Autoimmune hepatitis displays distinctively high multi-antennary sialylation on plasma N-glycans compared to other liver diseases.

BACKGROUND: Changes in plasma protein glycosylation are known to functionally affect proteins and to associate with liver diseases, including cirrhosis and hepatocellular carcinoma. Autoimmune hepatitis (AIH) is a liver disease characterized by liver inflammation and raised serum levels of IgG, and is difficult to distinguish from other liver diseases. The aim of this study was to examine plasma and IgG-specific N-glycosylation in AIH and compare it with healthy controls and other liver diseases. METHODS: In this cross-sectional cohort study, total plasma N-glycosylation and IgG Fc glycosylation analysis was performed by mass spectrometry for 66 AIH patients, 60 age- and sex-matched healthy controls, 31 primary biliary cholangitis patients, 10 primary sclerosing cholangitis patients, 30 non-alcoholic fatty liver disease patients and 74 patients with viral or alcoholic hepatitis. A total of 121 glycans were quantified per individual. Associations between glycosylation traits and AIH were investigated as compared to healthy controls and other liver diseases. RESULTS: Glycan traits bisection (OR: 3.78 [1.88-9.35], p-value: 5.88 × 10- 3), tetraantennary sialylation per galactose (A4GS) (OR: 2.88 [1.75-5.16], p-value: 1.63 × 10- 3), IgG1 galactosylation (OR: 0.35 [0.2-0.58], p-value: 3.47 × 10- 5) and hybrid type glycans (OR: 2.73 [1.67-4.89], p-value: 2.31 × 10- 3) were found as discriminators between AIH and healthy controls. High A4GS differentiated AIH from other liver diseases, while bisection associated with cirrhosis severity. CONCLUSIONS: Compared to other liver diseases, AIH shows distinctively high A4GS levels in plasma, with potential implications on glycoprotein function and clearance. Plasma-derived glycosylation has potential to be used as a diagnostic marker for AIH in the future. This may alleviate the need for a liver biopsy at diagnosis. Glycosidic changes should be investigated further in longitudinal studies and may be used for diagnostic and monitoring purposes in the future.

Anti-TNF therapy impairs both short- and long-term IgG responses after repeated vaccination.

BACKGROUND: Recently, it has been questioned whether vaccination of patients with inflammatory (auto)immune diseases under anti-tumor necrosis factor (TNF) treatment leads to impaired vaccine-induced immune responses and protection against breakthrough infections. However, the effects of TNF blockade on short- and long-term immune responses after repeated vaccination remain unclear. Vaccination studies have shown that initial short-term IgG antibodies (Abs) carry highly galactosylated and sialylated Fc glycans, whilst long-term IgG Abs have low levels of galactosylation and sialylation and are most likely generated by long-lived plasma cells (PCs) derived primarily from the germinal center (GC) response. Thus, IgG Fc glycosylation patterns may be applicable to distinguish short- and long-term vaccine responses after repeated vaccination under the influence of anti-TNF treatment. METHODS: We used COVID-19 vaccination as a model to investigate vaccine-induced IgG subclass levels and Fc glycosylation patterns, B cell subsets, and effector functions of short- and long-term Ab responses after up to three vaccinations in patients on anti-TNF or other immunosuppressive treatments and in healthy individuals. Using TriNetX, a global healthcare database, we determined the risk of SARS-CoV-2 breakthrough infections in vaccinated patients treated with anti-TNF or other immunosuppressive drugs. RESULTS: Anti-TNF treatment reduced the long-term abundance of all anti-S IgG subclasses with low levels of galactosylation and sialylation. Re-activation of potential memory B cells initially generated highly galactosylated and sialylated IgG antibodies, which were progressively reduced after each booster dose in anti-TNF-treated patients, especially in the elderly. The reduced short- and long-term IgG (1) levels in anti-TNF-treated patients correlated with diminished functional activity and an increased risk for the development of COVID-19. CONCLUSIONS: The data suggest that anti-TNF treatment reduces both GC-dependent long-lived PCs and GC-dependent memory B cell-derived short-lived PCs, hence both the long- and short-term IgG subclass responses, respectively, after repeated vaccination. We propose that anti-TNF therapy, especially in the elderly, reduces the benefit of booster vaccination.

Antibody glycosylation in COVID-19.

Antibody glycosylation has received considerable attention in coronavirus disease 2019 (COVID-19) infections and recently also in vaccination. Antibody glycosylation and in particular immunoglobulin G1 fucosylation levels influence effector functions and are therefore key parameters for assessing the efficacy and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directed immune responses. This review article summarizes and interprets recent research into antibody glycosylation in COVID-19. Experimental approaches for analyzing the glycosylation of SARS-CoV-2-directed antibody responses are evaluated. The pronounced dynamics, effector functions, clinical utility, and regulation of antibody glycosylation in COVID-19 are assessed. Future research on the role of antibody glycosylation in COVID may cover the glycosylation of other antibody classes beyond immunoglobulin G, the regulation of antibody glycosylation, and the role of non-canonical antibody receptors in determining effector functions.

The structure and role of lactone intermediates in linkage-specific sialic acid derivatization reactions.

Sialic acids occur ubiquitously throughout vertebrate glycomes and often endcap glycans in either α2,3- or α2,6-linkage with diverse biological roles. Linkage-specific sialic acid characterization is increasingly performed by mass spectrometry, aided by differential sialic acid derivatization to discriminate between linkage isomers. Typically, during the first step of such derivatization reactions, in the presence of a carboxyl group activator and a catalyst, α2,3-linked sialic acids condense with the subterminal monosaccharides to form lactones, while α2,6-linked sialic acids form amide or ester derivatives. In a second step, the lactones are converted into amide derivatives. Notably, the structure and role of the lactone intermediates in the reported reactions remained ambiguous, leaving it unclear to which extent the amidation of α2,3-linked sialic acids depended on direct aminolysis of the lactone, rather than lactone hydrolysis and subsequent amidation. In this report, we used mass spectrometry to unravel the role of the lactone intermediate in the amidation of α2,3-linked sialic acids by applying controlled reaction conditions on simple and complex glycan standards. The results unambiguously show that in common sialic acid derivatization protocols prior lactone formation is a prerequisite for the efficient, linkage-specific amidation of α2,3-linked sialic acids, which proceeds predominantly via direct aminolysis. Furthermore, nuclear magnetic resonance spectroscopy confirmed that exclusively the C2 lactone intermediate is formed on a sialyllactose standard. These insights allow a more rationalized method development for linkage-specific sialic derivatization in the future.

Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization.

The human glycome is characterized by a high degree of sialylation, affecting, amongst others, cell-cell interactions and protein half-life. An established method for the linkage isomer-specific characterization of N-glycan sialylation is based on the linkage-specific derivatization of sialylated glycoconjugates, inducing ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked sialic acids. While the carboxylic acid activator and nucleophile used in this reaction received extensive investigation, the role of the catalyst was never thoroughly explored. A frequently used catalyst for the linkage-specific esterification of sialic acids is 1-hydroxybenzotriazole (HOBt). Here, a systematic evaluation was performed of five HOBt alternatives in combination with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in ethanol for the linkage-specific derivatization of sialic acids. Derivatized glycans were analyzed by MALDI-TOF-MS and the catalyst performance was evaluated based on the completeness of the reactions and the linkage-specificity obtained. The use of both 6-Cl-HOBt and 6-CF3-HOBt resulted in high linkage-specificity and minimal byproduct formation, similar to the benchmark method using HOBt. Performing the reaction with these catalysts at neutral or acidic pH showed comparable efficiencies on both sialyllactose and complex-type N-glycans. The reported investigations resulted in an expansion of the reaction space for linkage-specific sialic acid derivatization.

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection.

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low-level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.

The Human Blood N-Glycome: Unraveling Disease Glycosylation Patterns.

Most of the proteins in the circulation are N-glycosylated, shaping together the total blood N-glycome (TBNG). Glycosylation is known to affect protein function, stability, and clearance. The TBNG is influenced by genetic, environmental, and metabolic factors, in part epigenetically imprinted, and responds to a variety of bioactive signals including cytokines and hormones. Accordingly, physiological and pathological events are reflected in distinct TBNG signatures. Here, we assess the specificity of the emerging disease-associated TBNG signatures with respect to a number of key glycosylation motifs including antennarity, linkage-specific sialylation, fucosylation, as well as expression of complex, hybrid-type and oligomannosidic N-glycans, and show perplexing complexity of the glycomic dimension of the studied diseases. Perspectives are given regarding the protein- and site-specific analysis of N-glycosylation, and the dissection of underlying regulatory layers and functional roles of blood protein N-glycosylation.

Total plasma N-glycomic signature of SARS-CoV-2 infection.

Total plasma protein N-glycosylation (TPNG) changes are a hallmark of many diseases. Here, we analyzed the TPNG of 169 COVID-19 patients and 12 healthy controls, using mass spectrometry, resulting in the relative quantification of 85 N-glycans. We found a COVID-19 N-glycomic signature, with 59 glycans differing between patients and controls, many of them additionally differentiating between severe and mild COVID-19. Tri- and tetra-antennary N-glycans were increased in patients, showing additionally elevated levels of antennary α2,6-sialylation. Conversely, bisection of di-antennary, core-fucosylated, nonsialylated glycans was low in COVID-19, particularly in severe cases, potentially driven by the previously observed low levels of bisection on antibodies of severely diseased COVID-19 patients. These glycomic changes point toward systemic changes in the blood glycoproteome, particularly involvement of acute-phase proteins, immunoglobulins and the complement cascade. Further research is needed to dissect glycosylation changes in a protein- and site-specific way to obtain specific functional leads.

Comparative analysis of spike-specific IgG Fc glycoprofiles elicited by adenoviral, mRNA, and protein-based SARS-CoV-2 vaccines.

IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.

The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylated IgG1 in naive but not in antigen-experienced vaccinees.

BACKGROUND: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced FcγRIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. METHODS: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). FINDINGS: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen-experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen-experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. INTERPRETATION: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. FUNDING: LSBR1721, 1908; ZonMW10430012010021, 09150161910033, 10430012010008; DFG398859914, 400912066, 390884018; PMI; DOI4-Nr. 3; H2020-MSCA-ITN 721815.

Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation.

Antibody-mediated rejection (AMR) is the leading cause of graft failure. While donor-specific antibodies (DSAs) are associated with a higher risk of AMR, not all patients with DSAs develop rejection, suggesting that the characteristics of alloantibodies determining their pathogenicity remain undefined. Using human leukocyte antigen (HLA)-A2-specific antibodies as a model, we apply systems serology tools to investigate qualitative features of immunoglobulin G (IgG) alloantibodies including Fc-glycosylation patterns and FcγR-binding properties. Levels of afucosylated anti-A2 antibodies are elevated in seropositive patients, especially those with AMR, suggesting potential cytotoxicity via FcγRIII-mediated mechanisms. Afucosylation of both glycoengineered monoclonal and naturally glycovariant polyclonal serum IgG specific to HLA-A2 drives potentiated binding to, slower dissociation from, and enhanced signaling through FcγRIII, a receptor widely expressed on innate effector cells, and greater cytotoxicity against HLA-A2+ cells mediated by natural killer (NK) cells. Collectively, these results suggest that afucosylated DSA may be a biomarker of AMR and contribute to pathogenesis.

The IgG glycome of SARS-CoV-2 infected individuals reflects disease course and severity.

Immunoglobulin G (IgG) antibodies play an important role in the immune response against viruses such as SARS-CoV-2. As the effector functions of IgG are modulated by N-glycosylation of the Fc region, the structure and possible function of the IgG N-glycome has been under investigation in relation to divergent COVID-19 disease courses. Through LC-MS analysis we studied both total IgG1 and spike protein-specific IgG1 Fc glycosylation of 129 German and 163 Brazilian COVID-19 patients representing diverse patient populations. We found that hospitalized COVID-19 patients displayed decreased levels of total IgG1 bisection and galactosylation and lowered anti-S IgG1 fucosylation and bisection as compared to mild outpatients. Anti-S IgG1 glycosylation was dynamic over the disease course and both anti-S and total IgG1 glycosylation were correlated to inflammatory markers. Further research is needed to dissect the possible role of altered IgG glycosylation profiles in (dys)regulating the immune response in COVID-19.

mRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine.

Background: The new types of mRNA-containing lipid nanoparticle vaccines BNT162b2 and mRNA-1273 and the adenovirus-based vaccine AZD1222 were developed against SARS-CoV-2 and code for its spike (S) protein. Several studies have investigated short-term antibody (Ab) responses after vaccination. Objective: However, the impact of these new vaccine formats with unclear effects on the long-term Ab response - including isotype, subclass, and their type of Fc glycosylation - is less explored. Methods: Here, we analyzed anti-S Ab responses in blood serum and the saliva of SARS-CoV-2 naïve and non-hospitalized pre-infected subjects upon two vaccinations with different mRNA- and adenovirus-based vaccine combinations up to day 270. Results: We show that the initially high mRNA vaccine-induced blood and salivary anti-S IgG levels, particularly IgG1, markedly decrease over time and approach the lower levels induced with the adenovirus-based vaccine. All three vaccines induced, contrary to the short-term anti-S IgG1 response with high sialylation and galactosylation levels, a long-term anti-S IgG1 response that was characterized by low sialylation and galactosylation with the latter being even below the corresponding total IgG1 galactosylation level. Instead, the mRNA, but not the adenovirus-based vaccines induced long-term IgG4 responses - the IgG subclass with inhibitory effector functions. Furthermore, salivary anti-S IgA levels were lower and decreased faster in naïve as compared to pre-infected vaccinees. Predictively, age correlated with lower long-term anti-S IgG titers for the mRNA vaccines. Furthermore, higher total IgG1 galactosylation, sialylation, and bisection levels correlated with higher long-term anti-S IgG1 sialylation, galactosylation, and bisection levels, respectively, for all vaccine combinations. Conclusion: In summary, the study suggests a comparable "adjuvant" potential of the newly developed vaccines on the anti-S IgG Fc glycosylation, as reflected in relatively low long-term anti-S IgG1 galactosylation levels generated by the long-lived plasma cell pool, whose induction might be driven by a recently described TH1-driven B cell response for all three vaccines. Instead, repeated immunization of naïve individuals with the mRNA vaccines increased the proportion of the IgG4 subclass over time which might influence the long-term Ab effector functions. Taken together, these data shed light on these novel vaccine formats and might have potential implications for their long-term efficacy.

Altered Fc glycosylation of anti-HLA alloantibodies in hemato-oncological patients receiving platelet transfusions.

BACKGROUND: The formation of alloantibodies directed against class I human leukocyte antigens (HLA) continues to be a clinically challenging complication after platelet transfusions, which can lead to platelet refractoriness (PR) and occurs in approximately 5%-15% of patients with chronic platelet support. Interestingly, anti-HLA IgG levels in alloimmunized patients do not seem to predict PR, suggesting functional or qualitative differences among anti-HLA IgG. The binding of these alloantibodies to donor platelets can result in rapid clearance after transfusion, presumably via FcγR-mediated phagocytosis and/or complement activation, which both are affected by the IgG-Fc glycosylation. OBJECTIVES: To characterize the Fc glycosylation profile of anti-HLA class I antibodies formed after platelet transfusion and to investigate its effect on clinical outcome. PATIENTS/METHODS: We screened and captured anti-HLA class I antibodies (anti-HLA A2, anti-HLA A24, and anti-HLA B7) developed after platelet transfusions in hemato-oncology patients, who were included in the PREPAReS Trial. Using liquid chromatography-mass spectrometry, we analyzed the glycosylation profiles of total and anti-HLA IgG1 developed over time. Subsequently, the glycosylation data was linked to the patients' clinical information and posttransfusion increments. RESULTS: The glycosylation profile of anti-HLA antibodies was highly variable between patients. In general, Fc galactosylation and sialylation levels were elevated compared to total plasma IgG, which correlated negatively with the platelet count increment. Furthermore, high levels of afucosylation were observed for two patients. CONCLUSIONS: These differences in composition of anti-HLA Fc-glycosylation profiles could potentially explain the variation in clinical severity between patients.

Immunoglobulin G1 Fc glycosylation as an early hallmark of severe COVID-19.

BACKGROUND: Immunoglobulin G1 (IgG1) effector functions are impacted by the structure of fragment crystallizable (Fc) tail-linked N-glycans. Low fucosylation levels on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-specific IgG1 has been described as a hallmark of severe coronavirus disease 2019 (COVID-19) and may lead to activation of macrophages via immune complexes thereby promoting inflammatory responses, altogether suggesting involvement of IgG1 Fc glycosylation modulated immune mechanisms in COVID-19. METHODS: In this prospective, observational single center cohort study, IgG1 Fc glycosylation was analyzed by liquid chromatography-mass spectrometry following affinity capturing from serial plasma samples of 159 SARS-CoV-2 infected hospitalized patients. FINDINGS: At baseline close to disease onset, anti-S IgG1 glycosylation was highly skewed when compared to total plasma IgG1. A rapid, general reduction in glycosylation skewing was observed during the disease course. Low anti-S IgG1 galactosylation and sialylation as well as high bisection were early hallmarks of disease severity, whilst high galactosylation and sialylation and low bisection were found in patients with low disease severity. In line with these observations, anti-S IgG1 glycosylation correlated with various inflammatory markers. INTERPRETATION: Association of low galactosylation, sialylation as well as high bisection with disease severity and inflammatory markers suggests that further studies are needed to understand how anti-S IgG1 glycosylation may contribute to disease mechanism and to evaluate its biomarker potential. FUNDING: This project received funding from the European Commission's Horizon2020 research and innovation program for H2020-MSCA-ITN IMforFUTURE, under grant agreement number 721815, and supported by Crowdfunding Wake Up To Corona, organized by the Leiden University Fund.

Profiling the proteoforms of urinary prostate-specific antigen by capillary electrophoresis - mass spectrometry.

Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients' urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA. SIGNIFICANCE: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and non-cleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies.

Recombinant human monoclonal HLA antibodies of different IgG subclasses recognising the same epitope: Excellent tools to study differential effects of donor-specific antibodies.

In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.