ERAP1 Expression Research Articles

The effect of rs2910686 on ERAP2 expression in IBD and epithelial inflammatory response

BackgroundERAP2 is an aminopeptidase involved in antigen processing and presentation, and harbor genetic variants linked to several inflammatory diseases such as Inflammatory Bowel Disease (IBD). The lack of an ERAP2 gene homologue in mice has hampered functional studies, and most human studies have focused on cells of hematopoietic origin. Using an IBD biobank as vantage point, this study explores how genetic variation in ERAP2 affects gene expression in human-derived epithelial organoids upon proinflammatory stimulation.MethodsAn IBD patient cohort was genotyped with regards to two single nucleotide polymorphisms (SNP) (rs2910686/rs2248374) associated with ERAP2 expression levels, and we examined the correlation between colon gene expression and genotype, specifically aiming to establish a relationship with ERAP2 expression proficiency. Human-derived colon organoids (colonoids) with known ERAP2 genotype were established and used to explore differences in whole genome gene expression between ERAP2-deficient (n = 4) and -proficient (n = 4) donors upon pro-inflammatory encounter.ResultsWhen taking rs2910686 genotype into account, ERAP2 gene expression is upregulated in the inflamed colon of IBD patients. Colonoids upregulate ERAP2 upon IFNɣ stimulation, and ERAP2 expression proficiency is dependent on rs2910686 genotype. Colonoid genotyping confirms that mechanisms independent of the frequently studied SNP rs2248374 can cause ERAP2-deficiency. A total of 586 genes involved in various molecular mechanisms are differentially expressed between ERAP2 proficient- and deficient colonoids upon proinflammatory stimulation, including genes encoding proteins with the following molecular function: catalytic activity (AOC1, CPE, ANPEP and MEP1A), regulator activity (TNFSF9, MDK, GDF15, ILR6A, LGALS3 and FLNA), transmembrane transporter activity (SLC40A1 and SLC5A1), and extracellular matrix structural constituents (FGL2, HMCN2, and MUC17).ConclusionsERAP2 is upregulated in the inflamed IBD colon mucosa, and expression proficiency is highly correlated with genotype of rs2910686. While the SNP rs2248374 is commonly used to determine ERAP2 expressional proficiency, our data confirms that mechanisms independent of this SNP can lead to ERAP2 deficiency. Our data demonstrates that epithelial ERAP2 presence affects the inflammatory response in colonoids, suggesting a pleiotropic role of ERAP2 beyond MHC class I antigen processing.

P1197 ERAP2 genetic variant rs2248374 is linked to paradoxical skin reactions induced by TNF-alpha inhibitors in Romanian patients with Inflammatory Bowel Diseases

Abstract Background The endoplasmic reticulum aminopeptidases (ERAPs) are responsible for trimming protein residues to optimal length for major histocompatibility complex class-1 mediated antigen presentation process. Genome-wide association studies have shown the influence of ERAP1 and ERAP2 genetic variants in disease susceptibility for autoimmune disorders. Their role in inflammatory bowel diseases (IBD) however remains obscure due to literature data paucity. The aim of this study was to evaluate the role of a single nucleotide polymorphism (SNP) rs2248374 (G/A) of ERAP2 gene in disease susceptibility and response to treatment in Romanian population with IBD. This SNP is of particular importance because the G allele produces a truncated ERAP2 transcript undergoing nonsense-mediated decay and therefore individuals with GG genotype have no functional ERAP2 enzyme. Methods The study involved 186 IBD patients (87/99 UC/CD, 91M/69F) and 150 healthy controls (77M/73F) all unrelated and of Romanian ethnicity. Extraintestinal manifestations (EIM) were documented in 43 IBD patients (19UC/24BC). Among patients with biological treatment against TNF-alpha (N=113) there were 31 (27.4%) primary non-responders and 13 (11.5%) with paradoxical skin reactions. All subjects were genotyped for ERAP2 rs2248374 (G/A) using TaqMan Allelic Discrimination Assay (C_25649529_10 Real-Time PCR System, Applied Biosystems by Thermo Fisher Scientific, USA). Association tests were performed with OpenEpi online software using Two by Two Table statistics. P-values (2-tail from Mid-P exact test) were considered significant if <0.05. Results Controls and patients were in Hardy-Weinberg equilibrium for the investigated SNP. No significant differences were observed in relation with the risk of disease or with the therapy response. The minor allele A frequency was significantly lower in IBD patients with paradoxical skin reactions (27%) versus patients without these manifestations (50%, p=0.02, OR 0.36). The genotype AA, responsible for full ERAP2 expression, had 0% frequency in the group with adverse skin reactions compared with 23.2% in patients without (p=0.04, OR 0.0). The GG genotype which is related with no ERAP2 protein expression was more frequent in the subgroup of patients with EIM (41.8%) than in patients without EIM (26.7%), showing a marginal association (p=0.06, OR 1.97). Conclusion Our results point to the fact that ERAP2 rs2248374 AA genotype exerts a protective effect over paradoxical skin reaction induced by TNF antagonist agents, and GG genotype shows marginal association with the presence of EIM in Romanian patients with IBD. We consider these results worthy of replication in larger cohorts of patients.

A cis-regulatory element regulates ERAP2 expression through autoimmune disease risk SNPs

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.

Down-regulation of ERAP1 mRNA expression in non-small cell lung cancer

BackgroundERAP1 is a major aminopeptidase that serves as an editor of the peptide repertoire by trimming N-terminal residues of antigenic peptides, creating a pool of peptides with the optimal length for MHC-I binding. As an important component of the antigen processing and presenting machinery – APM, ERAP1 is frequently down-regulated in many cancers. Since ERAP1 expression has not yet been thoroughly investigated in non-small cell lung cancer (NSCLC), we decided to analyze ERAP1 mRNA levels in tissues collected from NSCLC patients.MethodsUsing real-time qPCR, we evaluated ERAP1 mRNA expression in samples of tumor and adjacent non-tumor tissue (serving as control tissue) from 61 NSCLC patients.ResultsWe observed a significantly lower level of ERAP1 mRNA expression in tumor tissue (MedTumor = 0.75) in comparison to non-tumor tissue (MedNon-tumor = 1.1), p = 0.008. One of the five tested polymorphisms, namely rs26653, turned out to be significantly associated with ERAP1 expression in non-tumor tissue (difference [d] = 0.59 CI95% (0.14;1.05), p = 0.0086), but not in tumor tissue.The levels of ERAP1 mRNA expression did not affect the overall survival of NSCLC patients, either in the case of the tumor (p = 0.788) or in non-tumor (p = 0.298) tissue.We did not detect any association between mRNA ERAP1 expression level in normal tissue and: (i) age at diagnosis (p = 0.8386), (ii) patient’s sex (p = 0.3616), (iii) histological type of cancer (p = 0.7580) and (iv) clinical stage of NSCLC (p = 0.7549). Furthermore, in the case of tumor tissue none of the abovementioned clinical parameters were associated with ERAP1 expression (p = 0.76).ConclusionDown-regulation of ERAP1 mRNA observed in NSCLC tissue may be related to tumor immune evasion strategy. The rs26653 polymorphism can be considered an expression quantitative trait locus (eQTL) associated with ERAP1 expression in normal lung tissue.

005 Cell-specific, disease-associated and variant-linked alterations in expression of ERAP1, ERAP2 And LNPEP aminopeptidases

005 Cell-specific, disease-associated and variant-linked alterations in expression of ERAP1, ERAP2 And LNPEP aminopeptidases

Characterization of immune responses associated with ERAP-1 expression in HSV-induced Behçet's disease mouse model

Behçet's disease (BD) is a chronic multisystem inflammatory disorder. Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphism has been reported as a risk factor for BD. However, the immunological role of ERAP1 in BD remains unclear. Therefore, the purpose of this study was to investigate the immunological role of ERAP1 in BD using a mouse model. ERAP1 incomplete expressing mice (ERAP1 hetero, +/−) were generated and inoculated with herpes simplex virus 1 to produce a BD mouse model. In these mice, dendritic cell activation markers and other immune response-related markers were analyzed. Among them, the factor showing a significant difference between ERAP1+/− BD mice and WT BD mice was IL-17. In ERAP1+/−, BD had significantly different expression levels of CD80, CD11b, Ly6G, RORγt, IFNγ, and IL-17 compared to asymptomatic controls. This study demonstrates ERAP1 defective expressions play an important role in BD development through inappropriate regulation of Th17.

Variation in ERAP2 has opposing effects on severe respiratory infection and autoimmune disease

ERAP2 is an aminopeptidase involved in immunological antigen presentation. Genotype data in human samples from before and after the Black Death, an epidemic due to Yersinia pestis, have marked changes in allele frequency of the single-nucleotide polymorphism (SNP) rs2549794, with the T allele suggested to be deleterious during this period, while ERAP2 is also implicated in autoimmune diseases. This study explored the association between variation at ERAP2 and (1) infection, (2) autoimmune disease, and (3) parental longevity. Genome-wide association studies (GWASs) of these outcomes were identified in contemporary cohorts (UK Biobank, FinnGen, and GenOMICC). Effect estimates were extracted for rs2549794 and rs2248374, a haplotype tagging SNP. Additionally, cis expression and protein quantitative trait loci (QTLs) for ERAP2 were used in Mendelian randomization (MR) analyses. Consistent with decreased survival in the Black Death, the T allele of rs2549794 showed evidence of association with respiratory infection (odds ratio; OR for pneumonia 1.03; 95% CI 1.01-1.05). Effect estimates were larger for more severe phenotypes (OR for critical care admission with pneumonia 1.08; 95% CI 1.02-1.14). In contrast, opposing effects were identified for Crohn disease (OR 0.86; 95% CI 0.82-0.90). This allele was shown to associate with decreased ERAP2 expression and protein levels, independent of haplotype. MR analyses suggest that ERAP2 expression may be mediating disease associations. Decreased ERAP2 expression is associated with severe respiratory infection with an opposing association with autoimmune diseases. These data support the hypothesis of balancing selection at this locus driven by autoimmune and infectious disease.

Cell-Specific and Variant-Linked Alterations in Expression of ERAP1, ERAP2, and LNPEP Aminopeptidases in Psoriasis

ERAP1, ERAP2, and LNPEP are aminopeptidases implicated in autoimmune pathophysiology. In this study, we show that ERAP2 is upregulated and ERAP1 is downregulated in patients with psoriasis who are homozygous for autoimmune-linked variants of ERAP. We also demonstrate that aminopeptidase expression is not uniform in the skin. Specifically, the intracellular antigen-processing aminopeptidases ERAP1 and ERAP2 are strongly expressed in basal and early spinous layer keratinocytes, whereas granular layer keratinocytes expressed predominantly LNPEP, an aminopeptidase specialized in the processing of extracellular antigens for presentation to T cells. In psoriasis, basal keratinocytes also expressed the T-cell- and monocyte-attracting chemokine, CCL2, and the T-cell-supporting cytokine, IL-15. In contrast, TGF-β1 was the major cytokine expressed by healthy control basal keratinocytes. SFRP2-high dermal fibroblasts were also noted to have an ERAP2-high expression phenotype and elevated HLA-C. In psoriasis, the SFRP2-high fibroblast subpopulation also expressed elevated CXCL14. From these results, we postulate that (i) an increased ERAP2/ERAP1 ratio results in altered antigen processing, a potential mechanism by which ERAP risk alleles predispose individuals to autoimmunity; (ii) ERAP2-high expressing cells display a unique major histocompatibility complex-bound peptidome generated from intracellular antigens; and (iii) the granular layer peptidome is skewed toward extracellular antigens.

ERAP2 supports TCR recognition of three immunotherapy targeted tumor epitopes

The therapy of cancer by adoptive T cell transfer (ACT) requires T cell receptors (TCRs) with optimal affinity for HLA class I-bound peptides (pHLA-I). But not every patient responds to ACT. Therefore, it is critical to understand the individual factors influencing the recognition of HLA class I-bound peptides (pHLA-I) by TCRs. Focusing on three immunotherapy-targeted human HLA-A* 02:01-presented T cell epitopes we investigated the contribution of the ER-resident aminopeptidases ERAP1 and ERAP2 to TCR recognition of cancer cells. We found that ERAP2 on its own, when expressed in ERAP-deficient cells, elicited a strong CTL response towards the Tyrosinase368–376 epitope. In vitro generated TAP-dependent N-terminally extended epitope precursor peptides were differently customized by ERAP1 and ERAP2 and thus may serve as potential source for the Tyrosinase368–376 epitope. ERAP2 also influenced recognition of the gp100209–217 tumor epitope and enhanced T cell recognition of the MART-126/27–35 epitope in the absence of ERAP1 expression. Our results underline the relevance of ERAP2 for tumor epitope presentation and TCR recognition and may need to be considered when designing ACT in the future.

Identification of Reduced ERAP2 Expression and a Novel HLA Allele as Components of a Risk Score for Susceptibility to Liver Injury Due to Amoxicillin-Clavulanate

Drug-induced liver injury (DILI) due to amoxicillin-clavulanate (AC) has been associated with HLA-A∗02:01, HLA-DRB1∗15:01, and rs2476601, a missense variant in PTPN22. The aim of this study was to identify novel risk factors for AC-DILI and to construct a genetic risk score (GRS). Transcriptome-wide association study and genome-wide association study analyses were performed on 444 AC-DILI cases and 10,397 population-based controls of European descent. Associations were confirmed in a validation cohort (n= 133 cases and 17,836 population-based controls). Discovery and validation AC-DILI cases were also compared with 1358 and 403 non-AC-DILI cases. Transcriptome-wide association study revealed a significant association of AC-DILI risk with reduced liver expression of ERAP2 (P= 3.7× 10-7), coding for an aminopeptidase involved in antigen presentation. The lead eQTL single nucleotide polymorphism, rs1363907 (G), was associated with AC-DILI risk in the discovery (odds ratio [OR], 1.68; 95% CI, 1.23-1.66; P= 1.7× 10-7) and validation cohorts (OR, 1.2; 95% CI, 1.04-2.05; P= .03), following a recessive model. We also identified HLA-B∗15:18 as a novel AC-DILI risk factor in both discovery (OR, 4.19; 95% CI, 2.09-8.36; P= 4.9× 10-5) and validation (OR, 7.78; 95% CI, 2.75-21.99; P= .0001) cohorts. GRS, incorporating rs1363907, rs2476601, HLA-B∗15:18, HLA-A∗02:01, and HLA-DRB1∗15:01, was highly predictive of AC-DILI risk when cases were analyzed against both general population and non-AC-DILI control cohorts. GRS was the most significant predictor in a regression model containing known AC-DILI clinical risk characteristics and significantly improved the predictive model. We identified novel associations of AC-DILI risk with ERAP2 low expression and with HLA-B∗15:18. GRS based on the 5 risk variants may assist AC-DILI causality assessment and risk management.

Pitch session 2Functional interation of endoplasmic reticulum aminopeptidase ERAP2 and the peptidome of the main MHC-I-associated inflammatory disorders

Several inflammatory diseases are strongly associated with Major Histocompatibility Complex class I (MHC-I) molecules: Among them birdshot chorioretinopathy (HLA-A*29:02) and ankylosing spondylitis (HLA-B*27) have the strongest association with about 100 and 90% of patients being A*29:02 and B*27 positive, respectively. Genome –wide associated studies (GWAS) showed the significance of the endoplasmatic reticulum aminopeptidasas ERAP1 and ERAP2 in this MHC-I associated inflammatory diseases. Although ERAP2 has been primarily described as an accessory and complementary enzyme to the homologous ERAP1, the aim of this work was to study whether it could play distinct and important roles in processing antigenic peptides. HLA-B*27 and B*29-bound peptides were isolated from cell lines that differ in the expression or abscence of ERAP2, by immunoaffinity chromatography and acid extraction. Over 2000-4000 ligands were identified from each cell line and their relative abundance was established by quantitative tandem mass spectrometry and MaxQuant-based analyses. The comparison of HLA-B*27 and A*29-bound peptidomes from cells expressing ERAP2 or not revealed that ERAP2 expression induces significant changes in multiple MHC-I-bound peptidomes. These changes are MHC allotype-specific and, without excluding a degree of functional inter-dependence with ERAP1, reflect largely separate roles in their processing of MHC-I ligands. The magnitude of the effects in different peptidomes of ERAP2 suggests that it could affect not only specific antigen presentation, but also the pro-inflammatory and autoimmune potential of MHC-I molecules.

Abstract 2065: A small molecule approach to drive novel neoantigen generation: First-in-class inhibitors of ERAP1 generate novel neoantigens driving anti-tumor effects

Abstract Immune checkpoint inhibition (ICI) therapy has changed the cancer treatment paradigm; however, most patients respond poorly to ICI alone. In order to be effective, ICI requires neoantigen expression to drive anti-tumor T cell responses and numerous clinical studies have shown that high neoantigen load and tumor mutation burden correlate with improved patient response. Given the central importance of neoantigens in ICI response there is an urgent need for therapies that generate new neoantigens to drive novel anti-tumour cytotoxic T cell responses thereby increasing the number of patients achieving durable benefit. Grey Wolf are developing inhibitors of endoplasmic reticulum aminopeptidases (ERAP1 and ERAP2) to generate novel neoantigens. ERAP enzymes are members of the antigen presentation pathway, trimming a subset of peptides (15-20%) prior to their loading onto MHC Class I. Strikingly, reduced tumoral expression of ERAP1 in cancer correlates with a 50% increase in overall survival. Independent groups have also shown that ablation of ERAP1 generates neoantigens, causing tumor growth inhibition in various syngeneic models in combination with ICI. Grey Wolf have generated and profiled highly potent and selective ERAP1 inhibitors, leading to the nomination of the first-in-class ERAP1 inhibitor development candidate, GRWD5769. Extensive analysis of the immunopeptidomes of diverse cancer cell lines robustly show that ERAP1 inhibition consistently generates novel neoantigens in vitro and in vivo. Analysis of syngeneic tumor models treated with an orally administered ERAP1 inhibitor in combination with anti-PD-1 show that neoantigen generation produces a differentiated T cell response, including: 1) diversification of the tumoral T cell repertoire; 2) increased infiltration of T cells into tumors; and 3) synergistic upregulation of translationally relevant immune gene markers shown to correlate with response to anti-PD-1 in patients. In addition, elevations in immune mediated effects caused by ERAP1 inhibition in combination with anti-PD-1 correlate with tumor growth inhibition in mouse syngeneic studies. Importantly, ERAP1 inhibitors are well tolerated in mouse, rat, and non-human primates, which aligns with the observations that increases in T cell effects are tumor specific and not observed in peripheral tissue. Our investigation of the effects of ERAP1 inhibitors in mouse tumor models and ex vivo primary human T cell co-cultures have provided proof of mechanism and proof of principle biomarkers that will be used to explore the effects of GRWD5769 during Phase 1 clinical development in the second half of 2022. Citation Format: Andrew Leishman, Wayne Paes, Richard Coulson, Michael Pinggera, Jessica Sette, Kate Anderton, Nicola Ternette, Juliet Morgan, Jason Shiers, Martin Quibell, Peter Ian Joyce. A small molecule approach to drive novel neoantigen generation: First-in-class inhibitors of ERAP1 generate novel neoantigens driving anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2065.

Functional antigen processing and presentation mechanism as a prerequisite factor of response to treatment with dendritic cell vaccines and anti-PD-1 in preclinical murine LLC1 and GL261 tumor models.

Low efficacy of cancer immunotherapy encourages the search for possible resistance mechanisms and biomarkers that would predict the outcome of immunotherapy in oncology patients. Most cancer immunotherapies act on T lymphocytes, which can specifically recognize and kill tumor cells. However, for immunotherapy-activated T lymphocytes to be able to perform these functions, proper tumor Ag processing and surface presentation by MHC-I molecule is important. Knowing the significance of Ag processing and presentation mechanism (APM) in anti-tumor immune response, we sought to evaluate how the functionality of APM affects tumor immune microenvironment and response to dendritic cell vaccines (DCV) and anti-PD-1. By comparing murine Lewis lung carcinoma LLC1 and glioma GL261 models a decreased expression of APM-related genes, such as Psmb8, Psmb9, Psmb10, Tap1, Tap2, Erap1, B2m, and low expression of surface MHC-I molecule were found in LLC1 cells. Changes in APM-related gene expression affected the ability of T lymphocytes to recognize and kill LLC1 cells, resulting in the absence of cytotoxic immune response and resistance to DCV and anti-PD-1. An emerging cytotoxic immune reaction and sensitivity to DCV and anti-PD-1 were observed in GL261 tumors where APM remained functional. This study demonstrates that one of the possible mechanisms of tumor resistance to immunotherapy is a dysfunctional APM and reveals a predictive potential of APM-related gene set expression for the personalization of dendritic cell vaccine and anti-PD-1 therapies in murine pre-treated tumors.

CD158a+ /CD158b+ NK cell imbalance correlates with hypertension in patients with pre-eclampsia.

Preeclampsia, a pregnancy complication with hypertension and proteinuria, seriously threats the health and lives of the mother and the baby. The pathogenesis of pre-eclampsia remains incompletely understood. The role of peripheral natural killer cells (NK cells) in the pre-eclampsia is unclear. Flow cytometry was performed to detect the expression of CD158a (KIR2DL1) and CD158b (KIR2DL2/3) in peripheral NK cells of healthy pregnant women (HP) and patients with pre-eclampsia (PE). Differentially expressed genes (DEGs) in CD158a+ and CD158b+ NK cells were identified by RNA-sequencing and real-time PCR. Protein array analysis was used to identify altered protein levels in the serum of study participants. CD158a+ NK cell numbers were increased in the peripheral blood of patients while the number of CD158b+ NK cells was reduced. In addition, the percentage of CD158a+ NK cells within the peripheral NK subset was positively correlated with systolic blood pressure while the percentage of CD158b+ NK cells was negatively correlated with systolic blood pressure. RNA-seq and real-time PCR showed that the expression of ERAP2 and GCH1, the genes that regulate blood pressure and angiogenesis, was decreased in CD158a+ compared to CD158b+ NK cells. Consistently, the level of proteins involved in angiogenesis was altered in the serum of pre-eclampsia patients compared to healthy individuals. CD158a+ NK cells increased while CD158b+ NK cells decreased in the peripheral blood of patients with pre-eclampsia compared to healthy individuals. The change in the frequency of CD158a+ /CD158b+ NK cells is related to the increase in blood pressure.

Genetic contributions of MHC class I antigen processing and presentation pathway to bladder cancer risk and recurrence.

Human leukocyte antigen class I (HLA class I) antigen processing and presentation pathway (APP) defines anti-tumor immune response. ERAP, TAP, tapasin (TAPBP), and IFNγ modulate APP: control HLA class I expression in the tumor and the repertoire of presented tumor antigens. At the same time, vascular endothelial growth factor (VEGF) acts as an immunomodulator in the tumor microenvironment. The objective of the current study was to examine the association of single nucleotide polymorphisms (SNPs) in the ERAP1, ERAP2, TAP1, TAP2, TAPBP, IFNG genes with the corresponding mRNA expression in bladder cancer (BC) risk and recurrence after transurethral resection of BC. Moreover, we assessed the relationship between HLA class I and VEGF plasma levels and BC recurrence. We analyzed 9 SNPs in 124 BC patients using TaqMan genotyping and compared them with the data from 503 healthy individuals from the 1000 Genomes Project. In addition, we quantified the effects of SNPs on the corresponding mRNA expression in tumor and non-tumor adjacent tissue in 60 BC patients with primary and 30 with recurrent tumor by quantitative real-time PCR. Furthermore, the plasma HLA class I and VEGF levels were analyzed in BC patients and healthy controls by ELISA. IFNG (rs1861493) was associated with BC risk, TAPBP (rs3106189, rs2071888) with recurrence-free survival (RFS). Moreover, TAPBP mRNA expression was lower in tumors than in the adjacent tissue. The SNPs ERAP2 (rs251339) and TAP2 (rs241447, rs241448) variants affected mRNA expression in BC tissue. In tumor tissue, the high mRNA expression of ERAP1 was more common in BC patients with single tumors, ERAP2 in non-smokers, and TAP2 mRNA in recurrence. The lower HLA and higher VEGF plasma levels were observed in BC patients compared with healthy controls. We conclude that the genetic elements responsible for MHC class I APP may influence the BC risk, risk of recurrence, and RFS.

ERAP2 Is Associated With Immune Infiltration and Predicts Favorable Prognosis in SqCLC.

BackgroundImmunotherapy has been proven effective among several human cancer types, including Squamous cell lung carcinoma (SqCLC). ERAP2 plays a pivotal role in peptide trimming of many immunological processes. However, the prognostic role of ERAP2 and its relationship with immune cell infiltration in SqCLC remains unclear.MethodsThe differential expression of ERAP2 was identified via GEO and TCGA databases. We calculated the impact of ERAP2 on clinical prognosis using the Kaplan-Meier plotter. TIMER was applied to evaluate the abundance of immune cells infiltration and immune markers. SqCLC tissue microarrays containing 190 patients were constructed, and we performed immunohistochemical staining for ERAP2, CD8, CD47, CD68, and PD-L1 to validate our findings in public data.ResultsIn the GEO SqCLC database, ERAP2 was upregulated in patients with better survival (p=0.001). ERAP2 expression in SqCLC was significantly lower than that of matched normal samples (p<0.05) based on TCGA SqCLC data. Higher expression of ERAP2 was significantly associated with better survival in SqCLC patients from TCGA (p=0.007), KM-plotter (p=0.017), and our tissue microarrays (TMAs) (p=0.026). In univariate and multivariate Cox analysis of SqCLC TMAs, high ERAP2 expression was identified as an independent protective factor for SqCLC patients (Univariate Cox, HR=0.659, range 0.454-0.956, p<0.05. Multivariate Cox, HR=0.578, range 0.385-0.866, p<0.05). In TIMER, ERAP2 was positively correlated with several immune markers (CD274, p=1.27E-04; CD68, p=5.88E-08) and immune infiltrating cells (CD8+ T cell, p=4.09E-03; NK cell, p=1.00E-04). In our cohort, ERAP2 was significantly correlated with CD8+ tumor-infiltrating lymphocytes (TILs) (p=0.0029), and patients with higher ERAP2 expression had a higher percentage of PD-L1 positive patients (p=0.049) and a higher CD8+ TILs level (p=0.036).ConclusionsFor the first time, our study demonstrates that higher expression of ERAP2 is tightly associated with the immuno-supportive microenvironment and can predict a favorable prognosis in SqCLC. Meanwhile, ERAP2 may be a promising immunotherapeutic target for patients with SqCLC.

ERAP1 Controls the Interaction of the Inhibitory Receptor KIR3DL1 With HLA-B51:01 by Affecting Natural Killer Cell Function.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by major histocompatibility complex (MHC) class I molecules. Previously, we have shown that genetic or pharmacological inhibition of ERAP1 on murine and human tumor cell lines perturbs the engagement of NK cell inhibitory receptors Ly49C/I and Killer-cell Immunoglobulin-like receptors (KIRs), respectively, by their specific ligands (MHC class I molecules), thus leading to NK cell killing. However, the effect of ERAP1 inhibition in tumor cells was highly variable, suggesting that its efficacy may depend on several factors, including MHC class I typing. To identify MHC class I alleles and KIRs that are more sensitive to ERAP1 depletion, we stably silenced ERAP1 expression in human HLA class I-negative B lymphoblastoid cell line 721.221 (referred to as 221) transfected with a panel of KIR ligands (i.e. HLA-B*51:01, -Cw3, -Cw4 and -Cw7), or HLA-A2 which does not bind any KIR, and tested their ability to induce NK cell degranulation and cytotoxicity. No change in HLA class I surface expression was detected in all 221 transfectant cells after ERAP1 depletion. In contrast, CD107a expression levels were significantly increased on NK cells stimulated with 221-B*51:01 cells lacking ERAP1, particularly in the KIR3DL1-positive NK cell subset. Consistently, genetic or pharmacological inhibition of ERAP1 impaired the recognition of HLA-B*51:01 by the YTS NK cell overexpressing KIR3DL1*001, suggesting that ERAP1 inhibition renders HLA-B*51:01 molecules less eligible for binding to KIR3DL1. Overall, these results identify HLA-B*51:01/KIR3DL1 as one of the most susceptible combinations for ERAP1 inhibition, suggesting that individuals carrying HLA-B*51:01-like antigens may be candidates for immunotherapy based on pharmacological inhibition of ERAP1.

ERAP1, ERAP2, and Two Copies of HLA-Aw19 Alleles Increase the Risk for Birdshot Chorioretinopathy in HLA-A29 Carriers.

PurposeBirdshot chorioretinopathy (BSCR) is strongly associated with HLA-A29. This study was designed to elucidate the genetic modifiers of BSCR in HLA-A29 carriers.MethodsWe sequenced the largest BSCR cohort to date, including 286 cases and 108 HLA-A29–positive controls to determine genome-wide common and rare variant associations. We further typed the HLA alleles of cases and 45,386 HLA-A29 controls of European ancestry to identify HLA alleles that associate with BSCR risk.ResultsCarrying a second allele that belongs to the HLA-Aw19 broad antigen family (including HLA-A29, -A30, -A31, and -A33) increases the risk for BSCR (odds ratio [OR] = 4.44; P = 2.2e-03). This result was validated by comparing allele frequencies to large HLA-A29-controlled cohorts (n = 45,386; OR > 2.5; P < 1.3e-06). We also confirm that ERAP1 and ERAP2 haplotypes modulate disease risk. A meta-analysis with an independent dataset confirmed that ERAP1 and ERAP2 haplotypes modulate the risk for disease at a genome-wide significant level: ERAP1-rs27432 (OR = 2.46; 95% confidence interval [CI], 1.85–3.26; P = 4.07e-10), an expression quantitative trait locus (eQTL) decreasing ERAP1 expression; and ERAP2-rs10044354 (OR = 1.95; 95% CI, 1.55–2.44; P = 6.2e-09), an eQTL increasing ERAP2 expression. Furthermore, ERAP2-rs2248374 that disrupts ERAP2 expression is protective (OR = 0.56; 95% CI, 0.45–0.70; P = 2.39e-07). BSCR risk is additively increased when combining ERAP1/ERAP2 risk genotypes with two copies of HLA-Aw19 alleles (OR = 13.53; 95% CI, 3.79–54.77; P = 1.17e-05).ConclusionsThe genetic factors increasing BSCR risk demonstrate a pattern of increased processing, as well as increased presentation of ERAP2-specific peptides. This suggests a mechanism in which exceeding a peptide presentation threshold activates the immune response in choroids of A29 carriers.

Interaction between ERAP Alleles and HLA Class I Types Support a Role of Antigen Presentation in Hodgkin Lymphoma Development.

Simple SummaryHodgkin lymphoma (HL) is a common lymphoma in young adults derived from B cells. Emerging evidence suggests that antigen presentation by the malignant B cells is critically involved in HL pathogenesis. In fact, genetic variants of the antigen presenting Human Leukocyte Antigens (HLA) are strongly associated with HL susceptibility. Interestingly, the endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP2 genes, that code for enzymes that process antigens, also appear to be associated. In this study, we show that genetic variants of ERAP genes strongly affect expression levels of ERAP1 and ERAP2. In addition, we find that certain ERAP variants interact with specific HLA class I types in HL patients. This suggests that mechanisms that determine the repertoire of antigens that are presented to the immune system, affect the chance of developing HL. Our findings therefore support a prominent role of antigen presentation in HL susceptibility.Genetic variants in the HLA region are the strongest risk factors for developing Hodgkin lymphoma (HL), suggesting an important role for antigen presentation. This is supported by another HL-associated genomic region which contains the loci of two enzymes that process endogenous proteins to peptides to be presented by HLA class I, i.e., endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2. We hypothesized that ERAP and HLA class I type interact in HL susceptibility, as shown previously for several autoimmune diseases. We detected ERAP1 and ERAP2 expression in tumor cells and cells in the microenvironment in primary HL tissue samples. Seven ERAP SNPs and ERAP1 haplotypes showed strong associations with RNA and protein levels of ERAP1 and ERAP2 in LCLs and HL cell lines. Analysis of HLA class I types, ERAP SNPs and ERAP haplotypes by direct genotyping or imputation from genome-wide association data in 390 HL patients revealed significant interactions between HLA-A11, rs27038 and the rs27038 associated ERAP haplotype, as well as between HLA-Cw2 and rs26618. In conclusion, our results show that ERAP and HLA class I interact in genetic susceptibility to HL, providing further evidence that antigen presentation is an important process in HL susceptibility and pathogenesis.

Abstract IA11: Overcoming immune evasion in pediatric brain tumors

Abstract Many immunotherapies act by enhancing T-cell killing of tumor cells. Cytotoxic T cells recognize antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. Our studies suggest that medulloblastomas and high-grade gliomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. Treatment with tumor necrosis factor or lymphotoxin beta receptor agonist rescues expression of Erap1, Tap1, and MHC-I on p53 mutant tumor cells. In vivo, TNF treatment prolongs survival and markedly augments the efficacy of the immune checkpoint inhibitor anti-PD-1. These studies identify p53 as a key regulator of immune evasion in vivo and suggest that TNF could be used to enhance sensitivity of p53-mutant tumors to immunotherapy. Citation Format: Alexandra Garancher, Hiromichi Suzuki, Svasti Haricharan, Meher B. Masihi, Jessica M. Rusert, Paula S. Norris, Florent Carrette, Megan M. Romero, Sorana A. Morrissy, Patryk Skowron, Florence M.G. Cavalli, Hamza Farooq, Vijay Ramaswamy, Alaide Morcavallo, Jacob J. Henderson, James M. Olson, Yoon-Jae Cho, Xiao-Nan Li, Louis Chesler, Marco A. Marra, Oren J. Becher, Linda M. Bradley, Carl F. Ware, Michael D. Taylor, Robert J. Wechsler-Reya. Overcoming immune evasion in pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA11.