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1.
Life Sci ; 246: 117396, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32035130

RESUMO

AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Decrease in NKG2D ligand levels and exhaustion of NK cells in HCC patients are major causes of immune escape, high recurrence, poor prognosis, and low overall survival. Enhancing the susceptibility of HCC to NK cells by upregulating NKG2DLs on tumor cells is an effective treatment strategy. This study aimed to identify the effect of the Anterior gradient 2 (AGR2)-derived peptide P1, which was reported to bind to HLA-A*0201 as an epitope, on both the expression of major histocompatibility complex class I-related chains A/B (MICA/B) on HCC cells and the cytotoxicity of NK cells. MAIN METHODS: The effect of P1 on MICA/B expression on HCC cells was determined by qRT-PCR, western blotting, and flow cytometry analysis. HCC cells were pre-treated with various pathway inhibitors to identify the molecular pathways associated with P1 treatment. The cytotoxicity of NK cells toward HCC was investigated by LDH cytotoxicity assay. The tumor-suppression effect of P1 was determined in vivo using a NOD/SCID mice HCC model. KEY FINDINGS: P1 significantly increased MICA/B expression on HCC cells, thereby enhancing their susceptibility to the cytotoxicity of NK cells in vitro and in vivo. Further, p38 MAPK cell signaling pathway inhibitor SB203580 significantly attenuated the effects of P1 in vivo and in vitro. SIGNIFICANCE: P1 upregulates MICA and MICB expression on HCC cells, thereby promoting their recognition and elimination by NK cells, which makes P1 an attractive novel immunotherapy agent.


Assuntos
Carcinoma Hepatocelular/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Hepáticas/metabolismo , Mucoproteínas/fisiologia , Proteínas Oncogênicas/fisiologia , Animais , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Mucoproteínas/metabolismo , Transplante de Neoplasias , Proteínas Oncogênicas/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
2.
PLoS One ; 14(12): e0226245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887144

RESUMO

Antibody therapies for Alzheimer's Disease (AD) hold promise but have been limited by the inability of these proteins to migrate efficiently across the blood brain barrier (BBB). Central nervous system (CNS) gene transfer by vectors like adeno-associated virus (AAV) overcome this barrier by allowing the bodies' own cells to produce the therapeutic protein, but previous studies using this method to target amyloid-ß have shown success only with truncated single chain antibodies (Abs) lacking an Fc domain. The Fc region mediates effector function and enhances antigen clearance from the brain by neonatal Fc receptor (FcRn)-mediated reverse transcytosis and is therefore desirable to include for such treatments. Here, we show that single chain Abs fused to an Fc domain retaining FcRn binding, but lacking Fc gamma receptor (FcγR) binding, termed a silent scFv-IgG, can be expressed and released into the CNS following gene transfer with AAV. While expression of canonical IgG in the brain led to signs of neurotoxicity, this modified Ab was efficiently secreted from neuronal cells and retained target specificity. Steady state levels in the brain exceeded peak levels obtained by intravenous injection of IgG. AAV-mediated expression of this scFv-IgG reduced cortical and hippocampal plaque load in a transgenic mouse model of progressive ß-amyloid plaque accumulation. These findings suggest that CNS gene delivery of a silent anti-Aß scFv-IgG was well-tolerated, durably expressed and functional in a relevant disease model, demonstrating the potential of this modality for the treatment of Alzheimer's disease.


Assuntos
Doença de Alzheimer/terapia , Sistema Nervoso Central/metabolismo , Vetores Genéticos/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Anticorpos de Cadeia Única/genética , Doença de Alzheimer/genética , Animais , Barreira Hematoencefálica , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Progressão da Doença , Terapia Genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Camundongos Transgênicos , Domínios Proteicos , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo
3.
Pathologe ; 40(Suppl 3): 259-264, 2019 Dec.
Artigo em Alemão | MEDLINE | ID: mdl-31720747

RESUMO

Hyperosmolar micromilieu has been observed in physiologic (kidney medulla, lymphatic tissue) and pathologic (renal allorejection, solid tumors) conditions. Hyperosmolarity can modulate gene expression and alter the stimulatory profile of macrophages and dendritic cells. We have reported that dendritic cells upon exposure to hypertonic stimuli shift their profile towards a macrophage-M2-like phenotype, resulting in attenuated local alloreactivity during acute kidney graft rejection. Moreover, we showed that a hyperosmotic microenvironment affects the cross-priming capacity of dendritic cells. Using ovalbumin as a model antigen, we showed that exposure of dendritic cells to hyperosmolarity strongly inhibits activation of antigen-specific T cells despite enhancement of antigen uptake, processing, and presentation; it can reduce dendritic cell-T cell contact time. We have identified TRIF as key mediator of this phenomenon. Moreover, we detected a hyperosmolarity-triggered, TRIF-dependent clustering of MHC class I­antigen complexes, but not of unloaded MHCI molecules, providing a possible explanation for a reduced T cell activation. Our findings identify dendritic cells as important players in hyperosmolarity-triggered immune imbalance and suggest that targeting local hyperosmolarity in tumor micromilieu may contribute to an enhanced specific anti-tumor immune response.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células Dendríticas , Antígenos de Histocompatibilidade Classe I , Apresentação Cruzada , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ativação Linfocitária , Osmorregulação , Ovalbumina/imunologia , Linfócitos T
4.
J Immunol Res ; 2019: 5370706, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583257

RESUMO

MHC class I molecules are key in the presentation of antigen and initiation of adaptive CD8+ T cell responses. In addition to its classical activity, MHC I may possess nonclassical functions. We have previously identified a regulatory role of MHC I in TLR signaling and antibacterial immunity. However, its role in innate antiviral immunity remains unknown. In this study, we found a reduced viral load in MHC I-deficient macrophages that was independent of type I IFN production. Mechanically, MHC I mediated viral suppression by inhibiting the type I IFN signaling pathway, which depends on SHP2. Cross-linking MHC I at the membrane increased SHP2 activation and further suppressed STAT1 phosphorylation. Therefore, our data revealed an inhibitory role of MHC I in type I IFN response to viral infection and expanded our understanding of MHC I and antigen presentation.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Transdução de Sinais , Viroses/metabolismo , Viroses/virologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon Tipo I/metabolismo , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Viroses/imunologia , Replicação Viral
5.
Immunology ; 158(4): 255-266, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31509607

RESUMO

Tumours can escape T-cell responses by losing major histocompatibility complex (MHC)/ human leucocyte antigen (HLA) class I molecules. In the early stages of cancer development, primary tumours are composed of homogeneous HLA class I-positive cancer cells. Subsequently, infiltration of the tumour by T cells generates a vast diversity of tumour clones with different MHC class I expressions. A Darwinian type of T-cell-mediated immune selection results in a tumour composed solely of MHC class I-negative cells. Metastatic colonization is a highly complex phenomenon in which T lymphocytes and natural killer cells play a major role. We have obtained evidence that the MHC class I phenotype of metastatic colonies can be highly diverse and is not necessarily the same as that of the primary tumour. The molecular mechanisms responsible for MHC/HLA class I alterations are an important determinant of the clinical response to cancer immunotherapy. Hence, immunotherapy can successfully up-regulate MHC/HLA class I expression if the alteration is reversible ('soft'), leading to T-cell-mediated tumour regression. In contrast, it cannot recover this expression if the alteration is irreversible ('hard'), when tumour cells escape T-cell-mediated destruction with subsequent cancer progression. This review summarizes clinical and experimental data on the complexity of immune escape mechanisms used by tumour cells to avoid T and natural killer cell responses. We also provide in-depth analysis of the nature of MHC/HLA class I changes during metastatic colonization and contribute evidence of the enormous diversity of MHC/HLA class I phenotypes that can be produced by tumour cells during this process.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Metástase Neoplásica/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Evasão Tumoral , Animais , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Humanos
6.
Org Biomol Chem ; 17(40): 8992-9000, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31497838

RESUMO

Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2'-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2'-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariáveis Associadas à Mucosa/efeitos dos fármacos , Pteridinas/farmacologia , Ribitol/análogos & derivados , Uracila/análogos & derivados , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Células T Invariáveis Associadas à Mucosa/metabolismo , Pteridinas/síntese química , Pteridinas/química , Receptores de Antígenos de Linfócitos T , Ribitol/síntese química , Ribitol/química , Ribitol/farmacologia , Uracila/síntese química , Uracila/química , Uracila/farmacologia
7.
Fish Shellfish Immunol ; 94: 122-131, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31491527

RESUMO

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.


Assuntos
Proteínas de Peixes/genética , Linguados/genética , Linguados/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Transcriptoma/imunologia , Animais , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Linguado/genética , Linguado/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo
8.
Pharm Res ; 36(11): 157, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31493066

RESUMO

PURPOSE: Although pharmacokinetic (PK) interaction effects of methotrexate (MTX) on adalimumab have been found, the mechanism of these effects is still unclear. In this work, effects of MTX on the concentration of neonatal Fc receptor (FcRn) and the role of FcRn in the interaction between MTX and adalimumab were investigated. METHODS: The experiment was performed in rats whose FcRn had normal physiological function and also in rats whose FcRn was blocked with FcRn antibody. Rats were randomly assigned to receive placebo or 0.2 mg/kg MTX orally every week while taking one abdominal subcutaneous injection of 0.5 mg/kg adalimumab. The FcRn concentration in tissues and the PK parameters of adalimumab were compared between MTX-treated and placebo groups. RESULTS: In rats with normally functioning FcRn, the concentrations of FcRn were significantly increased in the liver (F=105.5, p=0.000) and kidney (F=996.312, p=0.000) after treatment with MTX, and the clearance (CL/F) of adalimumab was decreased accordingly (F=4.423, p=0.048). However, in rats injected with FcRn antibody, the concentrations of FcRn in MTX-treated rats were close to that of the placebo rats in the tissues of the liver (F=1.279, p=0.268) and kidney (F=0.661, p=0.424). The CL/F of adalimumab in rats was also not affected by MTX (F=0.002, p=0.961). CONCLUSIONS: FcRn may play a vital role in the interaction between adalimumab and MTX.


Assuntos
Adalimumab/farmacocinética , Antígenos de Histocompatibilidade Classe I/metabolismo , Metotrexato/metabolismo , Receptores Fc/metabolismo , Adalimumab/administração & dosagem , Animais , Feminino , Humanos , Injeções Subcutâneas , Rim/metabolismo , Fígado/metabolismo , Masculino , Metotrexato/administração & dosagem , Metotrexato/farmacologia , Ratos Sprague-Dawley
9.
Nat Immunol ; 20(9): 1244-1255, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31431722

RESUMO

Mucosal-associated invariant T cells (MAIT cells) recognize the microbial metabolite 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) presented by the MHC class Ib molecule, MR1. MAIT cells acquire effector functions during thymic development, but the mechanisms involved are unclear. Here we used single-cell RNA-sequencing to characterize the developmental path of 5-OP-RU-specific thymocytes. In addition to the known MAIT1 and MAIT17 effector subsets selected on bone-marrow-derived hematopoietic cells, we identified 5-OP-RU-specific thymocytes that were selected on thymic epithelial cells and differentiated into CD44- naive T cells. MAIT cell positive selection required signaling through the adapter, SAP, that controlled the expression of the transcription factor, ZBTB16. Pseudotemporal ordering of single cells revealed transcriptional trajectories of 5-OP-RU-specific thymocytes selected on either thymic epithelial cells or hematopoietic cells. The resulting model illustrates T cell lineage decisions.


Assuntos
Linhagem da Célula/imunologia , Células T Invariáveis Associadas à Mucosa/citologia , Células T Invariáveis Associadas à Mucosa/imunologia , Ribitol/análogos & derivados , Timócitos/citologia , Timócitos/imunologia , Uracila/análogos & derivados , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Hialuronatos/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Ribitol/imunologia , Análise de Sequência de RNA , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Timo/citologia , Timo/imunologia , Uracila/imunologia
10.
Cell Host Microbe ; 26(3): 359-368.e8, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31447307

RESUMO

Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (cryo-EM). Nef refolds the first α-helix of the ß2 subunit of AP-2 to a ß hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antígeno 2 do Estroma da Médula Óssea/química , Antígeno 2 do Estroma da Médula Óssea/farmacologia , Infecções por Lentivirus/prevenção & controle , Infecções por Lentivirus/transmissão , Zoonoses/virologia , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/química , Animais , Sítios de Ligação , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Membrana Celular/efeitos dos fármacos , Microscopia Crioeletrônica , Regulação para Baixo , Produtos do Gene nef/química , Produtos do Gene nef/metabolismo , Células HEK293 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Infecções por Lentivirus/virologia , Proteínas de Membrana/metabolismo , Modelos Moleculares , Cultura Primária de Células , Conformação Proteica , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/metabolismo , Vírion/efeitos dos fármacos
11.
Nat Commun ; 10(1): 3020, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289263

RESUMO

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with ß2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune , Imunidade Humoral , Proteínas de Ligação a RNA/metabolismo , Receptores Fc/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/virologia , Degradação Associada com o Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Fc/imunologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia
12.
Cells ; 8(7)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269655

RESUMO

BACKGROUND: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood. Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes. Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance. METHODS: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs. The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions. RESULTS: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs. Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced. TGF-ß transported by sEVs contributed to the promotion of a suppressive phenotype of APCs. CONCLUSION: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Vesículas Extracelulares/imunologia , Melanoma/imunologia , Evasão Tumoral/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Vesículas Extracelulares/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Melanoma/patologia
13.
Immunogenetics ; 71(7): 479-487, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270568

RESUMO

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.


Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Antígenos Heterófilos/genética , Arginina/genética , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Mutagênese Sítio-Dirigida , Mutação Puntual , Suínos
14.
PLoS Genet ; 15(7): e1008227, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31344031

RESUMO

Somatic mutations in protein-coding regions can generate 'neoantigens' causing developing cancers to be eliminated by the immune system. Quantitative estimates of the strength of this counterselection phenomenon have been lacking. We quantified the extent to which somatic mutations are depleted in peptides that are predicted to be displayed by major histocompatibility complex (MHC) class I proteins. The extent of this depletion depended on expression level of the neoantigenic gene, and on whether the patient had one or two MHC-encoding alleles that can display the peptide, suggesting MHC-encoding alleles are incompletely dominant. This study provides an initial quantitative understanding of counter-selection of identifiable subclasses of neoantigenic somatic variation.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Mutação de Sentido Incorreto , Peptídeos/genética , Alelos , Apresentação do Antígeno , Antígenos de Neoplasias/genética , Humanos
15.
Cells ; 8(6)2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226820

RESUMO

Metastasis is the leading cause of cancer death worldwide. Circulating tumor cells (CTCs) are a critical step in the metastatic cascade and a good tool to study this process. We isolated CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) and a human xenograft mouse model of castration-resistant prostate cancer (CRPC). From these models, novel primary tumor and CTC cell lines were established. CTCs exhibited greater migration than primary tumor-derived cells, as well as epithelial-to-mesenchymal transition (EMT), as observed from decreased E-cadherin and increased SLUG and fibronectin expression. Additionally, when fibronectin was knocked down in CTCs, integrin B1 and SLUG were decreased, indicating regulation of these molecules by fibronectin. Investigation of cell surface molecules and secreted cytokines conferring immunomodulatory advantage to CTCs revealed decreased major histocompatibility complex class I (MHCI) expression and decreased endostatin, C-X-C motif chemokine 5 (CXCL5), and proliferin secretion by CTCs. Taken together, these findings indicate that CTCs exhibit distinct characteristics from primary tumor-derived cells. Furthermore, CTCs demonstrate enhanced migration in part through fibronectin regulation of integrin B1 and SLUG. Further study of CTC biology will likely uncover additional important mechanisms of cancer metastasis.


Assuntos
Fibronectinas/metabolismo , Integrina beta1/metabolismo , Células Neoplásicas Circulantes/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Biomarcadores Tumorais/sangue , Caderinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Endostatinas , Transição Epitelial-Mesenquimal , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunomodulação , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/patologia
16.
Histopathology ; 75(5): 672-682, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31237963

RESUMO

AIMS: Salivary duct carcinoma (SDC) is an aggressive salivary malignancy that results in high mortality rates and is often resistant to chemotherapy. Anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint inhibitors have led to dramatic improvements in patients with various cancers. Other immunotherapeutic approaches, e.g. cancer vaccines, have shown promising results. Cancer testis antigens, e.g. preferentially expressed antigen in melanoma (PRAME), are regarded as promising vaccine targets because of their tumour-specific expression pattern. METHODS AND RESULTS: We analysed the immunoexpression of PD-L1, PD-1, major histocompatibility complex class I (MHC I) and PRAME in 53 SDCs. The immunoexpression levels of PD-L1 in tumour cells (TCs) and immune cells (ICs), PD-1 in ICs, PRAME in TCs and MHC I in TCs were analysed, and were correlated with outcome. PRAME expression was seen in 83% of SDCs. No PRAME staining was present in normal salivary gland tissue. With the three established diagnostic algorithms proposed for head and neck squamous cell carcinoma, the criteria being a combined positive score of ≥1, TC% ≥1%, and TC% ≥25%, 35 (66%), 17 (32%) and three cases (6%), respectively, were deemed to be positive for PD-L1. PD-1-positive ICs were seen in 35 (66%) cases. MHC I down-regulation was seen in 82% of SDCs. There was a significant correlation among PD-L1 expression in ICs, PD-1 expression in ICs, and PRAME expression in TCs. PD-L1 expression in TCs and lack of PD-1 expression in ICs were associated with decreased disease-specific survival in SDC patients. CONCLUSIONS: Alterations of the tumour immune microenvironment are common in SDCs, including expression of PD-1/PD-L1 and PRAME, which opens the way to potential novel immune therapies, such as cancer vaccination and PD-1/PD-L1 blockade, in these tumours.


Assuntos
Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/metabolismo , Carcinoma Ductal/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral/imunologia , Carcinoma Ductal/metabolismo , Histocitoquímica , Humanos , Neoplasias das Glândulas Salivares
17.
J Immunol Res ; 2019: 1242979, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31198791

RESUMO

Cervical cancer is the second most frequent cancer in women in Mexico, and its development depends on the presence of human papillomaviruses in the uterine cervix. These oncogenic viruses transform cells where the control over cell cycle disappears, and the capacity to induce apoptosis is absent. On the other hand, some mutations confer to the transformed cells the ability to evade recognition by the immune system. The expression of markers of the immune system such as CD95, MICA/B, CD39, CD73, NKp30, NKp46, CD44, CD24, NKG2A, and CTLA-4 was analysed by flow cytometry on cervical cancer cells INBL (HPV 18, stage IVB), HeLa (HPV 18), CaSki (HPV 16), and C33A (HPV-). Our results showed the presence of atypical markers on cervical cancer cells; some of them are molecules involved in tumour cell recognition such as MICA/B and CD95. Other markers associated with immune system escape, such as CD39, CD73, and CTLA-4, were also present. Furthermore, we found that some cervical cancer cells expressed typical markers of NK cells like NKp30, NKp46, NKG2A, and KIR3DL1. It is not clear whether these molecules confer any gain to the tumour cells or if they represent a disadvantage, but we hypothesise that these molecules that are present in cervical cancer cells allow them to mimic in front of the immune system.


Assuntos
Papillomavirus Humano 16/fisiologia , Papillomavirus Humano 18/fisiologia , Células Matadoras Naturais/imunologia , Infecções por Papillomavirus/metabolismo , Neoplasias do Colo do Útero/metabolismo , 5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Antígeno CTLA-4/metabolismo , Feminino , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vigilância Imunológica , Receptores de Células Matadoras Naturais/metabolismo , Evasão Tumoral , Receptor fas/metabolismo
18.
Methods Mol Biol ; 1988: 31-43, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147930

RESUMO

Studies over the last decade on characterization of the major histocompatibility complex (MHC) class I antigen presentation pathway have highlighted the importance of antigen processing, peptide transport, peptide trimming, and peptide selection as key stages for the development of optimal peptide repertoires that are presented by MHC class I molecules to cytotoxic T lymphocytes (CTLs). The study of these stages and how they are regulated, is fundamental for progress in understanding the adaptive immune system. Here we describe an in vitro assay monitoring peptide trimming by the human endoplasmic reticulum amino peptidases 1 (ERAP1) and ERAP2 (ERAPs) as a tool to characterize trimming events and gain a better understanding of the role and function of ERAPs in peptide repertoire development. Specifically, our assay allows for monitoring trimming of free but also of MHC I-bound peptides which may reflect the physiological situation best.


Assuntos
Aminopeptidases/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Biologia Molecular/métodos , Sequência de Aminoácidos , Animais , Baculoviridae/metabolismo , Humanos , Ligantes , Peptídeos/química , Peptídeos/metabolismo , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Células Sf9
19.
Methods Mol Biol ; 1988: 45-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147931

RESUMO

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 have recently emerged as important players in regulating innate and adaptive immune responses by trimming peptide ligands for MHC class I molecules. Functional polymorphisms in ERAP1 and ERAP2 genes have been associated with predisposition to several diseases including autoimmune diseases, viral infections, and virally induced cancers. In this chapter, we describe two basic methods for monitoring peptide-trimming activity by ER aminopeptidases and screening potential chemical inhibitors.


Assuntos
Aminopeptidases/metabolismo , Apresentação do Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Biologia Molecular/métodos , Peptídeos/metabolismo , Aminopeptidases/antagonistas & inibidores , Fluorescência , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Sefarose/química , Especificidade por Substrato
20.
Methods Mol Biol ; 1988: 59-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147932

RESUMO

The major histocompatibility complex (MHC) class I restricted pathway of antigen processing allows the presentation of intracellular antigens to cytotoxic T lymphocytes. The proteasome is the main protease in the cytoplasm and the nucleus, which is responsible for the generation of most peptide ligands of MHC-I molecules. Peptides produced by the proteasome can be further trimmed or destroyed by numerous cytosolic or endoplasmic reticulum (ER) luminal proteases. Small molecule inhibitors are useful tools for probing the role of proteases in MHC class I antigen processing. Here, we describe different methods to test the impact of protease inhibitors in antigen presentation assays.


Assuntos
Apresentação do Antígeno/efeitos dos fármacos , Imunoensaio/métodos , Inibidores de Proteases/farmacologia , Animais , Linhagem Celular , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Hibridomas/metabolismo , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Coloração e Rotulagem , Linfócitos T/metabolismo , beta-Galactosidase/metabolismo
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