RESUMO
Background: Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. Methods and Results: Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded in vitro, and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IAd). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25high, CD127low). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. Conclusions: Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease.
Assuntos
Doença de Graves/imunologia , Antígeno HLA-DR3/genética , Hipertireoidismo/imunologia , Receptores da Tireotropina/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/imunologia , Ligante de CD40/imunologia , Modelos Animais de Doenças , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunização , Imunofenotipagem , Interferon gama/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Transgênicos , Peptídeos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Purpose: The causative agent of most cases of Merkel cell carcinoma (MCC) has been identified as the Merkel cell polyomavirus (MCV). MCV-encoded T antigens (Tag) are essential not only for virus-mediated tumorigenesis but also for maintaining MCC cell lines in vitro MCV Tags are thus an appealing target for viral oncoprotein-directed T-cell therapy for MCC. With this study, we aimed to isolate and characterize Tag-specific T-cell receptors (TCR) for potential use in gene therapy clinical trials.Experimental Design: T-cell responses against MCV Tag epitopes were investigated by immunizing transgenic mice that express a diverse human TCR repertoire restricted to HLA-A2. Human lymphocytes genetically engineered to express Tag-specific TCRs were tested for specific reactivity against MCC cell lines. The therapeutic potential of Tag-specific TCR gene therapy was tested in a syngeneic cancer model.Results: We identified naturally processed epitopes of MCV Tags and isolated Tag-specific TCRs. T cells expressing these TCRs were activated by HLA-A2-positive cells loaded with cognate peptide or cells that stably expressed MCV Tags. We showed cytotoxic potential of T cells engineered to express these TCRs in vitro and demonstrated regression of established tumors in a mouse model upon TCR gene therapy.Conclusions: Our findings demonstrate that MCC cells can be targeted by MCV Tag-specific TCRs. Although recent findings suggest that approximately half of MCC patients benefit from PD-1 pathway blockade, additional patients may benefit if their endogenous T-cell response can be augmented by infusion of transgenic MCV-specific T cells such as those described here. Clin Cancer Res; 24(15); 3644-55. ©2018 AACR.
Assuntos
Antígenos Virais de Tumores/uso terapêutico , Carcinoma de Célula de Merkel/terapia , Terapia Genética , Linfócitos T/imunologia , Animais , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/imunologia , Carcinogênese/genética , Carcinogênese/imunologia , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/imunologia , Carcinoma de Célula de Merkel/virologia , Citotoxicidade Imunológica/genética , Epitopos/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/uso terapêutico , Humanos , Imunoterapia , Linfócitos/imunologia , Poliomavírus das Células de Merkel/imunologia , Poliomavírus das Células de Merkel/patogenicidade , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/uso terapêuticoRESUMO
Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer+ CD8+ T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRß (TRB) sequencing was performed on tetramer+ cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer+ T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137-47. ©2017 AACR.
Assuntos
Carcinoma de Célula de Merkel/etiologia , Carcinoma de Célula de Merkel/mortalidade , Linfócitos do Interstício Tumoral/imunologia , Poliomavírus das Células de Merkel/imunologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/mortalidade , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Célula de Merkel/patologia , Evolução Clonal/genética , Evolução Clonal/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Variação Genética , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Prognóstico , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Sequência de DNA , Neoplasias Cutâneas/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⺠T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antígenos de Neoplasias/imunologia , Imunoensaio/métodos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia Adotiva/métodos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas/instrumentação , Mapeamento de Interação de Proteínas/métodos , Especificidade da EspécieRESUMO
Biallelic mutations in MCPH1 cause primary microcephaly (MCPH) with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL) and a second transcript lacking the six 3' exons (MCPH1Δe9-14). Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1Δe9-14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level.
Assuntos
Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Motivos de Aminoácidos , Ciclo Celular , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Centrossomo/ultraestrutura , Cromatina/química , Cromossomos/ultraestrutura , Proteínas do Citoesqueleto , Éxons , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Peptídeos/química , Isoformas de Proteínas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Radiação Ionizante , Distribuição TecidualRESUMO
Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, which is characterized by a marked reduction of brain size. Interestingly, hMCPH1 mutant patient cells display unique cellular phenotypes, including premature chromosome condensation (PCC), in G2 phase. To test whether hMCPH1 might directly participate in the regulation of chromosome condensation and, if so, how, we developed a cell-free assay using Xenopus laevis egg extracts. Our results demonstrate that an N-terminal domain of hMCPH1 specifically inhibits the action of condensin II by competing for its chromosomal binding sites in vitro. This simple and powerful assay allows us to dissect mutations causing primary microcephaly in vivo and evolutionary substitutions among different species. A complementation assay using patient cells revealed that, whereas the N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, its central domain plays an auxiliary role in shaping metaphase chromosomes by physically interacting with condensin II. Thus, hMCPH1 acts as a composite modulator of condensin II to regulate chromosome condensation and shaping.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas do Citoesqueleto , Humanos , Metáfase , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , XenopusRESUMO
Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G2/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.
Assuntos
Ciclo Celular/fisiologia , Dano ao DNA , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ciclo Celular , Quebra Cromossômica , Proteínas do Citoesqueleto , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Reparo do DNA , Predisposição Genética para Doença , Células HeLa , Humanos , Microcefalia/genética , Mutação , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Interferência de RNARESUMO
Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1(gt/gt) mice, the overall survival rates of the Mcph1(gt/gt) animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function.