Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells

Bailey, A.G.; Nicholas, Ben; Darley, Rachel; Parkinson, Erika; Teo, Ying; Aleksić, Maja; Maxwell, Gavin; Elliott, Tim; Ardern‐Jones, Michael R.; Skipp, Paul

doi:10.1093/toxsci/kfaa184

Cited by 12 publications

(15 citation statements)

References 89 publications

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“…Protein-A sepharose beads (Repligen, Waltham, Mass. USA) were covalently conjugated to 10 mg/mL W6/32 (pan-anti-HLA-I) or 5 mg/mL HB145 (pan-anti-HLA-II) monoclonal antibodies (SAL Scientific, Hampshire, UK) using DMP as previously described [ 53 ]. Snap frozen tissue samples were briefly thawed and weighed prior to 30 S of mechanical homogenization using a 150W handheld mechanical homogeniser with disposable probes (Thermo Fisher Scientific) in 4 mL lysis buffer (0.02M Tris, 0.5% (w/v) IGEPAL, 0.25% (w/v) sodium deoxycholate, 0.15mM NaCl, 1mM EDTA, 0.2mM iodoacetamide supplemented with EDTA-free protease inhibitor mix).…”

Section: Methodsmentioning

confidence: 99%

Immunopeptidomic analysis of influenza A virus infected human tissues identifies internal proteins as a rich source of HLA ligands

Nicholas

Bailey²,

Staples³

et al. 2022

PLoS Pathog

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CD8+ and CD4+ T cells provide cell-mediated cross-protection against multiple influenza strains by recognising epitopes bound as peptides to human leukocyte antigen (HLA) class I and -II molecules respectively. Two challenges in identifying the immunodominant epitopes needed to generate a universal T cell influenza vaccine are: A lack of cell models susceptible to influenza infection which present population-prevalent HLA allotypes, and an absence of a reliable in-vitro method of identifying class II HLA peptides. Here we present a mass spectrometry-based proteomics strategy for identifying viral peptides derived from the A/H3N2/X31 and A/H3N2/Wisconsin/67/2005 strains of influenza. We compared the HLA-I and -II immunopeptidomes presented by ex-vivo influenza challenged human lung tissues. We then compared these with directly infected immortalised macrophage-like cell line (THP1) and primary dendritic cells fed apoptotic influenza-infected respiratory epithelial cells. In each of the three experimental conditions we identified novel influenza class I and II HLA peptides with motifs specific for the host allotype. Ex-vivo infected lung tissues yielded few class-II HLA peptides despite significant numbers of alveolar macrophages, including directly infected ones, present within the tissues. THP1 cells presented HLA-I viral peptides derived predominantly from internal proteins. Primary dendritic cells presented predominantly viral envelope-derived HLA class II peptides following phagocytosis of apoptotic infected cells. The most frequent viral source protein for HLA-I and -II was matrix 1 protein (M1). This work confirms that internal influenza proteins, particularly M1, are a rich source of CD4+ and CD8+ T cell epitopes. Moreover, we demonstrate the utility of two ex-vivo fully human infection models which enable direct HLA-I and -II immunopeptide identification without significant viral tropism limitations. Application of this epitope discovery strategy in a clinical setting will provide more certainty in rational vaccine design against influenza and other emergent viruses.

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Section: Methodsmentioning

confidence: 99%

Immunopeptidomic analysis of influenza A virus infected human tissues identifies internal proteins as a rich source of HLA ligands

Nicholas

Bailey²,

Staples³

et al. 2022

PLoS Pathog

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“…Hence, epidermal responses to hapten encounter, e.g., by keratinocytes and dendritic epidermal T cells (DETC) [ 259 , 264 , 275 ], seem to precede MC activation. In vitro, the formation and internalization of hapten–protein complexes by human keratinocytes has been shown to result in neoepitope formation [ 276 , 277 , 278 ]. While in vivo, the sequence of events is less understood, epidermal stress responses include inflammasome activation and release of IL-1β and IL-18 [ 279 , 280 ], production of reactive oxygen species (ROS) [ 281 ] and alarmins, e.g., ATP [ 282 , 283 ] and IL-33 [ 284 , 285 , 286 , 287 ].…”

Section: Mast Cell Contribution To Inflammatory Skin Disordersmentioning

confidence: 99%

Mast Cells in the Skin: Defenders of Integrity or Offenders in Inflammation?

Voß

Kotrba

Gaffal

et al. 2021

IJMS

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Mast cells (MCs) are best-known as key effector cells of immediate-type allergic reactions that may even culminate in life-threatening anaphylactic shock syndromes. However, strategically positioned at the host–environment interfaces and equipped with a plethora of receptors, MCs also play an important role in the first-line defense against pathogens. Their main characteristic, the huge amount of preformed proinflammatory mediators embedded in secretory granules, allows for a rapid response and initiation of further immune effector cell recruitment. The same mechanism, however, may account for detrimental overshooting responses. MCs are not only detrimental in MC-driven diseases but also responsible for disease exacerbation in other inflammatory disorders. Focusing on the skin as the largest immune organ, we herein review both beneficial and detrimental functions of skin MCs, from skin barrier integrity via host defense mechanisms to MC-driven inflammatory skin disorders. Moreover, we emphasize the importance of IgE-independent pathways of MC activation and their role in sustained chronic skin inflammation and disease exacerbation.

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“…Homogenates were clarified for 10 min at 2,000g, 4°C and then for a further 60 min at 13,500g, 4°C. 2 mg of anti-MHC-I mouse monoclonal antibodies (W6/32) covalently conjugated to Protein A sepharose (Repligen) using DMP as previously described [42,43] were added to the clarified supernatants and incubated with constant agitation for 2 h at 4°C. The captured MHC-I/□~2~m/immunopeptide complex on the beads was washed sequentially with 10 column volumes of low (isotonic, 0.15M NaCl) and high (hypertonic, 0.4M NaCl) TBS washes prior to elution in 10% acetic acid and dried under vacuum.…”

Section: Methodsmentioning

confidence: 99%

Identification of neoantigens in esophageal adenocarcinoma

Nicholas

Bailey

McCann

et al. 2022

Preprint

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Esophageal adenocarcinoma (EAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumor mutations, presented to cytotoxic T-cells by human leukocyte antigen molecules (HLA). Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumors from a cohort of seven patients with EAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

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Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells

Cited by 12 publications

References 89 publications

Immunopeptidomic analysis of influenza A virus infected human tissues identifies internal proteins as a rich source of HLA ligands

Immunopeptidomic analysis of influenza A virus infected human tissues identifies internal proteins as a rich source of HLA ligands

Mast Cells in the Skin: Defenders of Integrity or Offenders in Inflammation?

Identification of neoantigens in esophageal adenocarcinoma

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