HLA-class II specificity assessed by high-density peptide microarray interactions

AbstractThe ability to predict and/or identify MHC binding peptides is an essential component of T cell epitope discovery; something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I (MHC-I) prediction tools have matured to a point where accurate selection of optimal peptide epitopes is possible for virtually all MHC-I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Since MHC-II restricted CD4+ T cells control and orchestrate most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of peptides and subsequently large bodies of peptide-MHC-II interaction data is key to the solution of this problem; a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse and representative data. Here, we have used recombinant HLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density peptide arrays, in casu containing 70,000 random peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated. Collectively, with a cost per peptide reduced to a few cents combined with the flexibility of recombinant HLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating peptide-MHC-II binding data as well as improving MHC-II prediction tools.

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CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex

Genes & Development ◽

10.1101/gad.14.9.1156 ◽

2000 ◽

Vol 14 (9) ◽

pp. 1156-1166

Author(s):

Krzysztof Masternak ◽

Annick Muhlethaler-Mottet ◽

Jean Villard ◽

Madeleine Zufferey ◽

Viktor Steimle ◽

...

Keyword(s):

Mhc Class Ii ◽

Protein Interactions ◽

Class Ii ◽

Hereditary Disease ◽

Transcriptional Coactivator ◽

Protein Protein Interactions ◽

Mhc I ◽

Mhc Ii ◽

Class Ii Mhc

By virtue of its control over major histocompatibility complex class II (MHC-II) gene expression, CIITA represents a key molecule in the regulation of adaptive immune responses. It was first identified as a factor that is defective in MHC-II deficiency, a hereditary disease characterized by the absence of MHC-II expression. CIITA is a highly regulated transactivator that governs all spatial, temporal, and quantitative aspects of MHC-II expression. It has been proposed to act as a non-DNA-binding transcriptional coactivator, but evidence that it actually functions at the level of MHC-II promoters was lacking. By means of chromatin immunoprecipitation assays, we show here for the first time that CIITA is physically associated with MHC-II, as well asHLA–DM, Ii, MHC-I, and β2mpromoters in vivo. To dissect the mechanism by which CIITA is recruited to the promoter, we have developed a DNA-dependent coimmunoprecipitation assay and a pull-down assay using immobilized promoter templates. We demonstrate that CIITA recruitment depends on multiple, synergistic protein–protein interactions with DNA-bound factors constituting the MHC-II enhanceosome. CIITA therefore represents a paradigm for a novel type of regulatory and gene-specific transcriptional cofactor.

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The MHC Class II Transactivator CIITA: Not (Quite) the Odd-One-Out Anymore among NLR Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms22031074 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1074

Author(s):

Jorge Alfonso León Machado ◽

Viktor Steimle

Keyword(s):

Gene Expression ◽

Mhc Class Ii ◽

Protein Interactions ◽

Transcriptional Regulator ◽

Class Ii ◽

Class Ii Transactivator ◽

Mhc I ◽

Mhc Ii ◽

Mhc Class Ii Transactivator

In this review, we discuss the major histocompatibility complex (MHC) class II transactivator (CIITA), which is the master regulator of MHC class II gene expression. CIITA is the founding member of the mammalian nucleotide-binding and leucine-rich-repeat (NLR) protein family but stood apart for a long time as the only transcriptional regulator. More recently, it was found that its closest homolog, NLRC5 (NLR protein caspase activation and recruitment domain (CARD)-containing 5), is a regulator of MHC-I gene expression. Both act as non-DNA-binding activators through multiple protein–protein interactions with an MHC enhanceosome complex that binds cooperatively to a highly conserved combinatorial cis-acting module. Thus, the regulation of MHC-II expression is regulated largely through the differential expression of CIITA. In addition to the well-defined role of CIITA in MHC-II GENE regulation, we will discuss several other aspects of CIITA functions, such as its role in cancer, its role as a viral restriction element contributing to intrinsic immunity, and lastly, its very recently discovered role as an inhibitor of Ebola and SARS-Cov-2 virus replication. We will briefly touch upon the recently discovered role of NLRP3 as a transcriptional regulator, which suggests that transcriptional regulation is, after all, not such an unusual feature for NLR proteins.

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Immunogenetic Variation Shapes the Gut Microbiome in a Natural Vertebrate Population

10.21203/rs.3.rs-703361/v1 ◽

2021 ◽

Author(s):

Charli S. Davies ◽

Sarah F. Worsley ◽

Kathryn H. Maher ◽

Jan Komdeur ◽

Terry Burke ◽

...

Keyword(s):

Natural Population ◽

Mhc Class Ii ◽

Gut Microbiome ◽

Host Genotype ◽

Class I Mhc ◽

Mhc I ◽

Mhc Ii ◽

Individual Fitness ◽

In The Wild ◽

Seychelles Warbler

Abstract Background: The gut microbiome (GM) can influence many biological processes in the host, impacting its health and survival, but the GM can also be influenced by the host’s traits. In vertebrates, Major Histocompatibility Complex (MHC) genes play a pivotal role in combatting pathogens and are thought to shape the host’s GM. However, despite this - and the documented importance of both GM and MHC variation to individual fitness - few studies have investigated the association between the GM and MHC in the wild. Results: We characterised MHC class I (MHC-I), MHC class II (MHC-II), and GM variation in individuals within a natural population of the Seychelles warbler, Acrocephalus sechellensis. We determined how the diversity and composition of the GM varied with MHC characteristics, in addition to environmental factors and other host intrinsic traits. Our results show that the presence of specific MHC alleles, but not MHC diversity, influences both the diversity and composition of the GM in this population. We found that MHC-I alleles, rather than MHC-II alleles, had the greatest impact on the GM. GM diversity was negatively associated with the presence of three MHC-I alleles (Ase-ua3, Ase-ua4, Ase-ua5), and one MHC-II allele (Ase-dab4), while changes in GM composition were associated with the presence of four different MHC-I alleles (Ase-ua1, Ase-ua7, Ase-ua10, Ase-ua11). GM diversity was also positively correlated with genome-wide heterozygosity and varied with host age and field period.Conclusions: These results suggest that components of the host’s immune system play a role in shaping the GM of wild animals. Host genotype – specifically, adaptive immune receptor MHC-I alleles – can modulate the GM, although whether this occurs directly, or indirectly through effects on host health is unclear. To our knowledge this is the first-time individual MHC-I variation has been shown to be associated with differences in GM characteristics in a natural population. Importantly, it suggests that host–microbiome coevolution may play a role in maintaining functional immunogenetic variation within natural vertebrate populations.

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T-cell Epitope-based Vaccine Design for Nipah Virus by Reverse Vaccinology Approach

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207323666200427114343 ◽

2020 ◽

Vol 23 (8) ◽

pp. 788-796

Author(s):

Praveen K.P. Krishnamoorthy ◽

Sekar Subasree ◽

Udhayachandran Arthi ◽

Mohammad Mobashir ◽

Chirag Gowda ◽

...

Keyword(s):

T Cell ◽

Mhc Class Ii ◽

Vaccine Development ◽

Nipah Virus ◽

3D Structure ◽

Cell Epitope ◽

Class Ii ◽

Class I ◽

Reverse Vaccinology ◽

Stable Complex

Aim and Objective: Nipah virus (NiV) is a zoonotic virus of the paramyxovirus family that sporadically breaks out from livestock and spreads in humans through breathing resulting in an indication of encephalitis syndrome. In the current study, T cell epitopes with the NiV W protein antigens were predicted. Materials and Methods: Modelling of unavailable 3D structure of W protein followed by docking studies of respective Human MHC - class I and MHC - class II alleles predicted was carried out for the highest binding rates. In the computational analysis, epitopes were assessed for immunogenicity, conservation, and toxicity analysis. T – cell-based vaccine development against NiV was screened for eight epitopes of Indian - Asian origin. Results: Two epitopes, SPVIAEHYY and LVNDGLNII, have been screened and selected for further docking study based on toxicity and conservancy analyses. These epitopes showed a significant score of -1.19 kcal/mol and 0.15 kcal/mol with HLA- B*35:03 and HLA- DRB1 * 07:03, respectively by using allele - Class I and Class II from AutoDock. These two peptides predicted by the reverse vaccinology approach are likely to induce immune response mediated by T – cells. Conclusion: Simulation using GROMACS has revealed that LVNDGLNII epitope forms a more stable complex with HLA molecule and will be useful in developing the epitope-based Nipah virus vaccine.

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NetCleave: an open-source algorithm for predicting C-terminal antigen processing for MHC-I and MHC-II

Scientific Reports ◽

10.1038/s41598-021-92632-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pep Amengual-Rigo ◽

Victor Guallar

Keyword(s):

Open Source ◽

Antigen Processing ◽

High Accuracy ◽

Biological Processes ◽

Mhc I ◽

Mhc Ii ◽

Prediction Tools ◽

Immunogenic Peptides ◽

Physicochemical Descriptors ◽

Peptide Presentation

AbstractAntigens presented on the cell surface have been subjected to multiple biological processes. Among them, C-terminal antigen processing constitutes one of the main bottlenecks of the peptide presentation pathways, as it delimits the peptidome that will be subjected downstream. Here, we present NetCleave, an open-source and retrainable algorithm for the prediction of the C-terminal antigen processing for both MHC-I and MHC-II pathways. NetCleave architecture consists of a neural network trained on 46 different physicochemical descriptors of the cleavage site amino acids. Our results demonstrate that prediction of C-terminal antigen processing achieves high accuracy on MHC-I (AUC of 0.91), while it remains challenging for MHC-II (AUC of 0.66). Moreover, we evaluated the performance of NetCleave and other prediction tools for the evaluation of four independent immunogenicity datasets (H2-Db, H2-Kb, HLA-A*02:01 and HLA-B:07:02). Overall, we demonstrate that NetCleave stands out as one of the best algorithms for the prediction of C-terminal processing, and we provide one of the first evidence that C-terminal processing predictions may help in the discovery of immunogenic peptides.

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Synaptic Clusters of MHC Class II Molecules Induced on DCs by Adhesion Molecule–mediated Initial T-Cell Scanning

Molecular Biology of the Cell ◽

10.1091/mbc.e05-01-0005 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3314-3322 ◽

Cited By ~ 54

Author(s):

Hortensia de la Fuente ◽

María Mittelbrunn ◽

Lorena Sánchez-Martín ◽

Miguel Vicente-Manzanares ◽

Amalia Lamana ◽

...

Keyword(s):

T Cells ◽

Mhc Class Ii ◽

Class Ii ◽

Regulatory Pathway ◽

Immune Synapse ◽

Mhc Ii ◽

Cytoskeleton Reorganization ◽

Adhesive Contacts ◽

Enhance Antigen Presentation ◽

Mhc Class Ii Molecules

Initial adhesive contacts between T lymphocytes and dendritic cells (DCs) facilitate recognition of peptide-MHC complexes by the TCR. In this report, we studied the dynamic behavior of adhesion and Ag receptors on DCs during initial contacts with T-cells. Adhesion molecules LFA-1- and ICAM-1,3-GFP as well as MHC class II-GFP molecules were very rapidly concentrated at the DC contact area. Binding of ICAM-3, and ICAM-1 to a lesser extent, to LFA-1 expressed by mature but not immature DC, induced MHC-II clustering into the immune synapse. Also, ICAM-3 binding to DC induced the activation of the Vav1-Rac1 axis, a regulatory pathway involved in actin cytoskeleton reorganization, which was essential for MHC-II clustering on DCs. Our results support a model in which ICAM-mediated MHC-II clustering on DC constitutes a priming mechanism to enhance antigen presentation to T-cells.

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Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

Molecular & Cellular Proteomics ◽

10.1074/mcp.m112.019232 ◽

2012 ◽

Vol 11 (11) ◽

pp. 1457-1467 ◽

Cited By ~ 12

Author(s):

Olesya Chornoguz ◽

Alexei Gapeev ◽

Michael C. O'Neill ◽

Suzanne Ostrand-Rosenberg

Keyword(s):

Breast Cancer ◽

T Cells ◽

Cancer Cells ◽

Cancer Patients ◽

Mhc Class Ii ◽

Breast Cancer Cells ◽

Class Ii ◽

Breast Cancer Patients ◽

Mhc Ii ◽

Peptide Loading

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4+ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii− cells may present peptides not presented by Ii+ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii+ cells, but will not be tolerant to novel peptides presented by Ii− cells, we generated MHC II vaccines to activate cancer patients' T cells. The vaccines are Ii− tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii+ and Ii− MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii− cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii− and Ii+ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii− cells are not presented by Ii+ cells, and are derived from source proteins not used by Ii+ cells. Peptides from Ii− cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7+ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II+ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.

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MPYS, a Novel Membrane Tetraspanner, Is Associated with Major Histocompatibility Complex Class II and Mediates Transduction of Apoptotic Signals

Molecular and Cellular Biology ◽

10.1128/mcb.00640-08 ◽

2008 ◽

Vol 28 (16) ◽

pp. 5014-5026 ◽

Cited By ~ 243

Author(s):

Lei Jin ◽

Paul M. Waterman ◽

Karen R. Jonscher ◽

Cindy M. Short ◽

Nichole A. Reisdorph ◽

...

Keyword(s):

Major Histocompatibility Complex ◽

Mhc Class Ii ◽

Cell Activation ◽

Cytoplasmic Tail ◽

Class Ii ◽

Antigenic Peptides ◽

Major Histocompatibility ◽

Mhc Ii ◽

Histocompatibility Complex ◽

Death Signals

ABSTRACT Although the best-defined function of type II major histocompatibility complex (MHC-II) is presentation of antigenic peptides to T lymphocytes, these molecules can also transduce signals leading alternatively to cell activation or apoptotic death. MHC-II is a heterodimer of two transmembrane proteins, each containing a short cytoplasmic tail that is dispensable for transduction of death signals. This suggests the function of an undefined MHC-II-associated transducer in signaling the death response. Here we describe a novel plasma membrane tetraspanner (MPYS) that is associated with MHC-II and mediates its transduction of death signals. MPYS is unusual among tetraspanners in containing an extended C-terminal cytoplasmic tail (∼140 amino acids) with multiple embedded signaling motifs. MPYS is tyrosine phosphorylated upon MHC-II aggregation and associates with inositol lipid and tyrosine phosphatases. Finally, MHC class II-mediated cell death signaling requires MPYS-dependent activation of the extracellular signal-regulated kinase signaling pathway.

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