In Vitro Generation of Human Tolerogenic Monocyte-Derived Dendritic Cells.

Human monocyte-derived dendritic cells (moDC) are commonly used as a research tool to investigate interactions between antigen-presenting cells and T cells. Generation of these cells involves the isolation of CD14 positive monocytes from peripheral blood and their in vitro differentiation into immature moDC by the cytokines GM-CSF and IL-4. Their functional characteristics can then be manipulated by maturing these cells with a cocktail of agents, which can be tailored to induce either immune activating or tolerogenic properties. Here, we describe a protocol for the generation of moDC with stable tolerogenic function, referred to as tolerogenic dendritic cells. These cells have been developed as an immunotherapeutic tool for the treatment of autoimmune disease but have also proven useful to dissect mechanisms of T cell tolerance induction in vitro.

Fluorine labelling of therapeutic human tolerogenic dendritic cells for 19F-magnetic resonance imaging.

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.

Interferon-α-mediated therapeutic resistance in early rheumatoid arthritis implicates epigenetic reprogramming.

OBJECTIVES: An interferon (IFN) gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients where it has been shown to negatively impact initial response to treatment. We wished to validate this effect and explore potential mechanisms of action. METHODS: In a multicentre inception cohort of eRA patients (n=191), we examined the whole blood IGS (MxA, IFI44L, OAS1, IFI6, ISG15) with reference to circulating IFN proteins, clinical outcomes and epigenetic influences on circulating CD19+ B and CD4+ T lymphocytes. RESULTS: We reproduced our previous findings demonstrating a raised baseline IGS. We additionally showed, for the first time, that the IGS in eRA reflects circulating IFN-α protein. Paired longitudinal analysis demonstrated a significant reduction between baseline and 6-month IGS and IFN-α levels (p<0.0001 for both). Despite this fall, a raised baseline IGS predicted worse 6-month clinical outcomes such as increased disease activity score (DAS-28, p=0.025) and lower likelihood of a good EULAR clinical response (p=0.034), which was independent of other conventional predictors of disease activity and clinical response. Molecular analysis of CD4+ T cells and CD19+ B cells demonstrated differentially methylated CPG sites and dysregulated expression of disease relevant genes, including PARP9, STAT1, and EPSTI1, associated with baseline IGS/IFNα levels. Differentially methylated CPG sites implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1α). CONCLUSIONS: Our data suggest that, in eRA, IFN-α can cause a sustained, epigenetically mediated, pathogenic increase in lymphocyte activation and proliferation, and that the IGS is, therefore, a robust prognostic biomarker. Its persistent harmful effects provide a rationale for the initial therapeutic targeting of IFN-α in selected patients with eRA.

Lymphocyte DNA methylation mediates genetic risk at shared immune-mediated disease loci.

BACKGROUND: Defining regulatory mechanisms through which noncoding risk variants influence the cell-mediated pathogenesis of immune-mediated disease (IMD) has emerged as a priority in the post-genome-wide association study era. OBJECTIVES: With a focus on rheumatoid arthritis, we sought new insight into genetic mechanisms of adaptive immune dysregulation to help prioritize molecular pathways for targeting in this and related immune pathologies. METHODS: Whole-genome methylation and transcriptional data from isolated CD4+ T cells and B cells of more than 100 genotyped and phenotyped patients with inflammatory arthritis, all of whom were naive to immunomodulatory treatments, were obtained. Analysis integrated these comprehensive data with genome-wide association study findings across IMDs and other publicly available resources. RESULTS: We provide strong evidence that disease-associated DNA variants regulate cis-CpG methylation in CD4+ T and/or B cells at 37% RA loci. Using paired, cell-specific transcriptomic data and causal inference testing, we identify examples where site-specific DNA methylation in turn mediates gene expression, including FCRL3 in both cell types and ORMDL3/GSDMB, IL6ST/ANKRD55, and JAZF1 in CD4+ T cells. A number of genes regulated in this way highlight mechanisms common to RA and other IMDs including multiple sclerosis and asthma, in turn distinguishing them from osteoarthritis, a primarily degenerative disease. Finally, we corroborate the observed effects experimentally. CONCLUSIONS: Our observations highlight important mechanisms of genetic risk in RA and the wider context of immune dysregulation. They confirm the utility of DNA methylation profiling as a tool for causal gene prioritization and, potentially, therapeutic targeting in complex IMD.

Expression of STAT3-regulated genes in circulating CD4+ T cells discriminates rheumatoid arthritis independently of clinical parameters in early arthritis.

OBJECTIVES: Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance. METHODS: Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis. RESULTS: In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature's ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response. CONCLUSION: The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.

CD4+ and B Lymphocyte Expression Quantitative Traits at Rheumatoid Arthritis Risk Loci in Patients With Untreated Early Arthritis: Implications for Causal Gene Identification.

OBJECTIVE: Rheumatoid arthritis (RA) is a genetically complex disease of immune dysregulation. This study sought to gain further insight into the genetic risk mechanisms of RA by conducting an expression quantitative trait locus (eQTL) analysis of confirmed genetic risk loci in CD4+ T cells and B cells from carefully phenotyped patients with early arthritis who were naive to therapeutic immunomodulation. METHODS: RNA and DNA were isolated from purified B and/or CD4+ T cells obtained from the peripheral blood of 344 patients with early arthritis. Genotyping and global gene expression measurements were carried out using Illumina BeadChip microarrays. Variants in linkage disequilibrium (LD) with non-HLA RA single-nucleotide polymorphisms (defined as r2 ≥ 0.8) were analyzed, seeking evidence of cis- or trans-eQTLs according to whether the associated probes were or were not within 4 Mb of these LD blocks. RESULTS: Genes subject to cis-eQTL effects that were common to both CD4+ and B lymphocytes at RA risk loci were FADS1, FADS2, BLK, FCRL3, ORMDL3, PPIL3, and GSDMB. In contrast, those acting on METTL21B, JAZF1, IKZF3, and PADI4 were unique to CD4+ lymphocytes, with the latter candidate risk gene being identified for the first time in this cell subset. B lymphocyte-specific eQTLs for SYNGR1 and CD83 were also found. At the 8p23 BLK-FAM167A locus, adjacent genes were subject to eQTLs whose activity differed markedly between cell types; in particular, the FAM167A effect displayed striking B lymphocyte specificity. No trans-eQTLs approached experiment-wide significance, and linear modeling did not identify a significant influence of biologic covariates on cis-eQTL effect sizes. CONCLUSION: These findings further refine the understanding of candidate causal genes in RA pathogenesis, thus providing an important platform from which downstream functional studies, directed toward particular cell types, may be prioritized.

Immune reconstitution 20 years after treatment with alemtuzumab in a rheumatoid arthritis cohort: implications for lymphocyte depleting therapies.

BACKGROUND: Alemtuzumab, an anti-CD52 monoclonal antibody, was administered to patients with RA between 1991 and 1994. We have followed a cohort of recipients since that time and previously reported significant delays in immune reconstitution. Here we report >20 years of follow-up data from this unique cohort. METHOD: Surviving alemtuzumab recipients were age, sex and disease duration matched with RA controls. Updated mortality and morbidity data were collected for alemtuzumab recipients. For both groups antigenic responses were assessed following influenza, Pneumovax II and combined diphtheria/tetanus/poliovirus vaccines. Circulating cytokines and lymphocyte subsets were also quantified. RESULTS: Of 16 surviving alemtuzumab recipients, 13 were recruited: 9 recipients underwent a full clinical assessment and 4 had case notes review only. Since our last review 10 patients had died from causes of death consistent with long-standing RA, and no suggestion of compromised immune function. Compared with controls the alemtuzumab cohort had significantly reduced CD4+ and CD8+ central memory T-cells, CD5+ B cells, naïve B cells and CD19+CD24hiCD38hi transitional (putative regulatory) B cells. Nonetheless vaccine responses were comparable between groups. There were significantly higher serum IL-15 and IFN-γ levels in the alemtuzumab cohort. IL-15 levels were inversely associated with CD4+ total memory and central memory T cells. CONCLUSION: After 20 years the immune system of alemtuzumab recipients continues to show differences from disease controls. Nonetheless mortality and morbidity data, alongside vaccination responses, do not suggest clinical immune compromise. As lymphodepleting therapies, including alemtuzumab, continue to be administered this work is important with regard to long-term immune monitoring and stages of immune recovery.

Capture Hi-C identifies a novel causal gene, IL20RA, in the pan-autoimmune genetic susceptibility region 6q23.

BACKGROUND: The identification of causal genes from genome-wide association studies (GWAS) is the next important step for the translation of genetic findings into biologically meaningful mechanisms of disease and potential therapeutic targets. Using novel chromatin interaction detection techniques and allele specific assays in T and B cell lines, we provide compelling evidence that redefines causal genes at the 6q23 locus, one of the most important loci that confers autoimmunity risk. RESULTS: Although the function of disease-associated non-coding single nucleotide polymorphisms (SNPs) at 6q23 is unknown, the association is generally assigned to TNFAIP3, the closest gene. However, the DNA fragment containing the associated SNPs interacts through chromatin looping not only with TNFAIP3, but also with IL20RA, located 680 kb upstream. The risk allele of the most likely causal SNP, rs6927172, is correlated with both a higher frequency of interactions and increased expression of IL20RA, along with a stronger binding of both the NFκB transcription factor and chromatin marks characteristic of active enhancers in T-cells. CONCLUSIONS: Our results highlight the importance of gene assignment for translating GWAS findings into biologically meaningful mechanisms of disease and potential therapeutic targets; indeed, monoclonal antibody therapy targeting IL-20 is effective in the treatment of rheumatoid arthritis and psoriasis, both with strong GWAS associations to this region.

Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies.

Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

The c-Rel subunit of NF-κB regulates epidermal homeostasis and promotes skin fibrosis in mice.

The five subunits of transcription factor NF-κB have distinct biological functions. NF-κB signaling is important for skin homeostasis and aging, but the contribution of individual subunits to normal skin biology and disease is unclear. Immunohistochemical analysis of the p50 and c-Rel subunits within lesional psoriatic and systemic sclerosis skin revealed abnormal epidermal expression patterns, compared with healthy skin, but RelA distribution was unaltered. The skin of Nfkb1(-/-) and c-Rel(-/-) mice is structurally normal, but epidermal thickness and proliferation are significantly reduced, compared with wild-type mice. We show that the primary defect in both Nfkb1(-/-) and c-Rel(-/-) mice is within keratinocytes that display reduced proliferation both in vitro and in vivo. However, both genotypes can respond to proliferative stress, with 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation and closure rates of full-thickness skin wounds being equivalent to those of wild-type controls. In a model of bleomycin-induced skin fibrosis, Nfkb1(-/-) and c-Rel(-/-) mice displayed opposite phenotypes, with c-Rel(-/-) mice being protected and Nfkb1(-/-) developing more fibrosis than wild-type mice. Taken together, our data reveal a role for p50 and c-Rel in regulating epidermal proliferation and homeostasis and a profibrogenic role for c-Rel in the skin, and identify a link between epidermal c-Rel expression and systemic sclerosis. Modulating the actions of these subunits could be beneficial for treating hyperproliferative or fibrogenic diseases of the skin.

Immunity 12 years after alemtuzumab in RA: CD5⁺ B-cell depletion, thymus-dependent T-cell reconstitution and normal vaccine responses.

OBJECTIVES: Lymphocyte depleting therapies have been used to treat refractory autoimmune disease, including RA, but treatment may be associated with long-term lymphopenia. It is unclear whether delayed reconstitution preferentially affects lymphocyte subsets, how this modulates immune challenges and whether thymic function influences the outcome. These questions are now addressed in a detailed analysis of RA patients 12 years after alemtuzumab (anti-CD52) treatment. METHODS: Blood was obtained from 20 RA patients 12 years after alemtuzumab treatment. Lymphocyte subsets were enumerated by flow cytometry. T-cell receptor excision circles (TRECs)/ml were determined to quantify thymic function, and serological responses to neoantigens and recall antigens were assessed. RESULTS: RA patients remained lymphopenic 12 years after their first dose of alemtuzumab. CD5(+) B cells, which may be associated with autoantibody production, were significantly reduced in alemtuzumab-treated patients compared with age-matched disease controls. In addition, naïve and memory CD4(+) T-cell subsets were present in altered proportions in patients who had received alemtuzumab, with increased effector memory CD4(+) T cells, and decreased naïve and central memory CD4(+) T cells. TRECs were detectable in alemtuzumab-treated patients and correlated with CD4(+) lymphocyte counts. Vaccine responses to neoantigens and recall antigens fell within the normal range for an ageing population. CONCLUSIONS: Alemtuzumab therapy resulted in long-term alterations in lymphocyte subsets. The significance of these changes remains uncertain but patients respond normally to antigenic challenges. Thymic function remains an important determinant of T-cell reconstitution even several years after lymphocytotoxic therapy.

Differential regulation of naïve and memory CD4+ T cells by alternatively activated dendritic cells.

Promising immunotherapeutic tools for T cell-mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As naïve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of naïve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by naïve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Naïve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN-gamma/high IL-10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL-2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL-12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in naïve T cells, indicating that the lack of IL-12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate naïve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.