Background Therapeutic regimens designed to augment the immunological response of a patient with breast cancer (BC) to tumor tissue are critically informed by tumor mutational burden and the... Show moreBackground Therapeutic regimens designed to augment the immunological response of a patient with breast cancer (BC) to tumor tissue are critically informed by tumor mutational burden and the antigenicity of expressed neoepitopes. Herein we describe a neoepitope and cognate neoepitope-reactive T-cell identification and validation program that supports the development of next-generation immunotherapies. Methods Using GPS Cancer, NantOmics research, and The Cancer Genome Atlas databases, we developed a novel bioinformatic-based approach which assesses mutational load, neoepitope expression, human leukocyte antigen (HLA)-binding prediction, and in vitro confirmation of T-cell recognition to preferentially identify targetable neoepitopes. This program was validated by application to a BC cell line and confirmed using tumor biopsies from two patients with BC enrolled in the Tumor-Infiltrating Lymphocytes and Genomics (TILGen) study. Results The antigenicity and HLA-A2 restriction of the BC cell line predicted neoepitopes were determined by reactivity of T cells from HLA-A2-expressing healthy donors. For the TILGen subjects, tumor-infiltrating lymphocytes (TILs) recognized the predicted neoepitopes both as peptides and on retroviral expression in HLA-matched Epstein-Barr virus-lymphoblastoid cell line and BC cell line MCF-7 cells; PCR clonotyping revealed the presence of T cells in the periphery with T-cell receptors for the predicted neoepitopes. These high-avidity immune responses were polyclonal, mutation-specific and restricted to either HLA class I or II. Interestingly, we observed the persistence and expansion of polyclonal T-cell responses following neoadjuvant chemotherapy. Conclusions We demonstrate our neoepitope prediction program allows for the successful identification of neoepitopes targeted by TILs in patients with BC, providing a means to identify tumor-specific immunogenic targets for individualized treatment, including vaccines or adoptively transferred cellular therapies. Show less
Fuchs, K.J.; Honders, M.W.; Meijden, E.D. van der; Adriaans, A.E.; Lee, D.I. van der; Pont, M.J.; ... ; Griffioen, M. 2020
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... Show morePatients 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. Show less
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... Show moreAcute 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 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. Show less
Recent studies have demonstrated that CD4(+) T cells can efficiently reject MHC-II-negative tumors. This requires indirect presentation of tumor-associated antigens on surrounding antigen... Show moreRecent studies have demonstrated that CD4(+) T cells can efficiently reject MHC-II-negative tumors. This requires indirect presentation of tumor-associated antigens on surrounding antigen-presenting cells. We hypothesized that intercellular transfer of proteins is not the sole consequence of cell death-mediated protein release, but depends on heat-shock cognate protein 70 (HSC70) and its KFERQ-like binding motif on substrate proteins. Using human Y chromosome antigen DBY, we showed that mutation of one of its 2 putative binding motifs markedly diminished T cell activation after indirect presentation and reduced protein-protein interaction with HSC70. Intercellular antigen transfer was shown to be independent of cell-cell contact, but relied on engulfment within secreted microvesicles. In vivo, alterations of the homologous KFERQ-like motif in murine DBY hampered tumor rejection, T cell activation, and migration into the tumor and substantially impaired survival. Collectively, we show that intercellular antigen transfer of DBY is tightly regulated via binding to HSC70 and that this mechanism influences recognition and rejection of MHC-II-negative tumors in vivo. Show less
Griffioen, M.; Arindrarto, W.; Borras, D.M.; Locher, I.J.; Diessen, S.A.M.E. van; Holst, R. van der; ... ; Veelken, H. 2018
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... Show morePatients 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. Cancer Res; 70(22); 9073-83. (C) 2010 AACR. Show less
Rutten, C.E.; Luxemburg-Heijs, S.A.P. van; Meijden, E.D. van der; Griffioen, M.; Oudshoorn, M.; Willemze, R.; Falkenburg, J.H.F. 2010
Clinical studies have indicated that HLA-DPBI functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPBI was associated with an increased... Show moreClinical studies have indicated that HLA-DPBI functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPBI was associated with an increased risk of graft-versus-host disease (GVHD), but also a decreased risk of disease relapse. However, specific HLA-DPBI mismatches were associated with poor clinical outcome. It was suggested that this unfavorable effect was caused by a difference in immunogenicity between HLA-DPBI alleles. To analyze whether immunogenicity of HLA-DPBI mismatches could be predicted based on the presence or absence of specific amino acid sequences we developed a model to generate allo-HLA-DPBI responses in vitro. We tested in total 48 different stimulator/responder combinations by stimulating CD4(+) T cells from 5 HLA-DPBI homozygous individuals with the same antigen-presenting cells transduced with different allo-HLA-DPBI molecules. HLA-DPBI molecules used for stimulation comprised 76% to 99% of HLA-DPBI molecules present in different ethnic populations. We show that all HLA-DPBI mismatches as defined by allele typing resulted in high-frequency immune responses. Furthermore, we show that crossrecognition of different HLA-DPBI molecules is a broadly observed phenomenon. We confirm previously described patterns in crossrecognition, and demonstrate that a high degree in similarity between HLA-DPBI molecules is predictive for crossrecognition, but not for immunogenicity. Biol Blood Marrow Transplant 16: 1282-1292 (2010) (C) 2010 American Society for Blood and Marrow Tramplantation Show less
Stumpf, A.N.; Meijden, E.D. van der; Willemze, R.; Falkenburg, J.H.F.; Griffioen, M. 2010
