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1.
Front Immunol ; 11: 381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218783

RESUMO

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.


Assuntos
Cisteína Endopeptidases/metabolismo , Leucemia Mieloide Aguda , Antígenos de Histocompatibilidade Menor , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Variação Genética , Efeito Enxerto vs Leucemia/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
2.
Cancer Immunol Res ; 7(5): 797-804, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30890530

RESUMO

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.


Assuntos
Epitopos de Linfócito T/imunologia , Leucemia/imunologia , Linfócitos T Citotóxicos/imunologia , Humanos
3.
J Clin Invest ; 129(2): 774-785, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640174

RESUMO

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.


Assuntos
Transferência Adotiva , Leucemia Mieloide Aguda , Mutação , Proteínas de Neoplasias , Proteínas Nucleares , Peptídeos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Nucleofosmina , Peptídeos/genética , Peptídeos/imunologia , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Clin Cancer Res ; 22(16): 4185-96, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26964570

RESUMO

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.


Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , Integrina beta3/genética , Antígenos de Histocompatibilidade Menor/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos , Sequência de Bases , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Antígeno HLA-B15/genética , Antígeno HLA-B15/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Integrina beta3/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/imunologia , Peptídeos/genética , Peptídeos/imunologia , Linfócitos T , Transplante Homólogo
6.
Blood ; 120(16): 3246-55, 2012 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22889757

RESUMO

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.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Antígenos HLA-D/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Células Apresentadoras de Antígenos/imunologia , Biomarcadores/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Antígenos HLA-D/química , Antígenos HLA-D/metabolismo , Antígenos HLA-D/farmacologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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