid tumors, ECGF-1 can be expressed by the tumor cells themselves or by tumor-infiltrating monocytes. ECGF-1 expression is positively correlated with microvessel density and seems to be an unfavorable prognostic factor. The ECGF-1-specific CTL clone recognized mHag-positive, HLA-B7-expressing CML as well as melanoma cells, demonstrating that the ECGF-1-specific T cells are not only reactive against hematological malignancies but also against solid tumors. Therefore, ECGF-1 is an interesting target for immunotherapy of both hematological and solid tumors.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated cytotoxic CD8+T cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma (MM) after allogeneic HLA-matched SCT who suffered only from mild graft-versus-host disease (GVHD). The CTL clones were isolated by direct cloning of T cells producing IFN-γ upon stimulation with irradiated bone marrow cells harvested from the patient before SCT. The tumor-reactivity of the CTL clones was demonstrated by the recognition of MM cells in the bone marrow using the CFSE-based cytotoxicity assay. In addition to tumor cells, the CTL clones also lysed the patient-derived EBV-transformed lymphoblastoid cell line (EBV-LCL) and PHA-blasts, whereas donor-derived EBV-LCL cells and PHA blasts were not recognized demonstrating that these CTL clones were directed against a minor histocompatibility antigen (mHag). Using cDNA expression cloning, the target molecule of a HLA-B7-restricted CTL clone was identified. The CTL clone recognized a mHag produced by a non-synonymous single nucleotide polymorphism in the angiogenic endothelial cell growth factor-1 (ECGF-1) gene also known as thymidine phosphorylase. Analysis of the expression of the ECGF-1 gene in a micro array study demonstrated high levels of expression in hematopoietic cells, in particular CD14+monocytes and DC, but ECGF-1 expression could also be detected in lung, liver and heart. We confirmed the expression pattern of ECGF-1 using quantitative real-time RT-PCR. CD14+cells showed the highest levels of expression, although expression in other hematopoietic cells, like CD4+, CD8+and CD19+cells could also be detected. In agreement with the microarray study, expression of ECGF-1 in lung, liver and heart was found. By immunohistochemical analysis, it has been demonstrated that the expression of ECGF-1 in these tissues was mainly due to the presence of macrophages, although weak expression in stroma cells could also be detected. Although the patient from whom the CTL clone was isolated had more than 1% circulating ECGF-1-specific CD8+T cells as determined with tetramer staining, she only suffered from mild GVHD indicating that the low level of ECGF-1 expression in some normal tissues has no major detrimental side effects. ECGF-1 is not only expressed in hematological tumors like multiple myeloma, CML and AML, but is also expressed in various other tumors, like melanoma, breast carcinoma and renal cell carcinoma. In these solid tumors, ECGF-1 can be expressed by the tumor cells themselves or by tumor-infiltrating monocytes. ECGF-1 expression is positively correlated with microvessel density and seems to be an unfavorable prognostic factor. The ECGF-1-specific CTL clone recognized mHag-positive, HLA-B7-expressing CML as well as melanoma cells, demonstrating that the ECGF-1-specific T cells are not only reactive against hematological malignancies but also against solid tumors. Therefore, ECGF-1 is an interesting target for immunotherapy of both hematological and solid tumors.