Search

Supplementary Figure 2. The generation of the LB-TTK-1D peptide is proteasome-dependent. LB-TTK-1D antigen positive EBV-LCLs were treated with increasing concentrations of the proteasome inhibitor bortezomib for 48h, washed and exposed to LB-TTK-1D-specific T cells (clone 10-5). T cell-produced IFN-γ was determined in the supernatant of the cultures by ELISA. Depicted are OD450 values.

MHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been used to study their biological relevance. We here identify a naturally existing human T-cell epitope derived from a truncated protein. The antigenic peptide is derived from the gene TTK only through an alternative transcript containing a premature termination codon that may target the transcript for nonsense-mediated decay (NMD). This antigen is recognized by HLA-A*02:01–restricted CD8+ T cells derived from an allotransplanted leukemia patient. Functional analyses showed that these T cells failed to recognize several HLA-matched primary leukemic cells that expressed the alternative TTK transcript. Conventional antigen processing and presentation were not affected, suggesting that leukemic cells modify the generation of antigens processed from aberrant proteins. This natural TTK epitope provides insights in the source of transcripts producing antigenic epitopes in healthy and leukemic cells. Our data underscore potential pitfalls of targeting NMD-derived or other unconventionally generated epitopes as immunotherapeutic approach.

Supplementary Figure 1. The LB-TTK-1D-specific (clone 10-5) and USP11-specific T cell clones have a similar affinity for their cognate peptide. Synthetic peptides RLHDGRVFV (peptideTTK) or FTWEGLYNV (peptideUSP11) were loaded on T2 cells in a serial dilution. The depicted T cell clones were stimulated using these target cells followed by determination of IFN-γ secretion in the supernatant (ELISA). Calculated EC50 values are depicted.

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.

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.

The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin 1 (NPM1) gene. Mutated NPM1 (ΔNPM1) is an attractive target for immunotherapy, since it is an essential driver gene and 4 bp frameshift insertions occur in the same hotspot in 30%-35% of AMLs, resulting in a C-terminal alternative reading frame of 11 aa. By searching the HLA class I ligandome of primary AMLs, we identified multiple ΔNPM1-derived peptides. For one of these peptides, HLA-A*02:01-binding CLAVEEVSL, we searched for specific T cells in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and analyzed for reactivity against primary AMLs. From one clone with superior antitumor reactivity, we isolated the T cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive ΔNPM1 AML after retroviral transfer to CD8+ and CD4+ T cells. Antitumor efficacy of TCR-transduced T cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing ΔNPM1. In conclusion, the data show that ΔNPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. Immunotherapy targeting ΔNPM1 may therefore contribute to treatment of AML.

MHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been used to study their biological relevance. We here identify a naturally existing human T-cell epitope derived from a truncated protein. The antigenic peptide is derived from the gene TTK only through an alternative transcript containing a premature termination codon that may target the transcript for nonsense-mediated decay (NMD). This antigen is recognized by HLA-A*02:01–restricted CD8+ T cells derived from an allotransplanted leukemia patient. Functional analyses showed that these T cells failed to recognize several HLA-matched primary leukemic cells that expressed the alternative TTK transcript. Conventional antigen processing and presentation were not affected, suggesting that leukemic cells modify the generation of antigens processed from aberrant proteins. This natural TTK epitope provides insights in the source of transcripts producing antigenic epitopes in healthy and leukemic cells. Our data underscore potential pitfalls of targeting NMD-derived or other unconventionally generated epitopes as immunotherapeutic approach.

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.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

In HLA-matched allogeneic hematopoietic stem cell transplantation (SCT), donor T cells can mediate graft-versus-leukemia/lymphoma (GvL) reactivity and graft-versus-host disease (GvHD) by recognition of minor histocompatibility antigens (MiHA).1–4 Only a minority of MiHA shows hematopoiesis-restricted expression, and donor T cells for these MiHA may induce beneficial GvL reactivity without GvHD. The number of well-characterized MiHA with therapeutic relevance based on hematopoiesis-restricted expression remains limited and only 25% and 40% of recipients transplanted with grafts from sibling and unrelated donors, respectively, are eligible for therapies targeting known hematopoietic MiHA.3,4 Thus, in order to increase the efficacy and applicability of cellular therapy for selective GvL induction, more hematopoiesis-restricted MiHA with balanced population frequencies in common HLA molecules must be identified. Here, we investigated the therapeutic significance of a MiHA encoded by ARHGDIB.5 We demonstrated hematopoiesis-restricted gene expression with the exception of intermediate mRNA expression in endothelial cells and showed that T cells recognized LB-ARHGDIB-1R presented by HLA-B*07:02 on primary leukemic cells, but not on [interferon-gamma (IFN-γ)]-treated fibroblasts and keratinocytes. To evaluate potential toxicity against endothelial cells, we tested T cell recognition of LB-ARHGDIB-1R on human umbilical vein endothelial cells (HUVEC) and found only limited reactivity under inflammatory conditions. Furthermore, we demonstrated in vivo targeting of LB-ARHGDIB-1R in eight out of ten patients who were screened for post-transplant specific T-cell responses. In one patient with relapsed lymphoma, high T-cell frequencies were induced after donor lymphocyte infusion (DLI), coinciding with long-lasting anti-lymphoma immunity without GvHD. Our data thus support the relevance of LB-ARHGDIB-1R as a therapeutic target with the potential to induce selective GvL reactivity.

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.

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 Disclosures: No relevant conflicts of interest to declare.

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.