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1.
Nat Commun ; 11(1): 2908, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518267

RESUMO

Adoptive cell therapy (ACT) with tumor-specific T cells can mediate cancer regression. The main target of tumor-specific T cells are neoantigens arising from mutations in self-proteins. Although the majority of cancer neoantigens are unique to each patient, and therefore not broadly useful for ACT, some are shared. We studied oligoclonal T-cell receptors (TCRs) that recognize a shared neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by HLA-A2. Here we report structures of wild-type and mutant p53-HLA-A2 ligands, as well as structures of three tumor-specific TCRs bound to p53R175H-HLA-A2. These structures reveal how a driver mutation in p53 rendered a self-peptide visible to T cells. The TCRs employ structurally distinct strategies that are highly focused on the mutation to discriminate between mutant and wild-type p53. The TCR-p53R175H-HLA-A2 complexes provide a framework for designing TCRs to improve potency for ACT without sacrificing specificity.


Assuntos
Antígenos de Neoplasias/química , Antígeno HLA-A2/química , Mutação , Linfócitos T/imunologia , Proteína Supressora de Tumor p53/química , Sítios de Ligação , Biotinilação , Códon , Cristalografia por Raios X , Epitopos , Escherichia coli/metabolismo , Humanos , Imunoterapia Adotiva , Ligantes , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/metabolismo , Peptídeos/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Software , Ressonância de Plasmônio de Superfície
2.
PLoS Pathog ; 16(4): e1008477, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251475

RESUMO

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.


Assuntos
Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/virologia , Tacrolimo/farmacologia , Animais , Linfócitos B/metabolismo , DNA Viral , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Feminino , Perfilação da Expressão Gênica/métodos , Antígeno HLA-A2 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Humanos , Hospedeiro Imunocomprometido , Imunossupressores/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Transplante de Órgãos/efeitos adversos , Transcriptoma/genética , Carga Viral
3.
Nat Commun ; 11(1): 1314, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161266

RESUMO

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.


Assuntos
Apresentação do Antígeno , Dipeptídeos/metabolismo , Antígeno HLA-A2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dipeptídeos/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/ultraestrutura , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
4.
Cancer Immunol Immunother ; 69(7): 1217-1227, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32157447

RESUMO

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410-420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.


Assuntos
Apresentação do Antígeno/imunologia , Ciclina A1/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/imunologia , Neoplasias Ovarianas/imunologia , Linfócitos T Citotóxicos/imunologia , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Neoplasias Ovarianas/patologia , Fragmentos de Peptídeos/imunologia , Células Tumorais Cultivadas
5.
Mol Immunol ; 120: 101-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32113130

RESUMO

Histocompatibility Leukocyte Antigens, or HLAs, are one of the most polymorphic molecules in humans. This high degree of polymorphism endows HLA molecules with the ability to present a vast array of peptides, an essential trait for responding to ever-evolving pathogens. Unlike classical HLA molecules (HLA-Ia), some non-classical HLA-Ib molecules, including HLA-E, are almost monomorphic. Several studies show HLA-E can present self-peptides originating from the leader sequence of other HLA molecules, which signals to our immune system that the cell is healthy. Therefore, it was traditionally thought that the chief role of HLA-E in the body was in immune surveillance. However, there is emerging evidence that HLA-E is also able to present pathogen-derived peptides to the adaptive immune system, namely T cells, in a manner that is similar to classical HLA-Ia molecules. Here we describe the early findings of this less conventional role of HLA-E in the adaptive immune system and its importance for immunity.


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Imunidade Adaptativa , Sequência de Aminoácidos , Apresentação do Antígeno/imunologia , Sítios de Ligação , Infecções por Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por HIV/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Modelos Moleculares , Polimorfismo Genético , Conformação Proteica , Infecções por Salmonella/imunologia , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Tuberculose/imunologia
6.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31915283

RESUMO

The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.


Assuntos
Regulação para Baixo , Regulação da Expressão Gênica , Antígeno HLA-A2/metabolismo , Fator de Transcrição AP-1/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Microscopia Imunoeletrônica , Transporte Proteico , Linfócitos T/imunologia , Linfócitos T/virologia , Rede trans-Golgi/metabolismo
7.
Cancer Immunol Immunother ; 69(3): 449-463, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932876

RESUMO

Lactate dehydrogenase C (LDHC) is an archetypical cancer testis antigen with limited expression in adult tissues and re-expression in tumors. This restricted expression pattern together with the important role of LDHC in cancer metabolism renders LDHC a potential target for immunotherapy. This study is the first to investigate the immunogenicity of LDHC using T cells from healthy individuals. LDHC-specific T cell responses were induced by in vitro stimulation with synthetic peptides, or by priming with autologous peptide-pulsed dendritic cells. We evaluated T cell activation by IFN-γ ELISpot and determined cytolytic activity of HLA-A*0201-restricted T cells in breast cancer cell co-cultures. In vitro T cell stimulation induced IFN-γ secretion in response to numerous LDHC-derived peptides. Analysis of HLA-A*0201 responses revealed a significant T cell activation after stimulation with peptide pools 2 (PP2) and 8 (PP8). The PP2- and PP8-specific T cells displayed cytolytic activity against breast cancer cells with endogenous LDHC expression within a HLA-A*0201 context. We identified peptides LDHC41-55 and LDHC288-303 from PP2 and PP8 to elicit a functional cellular immune response. More specifically, we found an increase in IFN-γ secretion by CD8 + T cells and cancer-cell-killing of HLA-A*0201/LDHC positive breast cancer cells by LDHC41-55- and LDHC288-303-induced T cells, albeit with a possible antigen recognition threshold. The majority of induced T cells displayed an effector memory phenotype. To conclude, our findings support the rationale to assess LDHC as a targetable cancer testis antigen for immunotherapy, and in particular the HLA-A*0201 restricted LDHC41-55 and LDHC288-303 peptides within LDHC.


Assuntos
Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Imunoterapia/métodos , L-Lactato Desidrogenase/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Isoenzimas/imunologia , Masculino
8.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31852786

RESUMO

Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos HLA/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/virologia , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene pol/imunologia , Genótipo , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia , Interferon gama/imunologia , Peptídeos/imunologia , Ligação Proteica
9.
PLoS Pathog ; 15(11): e1008122, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765434

RESUMO

The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαß sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRß chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRß, in understanding the CD8 T cell receptor repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Proteínas Imediatamente Precoces/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Transativadores/imunologia , Sequência de Aminoácidos , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígeno HLA-A2/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transativadores/genética , Transativadores/metabolismo
10.
Nat Commun ; 10(1): 5387, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772172

RESUMO

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.


Assuntos
Anticorpos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/metabolismo , Imunoterapia/métodos , Linfócitos T/imunologia , Animais , Anticorpos/genética , Antineoplásicos Imunológicos/imunologia , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Efeito Espectador , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Medicina de Precisão/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Immunol Immunother ; 68(12): 1979-1993, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686124

RESUMO

5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17-25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and ß chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Vacinas Anticâncer/imunologia , Carcinoma de Células Renais/terapia , Epitopos de Linfócito T/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Glicoproteínas de Membrana/metabolismo , Linfócitos T CD8-Positivos/transplante , Carcinoma de Células Renais/imunologia , Células Clonais , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Neoplasias Renais/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética
12.
Proc Natl Acad Sci U S A ; 116(34): 16955-16960, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31375628

RESUMO

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.


Assuntos
Anoctaminas , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4 , Modelos Imunológicos , Mimetismo Molecular , Esclerose Múltipla , Anoctaminas/genética , Anoctaminas/imunologia , Autoanticorpos/imunologia , Reações Cruzadas/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Feminino , Antígeno HLA-A2/imunologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Haplótipos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Imunoglobulina G/imunologia , Masculino , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Fatores de Risco
13.
Cancer Sci ; 110(10): 3049-3060, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31390678

RESUMO

Heat shock protein 105 (HSP105) is overexpressed in many cancers, including colorectal cancer (CRC) and esophageal cancer (EC). We carried out a phase I clinical trial of HLA-A24- and HLA-A2-restricted HSP105 peptide vaccines in patients with CRC or EC. In this additional study of the trial, we examined the immunological efficacy of the novel vaccine. Thirty patients with advanced CRC or EC underwent HSP105 peptide vaccination. Immunological responses were evaluated by ex vivo and in vitro γ-interferon enzyme-linked immunospot assays and their correlation with patients' prognosis was analyzed. The HSP105 peptide vaccines induced peptide-specific CTLs in 15 of 30 patients. Among HLA-A24 patients (n = 15), 7 showed induction of CTLs only ex vivo, whereas among HLA-A2 patients (n = 15), 4 showed the induction ex vivo and 6 in vitro. Heat shock protein 105-specific CTL induction correlated with suppression of cancer progression and was revealed as a potential predictive biomarker for progression-free survival (P = .008; hazard ratio = 3.03; 95% confidence interval, 1.34-6.85) and overall survival (P = .025; hazard ratio = 2.72; 95% confidence interval, 1.13-6.52). Production of cytokines by HSP105 peptide-specific CTLs was observed at the injection sites (skin) and tumor tissues, suggesting that HSP105-specific CTLs not only accumulated at vaccination sites but also infiltrated tumors. Furthermore, we established 2 HSP105 peptide-specific CTL clones, which showed HSP105-specific cytokine secretion and cytotoxicity. Our results suggest that the HSP105 peptide vaccine could induce immunological effects in cancer patients and improve their prognosis.


Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Esofágicas/tratamento farmacológico , Proteínas de Choque Térmico HSP110/química , Proteínas de Choque Térmico HSP110/metabolismo , Adulto , Idoso , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Citocinas/metabolismo , Intervalo Livre de Doença , Neoplasias Esofágicas/imunologia , Feminino , Antígeno HLA-A2/metabolismo , Antígeno HLA-A24/metabolismo , Células Hep G2 , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Linfócitos T Citotóxicos/imunologia , Resultado do Tratamento , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia
14.
Mol Immunol ; 114: 389-394, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31454596

RESUMO

HLA-A2 is the most common serological HLA type among all ethnic groups. Through advances in DNA typing, more than 800 subtypes of HLA-A2 have been identified, and the existence of heterogeneity of antigen specificity among the HLA-A2 subtypes has been suggested by retrospective analyses of allogeneic transplantation patients and by studies of antigen amino acid structure. However, prior to this study, the antigenicity of a given subtype or the mismatch extent between two given subtypes could not be studied in vitro. Here, we used a modified autologous lymphocyte-monocyte coculture method to reveal heterogeneity of antigen specificity among HLA-A2 subtypes. The coculture was set up with HLA-A2 (non-A*02:01) lymphocytes and monocytes, and the monocytes were coated with an HLA-A*02:01/IgG1-Fc fusion protein (dimer) by high-affinity binding of the IgG1-Fc to FcgRI. Lymphocyte proliferation following coculture indicated that HLA-A*02:01 showed antigenicity against the HLA-A2 (non-A*02:01) subtype. Among the most frequent HLA-A2 subtypes in the Chinese population (HLA-A*02:01, -A*02:03, -A*02:06 and -A*02:07), we identified significant -A*02:01 antigenicity for T cells from -A*02:03 or -A*02:06 but not -A*02:07 individuals. Our findings were consistent with retrospective studies of allograft patients with a limited number of involved subtypes, indicating that this modified coculture method provides a practical and reliable means to study the antigenicity of HLA allele subtypes in vitro.


Assuntos
Especificidade de Anticorpos/imunologia , Antígeno HLA-A2/imunologia , Monócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Alelos , Técnicas de Cocultura/métodos , Humanos , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Estudos Retrospectivos
15.
Proc Natl Acad Sci U S A ; 116(34): 16943-16948, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31315981

RESUMO

The T cell receptor (TCR)-peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR-pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR-pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch-slip transitions and, second, 2D TCR-pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR-pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation.


Assuntos
Antígeno HLA-A2/química , Modelos Químicos , Receptores de Antígenos de Linfócitos T/química , Sistema Livre de Células , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Cinética , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia
16.
Zhonghua Zhong Liu Za Zhi ; 41(6): 429-434, 2019 Jun 23.
Artigo em Chinês | MEDLINE | ID: mdl-31216828

RESUMO

Objective: To predict the tumor neoantigen peptides in hepatocellular carcinoma (HCC), and examine their specific immune effects against the tumor cells without injury to normal cells. Methods: The data of whole-genome sequencing and exome sequencing of HCC tumor and matched non-tumor liver tissues were analyzed to confirm the HCC-associated somatic mutations. Based on the HLA phenotype of the patients, we used NetMHC software to predict the neoantigen epitopes with high binding affinity to their MHC-I molecules. The predicted peptides with mutation sites included were synthesized. GPL10687 platform was applied to examine the gene expression difference between tumor and normal tissues of the selected genes in GSE25097, one of the GEO databases. The quantitative real-time PCR (qRT-qPCR) and immunohistochemistry were used to confirm the expressions in tumors and normal tissues of the selected genes. By using the predicted peptides, we induced the generation of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) and examined their specific effects against tumor cells. Results: The mutation frequency of TP53 (tumor protein p53) was 40%, and LAMA3 (Laminin Subunit Alpha 3) was 8% in the analyzed HCC tissues. In GSE25097 database, TP53 and LAMA3 mRNA levels in tumors were 1.57±0.02 and 1.37±0.10, which were significantly increased than those in matched no-tumor tissue (0.54±0.01 and 0.36±0.01, P<0.05). The differences of expression levels of TP53 and LAMA3 in tumor and no-tumor tissues were validated by using qRT-qPCR and immunohistochemistry in 10 HCC tissues. The mRNA levels of TP53 and LAMA3 in tumors were 0.24±0.03 and 0.13±0.06, which were significantly elevated than those in matched no-tumor tissue (0.11±0.01 and 0.01±0.01, P<0.05). Among the Chinese population, HLA-A2 and HLA-A11 and HLA-A24 accounted for 70%, representing the major MHC-I molecules. The CTLs induced by predicted peptides showed cytotoxicity to the targets pulsed with mutated peptide, with no effect on the target pulsed with normal peptide and on normal cells. Conclusions: TP53 and LAMA3 existed relative higher mutation frequency in HCC, and expressed higher in tumor tissues. The induced CTLs by predicted peptides derived from mutation-associated protein could specific kill the target cells without injury to normal cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Antígeno HLA-A2 , Humanos , Linfócitos T Citotóxicos
17.
Medicine (Baltimore) ; 98(19): e15553, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31083216

RESUMO

RATIONALE: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are 2 rare but life-threatening diseases characterized by detachment of epidermis, bullous skin lesions, and mucous membrane erosions. Drugs are highly suspected to be the causative agents. We report a case of SJS/TEN induced by oseltamivir, which is a very rare event. PATIENT CONCERNS: A 9-year-old girl with upper respiratory tract infections presented with generalized maculopapular rash the second day after taking oseltamivir. DIAGNOSIS: The diagnosis of SJS/TEN was made based on cytotoxic skin lesions and mucous membrane involvement. INTERVENTIONS: After discontinuing of the drug and combination therapy of corticosteroid and human immunoglobulin initiation, the lesions were improved. Human leukocyte antigen (HLA) gene sequencing was done. OUTCOMES: The girl was followed-up for 1 year. The skin and mucous membranes symptoms were relieved. LESSONS: We report this case to attract attention to the rare but serious side effect of this antiviral drug.


Assuntos
Antivirais/efeitos adversos , Oseltamivir/efeitos adversos , Síndrome de Stevens-Johnson/etiologia , Antivirais/uso terapêutico , Criança , Feminino , Antígeno HLA-A2/genética , Humanos , Oseltamivir/uso terapêutico , Infecções Respiratórias/tratamento farmacológico , Síndrome de Stevens-Johnson/diagnóstico , Síndrome de Stevens-Johnson/genética , Síndrome de Stevens-Johnson/terapia
18.
Cytotherapy ; 21(6): 659-670, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31031152

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy. METHODS: We pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells. RESULTS: The Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity. DISCUSSION: This study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Vacinas Anticâncer/farmacologia , Células Dendríticas/imunologia , Leucemia/terapia , Receptores de Antígenos Quiméricos/imunologia , Antígenos CD19/genética , Antígenos CD19/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Citocinas/metabolismo , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia
19.
J Clin Invest ; 129(5): 2056-2070, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30835255

RESUMO

BACKGROUND: Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations. However, timely and efficient identification of neoantigens is still one of the major obstacles to using personalized neoantigen-based cancer immunotherapy. METHODS: Two different pipelines of neoantigens identification were established in this study: (1) Clinical grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were de novo synthesized. (2) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T-cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in six patients. RESULTS: Immunogenic neo-epitopes were recognized by autologous T cells in 3 of 4 patients who utilized the de novo synthesis mode and in 6 of 13 patients who performed shared neoantigen peptide library, respectively. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining four patients achieved the prolonged stabilization of disease with a median PFS of 8.6 months. CONCLUSIONS: The current study provided feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate the neoantigen-based translational immunotherapy research.TRIAL REGSITRATION. ChiCTR.org ChiCTR-OIC-16010092, ChiCTR-OIC-17011275, ChiCTR-OIC-17011913; ClinicalTrials.gov NCT03171220. FUNDING: This work was funded by grants from the National Key Research and Development Program of China (Grant No. 2017YFC1308900), the National Major Projects for "Major New Drugs Innovation and Development" (Grant No.2018ZX09301048-003), the National Natural Science Foundation of China (Grant No. 81672367, 81572329, 81572601), and the Key Research and Development Program of Jiangsu Province (No. BE2017607).


Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia/métodos , Mutação , Neoplasias/terapia , Adulto , Idoso , Vacinas Anticâncer/imunologia , Biologia Computacional , Intervalo Livre de Doença , Epitopos/imunologia , Feminino , Genômica , Antígeno HLA-A2/imunologia , Humanos , Sistema Imunitário , Fatores Imunológicos , Imunofenotipagem , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/imunologia , Neoplasias Pancreáticas/imunologia , Peptídeos/imunologia , Fenótipo , Reação em Cadeia da Polimerase , Medicina de Precisão/métodos , Linfócitos T/imunologia , Timoma/imunologia , Timoma/metabolismo , Pesquisa Médica Translacional , Adulto Jovem
20.
Biochem Biophys Res Commun ; 511(3): 711-717, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30827508

RESUMO

Immunogenicity of immature pluripotent stem cells is a topic of intense debate. Immunogenic antigens, which are specific in pluripotent states, have not been described previously. In this study, we identified glypican-3 (GPC3), a known carcinoembryonic antigen, as a pluripotent state-specific immunogenic antigen. Additionally, we validated the applicability of human leukocyte antigen (HLA)-class I-restricted GPC3-reactive cytotoxic T lymphocytes (CTLs) in the removal of undifferentiated pluripotent stem cells (PSCs) from human induced pluripotent stem cell (hiPSC)-derivatives. HiPSCs uniquely express GPC3 in pluripotent states and were rejected by GPC3-reactive CTLs, which were sensitized with HLA-class I-restricted GPC3 peptides. Furthermore, GPC3-reactive CTLs selectively removed undifferentiated PSCs from hiPSC-derivatives in vitro and inhibited tumor formation in vivo. Our results demonstrate that GPC3 works as a pluripotent state-specific immunogenic antigen in hiPSCs and is applicable to regenerative medicine as a method of removing undifferentiated PSCs, which are the main cause of tumor formation.


Assuntos
Glipicanas/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Diferenciação Celular , Linhagem Celular , Glipicanas/análise , Antígeno HLA-A2/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Neoplasias/imunologia
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