Edith D. van der Meijden

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.

Identification of 4 new HLA-DR-restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity

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.

Identification of phosphatidylinositol 4-kinase type II β as HLA class II-restricted target in graft versus leukemia reactivity

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because 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). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II β gene. A polyclonal CD4+ T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4+ T cells recognized and lysed the CD34+ CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-γ and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4+ T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8+ T cell immunity as helper cells.

Both permissive and nonpermissive HLA-DPB1 mismatches can induce polyclonal HLA-DPB1 specific immune responses in vivo and in vitro.

To the editor: Clinical studies have indicated that human leukocyte antigen (HLA)–DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation (SCT). Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD) but also a

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

Background 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. Design and Methods 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. Results 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. Conclusions 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.

HLA-DPB1 mismatching results in the generation of a full repertoire of HLA-DPB1-specific CD4+ T cell responses showing immunogenicity of all HLA-DPB1 alleles.

Clinical studies have indicated that HLA-DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD), but also a decreased risk of disease relapse. However, specific HLA-DPB1 mismatches were associated with poor clinical outcome. It was suggested that this unfavorable effect was caused by a difference in immunogenicity between HLA-DPB1 alleles. To analyze whether immunogenicity of HLA-DPB1 mismatches could be predicted based on the presence or absence of specific amino acid sequences we developed a model to generate allo-HLA-DPB1 responses in vitro. We tested in total 48 different stimulator/responder combinations by stimulating CD4(+) T cells from 5 HLA-DPB1 homozygous individuals with the same antigen-presenting cells transduced with different allo-HLA-DPB1 molecules. HLA-DPB1 molecules used for stimulation comprised 76% to 99% of HLA-DPB1 molecules present in different ethnic populations. We show that all HLA-DPB1 mismatches as defined by allele typing resulted in high-frequency immune responses. Furthermore, we show that crossrecognition of different HLA-DPB1 molecules is a broadly observed phenomenon. We confirm previously described patterns in crossrecognition, and demonstrate that a high degree in similarity between HLA-DPB1 molecules is predictive for crossrecognition, but not for immunogenicity.

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

Purpose: 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. Experimental Design: 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. Results: 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. Conclusions: 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.

Human Leukocyte Antigen–DO Regulates Surface Presentation of Human Leukocyte Antigen Class II–Restricted Antigens on B Cell Malignancies

Hematological malignancies often express surface HLA class II, making them attractive targets for CD4+ T cell therapy. We previously demonstrated that HLA class II ligands can be divided into DM-resistant and DM-sensitive antigens. In contrast to presentation of DM-resistant antigens, presentation of DM-sensitive antigens is suppressed by HLA-DM but can be rescued by HLA-DO. We also showed that HLA-DO expression remains low in nonhematopoietic cells under inflammatory conditions, suggesting that DM-sensitive antigens may be ideal T cell targets with a low risk for graft-versus-host disease. Here, we demonstrated that B cell malignancies often express HLA-DO and that levels are in particular high in chronic lymphocytic leukemia. Moreover, we showed that surface presentation of DM-sensitive antigens is regulated by HLA-DO, and that DM-sensitive antigens are relevant T cell targets for B cell malignancies and, especially, chronic lymphocytic leukemia. These data open the perspective to target HLA class II ligands with specific processing and presentation behavior for CD4+ T cell therapy of hematological malignancies.

Natural T-cell ligands that are created by genetic variants can be transferred between cells by extracellular vesicles

CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen‐presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II‐negative infected or transformed cells need to be transferred to surrounding antigen‐presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T‐cell epitope, but rather on characteristics of the full‐length protein. This mechanism of (neo)antigen transfer from HLA class II‐negative cells to surrounding antigen‐presenting cells may play a crucial role in induction of anti‐tumor immunity.

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.