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We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was enriched for prefibro- and fibro-, regulatory, reparative and prehypertrophic chondrocytes. A total of 61 cytokines and growth factors were predicted to regulate the 7 chondrocyte cell phenotypes. Based on production by > 1% of cells, 55% of the cytokines were produced by synovial cells (39% exclusive to synoviocytes and not expressed by chondrocytes) and their presence in osteoarthritic synovial fluid confirmed. The synoviocytes producing IL-1beta (a classic pathogenic cytokine in osteoarthritis), mainly inflammatory macrophages and dendritic cells, were characterized by co-expression of surface proteins corresponding to HLA-DQA1 , HLA-DQA2 , OLR1 or TLR2 . Strategies to deplete these pathogenic intra-articular cell subpopulations could be a therapeutic option for human osteoarthritis.

Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P  = 3.4 × 10 −12 ) and IRF4 (rs9405192, OR = 1.29, P = 1.4 × 10 −14 ), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P  = 4.7 × 10 −103 ) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P  = 2.0 × 10 −49 ), DQA1*0501 in Europeans (OR = 2.88, P  = 5.7 × 10 −93 ), and DRB1*0301 in both ethnicities (OR = 3.50, P  = 9.2 × 10 −23 and OR = 3.39, P  = 5.2 × 10 −82 , respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20–37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk. Membranous nephropathy (MN) is a rare autoimmune disease of podocyte-directed antibodies, such as anti-phospholipase A2 receptor. Here, the authors report a genome-wide association study for MN and identify two previously unreported loci encompassing the NFKB1 and IRF4 genes and additional ancestry-specific effects.

Most uterine cervical high-risk human papillomavirus (HPV) infections are transient, with only a small fraction developing into cervical cancer. Family aggregation studies and heritability estimates suggest a significant inherited genetic component. Candidate gene studies and previous genome-wide association studies (GWASs) report associations between the HLA region and cervical cancer. Adopting a genome-wide approach, we aimed to compare genetic variation in women with invasive cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 with that in healthy controls. We did a GWAS in a cohort of unrelated European individuals using data from UK Biobank, a population-based cohort including 273 377 women aged 40–69 years at recruitment between March 13, 2006, and Oct 1, 2010. We used an additive univariate logistic regression model to analyse genetic variants associated with invasive cervical cancer or CIN3. We sought replication of candidate associations in FinnGen, a large independent dataset of 128 123 individuals. We also did a two-sample mendelian randomisation approach to explore the role of risk factors in the genetic risk of cervical cancer. We included 4769 CIN3 and invasive cervical cancer case samples and 145 545 control samples in the GWAS. Of 9 600 464 assayed and imputed single-nucleotide polymorphisms (SNPs), six independent variants were associated with CIN3 and invasive cervical cancer. These included novel loci rs10175462 (PAX8; odds ratio [OR] 0·87, 95% CI 0·84–0·91; p=1·07 × 10−9) and rs27069 (CLPTM1L; 0·88, 0·84–0·92; p=2·51 × 10−9), and previously reported signals at rs9272050 (HLA-DQA1; 1·27, 1·21–1·32; p=2·51 × 10−28), rs6938453 (MICA; 0·79, 0·75–0·83; p=1·97 × 10−17), rs55986091 (HLA-DQB1; 0·66, 0·60–0·72; p=6·42 × 10−28), and rs9266183 (HLA-B; 0·73, 0·64–0·83; p=1·53 × 10−6). Three SNPs were replicated in the independent Finnish dataset of 1648 invasive cervical cancer cases: PAX8 (rs10175462; p=0·015), CLPTM1L (rs27069; p=2·54 × 10−7), and HLA-DQA1 (rs9272050; p=7·90 × 10−8). Mendelian randomisation further supported the complementary role of smoking (OR 2·46, 95% CI 1·64–3·69), older age at first pregnancy (0·80, 0·68–0·95), and number of sexual partners (1·95, 1·44–2·63) in the risk of developing cervical cancer. Our results provide new evidence for the genetic susceptibility to cervical cancer, specifically the PAX8, CLPTM1L, and HLA genes, suggesting disruption in apoptotic and immune function pathways. Future studies integrating host and viral, genetic, and epigenetic variation, could further elucidate complex host–viral interactions. NIHR Imperial BRC Wellcome 4i Clinician Scientist Training Programme.

BackgroundThere is emerging data on the continued efficacy of infliximab when switched from intravenous (IV) to subcutaneous (SC) route in Inflammatory bowel disease (IBD). The impact of cessation of concomitant immunomodulators (IMMs) following switch on trough levels or development of anti-drug antibodies (ADAs) is not evaluated in real world practice. We aimed to study the impact of cessation of IMMs following switch of infliximab from IV to SC route and determine relationship with HLA- DQA1 *05 status.MethodsConsecutive patients who participated in a planned switch programme were prospectively included. Clinical, biomarker and pharmacokinetic data were collected pre-switch, and at 8 weekly intervals post switch for 6 months. Trough levels and remission status in patients who discontinued or reduced the dose of IMMs were compared with those who remained or continued same dose on IMMs. All patients had HLA-DQA1*05 status determined at initiation of Infliximab and we evaluated the impact of HLA status on development of anti-Infliximab antibodies post switch.ResultsOne hundred and fifteen patients (M: F=1.4:1, Median age 44 years (13-76), UC/CD (48/67) who completed 6 month follow up were included. All patients were in clinical and biomarker remission prior to the switch. 72 patients (62.6%) were on concomitant IMMs and 46 (40.86%) was HLADQA1*05 positive. The mean infliximab trough level prior to switch was 5.54 µg/mL which increased 8 weeks post switch to 12.52 µg/mL (p=0.006). The enhancement of levels persisted at 16 weeks (Mean 16.28) and at week 24 (17.25). 36 patients (32%) were positive for ADAs before switch; in 8 patients (22%) the ADAs disappeared at 8 weeks and in further 4 (total 33%) by 6 months. In total antibody titre reduced or become negative in 17 patients (48.6%), static in 14 patients (40%) but increased in 5 patients (14%) without significant lowering of trough levels. 18 of the 72 patients (25%) had IMMs discontinued, and dose reduced in further 21 patients. The total de-escalation rate was 54% and none of these patients developed ADAs at 6 months irrespective of the HLADQA1*05 status (HR = 0.76, 95% = 0.64 to 1.73, p 0.12). One patient switched back to IV due to personal preference. At 6 months none of the patients discontinued SC infliximab or were switched within or out of class.Abstract O40 Figure 1ConclusionsSwitch from IV to SC infliximab is effective in maintaining and enhancing trough levels with positive impact on reducing ADAs. It is feasible to de-escalate concomitant IMMs in a considerable proportion of patients following switch. Economic analysis of switch should be incorporated in the feasibility of IMMs withdrawal.

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genome-wide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: a GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early- and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.

IntroductionCombination therapy using thiopurines or methotrexate is recommended to reduce the risk of immunogenicity in IBD patients treated with anti TNF agents. The PANTS study reported the association of HLA DQA1*05 carriage with development of anti-drug antibodies to anti TNFs in patients with Crohn`s disease. Since 2020, we have incorporated determination of HLA status in the pre-biologic screening of IBD patients. We aimed to evaluate persistence at 6 and 12 months in patients stratified to receive mono or combo therapy with anti TNFsMethodsA prospective database of all IBD patients starting biologics was interrogated to select patients initiating on anti TNF agents. We excluded patients Crohn`s disease perianal fistula who had specific indication for combo therapy, patients with acute severe UC receiving accelerated induction those who were already on imunomodulators prior to initiation of anti TNF agents. Data was collected on demographics, disease characteristics, HLA DQA1*05 status, drugs levels and antidrug antibody status at week 6, week 14 and at least one time point during a 1 year maintenance period. The primary outcome was drug persistence at 12 months.ResultsOne hundred and fifty six patients were initiated on anti TNFs during the study period. HLA DQA1*05 was positive in 59 (37.8%) of patients. These patients received combination therapy (Infliximab + thiopurines in 42, Infliximab plus methotrexate in 9, adalimumab plus azathioprine 6, Adalimumab plus methotrexate in 2). All patients negative for HLADQA1805 received monotherapy with infliximab (78) or adalimumab (19). Primary non response was identified in 30 patients (overall 19%, Monotherapy 17(17.5%), combo therapy 13 (22% ) and were switched to a non anti TNF agent Further 9% of patients had dose escalation due to suboptimal drug levels and partial response among whom 6 patients were switched to another class. Eleven patients stopped anti TNFs (4 and 7 in mono and combo therapy group respectively) due to intolerance and five patients discontinued immunomodulators in the combo therapy due to adverse effects.Anti-drug antibodies was detected at week 6 in 2 patients in the monotherapy group and 3 patients in combo therapy group (p=NS). There was no difference in rates of antidrug antibody development during maintenance period between the two groups. One hundred and twelve patients remained on anti TNFs across both groups at 12 months follow up. The overall drug persistence rates was similar in the mono and combination therapy group (66.1% vs 72.2%, p=NS).ConclusionsHLA DQA1*05 status incorporated anti TNF treatment strategy may alleviate the need for combination therapy in IBD patients with no impact on development of anti-drug antibodies and drug persistence. A biomarker stratified randomised trial is recommended.

BackgroundCombination therapy with immunomodulators and anti-TNFs were recommended following the results of the SONIC study. It was determined the main benefit for combination therapy was due to reduction in the risk of anti-drug antibodies. The PANTS study indicated that patients at higher risk of antidrug antibodies to anti-TNF agents could be identified with HLADQA1*05 allele carriage. A HLADQA1*05 stratified withdrawal of immunomodulators has not been evaluated before.MethodsWe included IBD patients on combination immunomodulaotrs and anti TNF agents from a prospective database who had HLADQA1 *05 status determined retrospectively after treatment of minimum 12 months. Patients with Crohn`s disease -perianal fistula or those on immunomodulators due to non-IBD indications were excluded. Immunomodulator withdrawal was offered to all patients in clinical and biomarker remission with a negative HLADQA1805 status. Patients were followed up with structured clinical and biomarker assessment. Therapeutic drug monitoring was performed at 3–6 month intervals. The primary outcome was development of anti-drug antibodies.ResultsThree hundred and eighteen patients on anti-TNF agents had HLA-DQA1*status determined. Among these 248 patients were on combination therapy. Withdrawal of immunomodulaotrs was suggested to 146 (59%) of the HLA-DQA1*05 negative patients. None of the patients declined attempt at withdrawal but five patients preferred dose reduction rather than complete cessation and these were not included in analysis. Median follow up among the remaining 141 patients was 13 months (range 3–27 months). Antidrug antibodies developed in 11 patients (7.8%) including in three patients with undetectable drug levels necessitating switch of agent. An additional 16 patients lost clinical response without development of antibodies.ConclusionIn anti TNF treated IBD patients in long-term remission, withdrawal of immunomodulators is feasible in majority of patients not carrying HLA-DQA1*05 with limited risk of development of antidrug antibodies.

Type 1 narcolepsy (T1N) is caused by hypocretin/orexin (HCRT) neuronal loss. Association with the HLA DQB1*06:02/DQA1*01:02 (98% vs. 25%) heterodimer (DQ0602), T cell receptors (TCR) and other immune loci suggest autoimmunity but autoantigens are unknown. Onset is seasonal and associated with influenza A, notably pandemic 2009 H1N1 (pH1N1) infection and vaccination (Pandemrix). Peptides derived from HCRT and influenza A, including pH1N1, were screened for DQ0602 binding and presence of cognate DQ0602 tetramer-peptide–specific CD4⁺ T cells tested in 35 T1N cases and 22 DQ0602 controls. Higher reactivity to influenza pHA273–287 (pH1N1 specific), PR8 (H1N1 pre-2009 and H2N2)-specific NP17–31 and C-amidated but not native version of HCRT54–66 and HCRT86–97 (HCRTNH2) were observed in T1N. Single-cell TCR sequencing revealed sharing of CDR3β TRBV4-2-CASSQETQGRNYGYTF in HCRTNH2 and pHA273–287-tetramers, suggesting molecular mimicry. This public CDR3β uses TRBV4-2, a segment modulated by T1N-associated SNP rs1008599, suggesting causality. TCR-α/β CDR3 motifs of HCRT54–66-NH2 and HCRT86–97-NH2 tetramers were extensively shared: notably public CDR3α, TRAV2-CAVETDSWGKLQF-TRAJ24, that uses TRAJ24, a chain modulated by T1N-associated SNPs rs1154155 and rs1483979. TCR-α/β CDR3 sequences found in pHA273–287, NP17–31, and HCRTNH2 tetramer-positive CD4⁺ cells were also retrieved in single INF-γ–secreting CD4⁺ sorted cells stimulated with Pandemrix, independently confirming these results. Our results provide evidence for autoimmunity and molecular mimicry with flu antigens modulated by genetic components in the pathophysiology of T1N.

Juvenile idiopathic arthritis (JIA) is a complex trait, the most common rheumatic disease in children. Considering clinical heterogeneity of the disease, the genetic background of particular JIA subtypes may also vary significantly. This work was aimed to reveal characteristic patterns of HLA associations within 11 loci for two clinically different forms of JIA in the Belarusian population. 24 patients diagnosed with systemic JIA, 24 patients with oligoarticular JIA and 24 healthy controls were included into the study. The JIA patients were divided into subgroups according to IIAR classification criteria. High-throughput HLA typing was performed using TruSight HLA v2 Sequencing Panel (Illumina) on MiSeq system. Sample analysis was performed using Assign TruSight HLA v2.0 software. DQA1*05:01:01 and DQB1*02:01:01 alleles showed protective effect against both systemic (p = 0.007; OR=0.08; 95% CI=[0.009–0.65] and p = 0.01; OR=0.09; 95% CI=[0.01–0.83]) and oliarticular JIA (p = 0.026; OR=0.16; 95% CI=[0.03–0.79] and p = 0.046; OR=0.2; 95% CI=[0.04–1.00], while for DRB1*03:01 negative association was revealed only for systemic JIA (p = 0.03; OR=0.11; 95% CI=[0.01–0.88]). At the same time the DQB1*04:02:01 (p=0.026; OR=5.88; 95% CI=[1.20–28.72]) and DRB1*08:01:01 (p=0.07; OR=3.94; 95% CI=[1.01–15.39]) allele frequencies were significantly higher in patients with oligoarthritis but not systemic JIA when compared with controls. High-throughput HLA typing revealed distinct HLA-alleles associated with different JIA subtypes in the Belarusian population as well as some alleles protective both for oligoartricular and systemic JIA. None declared

DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.