RESUMEN
Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Neoplasias , Escape del Tumor , Humanos , Presentación de Antígeno , Linfocitos T CD8-positivos , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos HLA , Neoplasias/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.
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Ganglios Linfáticos , Melanoma , Animales , Tolerancia Inmunológica , Inmunoterapia , Metástasis Linfática/patología , Melanoma/patología , RatonesRESUMEN
Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
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Toxinas Bacterianas/metabolismo , Enterococcus , Leucocitos Mononucleares , Factores de Virulencia/metabolismo , Animales , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidad , Caballos , Ratones , Pruebas de Sensibilidad Microbiana , PorcinosRESUMEN
The MHC-I antigen presentation (AP) pathway is key to shaping mammalian CD8+ T cell immunity, with its aberrant expression closely linked to low tumor immunogenicity and immunotherapy resistance. While significant attention has been given to genetic mutations and downregulation of positive regulators that are essential for MHC-I AP, there is a growing interest in understanding how tumors actively evade MHC-I expression and/or AP through the induction of MHC-I inhibitory pathways. This emerging field of study may offer more viable therapeutic targets for future cancer immunotherapy. Here, we explore potential mechanisms by which cancer cells evade MHC-I AP and function and propose therapeutic strategies that might target these MHC-I inhibitors to restore impaired T cell immunity within the tumor microenvironment (TME).
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Antígenos de Histocompatibilidad Clase I , Neoplasias , Animales , Humanos , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T CD8-positivos , Inmunoterapia , Antígenos de Neoplasias , Mamíferos , Microambiente TumoralRESUMEN
The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/biosíntesis , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Linfocitos T/metabolismo , Animales , Línea Celular Tumoral , Técnicas de Cocultivo , Células HEK293 , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno , Humanos , Vigilancia Inmunológica , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Melanoma/inmunología , Melanoma/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Linfocitos T/inmunología , Linfocitos T/virologíaRESUMEN
Phagocytes, particularly dendritic cells (DCs), generate peptide-major histocompatibility complex (MHC) I complexes from antigens they have collected from cells in tissues and report this information to CD8 T cells in a process called cross-presentation. This process allows CD8 T cells to detect, respond and eliminate abnormal cells, such as cancers or cells infected with viruses or intracellular microbes. In some settings, cross-presentation can help tolerize CD8 T cells to self-antigens. One of the principal ways that DCs acquire tissue antigens is by ingesting this material through phagocytosis. The resulting phagosomes are key hubs in the cross-presentation (XPT) process and in fact experimentally conferring the ability to phagocytize antigens can be sufficient to allow non-professional antigen presenting cells (APCs) to cross-present. Once in phagosomes, exogenous antigens can be cross-presented (XPTed) through three distinct pathways. There is a vacuolar pathway in which peptides are generated and then bind to MHC I molecules within the confines of the vacuole. Ingested exogenous antigens can also be exported from phagosomes to the cytosol upon vesicular rupture and/or possibly transport. Once in the cytosol, the antigen is degraded by the proteasome and the resulting oligopeptides can be transported to MHC I molecule in the endoplasmic reticulum (ER) (a phagosome-to-cytosol (P2C) pathway) or in phagosomes (a phagosome-to-cytosol-to-phagosome (P2C2P) pathway). Here we review how phagosomes acquire the necessary molecular components that support these three mechanisms and the contribution of these pathways. We describe what is known as well as the gaps in our understanding of these processes.
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Presentación de Antígeno , Reactividad Cruzada , Humanos , Antígenos de Histocompatibilidad Clase I , Células Dendríticas , Antígenos , Antígenos de Histocompatibilidad , Complejo Mayor de HistocompatibilidadRESUMEN
Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.
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Genes MHC Clase I , Neoplasias , Animales , Genes MHC Clase I/genética , Antígenos de Histocompatibilidad Clase I , Transactivadores/metabolismo , Neoplasias/genética , DesmetilaciónRESUMEN
Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation.
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Transportadoras de Casetes de Unión a ATP , Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Péptidos , Humanos , Péptidos/metabolismo , Péptidos/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Microscopía por Crioelectrón , Conformación Proteica , Unión Proteica , Modelos MolecularesRESUMEN
Evidence is mounting that the major histocompatibility complex (MHC) molecule HLA-F (human leukocyte antigen F) regulates the immune system in pregnancy, infection, and autoimmunity by signaling through NK cell receptors (NKRs). We present structural, biochemical, and evolutionary analyses demonstrating that HLA-F presents peptides of unconventional length dictated by a newly arisen mutation (R62W) that has produced an open-ended groove accommodating particularly long peptides. Compared to empty HLA-F open conformers (OCs), HLA-F tetramers bound with human-derived peptides differentially stained leukocytes, suggesting peptide-dependent engagement. Our in vitro studies confirm that NKRs differentiate between peptide-bound and peptide-free HLA-F. The complex structure of peptide-loaded ß2m-HLA-F bound to the inhibitory LIR1 revealed similarities to high-affinity recognition of the viral MHC-I mimic UL18 and a docking strategy that relies on contacts with HLA-F as well as ß2m, thus precluding binding to HLA-F OCs. These findings provide a biochemical framework to understand how HLA-F could regulate immunity via interactions with NKRs.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Imitación Molecular , Receptores de Células Asesinas Naturales/metabolismo , Proteínas Virales/química , Presentación de Antígeno , Antígenos/inmunología , Antígenos/metabolismo , Antígenos CD/metabolismo , Evolución Biológica , Cristalografía por Rayos X , Femenino , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Receptor Leucocitario Tipo Inmunoglobulina B1 , Mutación/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Embarazo , Unión Proteica , Conformación Proteica , Receptores Inmunológicos/metabolismo , Proteínas Virales/metabolismoRESUMEN
The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (ß2 microglobulin, ß2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/ß2m interface, to generate conformationally stable, peptide-receptive molecules named "open MHC-I." Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type when loaded with low- to moderate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in ß2m-interacting sites of the peptide-binding groove to long-range effects on the α2-1 helix and α3 domain. The interchain disulfide bond stabilizes MHC-I molecules in an open conformation to promote peptide exchange across multiple human leukocyte antigen (HLA) allotypes, covering representatives from five HLA-A supertypes, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structure-guided design, combined with conditional ß-peptide ligands, provides a universal platform to generate ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules.
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Antígenos de Histocompatibilidad Clase II , Antígenos de Histocompatibilidad , Humanos , Péptidos/genética , Complejo Mayor de Histocompatibilidad , Epítopos , DisulfurosRESUMEN
The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.
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Regulación hacia Abajo , Polarización de Fluorescencia , VIH-1 , Ensayos Analíticos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase I , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Humanos , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Regulación hacia Abajo/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/químicaRESUMEN
Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.
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Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Menor , Células T Invariantes Asociadas a Mucosa , Especificidad de la Especie , Animales , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Invariantes Asociadas a Mucosa/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Bovinos , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/química , Porcinos , Macaca , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Peptide-major histocompatibility complex I (MHC I) binding affinity prediction is crucial for vaccine development, but existing methods face limitations such as small datasets, model overfitting due to excessive parameters and suboptimal performance. Here, we present STMHCPan (STAR-MHCPan), an open-source package based on the Star-Transformer model, for MHC I binding peptide prediction. Our approach introduces an attention mechanism to improve the deep learning network architecture and performance in antigen prediction. Compared with classical deep learning algorithms, STMHCPan exhibits improved performance with fewer parameters in receptor affinity training. Furthermore, STMHCPan outperforms existing ligand benchmark datasets identified by mass spectrometry. It can also handle peptides of arbitrary length and is highly scalable for predicting T-cell responses. Our software is freely available for use, training and extension through Github (https://github.com/Luckysoutheast/STMHCPan.git).
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Algoritmos , Péptidos , Alelos , Péptidos/química , Unión Proteica , Programas InformáticosRESUMEN
MHC molecules are not randomly distributed on the plasma membrane but instead are present in discrete nanoclusters. The mechanisms that control formation of MHC I nanoclusters and the importance of such structures are incompletely understood. Here, we report a molecular association between tetraspanin-5 (Tspan5) and MHC I molecules that started in the endoplasmic reticulum and was maintained on the plasma membrane. This association was observed both in mouse dendritic cells and in human cancer cell lines. Loss of Tspan5 reduced the size of MHC I clusters without affecting MHC I peptide loading, delivery of complexes to the plasma membrane, or overall surface MHC I levels. Functionally, CD8 T cell responses to antigen presented by Tspan5-deficient dendritic cells were impaired but were restored by antibody-induced reclustering of MHC I molecules. In contrast, Tspan5 did not associate with two other plasma membrane proteins, Flotillin1 and CD55, with or the endoplasmic reticulum proteins Tapasin and TAP. Thus, our findings identify a mechanism underlying the clustering of MHC I molecules that is important for optimal T cell responses.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Animales , Linfocitos T CD8-positivos , Análisis por Conglomerados , Humanos , Proteínas de la Membrana/genética , Ratones , Tetraspaninas/genéticaRESUMEN
Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.
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Presentación de Antígeno , Linfocitos T CD8-positivos , COVID-19 , Antígenos de Histocompatibilidad Clase I , Proteínas Virales , Aminoácidos , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Complejo Mayor de Histocompatibilidad , Péptidos , SARS-CoV-2 , Proteínas Virales/inmunologíaRESUMEN
Tapasin (Tsn) plays a critical role in antigen processing and presentation by major histocompatibility complex class I (MHC-I) molecules. The mechanism of Tsn-mediated peptide loading and exchange hinges on the conformational dynamics governing the interaction of Tsn and MHC-I with recent structural and functional studies pinpointing the critical sites of direct or allosteric regulation. In this review, we highlight these recent findings and relate them to the extensive molecular and cellular data that are available for these evolutionary interdependent proteins. Furthermore, allotypic differences of MHC-I with regard to the editing and chaperoning function of Tsn are reviewed and related to the mechanistic observations. Finally, evolutionary aspects of the mode of action of Tsn will be discussed, a short comparison with the Tsn-related molecule TAPBPR (Tsn-related protein) will be given, and the impact of Tsn on noncanonical MHC-I molecules will be described.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Inmunoglobulinas , Proteínas de Transporte de Membrana , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismoRESUMEN
At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (ß2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for ß2m.
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Antígenos de Histocompatibilidad Clase I , Microglobulina beta-2 , Animales , Antígenos de Histocompatibilidad Clase I/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Péptidos/metabolismo , Unión Proteica , Microglobulina beta-2/metabolismoRESUMEN
In 2014, the Immune Epitope Database automated benchmark was created to compare the performance of the MHC class I binding predictors. However, this is not a straightforward process due to the different and non-standardized outputs of the methods. Additionally, some methods are more restrictive regarding the HLA alleles and epitope sizes for which they predict binding affinities, while others are more comprehensive. To address how these problems impacted the ranking of the predictors, we developed an approach to assess the reliability of different metrics. We found that using percentile-ranked results improved the stability of the ranks and allowed the predictors to be reliably ranked despite not being evaluated on the same data. We also found that given the rate new data are incorporated into the benchmark, a new method must wait for at least 4 years to be ranked against the pre-existing methods. The best-performing tools with statistically indistinguishable scores in this benchmark were NetMHCcons, NetMHCpan4.0, ANN3.4, NetMHCpan3.0 and NetMHCpan2.8. The results of this study will be used to improve the evaluation and display of benchmark performance. We highly encourage anyone working on MHC binding predictions to participate in this benchmark to get an unbiased evaluation of their predictors.
Asunto(s)
Benchmarking , Alelos , Epítopos , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Current cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5'-C; antisense strand, 3'-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy. METHODS: IsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5'-C; antisense strand, 3'-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-ß and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection. RESULTS: IsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-ß and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors. CONCLUSIONS: This study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5'-C; antisense strand, 3'-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.
Asunto(s)
Apoptosis , Glioma , ARN Interferente Pequeño , Humanos , Animales , Línea Celular Tumoral , Glioma/terapia , Glioma/patología , Glioma/genética , ARN Interferente Pequeño/metabolismo , Ratones Desnudos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Proliferación Celular , Movimiento Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Especies Reactivas de Oxígeno/metabolismo , Invasividad Neoplásica , Supervivencia CelularRESUMEN
BACKGROUND: Although immune checkpoint inhibitors (ICIs) have revolutionized the landscape of cancer treatment, only a minority of colorectal cancer (CRC) patients respond to them. Enhancing tumor immunogenicity by increasing major histocompatibility complex I (MHC-I) surface expression is a promising strategy to boost the antitumor efficacy of ICIs. METHODS: Dual luciferase reporter assays were performed to find drug candidates that can increase MHC-I expression. The effect of nilotinib on MHC-I expression was verified by dual luciferase reporter assays, qRT-PCR, flow cytometry and western blotting. The biological functions of nilotinib were evaluated through a series of in vitro and in vivo experiments. Using RNA-seq analysis, immunofluorescence assays, western blotting, flow cytometry, rescue experiments and microarray chip assays, the underlying molecular mechanisms were investigated. RESULTS: Nilotinib induces MHC-I expression in CRC cells, enhances CD8+ T-cell cytotoxicity and subsequently enhances the antitumor effects of anti-PDL1 in both microsatellite instability and microsatellite stable models. Mechanistically, nilotinib promotes MHC-I mRNA expression via the cGAS-STING-NF-κB pathway and reduces MHC-I degradation by suppressing PCSK9 expression in CRC cells. PCSK9 may serve as a potential therapeutic target for CRC, with nilotinib potentially targeting PCSK9 to exert anti-CRC effects. CONCLUSION: This study reveals a previously unknown role of nilotinib in antitumor immunity by inducing MHC-I expression in CRC cells. Our findings suggest that combining nilotinib with anti-PDL1 therapy may be an effective strategy for the treatment of CRC.