ERAP1 and ERAP2 Haplotypes Influence Suboptimal HLA-B*27:05-Restricted Anti-Viral CD8+ T Cell Responses Cross-Reactive to Self-Epitopes.

The human leukocyte antigen (HLA)-B*27 family of alleles is strongly associated with ankylosing spondylitis (AS), a chronic inflammatory disorder affecting the axial and peripheral joints, yet some HLA-B*27 variants not associated with AS have been shown. Since no major differences in the ligandome of associated compared to not-associated alleles have emerged, a plausible hypothesis is that the quantity rather than the quality of the presented epitopes makes the difference. In addition, the Endoplasmic Reticulum AminoPeptidases (ERAPs) 1 and 2, playing a crucial role in shaping the HLA class I epitopes, act as strong AS susceptibility factors, suggesting that an altered peptidome might be responsible for the activation of pathogenic CD8+ T cells. In this context, we have previously singled out a B*27:05-restricted CD8+ T cell response against pEBNA3A (RPPIFIRRL), an EBV peptide lacking the B*27 classic binding motif. Here, we show that a specific ERAP1/2 haplotype negatively correlates with such response in B*27:05 subjects. Moreover, we prove that the B*27:05 allele successfully presents peptides with the same suboptimal N-terminal RP motif, including the self-peptide, pDYNEIN (RPPIFGDFL). Overall, this study underscores the cooperation between the HLA-B*27 and ERAP1/2 allelic variants in defining CD8+ T cell reactivity to suboptimal viral and self-B*27 peptides and prompts further investigation of the B*27:05 peptidome composition.

Human Papillomavirus and Oral Lesions: What is the Best Diagnostic Method?

ABSTRACT: Oropharyngeal squamous cell carcinoma is the most common neoplasm of head and neck cancers related to the presence of human papillomavirus (HPV). in the dental, maxillofacial and ENT fields, the finding of mediated HPV lesions is quite common. The diagnostic techniques currently available are different and can be more or less invasive depending on the type of lesion and the need for the clinician. In this study, two clinical cases subjected to a double diagnostic technique were considered in order to exclude any possible risk of false negatives. The polymerase chain reaction (PCR) technique showed a lower sensitivity or in any case dictated by a limited number of HPV strains analyzed. Histological examination nowadays turns out to be the best diagnostic method despite requiring a surgical phase.

CD8+ T Cell Senescence: Lights and Shadows in Viral Infections, Autoimmune Disorders and Cancer.

CD8+ T lymphocytes are a heterogeneous class of cells that play a crucial role in the adaptive immune response against pathogens and cancer. During their lifetime, they acquire cytotoxic functions to ensure the clearance of infected or transformed cells and, in addition, they turn into memory lymphocytes, thus providing a long-term protection. During ageing, the thymic involution causes a reduction of circulating T cells and an enrichment of memory cells, partially explaining the lowering of the response towards novel antigens with implications in vaccine efficacy. Moreover, the persistent stimulation by several antigens throughout life favors the switching of CD8+ T cells towards a senescent phenotype contributing to a low-grade inflammation that is a major component of several ageing-related diseases. In genetically predisposed young people, an immunological stress caused by viral infections (e.g., HIV, CMV, SARS-CoV-2), autoimmune disorders or tumor microenvironment (TME) could mimic the ageing status with the consequent acceleration of T cell senescence. This, in turn, exacerbates the inflamed conditions with dramatic effects on the clinical progression of the disease. A better characterization of the phenotype as well as the functions of senescent CD8+ T cells can be pivotal to prevent age-related diseases, to improve vaccine strategies and, possibly, immunotherapies in autoimmune diseases and cancer.

The Impact of the 'Mis-Peptidome' on HLA Class I-Mediated Diseases: Contribution of ERAP1 and ERAP2 and Effects on the Immune Response.

The strong association with the Major Histocompatibility Complex (MHC) class I genes represents a shared trait for a group of autoimmune/autoinflammatory disorders having in common immunopathogenetic basis as well as clinical features. Accordingly, the main risk factors for Ankylosing Spondylitis (AS), prototype of the Spondyloarthropathies (SpA), the Behçet's disease (BD), the Psoriasis (Ps) and the Birdshot Chorioretinopathy (BSCR) are HLA-B*27, HLA-B*51, HLA-C*06:02 and HLA-A*29:02, respectively. Despite the strength of the association, the HLA pathogenetic role in these diseases is far from being thoroughly understood. Furthermore, Genome-Wide Association Studies (GWAS) have highlighted other important susceptibility factors such as Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and, less frequently, ERAP2 that refine the peptidome presented by HLA class I molecules to CD8+ T cells. Mass spectrometry analysis provided considerable knowledge of HLA-B*27, HLA-B*51, HLA-C*06:02 and HLA-A*29:02 immunopeptidome. However, the combined effect of several ERAP1 and ERAP2 allelic variants could generate an altered pool of peptides accounting for the "mis-immunopeptidome" that ranges from suboptimal to pathogenetic/harmful peptides able to induce non-canonical or autoreactive CD8+ T responses, activation of NK cells and/or garbling the classical functions of the HLA class I molecules. This review will focus on this class of epitopes as possible elicitors of atypical/harmful immune responses which can contribute to the pathogenesis of chronic inflammatory diseases.

The rs75862629 minor allele in the endoplasmic reticulum aminopeptidases intergenic region affects human leucocyte antigen B27 expression and protects from ankylosing spondylitis in Sardinia.

OBJECTIVES: HLA-B27 and the endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 genes are predisposing factors for AS. A single nucleotide polymorphism (SNP) in the ERAP2 promoter (rs75862629) coordinates the transcription of both ERAP genes. We investigated whether this SNP associates with AS and whether it affects the expression of the two major HLA-B27 alleles present in Sardinia, the AS-associated B*2705 and the non-AS-associated B*2709. METHODS: Four SNPs in the ERAP region were genotyped in HLA-B*2705-positive patients with AS (n = 145), B27-positive healthy subjects (n = 126) and B27-negative controls (n = 250) and the allele and haplotype frequencies were derived. The expression of ERAP1 and ERAP2 mRNAs in 36 HLA-B27-positive B lymphoblastoid cell lines was measured by quantitative PCR. An electrophoretic mobility shift assay was performed to search for a nuclear factor binding the DNA sequence encompassing rs75862629. The expression of HLA-B27 molecules related to the SNP at rs75862629 was determined by flow cytometry. RESULTS: The minor allele G at rs75862629 was found significantly increased in B27 healthy individuals, both B*2705 and B*2709, compared with B*2705-positive patients with AS and B27-negative controls. The electrophoretic mobility shift assay indicated the lack of binding of a transcription factor as the cause of the observed reduction in the ERAP2 concomitant with a higher ERAP1 expression. Of note, this occurs with a different cell surface expression of the HLA-B*2705 and HLA-B*2709 molecules. CONCLUSION: SNP rs75862629, by modulating simultaneously the expression of ERAP1 and ERAP2, provides protection from AS in HLA-B27-positive subjects in Sardinia. This has a functional impact on HLA-B27 expression and likely on disease onset.

The Peptide Repertoire of HLA-B27 may include Ligands with Lysine at P2 Anchor Position.

The HLA-B*27 peptidome has drawn significant attention due to the genetic association between some of the HLA-B*27 alleles and the inflammatory rheumatic disease ankylosing spondylitis (AS), for which a comprehensive biological explanation is still lacking. This study aims to expand the known limits of the HLA-B*27 peptidome to facilitate selection and testing of new peptides, possibly involved in the disease. The HLA peptidomes of HeLa and C1R cell lines stably transfected with the AS-associated HLA-B*27:05 allele, the nonassociated HLA-B*27:09 allele, or their cysteine 67 to serine mutants (C67S), are analyzed on a very large scale. In addition, the peptidomes of HLA-B*27:05 and HLA-B*27:05-C67S are analyzed from the spleens of rats transgenic for these alleles. The results indicate that C67S mutation increases the percentage of peptides with glutamine or lysine at their P2 position (P2-Lys), in both HLA-B*27:05 and HLA-B*27:09. Furthermore, a small fraction of HLA-B*27 peptides contains lysine at their second position (P2), in addition to the more commonly found peptides with arginine (P2-Arg) or the less common glutamine (P2-Gln) located at this anchor position. Overall these data indicate that peptides with P2-Lys should be considered as real ligands of HLA-B*27 molecules and taken into account while looking for putative peptides implicated in the AS.

The Ankylosing Spondylitis-associated HLA-B*2705 presents a B*0702-restricted EBV epitope and sustains the clonal amplification of cytotoxic T cells in patients.

HLA-B*27 is strongly associated with an inflammatory autoimmune disorder, the Ankylosing Spondylitis (AS) and plays a protective role in viral infections. The two aspects might be linked. In this work, we compared in B*2705/B*07 positive patients with AS, the CD8+ T cell responses to two immunodominant EBV-derived epitopes restricted for either the HLA-B*27 (pEBNA3C) or the HLA-B*07 (pEBNA3A). We have unexpectedly found that the HLA-B*07-restricted EBNA3A peptide is presented by both the B*0702 and the B*2705 but not by the non AS-associated B*2709, that differs from the AS-associated B*2705 for a single amino acid in the peptide-binding groove (His116Asp). We then analysed 38 B*2705-positive/B*07-negative (31 AS-patients and 7 healthy donors) and 8 B*2709-positive/B*07-negative subjects. EBNA3A-specific CD8+ T lymphocytes were present in 55.3% of the HLA-B*2705 but in none of the B*2709 donors (p=0.0049). TCR ß-chain analysis identified common TCRBV and TCRBJ gene segments and shared CDR3ß sequences in pEBNA3A-responsive CTLs of B*2705 carriers, suggesting the existence of a shared TCR repertoire for recognition of the uncanonical B*2705/pEBNA3A complex. These data highlight the plasticity of the AS-associated HLA-B*2705, which presents peptides with suboptimal binding motifs, possibly contributing both to its enhanced capacity to protect against pathogens and to predispose to autoimmunity.

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

Characterization of a proteasome and TAP-independent presentation of intracellular epitopes by HLA-B27 molecules.

Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

Corrigendum: Bivalent binding of staphylococcal superantigens to the TCR and CD28 triggers inflammatory signals independently of antigen presenting cells.

[This corrects the article DOI: 10.3389/fimmu.2023.1170821.].

Bivalent binding of staphylococcal superantigens to the TCR and CD28 triggers inflammatory signals independently of antigen presenting cells.

Staphylococcus aureus superantigens (SAgs) such as staphylococcal enterotoxin A (SEA) and B (SEB) are potent toxins stimulating T cells to produce high levels of inflammatory cytokines, thus causing toxic shock and sepsis. Here we used a recently released artificial intelligence-based algorithm to better elucidate the interaction between staphylococcal SAgs and their ligands on T cells, the TCR and CD28. The obtained computational models together with functional data show that SEB and SEA are able to bind to the TCR and CD28 stimulating T cells to activate inflammatory signals independently of MHC class II- and B7-expressing antigen presenting cells. These data reveal a novel mode of action of staphylococcal SAgs. By binding to the TCR and CD28 in a bivalent way, staphylococcal SAgs trigger both the early and late signalling events, which lead to massive inflammatory cytokine secretion.

Astrocytes and Inflammatory T Helper Cells: A Dangerous Liaison in Multiple Sclerosis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder of the central nervous system (CNS) characterized by the recruitment of self-reactive T lymphocytes, mainly inflammatory T helper (Th) cell subsets. Once recruited within the CNS, inflammatory Th cells produce several inflammatory cytokines and chemokines that activate resident glial cells, thus contributing to the breakdown of blood-brain barrier (BBB), demyelination and axonal loss. Astrocytes are recognized as key players of MS immunopathology, which respond to Th cell-defining cytokines by acquiring a reactive phenotype that amplify neuroinflammation into the CNS and contribute to MS progression. In this review, we summarize current knowledge of the astrocytic changes and behaviour in both MS and experimental autoimmune encephalomyelitis (EAE), and the contribution of pathogenic Th1, Th17 and Th1-like Th17 cell subsets, and CD8+ T cells to the morphological and functional modifications occurring in astrocytes and their pathological outcomes.

The emerging multifunctional roles of ERAP1, ERAP2 and IRAP between antigen processing and renin-angiotensin system modulation.

The Endoplasmic Reticulum Aminopeptidase 1 and 2 (ERAP1 and ERAP2) and Insulin Regulated Aminopeptidase (IRAP) are three M1 zinc metalloproteases whose role in antigen processing is the refining of peptidome either in the Endoplasmic reticulum (ERAP1 and ERAP2), or in the endosomes (IRAP). However, other novel and distinct functions are emerging. Here, we focus specifically on ERAP2. This gene has a peculiar evolutionary history, being absent in rodents and undergoing in humans to a balanced selection of two haplotypes, one of which not expressing the full length ERAP2. These observations suggest that its role in antigen presentation is not essential. An additional, less investigated role is in the regulation of the Renin Angiotensin System (RAS). ERAP1 and ERAP2 cleave Angiotensin II (Ang II) into Ang III and IV, which counteract the action of Ang II whereas IRAP is itself the receptor for Ang IV. We have recently reported that macrophages, independently from the haplotype, express and release a N-terminus ERAP2 "short" form which directly binds IRAP and the two molecules are co-expressed in the endosomes and on the cell membrane. This new evidence suggests that the maintenance of the ERAP2 gene in humans could be due to its activity in the regulation of the RAS system, possibly as an Ang IV agonist. Its role in the immune-mediated diseases as well as in disorders more specifically related to an imbalance of the RAS system, including hypertension, pre-eclampsia but also viral infections such as COVID-19, is discussed here.

The Double Game Played by Th17 Cells in Infection: Host Defense and Immunopathology.

T-helper 17 (Th17) cells represent a subpopulation of CD4+ T lymphocytes that play an essential role in defense against pathogens. Th17 cells are distinguished from Th1 and Th2 cells by their ability to produce members of the interleukin-17 (IL-17) family, namely IL-17A and IL-17F. IL-17 in turn induces several target cells to synthesize and release cytokines, chemokines, and metalloproteinases, thereby amplifying the inflammatory cascade. Th17 cells reside predominantly in the lamina propria of the mucosa. Their main physiological function is to maintain the integrity of the mucosal barrier against the aggression of infectious agents. However, in an appropriate inflammatory microenvironment, Th17 cells can transform into immunopathogenic cells, giving rise to inflammatory and autoimmune diseases. This review aims to analyze the complex mechanisms through which the interaction between Th17 and pathogens can be on the one hand favorable to the host by protecting it from infectious agents, and on the other hand harmful, potentially generating autoimmune reactions and tissue damage.

SARS-CoV-2 Spike Does Not Possess Intrinsic Superantigen-like Inflammatory Activity.

Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory disease occurring several weeks after SARS-CoV-2 infection. The clinical similarities between MIS-C and the toxic shock syndrome, together with the preferential expansion of T cells with a T-cell receptor variable ß chain (TCRVß) skewing, suggested a superantigen theory of MIS-C. For instance, recent in silico modelling evidenced the presence of a highly conserved motif within SARS-CoV-2 spike protein similar in structure to the superantigenic fragment of staphylococcal enterotoxin B (SEB). However, experimental data on the superantigenic activity of the SARS-CoV-2 spike have not yet been provided. Here, we assessed the superantigenic activity of the SARS-CoV-2 spike by analysing inflammatory cytokine production in both Jurkat cells and the peripheral blood CD4+ T cells stimulated with the SARS-CoV-2 spike or SEB as a control. We found that, unlike SEB, the SARS-CoV-2 spike does not exhibit an intrinsic superantigen-like activity.

The First Identification in Italy of SARS-CoV-2 Omicron BA.4 Harboring KSF141_del: A Genomic Comparison with Omicron Sub-Variants.

The rapid emergence and worldwide detection of the SARS-CoV-2 Omicron variant underscore the importance of robust genomic surveillance systems and prompt information sharing among global public health partners. The Omicron variant has rapidly replaced the Delta variant as a dominating SARS-CoV-2 variant because of natural selection, favoring the variant with higher infectivity and stronger vaccine breakthrough capability. The Omicron variant is also known as B.1.1.529. It has four sub-variants, indicated as BA.1, BA.2, BA.3 and BA.4. Among them, BA.1 is the currently prevailing sub-variant, and BA.2 has been found to be able to alarmingly re-infect patients initially infected by Omicron BA.1. The BA.3 sub-variant is a combination of mutations of BA.1 and BA.2, especially in the spike protein. Today, the BA.4 variant is emerging, which is herein described, and it was the first detected in Italy. Via bioinformatic analysis, we are reporting that the BA.4 that was identified harbors a new mutation, specifically a deletion in the ORF1ab gene, corresponding to KSF141_del in non-structural protein 1 (nsp1), a critical virulence factor able to suppress host translation. The bioinformatics comparison analysis with the other three sub-variants reveals that the deletion was not present before and was never reported until now. Therefore, we can speculate that Omicron BA.4 will become a new dominating "variant of concern" and may also break vaccine protection. Moreover, we show that other proteins are mutated in the BA.4. In particular, seven mutations are recognized in the nucleocapsid (N) protein, and the capability of five different types of rapid antigenic tests are used to identify it.

Binding of Staphylococcal Enterotoxin B (SEB) to B7 Receptors Triggers TCR- and CD28-Mediated Inflammatory Signals in the Absence of MHC Class II Molecules.

The inflammatory activity of staphylococcal enterotoxin B (SEB) relies on its capacity to trigger polyclonal T-cell activation by binding both T-cell receptor (TCR) and costimulatory receptor CD28 on T cells and MHC class II and B7 molecules on antigen presenting cells (APC). Previous studies highlighted that SEB may bind TCR and CD28 molecules independently of MHC class II, yet the relative contribution of these interactions to the pro-inflammatory function of SEB remained unclear. Here, we show that binding to MHC class II is dispensable for the inflammatory activity of SEB, whereas binding to TCR, CD28 and B7 molecules is pivotal, in both human primary T cells and Jurkat T cell lines. In particular, our finding is that binding of SEB to B7 molecules suffices to trigger both TCR- and CD28-mediated inflammatory signalling. We also provide evidence that, by strengthening the interaction between CD28 and B7, SEB favours the recruitment of the TCR into the immunological synapse, thus inducing lethal inflammatory signalling.

Human Papilloma Virus: Current Knowledge and Focus on Oral Health.

The human papilloma virus (HPV) is responsible for different pathological manifestations in humans. This agent gives rise to lesions of different types and in different areas of the organism, including the oral cavity. The aim of this study is to show which are the main diseases for which HPV is responsible and to bring to light some of the interceptive and therapeutic strategies. The analysis was conducted by consulting the major scientific databases with the aim of obtaining information on the characteristics of oral HPV and its management; furthermore, the literature was supported by some clinical cases proposed by the authors. The role of dentistry is essential in the early diagnosis of this type of pathologies and above all in knowing how to direct patients towards a path that can lead to patient management, especially in the event that these lesions have a malignant potential. Enhancing the knowledge and role of dentistry can lead to early diagnosis of this type of injury, intercepting a pathology that could have multiorgan implications.

Dual, HLA-B27 subtype-dependent conformation of a self-peptide.

The products of the human leukocyte antigen subtypes HLA-B*2705 and HLA-B*2709 differ only in residue 116 (Asp vs. His) within the peptide binding groove but are differentially associated with the autoimmune disease ankylosing spondylitis (AS); HLA-B*2705 occurs in AS-patients, whereas HLA-B*2709 does not. The subtypes also generate differential T cell repertoires as exemplified by distinct T cell responses against the self-peptide pVIPR (RRKWRRWHL). The crystal structures described here show that pVIPR binds in an unprecedented dual conformation only to HLA-B*2705 molecules. In one binding mode, peptide pArg5 forms a salt bridge to Asp116, connected with drastically different interactions between peptide and heavy chain, contrasting with the second, conventional conformation, which is exclusively found in the case of B*2709. These subtype-dependent differences in pVIPR binding link the emergence of dissimilar T cell repertoires in individuals with HLA-B*2705 or HLA-B*2709 to the buried Asp116/His116 polymorphism and provide novel insights into peptide presentation by major histocompatibility antigens.

Cardiovascular risk factors, nonalcoholic fatty liver disease, and carotid artery intima-media thickness in an adolescent population in southern Italy.

The objective of this study was to determine, in an adolescent population, the prevalence of nonalcoholic fatty liver disease (NAFLD) and the association of NAFLD and cardiovascular risk factors with carotid artery intima-media thickness (IMT), a marker of subclinical atherosclerosis. The authors conducted a population-based study among 642 randomly selected adolescents aged 11-13 years in Reggio Calabria, southern Italy, between November 2007 and October 2008. Prevalences of overweight and obesity were 30.5% and 13.5%, respectively. The overall prevalence of NAFLD was 12.5%, increasing to 23.0% in overweight/obese adolescents. In univariate analysis, increased IMT was positively associated with the presence of NAFLD, body mass index (BMI), waist circumference, systolic blood pressure (all P's < 0.001), diastolic blood pressure (P = 0.006), gamma-glutamyl transpeptidase (P = 0.006), alanine aminotransferase (P = 0.007), and C-reactive protein (P = 0.008) and was inversely associated with high density lipoprotein cholesterol (P < 0.001). In multivariate analysis, NAFLD (P = 0.002), BMI (P = 0.004), waist circumference (P = 0.003), and systolic blood pressure (P = 0.005) retained significant associations. The authors conclude that NAFLD, BMI, waist circumference, and systolic blood pressure are independent markers of increased IMT in a random sample of adolescents.