Keratinocyte-derived small extracellular vesicles supply antigens for CD1a-resticted T cells and promote their type 2 bias in the context of filaggrin insufficiency.

Introduction: Exosome-enriched small extracellular vesicles (sEVs) are nanosized organelles known to participate in long distance communication between cells, including in the skin. Atopic dermatitis (AD) is a chronic inflammatory skin disease for which filaggrin (FLG) gene mutations are the strongest genetic risk factor. Filaggrin insufficiency affects multiple cellular function, but it is unclear if sEV-mediated cellular communication originating from the affected keratinocytes is also altered, and if this influences peptide and lipid antigen presentation to T cells in the skin. Methods: Available mRNA and protein expression datasets from filaggrin-insufficient keratinocytes (shFLG), organotypic models and AD skin were used for gene ontology analysis with FunRich tool. sEVs secreted by shFLG and control shC cells were isolated from conditioned media by differential centrifugation. Mass spectrometry was carried out for lipidomic and proteomic profiling of the cells and sEVs. T cell responses to protein, peptide, CD1a lipid antigens, as well as phospholipase A2-digested or intact sEVs were measured by ELISpot and ELISA. Results: Data analysis revealed extensive remodeling of the sEV compartment in filaggrin insufficient keratinocytes, 3D models and the AD skin. Lipidomic profiles of shFLGsEV showed a reduction in the long chain (LCFAs) and polyunsaturated fatty acids (PUFAs; permissive CD1a ligands) and increased content of the bulky headgroup sphingolipids (non-permissive ligands). This resulted in a reduction of CD1a-mediated interferon-γ T cell responses to the lipids liberated from shFLG-generated sEVs in comparison to those induced by sEVs from control cells, and an increase in interleukin 13 secretion. The altered sEV lipidome reflected a generalized alteration in the cellular lipidome in filaggrin-insufficient cells and the skin of AD patients, resulting from a downregulation of key enzymes implicated in fatty acid elongation and desaturation, i.e., enzymes of the ACSL, ELOVL and FADS family. Discussion: We determined that sEVs constitute a source of antigens suitable for CD1a-mediated presentation to T cells. Lipids enclosed within the sEVs secreted on the background of filaggrin insufficiency contribute to allergic inflammation by reducing type 1 responses and inducing a type 2 bias from CD1a-restricted T cells, thus likely perpetuating allergic inflammation in the skin.

DIA-MS proteome analysis of formalin-fixed paraffin-embedded glioblastoma tissues.

Developments in quantitative proteomics and data-independent acquisition (DIA) methodology is enabling quantification of proteins in biological samples. Currently, there are a few reports on DIA mass spectrometry (MS) approaches for proteome analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, to facilitate detection and quantification of immune- and glioblastoma (GBM)-relevant proteins from FFPE patient materials, we established a simple and precise DIA-MS workflow. We first evaluated different lysis buffers for their efficiency in protein extractions from FFPE GBM tissues. Our results showed that more than 1700 proteins were detected and over 1400 proteins were quantified from GBM FFPE tissue microdissections. GBM-relevant proteins (e.g., GFAP, FN1, VIM, and MBP) were quantified with high precision (median coefficient of variation <12%). In addition, immune-related proteins (e.g., ILF2, MIF, and CD38) were consistently detected and quantified. The strategy holds great potential for routinizing protein quantification in FFPE tissue samples.

Structural determinants of peptide-dependent TAP1-TAP2 transit passage targeted by viral proteins and altered by cancer-associated mutations.

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8-10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform.

Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant technical gap of top-down proteomics is the inability to analyze a low amount of biological samples, which limits its access to isolated rare cells, fine needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (nanodroplet processing in one pot for trace samples), into a top-down proteomic workflow. A unique combination of a nonionic detergent dodecyl-ß-d-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ∼170 to ∼620 proteoforms from ∼70 to ∼770 HeLa cells containing ∼10 to ∼115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical research, particularly where clinical specimens are not available in amounts amenable to standard workflows.

Mass Spectrometry-Based Identification of MHC-Associated Peptides.

Neoantigen-based immunotherapies promise to improve patient outcomes over the current standard of care. However, detecting these cancer-specific antigens is one of the significant challenges in the field of mass spectrometry. Even though the first sequencing of the immunopeptides was done decades ago, today there is still a diversity of the protocols used for neoantigen isolation from the cell surface. This heterogeneity makes it difficult to compare results between the laboratories and the studies. Isolation of the neoantigens from the cell surface is usually done by mild acid elution (MAE) or immunoprecipitation (IP) protocol. However, limited amounts of the neoantigens present on the cell surface impose a challenge and require instrumentation with enough sensitivity and accuracy for their detection. Detecting these neopeptides from small amounts of available patient tissue limits the scope of most of the studies to cell cultures. Here, we summarize protocols for the extraction and identification of the major histocompatibility complex (MHC) class I and II peptides. We aimed to evaluate existing methods in terms of the appropriateness of the isolation procedure, as well as instrumental parameters used for neoantigen detection. We also focus on the amount of the material used in the protocols as the critical factor to consider when analyzing neoantigens. Beyond experimental aspects, there are numerous readily available proteomics suits/tools applicable for neoantigen discovery; however, experimental validation is still necessary for neoantigen characterization.

Proteome analysis of tissues by mass spectrometry.

Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.

The effect of epidermal levels of urocanic acid on 25-hydroxyvitamin D synthesis and inflammatory mediators upon narrowband UVB irradiation.

BACKGROUND/PURPOSE: Urocanic acid (UCA) absorbs ultraviolet (UV)B radiation in the epidermis which may interfere with phototherapy. Therefore, the influence of individual levels of UCA on immune reactivity and vitamin D synthesis induced by narrowband UVB radiation was assessed. METHODS: Twenty-eight subjects with irritant contact dermatitis of the hands were irradiated with suberythemal doses of narrowband UVB radiation on their unaffected lower forearms on three consecutive days. Stratum corneum tape strips and epidermal interstitial fluid (ISF) as well as blood samples were analyzed. RESULTS: Narrowband UVB irradiation led to the conversion of trans-UCA into its cis-isomer in the epidermis. The observed increase in 25-hydroxyvitamin D serum concentrations was inversely correlated with the baseline levels of trans-UCA. Furthermore, UVB irradiation induced significant changes in the levels of CXCL10/IP-10, CCL2/MCP-1, CCL4/MIP-1ß, and the IL-1RA/IL-1α ratio. The levels of IL-1α and CXCL9/MIG showed a trend toward increase. The changes in the levels of inflammatory and immunomodulatory mediators did not depend on baseline levels of trans-UCA. CONCLUSION: The results suggest that epidermal levels of trans-UCA affect vitamin D synthesis, but not cutaneous immune reactivity upon repeated exposure to suberythemal doses of narrowband UVB radiation. However, this requires further exploration.

Barrier function and natural moisturizing factor levels after cumulative exposure to a fruit-derived organic acid and a detergent: different outcomes in atopic and healthy skin and relevance for occupational contact dermatitis in the food industry.

BACKGROUND: Fruit-derived organic compounds and detergents are relevant exposure factors for occupational contact dermatitis in the food industry. Although individuals with atopic dermatitis (AD) are at risk for development of occupational contact dermatitis, there have been no controlled studies on the effects of repeated exposure to multiple irritants, relevant for the food industry, in atopic skin. OBJECTIVES: The aim of the study was to investigate the outcomes of repeated exposure to a fruit-derived organic acid and a detergent in AD compared to healthy volunteers. METHODS: The volunteers were exposed to 2.0% acetic acid (AcA) and/or 0.5% sodium lauryl sulfate (SLS) in controlled tandem repeated irritation test. The outcomes were assessed by measurements of erythema, transepidermal water loss (TEWL) and natural moisturizing factor (NMF) levels. RESULTS: In the AD volunteers, repeated AcA exposure led to barrier disruption and significant TEWL increase; no significant differences after the same exposure in the healthy controls were found. Repeated exposure to SLS and the irritant tandems enhanced the reactions and resulted in a significantly higher increase in TEWL in the AD compared to the control group. Cumulative irritant exposure reduced the NMF levels in both groups. CONCLUSIONS: Differences in the severity of irritant-induced barrier impairment in atopic individuals contribute to the risk for occupational contact dermatitis in result of multiple exposures to food-derived irritants and detergents.