• BCG Vaccination in Humans Elicits Trained Immunity via the Hematopoietic Progenitor Compartment.

      Cirovic, Branko; de Bree, L Charlotte J; Groh, Laszlo; Blok, Bas A; Chan, Joyce; van der Velden, Walter J F M; Bremmers, M E J; van Crevel, Reinout; Händler, Kristian; Picelli, Simone; et al. (Elsevier (Cell Press), 2020-06-09)
      Induction of trained immunity by Bacille-Calmette-Guérin (BCG) vaccination mediates beneficial heterologous effects, but the mechanisms underlying its persistence and magnitude remain elusive. In this study, we show that BCG vaccination in healthy human volunteers induces a persistent transcriptional program connected to myeloid cell development and function within the hematopoietic stem and progenitor cell (HSPC) compartment in the bone marrow. We identify hepatic nuclear factor (HNF) family members 1a and b as crucial regulators of this transcriptional shift. These findings are corroborated by higher granulocyte numbers in BCG-vaccinated infants, HNF1 SNP variants that correlate with trained immunity, and elevated serum concentrations of the HNF1 target alpha-1 antitrypsin. Additionally, transcriptomic HSPC remodeling was epigenetically conveyed to peripheral CD14+ monocytes, displaying an activated transcriptional signature three months after BCG vaccination. Taken together, transcriptomic, epigenomic, and functional reprogramming of HSPCs and peripheral monocytes is a hallmark of BCG-induced trained immunity in humans.
    • Comparative host transcriptome in response to pathogenic fungi identifies common and species-specific transcriptional antifungal host response pathways.

      Bruno, Mariolina; Dewi, Intan M W; Matzaraki, Vicky; Ter Horst, Rob; Pekmezovic, Marina; Rösler, Berenice; Groh, Laszlo; Röring, Rutger J; Kumar, Vinod; Li, Yang; et al. (Elsevier, 2020-12-26)
      Candidiasis, aspergillosis, and mucormycosis cause the majority of nosocomial fungal infections in immunocompromised patients. Using an unbiased transcriptional profiling in PBMCs exposed to the fungal species causing these infections, we found a core host response in healthy individuals that may govern effective fungal clearance: it consists of 156 transcripts, involving canonical and non-canonical immune pathways. Systematic investigation of key steps in antifungal host defense revealed fungal-specific signatures. As previously demonstrated, Candida albicans induced type I and Type II interferon-related pathways. In contrast, central pattern recognition receptor, reactive oxygen species production, and host glycolytic pathways were down-regulated in response to Rhizopus oryzae, which was associated with an ER-stress response. TLR5 was identified to be uniquely regulated by Aspergillus fumigatus and to control cytokine release in response to this fungus. In conclusion, our data reveals the transcriptional profiles induced by C. albicans, A. fumigatus, and R. oryzae, and describes both the common and specific antifungal host responses that could be exploited for novel therapeutic strategies.
    • COVID-19 immune signatures reveal stable antiviral T cell function despite declining humoral responses.

      Bonifacius, Agnes; Tischer-Zimmermann, Sabine; Dragon, Anna C; Gussarow, Daniel; Vogel, Alexander; Krettek, Ulrike; Gödecke, Nina; Yilmaz, Mustafa; Kraft, Anke R M; Hoeper, Marius M; et al.
      Cellular and humoral immunity to SARS-CoV-2 is critical to control primary infection and correlates with severity of disease. The role of SARS-CoV-2-specific T cell immunity, its relationship to antibodies, and pre-existing immunity against endemic coronaviruses (huCoV), which has been hypothesized to be protective, were investigated in 82 healthy donors (HDs), 204 recovered (RCs), and 92 active COVID-19 patients (ACs). ACs had high amounts of anti-SARS-CoV-2 nucleocapsid and spike IgG but lymphopenia and overall reduced antiviral T cell responses due to the inflammatory milieu, expression of inhibitory molecules (PD-1, Tim-3) as well as effector caspase-3, -7, and -8 activity in T cells. SARS-CoV-2-specific T cell immunity conferred by polyfunctional, mainly interferon-γ-secreting CD4+ T cells remained stable throughout convalescence, whereas humoral responses declined. Immune responses toward huCoV in RCs with mild disease and strong cellular SARS-CoV-2 T cell reactivity imply a protective role of pre-existing immunity against huCoV.
    • Evolution of cytokine production capacity in ancient and modern European populations.

      Domínguez-Andrés, Jorge; Kuijpers, Yunus; Bakker, Olivier B; Jaeger, Martin; Xu, Cheng-Jian; Van der Meer, Jos Wm; Jakobsson, Mattias; Bertranpetit, Jaume; Joosten, Leo Ab; Li, Yang; et al. (eLife Sciences Publications, 2021-09-07)
      As our ancestors migrated throughout different continents, natural selection increased the presence of alleles advantageous in the new environments. Heritable variations that alter the susceptibility to diseases vary with the historical period, the virulence of the infections, and their geographical spread. In this study we built polygenic scores for heritable traits that influence the genetic adaptation in the production of cytokines and immune-mediated disorders, including infectious, inflammatory, and autoimmune diseases, and applied them to the genomes of several ancient European populations. We observed that the advent of the Neolithic was a turning point for immune-mediated traits in Europeans, favoring those alleles linked with the development of tolerance against intracellular pathogens and promoting inflammatory responses against extracellular microbes. These evolutionary patterns are also associated with an increased presence of traits related to inflammatory and auto-immune diseases.
    • Integration of metabolomics, genomics, and immune phenotypes reveals the causal roles of metabolites in disease.

      Chu, Xiaojing; Jaeger, Martin; Beumer, Joep; Bakker, Olivier B; Aguirre-Gamboa, Raul; Oosting, Marije; Smeekens, Sanne P; Moorlag, Simone; Mourits, Vera P; Koeken, Valerie A C M; et al. (BMC, 2021-07-06)
      Background: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors. Result: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target. Conclusion: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.
    • IRF7 and RNH1 are modifying factors of HIV-1 reservoirs: a genome-wide association analysis.

      Zhang, Zhenhua; Trypsteen, Wim; Blaauw, Marc; Chu, Xiaojing; Rutsaert, Sofie; Vandekerckhove, Linos; van der Heijden, Wouter; Dos Santos, Jéssica Cristina; Xu, Cheng-Jian; Swertz, Morris A; et al. (BMC, 2021-11-16)
      The analysis resulted in one significant association with CA HIV-1 DNA (rs2613996, P < 5 × 10-8) and two suggestive associations with RNA:DNA ratio (rs7113204 and rs7817589, P < 5 × 10-7). Then, we prioritized PTDSS2, IRF7, RNH1, and DEAF1 as potential HIV-1 reservoir modifiers and validated that higher expressions of IRF7 and RNH1 were accompanied by rs7113204-G. Moreover, RNA:DNA ratio, indicating relative HIV-1 transcription activity, was lower in PLHIV carrying this variant.
    • Mendelian randomization while jointly modeling cis genetics identifies causal relationships between gene expression and lipids.

      van der Graaf, Adriaan; Claringbould, Annique; Rimbert, Antoine; Westra, Harm-Jan; Li, Yang; Wijmenga, Cisca; Sanna, Serena; CiiM, Zentrum für individualisierte Infektionsmedizin, Feodor-Lynen-Str.7, 30625 Hannover. (Nature publishing group (NPG), 2020-10-01)
      Inference of causality between gene expression and complex traits using Mendelian randomization (MR) is confounded by pleiotropy and linkage disequilibrium (LD) of gene-expression quantitative trait loci (eQTL). Here, we propose an MR method, MR-link, that accounts for unobserved pleiotropy and LD by leveraging information from individual-level data, even when only one eQTL variant is present. In simulations, MR-link shows false-positive rates close to expectation (median 0.05) and high power (up to 0.89), outperforming all other tested MR methods and coloc. Application of MR-link to low-density lipoprotein cholesterol (LDL-C) measurements in 12,449 individuals with expression and protein QTL summary statistics from blood and liver identifies 25 genes causally linked to LDL-C. These include the known SORT1 and ApoE genes as well as PVRL2, located in the APOE locus, for which a causal role in liver was not known. Our results showcase the strength of MR-link for transcriptome-wide causal inferences.
    • Multi-Omics Approaches in Immunological Research.

      Chu, Xiaojing; Zhang, Bowen; Koeken, Valerie A C M; Gupta, Manoj Kumar; Li, Yang; CiiM, Zentrum für individualisierte Infektionsmedizin, Feodor-Lynen-Str.7, 30625 Hannover. (2021-06-11)
      The immune system plays a vital role in health and disease, and is regulated through a complex interactive network of many different immune cells and mediators. To understand the complexity of the immune system, we propose to apply a multi-omics approach in immunological research. This review provides a complete overview of available methodological approaches for the different omics data layers relevant for immunological research, including genetics, epigenetics, transcriptomics, proteomics, metabolomics, and cellomics. Thereafter, we describe the various methods for data analysis as well as how to integrate different layers of omics data. Finally, we discuss the possible applications of multi-omics studies and opportunities they provide for understanding the complex regulatory networks as well as immune variation in various immune-related diseases.
    • Polymorphisms within Autophagy-Related Genes Influence the Risk of Developing Colorectal Cancer: A Meta-Analysis of Four Large Cohorts.

      Sainz, Juan; García-Verdejo, Francisco José; Martínez-Bueno, Manuel; Kumar, Abhishek; Sánchez-Maldonado, José Manuel; Díez-Villanueva, Anna; Vodičková, Ludmila; Vymetálková, Veronika; Martin Sánchez, Vicente; Da Silva Filho, Miguel Inacio; et al. (MDPI, 2021-03-12)
      The role of genetic variation in autophagy-related genes in modulating autophagy and cancer is poorly understood. Here, we comprehensively investigated the association of autophagy-related variants with colorectal cancer (CRC) risk and provide new insights about the molecular mechanisms underlying the associations. After meta-analysis of the genome-wide association study (GWAS) data from four independent European cohorts (8006 CRC cases and 7070 controls), two loci, DAPK2 (p = 2.19 × 10-5) and ATG5 (p = 6.28 × 10-4) were associated with the risk of CRC. Mechanistically, the DAPK2rs11631973G allele was associated with IL1 β levels after the stimulation of peripheral blood mononuclear cells (PBMCs) with Staphylococcus aureus (p = 0.002), CD24 + CD38 + CD27 + IgM + B cell levels in blood (p = 0.0038) and serum levels of en-RAGE (p = 0.0068). ATG5rs546456T allele was associated with TNF α and IL1 β levels after the stimulation of PBMCs with LPS (p = 0.0088 and p = 0.0076, respectively), CD14+CD16- cell levels in blood (p = 0.0068) and serum levels of CCL19 and cortisol (p = 0.0052 and p = 0.0074, respectively). Interestingly, no association with autophagy flux was observed. These results suggested an effect of the DAPK2 and ATG5 loci in the pathogenesis of CRC, likely through the modulation of host immune responses.
    • Polymorphisms within the and Loci Influence the Risk of Developing Invasive Aspergillosis: A Two-Stage Case Control Study in the Context of the aspBIOmics Consortium.

      ánchez-Maldonado, Jose Manuel; Moñiz-Díez, Ana; Rob Ter Horst, Daniele Campa; Cabrera-Serrano, Antonio José; Garrido-Collado, María Del Pilar; Hernández-Mohedo, Fracisca; Fernández-Puerta, Laura; López-Nevot, , Miguel Ángel; Cunha, Ctistina; González-Sierra, Pedro Antonio; et al. (MDPI, 2020-12-23)
      Here, we assessed whether 36 single nucleotide polymorphisms (SNPs) within the TNFSF4 and MAPKAPK2 loci influence the risk of developing invasive aspergillosis (IA). We conducted a two-stage case control study including 911 high-risk patients diagnosed with hematological malignancies that were ascertained through the aspBIOmics consortium. The meta-analysis of the discovery and replication populations revealed that carriers of the TNFSF4rs7526628T/T genotype had a significantly increased risk of developing IA (p = 0.00022). We also found that carriers of the TNFSF4rs7526628T allele showed decreased serum levels of TNFSF14 protein (p = 0.0027), and that their macrophages had a decreased fungicidal activity (p = 0.048). In addition, we observed that each copy of the MAPKAPK2rs12137965G allele increased the risk of IA by 60% (p = 0.0017), whereas each copy of the MAPKAPK2rs17013271T allele was estimated to decrease the risk of developing the disease (p = 0.0029). Mechanistically, we found that carriers of the risk MAPKAPK2rs12137965G allele showed increased numbers of CD38+IgM-IgD- plasmablasts in blood (p = 0.00086), whereas those harboring two copies of the allele had decreased serum concentrations of thymic stromal lymphopoietin (p = 0.00097). Finally, we also found that carriers of the protective MAPKAPK2rs17013271T allele had decreased numbers of CD27-IgM-IgD- B cells (p = 0.00087) and significantly lower numbers of CD14+ and CD14+CD16- cells (p = 0.00018 and 0.00023). Altogether, these results suggest a role of the TNFSF4 and MAPKAPK2 genes in determining IA risk.
    • Reprogramming of bone marrow myeloid progenitor cells in patients with severe coronary artery disease.

      Noz, Marlies P; Bekkering, Siroon; Groh, Laszlo; Nielen, Tim Mj; Lamfers, Evert Jp; Schlitzer, Andreas; El Messaoudi, Saloua; van Royen, Niels; Huys, Erik Hjpg; Preijers, Frank Wmb; et al. (elifesciences.org, 2020-11-10)
      Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.
    • Resolving trained immunity with systems biology.

      Koeken, Valerie A C M; van Crevel, Reinout; Netea, Mihai G; Li, Yang; CiiM, Zentrum für individualisierte Infektionsmedizin, Feodor-Lynen-Str.7, 30625 Hannover. (Wiley-VCH, 2021-02-11)
      Trained immunity is characterized by long-term functional reprogramming of innate immune cells following challenge with pathogens or microbial ligands during infection or vaccination. This cellular reprogramming leads to increased responsiveness upon re-stimulation, and is mediated through epigenetic and metabolic modifications. In this review, we describe how molecular mechanisms underlying trained immunity, for example induced by β-glucan or Bacille Calmette-Guérin (BCG) vaccination, can be investigated by using and integrating different layers of information, including genome, epigenome, transcriptome, proteome, metabolome, microbiome, immune cell phenotyping and function. We also describe the most commonly used experimental and computational techniques. Finally, we provide a number of examples of how a systems biology approach was applied to study trained immunity to understand inter-individual variation or the complex interplay between molecular layers. In conclusion, trained immunity represents an opportunity for regulating innate immune function, and understanding the complex interplay of mechanisms that mediate trained immunity might enable us to employ it as a clinical tool in the future. This article is protected by copyright. All rights reserved.
    • The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by β-Glucan.

      Keating, Samuel T; Groh, Laszlo; van der Heijden, Charlotte D C C; Rodriguez, Hanah; Dos Santos, Jéssica C; Fanucchi, Stephanie; Okabe, Jun; Kaipananickal, Harikrishnan; van Puffelen, Jelmer H; Helder, Leonie; et al.
      Trained immunity confers a sustained augmented response of innate immune cells to a secondary challenge, via a process dependent on metabolic and transcriptional reprogramming. Because of its previous associations with metabolic and transcriptional memory, as well as the importance of H3 histone lysine 4 monomethylation (H3K4me1) to innate immune memory, we hypothesize that the Set7 methyltransferase has an important role in trained immunity induced by β-glucan. Using pharmacological studies of human primary monocytes, we identify trained immunity-specific immunometabolic pathways regulated by Set7, including a previously unreported H3K4me1-dependent plasticity in the induction of oxidative phosphorylation. Recapitulation of β-glucan training in vivo additionally identifies Set7-dependent changes in gene expression previously associated with the modulation of myelopoiesis progenitors in trained immunity. By revealing Set7 as a key regulator of trained immunity, these findings provide mechanistic insight into sustained metabolic changes and underscore the importance of characterizing regulatory circuits of innate immune memory.
    • Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

      Schulte-Schrepping, Jonas; Reusch, Nico; Paclik, Daniela; Baßler, Kevin; Schlickeiser, Stephan; Zhang, Bowen; Krämer, Benjamin; Krammer, Tobias; Brumhard, Sophia; Bonaguro, Lorenzo; et al. (Elsevier /Cell Press), 2020-08-05)
      Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
    • Sex-Specific Regulation of Inflammation and Metabolic Syndrome in Obesity.

      Ter Horst, Rob; van den Munckhof, Inge C L; Schraa, Kiki; Aguirre-Gamboa, Raul; Jaeger, Martin; Smeekens, Sanne P; Brand, Tessa; Lemmers, Heidi; Dijkstra, Helga; Galesloot, Tessel E; et al. (Lippincott, Williams & Wilkins, 2020-05-28)
      Metabolic dysregulation and inflammation are important consequences of obesity and impact susceptibility to cardiovascular disease. Anti-inflammatory therapy in cardiovascular disease is being developed under the assumption that inflammatory pathways are identical in women and men, but it is not known if this is indeed the case. In this study, we assessed the sex-specific relation between inflammation and metabolic dysregulation in obesity. Approach and Results: Three hundred two individuals were included, half with a BMI 27 to 30 kg/m2 and half with a BMI>30 kg/m2, 45% were women. The presence of metabolic syndrome was assessed according to the National Cholesterol Education Program-ATPIII criteria, and inflammation was studied using circulating markers of inflammation, cell counts, and ex vivo cytokine production capacity of isolated immune cells. Additionally, lipidomic and metabolomic data were gathered, and subcutaneous fat biopsies were histologically assessed. Metabolic syndrome is associated with an increased inflammatory profile that profoundly differs between women and men: women with metabolic syndrome show a lower concentration of the anti-inflammatory adiponectin, whereas men show increased levels of several pro-inflammatory markers such as IL (interleukin)-6 and leptin. Adipose tissue inflammation showed similar sex-specific associations with these markers. Peripheral blood mononuclear cells isolated from men, but not women, with metabolic syndrome display enhanced cytokine production capacity.
    • Systematic Prioritization of Candidate Genes in Disease Loci Identifies as a Master Regulator of IFNγ Signaling in Celiac Disease.

      van der Graaf, Adriaan; Zorro, Maria M; Claringbould, Annique; Võsa, Urmo; Aguirre-Gamboa, Raúl; Li, Chan; Mooiweer, Joram; Ricaño-Ponce, Isis; Borek, Zuzanna; Koning, Frits; et al. (Frontiers, 2021-01-25)
      Celiac disease (CeD) is a complex T cell-mediated enteropathy induced by gluten. Although genome-wide association studies have identified numerous genomic regions associated with CeD, it is difficult to accurately pinpoint which genes in these loci are most likely to cause CeD. We used four different in silico approaches-Mendelian randomization inverse variance weighting, COLOC, LD overlap, and DEPICT-to integrate information gathered from a large transcriptomics dataset. This identified 118 prioritized genes across 50 CeD-associated regions. Co-expression and pathway analysis of these genes indicated an association with adaptive and innate cytokine signaling and T cell activation pathways. Fifty-one of these genes are targets of known drug compounds or likely druggable genes, suggesting that our methods can be used to pinpoint potential therapeutic targets. In addition, we detected 172 gene combinations that were affected by our CeD-prioritized genes in trans. Notably, 41 of these trans-mediated genes appear to be under control of one master regulator, TRAF-type zinc finger domain containing 1 (TRAFD1), and were found to be involved in interferon (IFN)γ signaling and MHC I antigen processing/presentation. Finally, we performed in vitro experiments in a human monocytic cell line that validated the role of TRAFD1 as an immune regulator acting in trans. Our strategy confirmed the role of adaptive immunity in CeD and revealed a genetic link between CeD and IFNγ signaling as well as with MHC I antigen processing, both major players of immune activation and CeD pathogenesis.
    • Tissue alarmins and adaptive cytokine induce dynamic and distinct transcriptional responses in tissue-resident intraepithelial cytotoxic T lymphocytes.

      Zorro, Maria Magdalena; Aguirre-Gamboa, Raul; Mayassi, Toufic; Ciszewski, Cezary; Barisani, Donatella; Hu, Shixian; Weersma, Rinse K; Withoff, Sebo; Li, Yang; Wijmenga, Cisca; et al. (Elsevier, 2020-02-04)
      The respective effects of tissue alarmins interleukin (IL)-15 and interferon beta (IFNβ), and IL-21 produced by T cells on the reprogramming of cytotoxic T lymphocytes (CTLs) that cause tissue destruction in celiac disease is poorly understood. Transcriptomic and epigenetic profiling of primary intestinal CTLs showed massive and distinct temporal transcriptional changes in response to tissue alarmins, while the impact of IL-21 was limited. Only anti-viral pathways were induced in response to all the three stimuli, albeit with differences in dynamics and strength. Moreover, changes in gene expression were primarily independent of changes in H3K27ac, suggesting that other regulatory mechanisms drive the robust transcriptional response. Finally, we found that IL-15/IFNβ/IL-21 transcriptional signatures could be linked to transcriptional alterations in risk loci for complex immune diseases. Together these results provide new insights into molecular mechanisms that fuel the activation of CTLs under conditions that emulate the inflammatory environment in patients with autoimmune diseases.
    • Validation of GWAS-Identified Variants for Anti-TNF Drug Response in Rheumatoid Arthritis: A Meta-Analysis of Two Large Cohorts.

      Sánchez-Maldonado, Jose Manuel; Cáliz, Rafael; López-Nevot, Miguel Ángel; Cabrera-Serrano, Antonio José; Moñiz-Díez, Ana; Canhão, Helena; Ter Horst, Rob; Quartuccio, Luca; Sorensen, Signe B; Glintborg, Bente; et al. (Frontiers, 2021-10-27)
      We aimed to validate the association of 28 GWAS-identified genetic variants for response to TNF inhibitors (TNFi) in a discovery cohort of 1361 rheumatoid arthritis (RA) patients monitored in routine care and ascertained through the REPAIR consortium and DANBIO registry. We genotyped selected markers and evaluated their association with response to TNFi after 6 months of treatment according to the change in disease activity score 28 (ΔDAS28). Next, we confirmed the most interesting results through meta-analysis of our data with those from the DREAM cohort that included 706 RA patients treated with TNFi. The meta-analysis of the discovery cohort and DREAM registry including 2067 RA patients revealed an overall association of the LINC02549 rs7767069 SNP with a lower improvement in DAS28 that remained significant after correction for multiple testing (per-allele ORMeta=0.83, P Meta=0.000077; P Het=0.61). In addition, we found that each copy of the LRRC55 rs717117G allele was significantly associated with lower improvement in DAS28 in rheumatoid factor (RF)-positive patients (per-allele ORMeta=0.67, P=0.00058; P Het=0.06) whereas an opposite but not significant effect was detected in RF-negative subjects (per-allele ORMeta=1.38, P=0.10; P Het=0.45; P Interaction=0.00028). Interestingly, although the identified associations did not survive multiple testing correction, the meta-analysis also showed overall and RF-specific associations for the MAFB rs6071980 and CNTN5 rs1813443 SNPs with decreased changes in DAS28 (per-allele ORMeta_rs6071980 = 0.85, P=0.0059; P Het=0.63 and ORMeta_rs1813443_RF+=0.81, P=0.0059; P Het=0.69 and ORMeta_rs1813443_RF-=1.00, P=0.99; P Het=0.12; P Interaction=0.032). Mechanistically, we found that subjects carrying the LINC02549 rs7767069T allele had significantly increased numbers of CD45RO+CD45RA+ T cells (P=0.000025) whereas carriers of the LINC02549 rs7767069T/T genotype showed significantly increased levels of soluble scavengers CD5 and CD6 in serum (P=0.00037 and P=0.00041). In addition, carriers of the LRRC55 rs717117G allele showed decreased production of IL6 after stimulation of PBMCs with B burgdorferi and E coli bacteria (P=0.00046 and P=0.00044), which suggested a reduced IL6-mediated anti-inflammatory effect of this marker to worsen the response to TNFi. In conclusion, this study confirmed the influence of the LINC02549 and LRRC55 loci to determine the response to TNFi in RA patients and suggested a weak effect of the MAFB and CNTN5 loci that need to be further investigated.