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.

No abstract

No abstract

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide riskadapted 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 EVI1 irrespective 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 FLT3 and KMT2A were 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. Supplementary informationThe online version of this article (https://

No abstract

The online version of this article has a Supplementary Appendix. BackgroundPatients 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 graftversus-host disease. Design and MethodsWe 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. ResultsThree 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. ConclusionsOur 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.Key words: HLA-matched transplantation, novel minor histocompatibility antigens, CD8 positive, graft-versus-leukemia effect, T-cell reactivity.Citation: Griffioen M, Honders MW, van der Meijden ED, van Luxemburg-Heijs SAP, Lurvink EGA, Kester MGD, van Bergen CAM, and Falkenburg JHF. Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity. Haematologica 2012;97(8):1196-1204. doi:10.3324/haematol.2011

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.

IntroductionCD4 ϩ T cells are important in antitumor immunity as helper cells for the induction and maintenance of cytotoxic CD8 ϩ T cells. [1][2][3] Moreover, evidence is emerging that CD4 ϩ T cells alone can provide efficient antitumor reactivity. [4][5][6][7] In a NOD/SCID mouse model for human acute lymphoblastic leukemia, infusion of human CD4 ϩ T cells led to eradication of the malignant cells. 8 Clinical trials in allogeneic stem cell transplantation (alloSCT) found that depletion of CD8 ϩ T cells from donor lymphocyte infusion preserved the ability of the T cells to induce conversion to donor chimerism, 9,10 highlighting the direct effector function of CD4 ϩ T cells.Antigen presentation to T cells by HLA surface molecules depends on protein degradation mediated by proteasomes and lysosomal proteases as the 2 major cellular breakdown mechanisms. Traditionally, it has been assumed that HLA class II molecules present exogenous antigens which are degraded by proteases within the endosomal/lysosomal system, whereas endogenous cytosolic antigens are degraded by the proteasome and presented by HLA class I molecules. In the past decade, however, evidence emerged that HLA class I molecules can also present exogenous antigens via a process called cross-presentation, 11 and that intracellular antigens can enter the HLA class II pathway. 12,13 In antitumor immunity, professional APCs such as dendritic cells (DCs) take up and present antigens derived from tumor cells. The encounter of these exogenous antigens presented on DCs may be sufficient for the induction of CD4 ϩ T cells and their function as helper cells. However, to serve as specific targets for cytolytic CD4 ϩ T cells, tumor cells need to present endogenous peptides in HLA class II molecules as expressed at their cell surface. We and others, however, observed that some HLA class II-positive target cells are not recognized by the respective CD4 ϩ T-cell clones, 14,15 despite appropriate HLA class II and antigen expression. Already in 1996, Harris et al provided evidence for cell type-specific differences in presentation of endogenous antigens in HLA class II. 16 By eluting peptides from HLA-DR, professional APCs were shown to present an extended repertoire of self-peptides 17,18 compared with nonprofessional APCs. The investigators also found that significant amounts of class II-associated invariant chain peptide (CLIP), the remnant of the invariant chain (Ii), were presented at the cell surface of professional APCs, whereas CLIP was absent on nonprofessional APCs. 16 Surface expression of CLIP on professional APCs may be attributed to a molecule called HLA-DO. Although its precise biologic role is still unclear, HLA-DO has been shown to increase CLIP surface presentation by inhibition of HLA-DM in a pH-dependent manner, 19,20 and its expression has been reported to be confined to professional APCs, such as B cells, DCs, and thymic epithelial cells. 21 Here, we investigated whether endogenous antigens identified as natural targets for CD4 ϩ T cells are diffe...