PURPOSE:The purpose of this study was to examine the reactivity of T cells from children with paediatric uveitis to the complete Epstein-Barr virus (EBV) EBNA-1 antigen and to the EBNA-1 AA 397-408 epitope in relation to the class II HLA-DRB1*15:01 genetic profile. METHODS:We used ELISPOT to measure the IFN-γ T cell response of eleven patients with paediatric non-infectious uveitis and three control patients to a peptide pool containing the complete EBV EBNA-1 antigen and to the EBV EBNA-1 AA 397-408 peptide motif. Next-generation sequencing data or, when unavailable, TaqMan assay was used to categorize patients based on HLA-DRB1*15:01 profile. RESULT:Overall, 9/11 (81.8%) of uveitis patients had a positive T cell response against the EBV EBNA-1 pool. Of the five HLA-DRB1*15:01 positive uveitis and control patients, four (80%) had a positive T cell response to the EBV EBNA-1 pool. No statistically significant differences were seen in number of spot forming units per 106 cells against the EBV EBNA-1 pool between HLA-DRB1*15:01 positive compared to HLA-DRB1*15:01 negative uveitis patients. One patient with intermediate uveitis who was homozygous positive for HLA-DRB1*15:01 had a positive T cell response to the EBNA-1 AA 397-408 peptide motif condition. CONCLUSION:In this exploratory study, we found that the majority of uveitis patients, regardless of HLA-DRB1*15:01 genetic background, had a measurable T cell response against the complete EBV EBNA-1 antigen. A positive T cell response against the EBNA-1 AA 397-408 epitope specifically was measured in one uveitis patient; a homozygote HLA-DRB1*15:01 positive patient with intermediate uveitis.

Manipulation of the activity of Endoplasmic Reticulum AminoPeptidase 1 (ERAP1) has emerged as a powerful tool for the modulation of the cellular antigenicity and concomitant adaptive immune responses, with potential applications in cancer immunotherapy and Major Histocompatibility class (MHC)-associated autoimmunity being explored. Small molecule inhibitors often target allosteric sites of ERAP1 to achieve superior selectivity. However, recent findings suggested that ERAP1 functional perturbation may also affect cellular metabolism, raising concerns on the safety of this approach. To address this, we treated a melanoma cancer cell line with near saturating doses of an allosteric ERAP1 inhibitor and compared metabolite homeostasis to untreated and knock-out cells. Analysis of 8625 detected metabolite features revealed non-significant changes in metabolite composition in both inhibitor concentrations tested and only small changes in the ERAP1 knockout (KO) cells, limited to up to 25 metabolite features, most notably choline. Our results suggest that pharmacological inhibition of ERAP1 that targets allosteric sites, does not induce sufficiently large metabolic shifts to alter metabolite homeostasis. This is distinct from genetic deletion of the protein, which induces additional effects, albeit limited. These results encourage the exploration of allosteric ERAP1 inhibitors as pharmacological agents for the modulation of immune responses in the clinic.

The Endoplasmic Reticulum AminoPeptidase 2 ( ERAP2 ) gene encodes an aminopeptidase involved in antigenic peptide processing for the MHC-I pathway. Genetic variants in the ERAP2 gene are associated with autoimmune conditions and infectious diseases. The linkage between genetic variants in the ERAP2 gene has given rise to the prevailing assumption that a single ERAP2 allotype with invariant amino acid sequence accounts for all immunological functions of ERAP2. Here, we show by analyzing exon-sequencing data from 160,000 individuals that 15 missense amino acid variants across the ERAP2 gene result in an array of different protein allotypes that are maintained in the European population. These allotypes can be divided into three haplotype groups, defined by the genotypes of two common splice-altering variants. We found novel associations between newly identified protein allotypes and immune-mediated diseases, suggesting that ERAP2 functional variation modifies disease susceptibility at the population level. An MHC class I antigen presentation assay revealed that disease-associated ERAP2 allotypes differ in their capacity to generate antigenic peptides for MHC-I presentation, resulting in differential activation of an antigen-specific T-cell receptor compared to non-disease-associated allotypes. These findings provide strong evidence that ERAP2 function is allotype-dependent and demonstrate that ERAP2 diversity shapes MHC-I antigen presentation and T-cell immunity. The ERAP2 enzyme modulates adaptive immunity and plays a role in autoimmunity, infection, and cancer. The authors discovered that a variety of protein allotypes of ERAP2 are maintained in the human population. Allotypes that increase disease risk for autoimmune and cardiovascular conditions are functionally distinct in their capacity to activate T-cells. The results of this study demonstrate that ERAP2 is a functionally diverse immune modulator that contributes to immune variation and influences susceptibility to immune-mediated diseases.

Abstract Age-related macular degeneration (AMD) shows substantial clinical heterogeneity that remains unexplained despite extensive genetic and clinical characterization. We evaluated whether proteomic stratification could provide insight beyond clinical phenotype and genetic risk. We performed 384-plex plasma proteomics in a cohort of 215 individuals, including patients with early and late neovascular AMD, other complement-associated retinal diseases, and age-matched controls. Proteome-based reclassification identified four disease-overarching clusters. Neovascular AMD cases were partitioned almost exclusively between two clusters (30/36). Early AMD cases were predominantly assigned to one of these clusters (10/18), whereas only two localized to the other (2/18). Both AMD-associated clusters shared elevated levels of a protein module enriched for lipoprotein-related functions compared to the other clusters. However, the cluster containing both early and neovascular AMD cases showed higher levels of additional protein modules enriched for complement pathways and cellular stress-response pathways compared with the other AMD-associated cluster. Importantly, this molecular divergence in neovascular AMD could not be explained by genetic predisposition (i.e., 52-variant AMD genetic risk score), signatures of biological ageing, nor by other clinical features. Together, these findings support two proteomic endotypes of neovascular AMD with distinct involvement of cellular stress pathways.

Purpose To compare the plasma metabolic profile of patients with a CRB1-associated inherited retinal degeneration (CRB1-IRD) with healthy controls (HCs). Design A case-control study. Participants A cohort of 30 Dutch CRB1-associated IRD patients and 29 Dutch healthy controls. Methods The MxP® Quant 500 Kit was used for measuring metabolite concentrations. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in μM (μmol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls. Main Outcome Measures Plasma concentration of 619 metabolites. Results Over-representation of pathways among metabolites associated strongest to CRB1-IRDs (P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes. Conclusions Lipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1-IRD.

Purpose:To determine the profile of inflammation-related proteins and complement system factors in the plasma of CRB1-associated inherited retinal dystrophies (CRB1-IRDs). Methods:We used the Olink Explore 384 Inflammation II panel for targeted proteomics in 30 cases and 29 controls (cohort I) to identify immune pathways involved in CRB1-IRDs. Genotyping was performed in cohort I and a second cohort of 123 patients from 14 countries and 1292 controls (cohort II). Results:A significant shift in complement cascade factors was observed in plasma proteomes of CRB1-IRD patients (enrichment for complement cascade, Padj = 3.03 × 10-15). We detected higher plasma levels of complement factor I and complement factor H [CFH] (q = 0.008 and q = 0.046, respectively, adjusted for age and sex), inhibitors of complement component 3 (C3), which correlated significantly (Pearson's coefficient >0.6) with elevated levels of C3 (q = 0.064). The CRB1 missense variants frequently found in patients showed a strong linkage disequilibrium with the common CFH variant rs7535263 (D' = 0.97 for p.(Cys948Tyr); D' = 1.0 for p.(Arg764Cys)), known to be linked with altered plasma CFH-related protein levels. Correction for the CFH genotype revealed significantly elevated plasma levels of CFH-related 2 (CFHR2) in CRB1-IRD patients (q = 0.041). Conclusions:CRB1-IRDs are characterized by changes in plasma levels of complement factors and proteins of the innate immune system, and linkage between CRB1 and CFH genes implicates functional variants of the CFH-CFHR locus with specific pathogenic variants of CRB1.

BACKGROUND:Non-infectious uveitis is an immune-mediated disease characterized by vision-threatening inflammation within the eye. Increasing evidence indicates that microbial agents promote non-infectious uveitis, but the natural history of immune responses to pathogens in patients remains unexplored. We determined intraocular antibodies against pathogens in paediatric uveitis. METHODS:We used peptide microarrays containing 3760 linear B-cell epitopes from 196 human pathogens to profile IgG levels in eye fluid biopsies and paired serum samples from 18 Dutch paediatric patients and 6 age-matched controls. We compared intensities of single epitopes and clusters based on overlapping amino acid sequence of peptides. Next-generation sequencing data was obtained to determine the HLA-DRB1∗15:01 genotype. FINDINGS:Intraocular antibody profiles largely matched serum profiles and were characterized by high IgG against the conserved PALTAVET-motif of enterovirus family members, as well as broad epitope reactivity against Epstein-Barr virus (EBV). The aqueous humour of patients showed elevated levels of antibodies against peptides containing the RRPFFHPV-motif of Epstein-Barr Virus Nuclear Antigen 1 [EBNA-1]. Antibody levels against the RRPFFHPV-motif of EBNA1 were significantly higher in individuals that carry the HLA-DRB1∗15:01 risk allele of paediatric uveitis. INTERPRETATION:Intraocular antibodies against an immunogenic epitope of EBV showed an association with paediatric uveitis, particularly HLA-DRB1∗15:01 positive uveitis, indicating a potential link between EBV-specific immune responses and autoimmune uveitis. FUNDING:Funding for this research was received from Fischer Stichting (UZ2022-3), ODAS (2021-02), LSBS and ANVVB.

Age-related macular degeneration (AMD) remains a leading cause of vision loss in the geriatric population. There are age-related changes in peripheral blood leukocyte composition, but their significance for AMD remains unclear. We aimed to determine changes in immune cell populations in the blood of AMD patients. A standardized 31-parameter flow cytometry analysis was conducted on peripheral blood mononuclear cells from 59 patients with early and advanced AMD and 39 controls without AMD, all older than 65 years. Fundus photography and optical coherence tomography were used to classify disease stages and a custom genotype array was used to compute an AMD genetic risk score based on 52 AMD disease risk variants (GRS-52). A generalized linear regression model corrected for age, sex, and smoking status revealed that AMD patients showed decreased frequencies of CD4+ T helper cell population expressing Integrin Alpha E (CD103) (Padj = 0.019). We further noted that early AMD was characterized by increased interleukin-4 (IL-4)-producing CD4+ T helper cells (Padj = 0.013; <0.001), as well as IL-4-producing cytotoxic CD8+ T cells (Padj = 0.016; <0.001). Reclassification of samples based on the GRS-52 revealed that IL-17-producing T cells decreased incrementally across GRS-52 categories. In AMD, alterations in peripheral blood leukocyte populations are associated with genetic risk score and disease stage and include specifically IL-4 and IL-17A cytokine-producing and CD103 integrin-expressing T cell populations.

Single-nucleotide polymorphisms (SNPs) near the ERAP2 gene are associated with various autoimmune conditions, as well as protection against lethal infections. Due to high linkage disequilibrium, numerous trait-associated SNPs are correlated with ERAP2 expression; however, their functional mechanisms remain unidentified. We show by reciprocal allelic replacement that ERAP2 expression is directly controlled by the splice region variant rs2248374. However, disease-associated variants in the downstream LNPEP gene promoter are independently associated with ERAP2 expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between the gene promoters of LNPEP and ERAP2 and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the SNPs in the LNPEP promoter by reference sequences lowered ERAP2 expression. These findings show that multiple SNPs act in concert to regulate ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

Purpose To compare the plasma metabolic profile of patients with a CRB1 -associated inherited retinal degeneration ( CRB1 -IRD) with healthy controls (HCs).Design A case-control study.Methods Plasma concentration of 619 metabolites was measured with the MxP® Quant 500 Kit in 30 patients with a CRB1 -IRD and 29 HCs. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in µM (µmol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls.Results Over-representation of pathways among metabolites associated strongest to CRB1 -IRDs ( P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes.Conclusions Lipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1 -IRD.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementBartimeus Foundation### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This study has the approval of the Institutional Review Board of the University Medical Center Utrecht (UMCU)I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Purpose: Nonanterior uveitis frequently involves the retinal vasculature; however, no molecular markers associated with the retinal vascular disease are currently known. In this study, we aimed to identify serum biomarker signatures associated with retinal vascular involvement in noninfectious pediatric uveitis. Methods: We performed a 384-plex targeted proteomic analysis of serum samples of 154 noninfectious pediatric uveitis patients diagnosed with nonanterior uveitis ( n = 74), idiopathic chronic anterior uveitis (iCAU, n = 36), or juvenile idiopathic arthritis- associated uveitis (JIA-U, n = 44), as well as 22 noninflammatory pediatric controls. Data on retinal vascular involvement (i.e., papillitis, cystoid macular edema, retinal vasculitis, or retinal capillary leakage on optical coherence tomography and/or fluorescein angiography) were used to stratify cases in the nonanterior uveitis group. Results: In the analysis of nonanterior uveitis, we identified nine proteins significantly associated with retinal vascular involvement, including F13B, MYOM3, and PTPN9. These proteins were enriched through pathway enrichment analysis for the coagulation cascade. Comparing cases and controls, we identified 63 differentially expressed proteins, notably proteins involved in platelet biology and complement cascades, which could be primarily attributed to differences in serum proteomes between anterior uveitis and nonanterior uveitis groups. Conclusions: Serum proteins related to the coagulation and complement cascade are associated with retinal vascular involvement in pediatric uveitis patients. Our results indicate involvement of mediators that could interact with the microcirculation in pediatric uveitis and might serve as potential biomarkers in personalized medicine in the future. Translational Relevance: Our targeted proteomics analysis in serum of pediatric uveitis patients indicates involvement of mediators that could interact with the microcirculation in pediatric uveitis and might serve as potential biomarkers in personalized medicine in the future.

Birdshot chorioretinopathy is an inflammatory eye condition strongly associated with MHC-I allele HLA-A29. The striking association with MHC-I suggests involvement of T cells, whereas natural killer (NK) cell involvement remains largely unstudied. Here we show that HLA-A29-positive birdshot chorioretinopathy patients have a skewed NK cell pool containing expanded CD16 positive NK cells which produce more proinflammatory cytokines. These NK cells contain populations that express CD8A which is involved in MHC-I recognition on target cells, display gene signatures indicative of high cytotoxic activity (GZMB, PRF1 and ISG15), and signaling through NK cell receptor CD244 (SH2D1B). Long-term monitoring of a cohort of birdshot chorioretinopathy patients with active disease identifies a population of CD8bright CD244bright NK cells, which rapidly declines to normal levels upon clinical remission following successful treatment. Collectively, these studies implicate CD8bright CD244bright NK cells in birdshot chorioretinopathy.

Aims: To analyse expression and genotype of Krüppel‐Like Factor 5 (KLF5) gene in the corneal epithelium of eyes with Keratoconus (KC) compared to normal eyes (controls); as well as, to determine the effect of several pro‐inflammatory cytokines on the KLF5 expression by an in vitro study. Methods: 64 corneal epithelium samples from 53 KC and 11 controls were included in this study. KC samples were collected from patients who received crosslinking epi‐off or penetrating keratoplasty for their mild to advanced KC. Control samples were collected from dead donors with healthy corneas. Molecular determinations to measure KLF5 expression and genotype on these samples were performed respectively by qPCR and TaqMan targeting the SNP of interest (rs17285550). In addition, using a cell line of healthy corneal epithelial cells, incubations with increasing concentrations of interleukin 6 (IL6), interleukin 1 beta (IL1β), and tumour necrosis factor alpha (TNFα) were performed to determine the KLF5 response to a pro‐inflammatory environment. KLF5 expression in the stimulated cell cultures was assessed by qPCR analysis. Results: KC samples evidenced significantly higher levels of KLF5 gene expression than the controls ( p < 0.0001). The GG genotypes showed an upward tendency for higher expression of KLF5 in the KC group. Higher IL6, IL1β, and TNFα concentrations contribute to a significatively KLF5 overexpression in corneal epithelial cell cultures. Conclusions: Overexpression of KLF5 in the corneal epithelium of KC patients and its upregulation against a proinflammatory microenvironment, could lead to an inadequate epithelial barrier function that destabilizes the surrounding corneal layers and contributes to the KC pathology. KLF5 represents a promising target for gene therapy in KC.

ER aminopeptidase 1 (ERAP1) is an ER-resident aminopeptidase that excises N-terminal residues of peptides that then bind onto Major Histocompatibility Complex I molecules (MHC-I) and indirectly modulates adaptive immune responses. ERAP1 contains an allosteric regulatory site that accommodates the C-terminus of at least some peptide substrates, raising questions about its exact influence on antigen presentation and the potential of allosteric inhibition for cancer immunotherapy. We used an inhibitor that targets this regulatory site to study its effect on the immunopeptidome of a human cancer cell line. The immunopeptidomes of allosterically inhibited and ERAP1 KO cells contain high-affinity peptides with sequence motifs consistent with the cellular HLA class I haplotypes but are strikingly different in peptide composition. Compared to KO cells, allosteric inhibition did not affect the length distribution of peptides and skewed the peptide repertoire both in terms of sequence motifs and HLA allele utilization, indicating significant mechanistic differences between the two ways of disrupting ERAP1 function. These findings suggest that the regulatory site of ERAP1 plays distinct roles in antigenic peptide selection, which should be taken into consideration when designing therapeutic interventions targeting the cancer immunopeptidome.

The Black Death, a notorious devastating pandemic caused by Yersinia pestis infection during the 14th century, posed a formidable challenge to human immune defenses. A new article by Klunk et al. reports that a variant in an antigen-processing gene may have favored survival during the plague and may have undergone genomic selection in Europeans at unprecedented speed.

Background:Type I interferons (IFNs) promote the expansion of subsets of CD1c+ conventional dendritic cells (CD1c+ DCs), but the molecular basis of CD1c+ DCs involvement in conditions not associated without elevated type I IFNs remains unclear.Methods:We analyzed CD1c+ DCs from two cohorts of non-infectious uveitis patients and healthy donors using RNA-sequencing followed by high-dimensional flow cytometry to characterize the CD1c+ DC populations.Results:We report that the CD1c+ DCs pool from patients with non-infectious uveitis is skewed toward a gene module with the chemokine receptor CX3CR1 as the key hub gene. We confirmed these results in an independent case–control cohort and show that the disease-associated gene module is not mediated by type I IFNs. An analysis of peripheral blood using flow cytometry revealed that CX3CR1+ DC3s were diminished, whereas CX3CR1− DC3s were not. Stimulated CX3CR1+ DC3s secrete high levels of inflammatory cytokines, including TNF-alpha, and CX3CR1+ DC3 like cells can be detected in inflamed eyes of patients.Conclusions:These results show that CX3CR1+ DC3s are implicated in non-infectious uveitis and can secrete proinflammatory mediators implicated in its pathophysiology.Funding:The presented work is supported by UitZicht (project number #2014-4, #2019-10, and #2021-4). The funders had no role in the design, execution, interpretation, or writing of the study.

AbstractSingle nucleotide polymorphisms (SNP) near theERAP2gene are associated with autoimmune conditions such asCrohn’s disease, andbirdshot chorioretinopathy, as well as protection against lethal infections, including theBlack Death. Due to high linkage disequilibrium (LD), a great number of trait-associated SNPs are correlated withERAP2expression, however their functional mechanisms remain unidentified. We used genome editing and functional genomics to identify causal variants that remain obscured by LD. We demonstrate by reciprocal allelic replacement thatERAP2expression is directly controlled by the genotype of splice region SNP rs2248374. However, we demonstrate that autoimmune disease-risk SNPs located near the downstreamLNPEPgene promoter are independently associated withERAP2expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between theLNPEPpromoter region and theERAP2promoter and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the disease-associated SNPs in theLNPEPpromoter by reference sequences loweredERAP2expression. These findings show that clustered GWAS signals associated with diverse autoimmune conditions and lethal infections act in concert to control ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

Background: HLA-A29-positive birdshot chorioretinitis (BCR) is an inflammatory eye disorder that is generally assumed to be caused by an autoimmune response to HLA-A29-presented peptides from retinal arrestin (SAG), yet the epitopes recognized by CD8+ T cells from patients remain to be identified. Objectives: The identification of natural ligands of SAG presented by HLA-A29. To quantify CD8+ T cells reactive to antigenic SAG peptides presented by HLA-A29 in patients and controls.Methods: We performed mass-spectrometry based immunopeptidomics of HLA-A29 of antigen-presenting cell lines from patients engineered to express SAG. MHC-I Dextramer technology was utilised to determine expansion of antigen-specific CD8+ T cells reactive to SAG peptides in complex with HLA-A29 in a cohort of BCR patients, HLA-A29-positive controls, and HLA-A29-negative controls.Results: We report on the naturally presented antigenic SAG peptides identified by sequencing the HLA-A29 immunopeptidome of antigen-presenting cells of patients. We show that the N-terminally extended SAG peptide precursors can be trimmed in vitro by the antigen-processing aminopeptidases ERAP1 and ERAP2. Unex-pectedly, no enhanced antigen engagement by CD8+ T cells upon stimulation with SAG peptides was observed in patients or HLA-A29-positive controls. Multiplexed HLA-A29-peptide dextramer profiling of a case-control cohort revealed that CD8+ T cells specific for these SAG peptides were neither detectable in peripheral blood nor in eye biopsies of patients.Conclusions: Collectively, these findings demonstrate that SAG is not a CD8+ T cell autoantigen and sharply contrast the paradigm in the pathogenesis of BCR. Therefore, the mechanism by which HLA-A29 is associated with BCR does not involve SAG.