Challenges in tolerogenic dendritic cell therapy for autoimmune diseases: the route of administration.

Tolerogenic dendritic cells (tolDCs) are a promising strategy to treat autoimmune diseases since they have the potential to re-educate and modulate pathological immune responses in an antigen-specific manner and, therefore, have minimal adverse effects on the immune system compared to conventional immunosuppressive treatments. TolDC therapy has demonstrated safety and efficacy in different experimental models of autoimmune disease, such as multiple sclerosis (MS), type 1 diabetes (T1D), and rheumatoid arthritis (RA). Moreover, data from phase I clinical trials have shown that therapy with tolDCs is safe and well tolerated by MS, T1D, and RA patients. Nevertheless, various parameters need to be optimized to increase tolDC efficacy. In this regard, one important parameter to be determined is the most appropriate route of administration. Several delivery routes, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranodal, and intraarticular routes, have been used in experimental models as well as in phase I clinical trials. This review summarizes data obtained from preclinical and clinical studies of tolDC therapy in the treatment of MS, T1D, and RA and their animal models, as well as data from the context of cancer immunotherapy using mature peptide-loaded DC, and data from in vivo cell tracking experiments, to define the most appropriate route of tolDC administration in relation to the most feasible, safest, and effective therapeutic use.

Epstein-Barr Virus and Multiple Sclerosis: A Convoluted Interaction and the Opportunity to Unravel Predictive Biomarkers.

Since the early 1980s, Epstein-Barr virus (EBV) infection has been described as one of the main risk factors for developing multiple sclerosis (MS), and recently, new epidemiological evidence has reinforced this premise. EBV seroconversion precedes almost 99% of the new cases of MS and likely predates the first clinical symptoms. The molecular mechanisms of this association are complex and may involve different immunological routes, perhaps all running in parallel (i.e., molecular mimicry, the bystander damage theory, abnormal cytokine networks, and coinfection of EBV with retroviruses, among others). However, despite the large amount of evidence available on these topics, the ultimate role of EBV in the pathogenesis of MS is not fully understood. For instance, it is unclear why after EBV infection some individuals develop MS while others evolve to lymphoproliferative disorders or systemic autoimmune diseases. In this regard, recent studies suggest that the virus may exert epigenetic control over MS susceptibility genes by means of specific virulence factors. Such genetic manipulation has been described in virally-infected memory B cells from patients with MS and are thought to be the main source of autoreactive immune responses. Yet, the role of EBV infection in the natural history of MS and in the initiation of neurodegeneration is even less clear. In this narrative review, we will discuss the available evidence on these topics and the possibility of harnessing such immunological alterations to uncover predictive biomarkers for the onset of MS and perhaps facilitate prognostication of the clinical course.

Vitamin C enhances NF-κB-driven epigenomic reprogramming and boosts the immunogenic properties of dendritic cells.

Dendritic cells (DCs), the most potent antigen-presenting cells, are necessary for effective activation of naïve T cells. DCs' immunological properties are modulated in response to various stimuli. Active DNA demethylation is crucial for DC differentiation and function. Vitamin C, a known cofactor of ten-eleven translocation (TET) enzymes, drives active demethylation. Vitamin C has recently emerged as a promising adjuvant for several types of cancer; however, its effects on human immune cells are poorly understood. In this study, we investigate the epigenomic and transcriptomic reprogramming orchestrated by vitamin C in monocyte-derived DC differentiation and maturation. Vitamin C triggers extensive demethylation at NF-κB/p65 binding sites, together with concordant upregulation of antigen-presentation and immune response-related genes during DC maturation. p65 interacts with TET2 and mediates the aforementioned vitamin C-mediated changes, as demonstrated by pharmacological inhibition. Moreover, vitamin C increases TNFß production in DCs through NF-κB, in concordance with the upregulation of its coding gene and the demethylation of adjacent CpGs. Finally, vitamin C enhances DC's ability to stimulate the proliferation of autologous antigen-specific T cells. We propose that vitamin C could potentially improve monocyte-derived DC-based cell therapies.

Vitamin D receptor, STAT3, and TET2 cooperate to establish tolerogenesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, induces a stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis in vitro. Differentiation to tolerogenic DCs associates with activation of the IL-6-JAK-STAT3 pathway. We show that JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of JAK2 reverts vitamin D-induced tolerogenic properties of DCs. This interplay among VDR, STAT3, and TET2 opens up possibilities for modulating DC immunogenic properties in clinics.

Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes.

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

Tolerogenic Dendritic Cells in Autoimmunity and Inflammatory Diseases.

Dendritic cells (DCs), the most efficient antigen-presenting cells, are necessary for the effective activation of naïve T cells. DCs can also acquire tolerogenic functions in vivo and in vitro in response to various stimuli, including interleukin (IL)-10, transforming growth factor (TGF)-ß, vitamin D3, corticosteroids, and rapamycin. In this review, we provide a wide perspective on the regulatory mechanisms, including crosstalk with other cell types, downstream signaling pathways, transcription factors, and epigenetics, underlying the acquisition of tolerogenesis by DCs, with a special focus on human studies. Finally, we present clinical assays targeting, or based on, tolerogenic DCs in inflammatory diseases. Our discussion provides a useful resource for better understanding the biology of tolerogenic DCs and their manipulation to improve the immunological fitness of patients with certain inflammatory conditions.

Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a Functional Hyporesponsiveness.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most robust approaches due to their immune regulatory properties, which are currently being tested in clinical trials. However, the mechanisms that vitD3-tolDC trigger for the induction of tolerance remain elusive. For this reason, we performed a full phenotypical, functional, and transcriptomic characterization of T cells upon their interaction with autologous, antigen-specific vitD3-tolDC. We observed a strong antigen-specific reduction of T cell proliferation, combined with a decrease in the relative prevalence of TH1 subpopulations and IFN-γ production. The analysis of the transcriptomic profile of T CD4+ cells evidenced a significant down-modulation of genes involved in cell cycle and cell response to mainly pro-inflammatory immune-related stimuli, highlighting the role of JUNB gene as a potential biomarker of these processes. Consequently, our results show the induction of a strong antigen-specific hyporesponsiveness combined with a reduction on the TH1 immune profile of T cells upon their interaction with vitD3-tolDC, which manifests the regulatory properties of these cells and, therefore, their therapeutic potential in the clinic.

Distinct pattern of peripheral lymphocyte subsets in Graves' disease with persistency of anti-TSHR autoantibodies.

Background: Graves' disease (GD) is characterized by the production of autoantibodies against the TSHR (TRAbs). With long-term treatment, serum concentrations of TRAbs decline but in some patients, despite being clinically stable, TRAbs persist for many years.Objective: To investigate whether GD patients with persistence of TRAbs constitute a subset of patients that could be identified by phenotypic analysis of circulating lymphocytes, suggesting disease heterogeneity.Materials and methods: Peripheral blood lymphocytes (including naïve, memory and effector T and B cells, Th17, regulatory T cells (Treg), recent thymic emigrants (RTEs) and double positive CD4+CD8+ (DP) cells) were analysed by flow cytometry in a cross-sectional study in 25 clinically stable GD patients, five patients at onset of GD disease and 40 healthy donors (HDs).Results: GD patients with persistence of TRAbs showed a lower percentage of Treg and lower absolute numbers of central and effector memory CD8+ T cells than HD. No differences in RTEs were found in peripheral blood from GD patients compared to HD. Stable GD patients had higher percentage of DP cells of effector phenotype than HD.Conclusions: Using extensive phenotypic analysis of lymphocyte subpopulations, it is possible to detect changes that help to identify patients with persistent TSHR antibodies and may contribute to understand why the autoimmune response is maintained.

MAP7 and MUCL1 Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients.

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both in vitro and in vivo. In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated CYP24A1, MAP7 and MUCL1 genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that MAP7 and MUCL1 genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that MAP7 and MUCL1 constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS.

Th1Th17CM Lymphocyte Subpopulation as a Predictive Biomarker of Disease Activity in Multiple Sclerosis Patients under Dimethyl Fumarate or Fingolimod Treatment.

Peripheral blood biomarkers able to predict disease activity in multiple sclerosis (MS) patients have not been identified yet. Here, we analyzed the immune phenotype of T lymphocyte subpopulations in peripheral blood samples from 66 RRMS patients under DMF (n = 22) or fingolimod (n = 44) treatment, by flow cytometry. A correlation study between the percentage and absolute cell number of each lymphocyte subpopulation with the presence of relapses or new MRI lesions during 12-month follow-up was performed. Patients who had undergone relapses showed at baseline higher percentage of Th1CM cells (relapsed: 11.60 ± 4.17%vs. nonrelapsed: 9.25 ± 3.17%, p < 0.05) and Th1Th17CM cells (relapsed: 15.65 ± 6.15%vs. nonrelapsed: 10.14 ± 4.05%, p < 0.01) before initiating DMF or fingolimod treatment. Kaplan-Meier analysis revealed that patients with Th1Th17CM (CD4+CCR7+CD45RA-CCR6+CXCR3+) cells > 11.48% had a 50% relapse-free survival compared to patients with Th1Th17CMcells < 11.48% whose relapse-free survival was 88% (p = 0.013, log-rank test). Additionally, a high percentage of Th1Th17CM cells was also found in patients with MRI activity (MRI activity: 14.02 ± 5.87%vs. no MRI activity: 9.82 ± 4.06%, p < 0.01). Our results suggest that the percentage of Th1Th17CM lymphocytes at baseline is a predictive biomarker of activity during the first 12 months of treatment, regardless of the treatment.

Ethyl Pyruvate Induces Tolerogenic Dendritic Cells.

Dendritic cells (DC) are professional antigen presenting cells that have a key role in shaping the immune response. Tolerogenic DC (tolDC) have immuno-regulatory properties and they are a promising prospective therapy for multiple sclerosis and other autoimmune diseases. Ethyl pyruvate (EP) is a redox analog of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that EP ameliorates experimental autoimmune encephalomyelitis, a multiple sclerosis murine model. Here, we expanded our study to its tolerogenic effects on DC. Phenotypic analysis has shown that DC obtained from mice or humans reduce expression of molecules required for T cell activation such as CD86, CD83, and HLA-DR under the influence of EP, while CD11c expression and viability of DC are not affected. Furthermore, EP-treated DC restrain proliferation and modulate cytokine production of allogeneic lymphocytes. These results demonstrate that EP has the ability to direct DC toward tolDC.

Heart⁻Lung⁻Muscle Anti-SAE Syndrome: An Atypical Severe Combination.

A 78-year-old man with 3 months of progressive dyspnea, dysphony, dysgeusia, and proximal muscle weakness was diagnosed of probably idiopathic inflammatory myopathy with nonspecific interstitial pneumonia. Variable degrees of atrioventricular block and persistently elevated cardiac enzymes indicated a diagnosis of myocarditis, confirmed with cardiac magnetic resonance imaging and endomyocardial biopsy. A comprehensive immune work-up revealed anti-small ubiquitin-like modifier-1 activating enzyme (anti-SAE) antibody, a novel myositis-specific antibody, previously described mainly with overt cutaneous dermatomyositis and late skeletal muscle manifestations. Here, heart⁻lung⁻muscle involvement combined with anti-SAE antibodies was a severe combination.

Comparative transcriptomic profile of tolerogenic dendritic cells differentiated with vitamin D3, dexamethasone and rapamycin.

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However, the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic, as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim, in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC), dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However, individually, CYP24A1, MUCL1 and MAP7 for vitD3-tolDC; CD163, CCL18, C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC, constituted good candidate biomarkers for each respective cellular product. In addition, a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways, while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes.

Searching for the Transcriptomic Signature of Immune Tolerance Induction-Biomarkers of Safety and Functionality for Tolerogenic Dendritic Cells and Regulatory Macrophages.

The last years have witnessed a breakthrough in the development of cell-based tolerance-inducing cell therapies for the treatment of autoimmune diseases and solid-organ transplantation. Indeed, the use of tolerogenic dendritic cells (tolDC) and regulatory macrophages (Mreg) is currently being tested in Phase I and Phase II clinical trials worldwide, with the aim of finding an effective therapy able to abrogate the inflammatory processes causing these pathologies without compromising the protective immunity of the patients. However, there exists a wide variety of different protocols to generate human tolDC and Mreg and, consequently, the characteristics of each product are heterogeneous. For this reason, the identification of biomarkers able to define their functionality (tolerogenicity) is of great relevance, on the one hand, to guarantee the safety of tolDC and Mreg before administration and, on the other hand, to compare the results between different cell products and laboratories. In this article, we perform an exhaustive review of protocols generating human tolDC and Mreg in the literature, aiming to elucidate if there are any common transcriptomic signature or potential biomarkers of tolerogenicity among the different approaches. However, and although several effectors seem to be induced in common in some of the most reported protocols to generate both tolDC or Mreg, the transcriptomic profile of these cellular products strongly varies depending on the approach used to generate them.

Predicting therapeutic response to fingolimod treatment in multiple sclerosis patients.

AIMS: Fingolimod, an orally active immunomodulatory drug for relapsing-remitting multiple sclerosis (RRMS), sequesters T cells in lymph nodes through functional antagonism of the sphingosine-1-phosphate receptor, reducing the number of potential autoreactive cells that migrate to the central nervous system. However, not all RRMS patients respond to this therapy. Our aim was to test the hypothesis that by immune-monitoring RRMS patient's leukocyte subpopulations it is possible to find biomarkers associated with clinical response to fingolimod. METHODS: Prospective study. Analysis of peripheral blood mononuclear cell subpopulations by multiparametric flow cytometry, at baseline and +1, +3, +6, +12 months of follow-up in 40 RRMS patients starting fingolimod therapy. RESULTS: Fingolimod treatment induced a severe lymphopenia affecting mainly T and B cells. A relative increase in Treg (memory Treg : 3.8 ± 1.0% baseline vs 8.8 ± 4.4% month +1; activated Treg : 1.5 ± 0.7% baseline vs 3.7 ± 2.1% month +1, P < 0.001) as well as transitional B cells (10.5 ± 12.3% baseline vs 18.7 ± 14.6% month +1, P < 0.001) was observed. Interestingly, lymphocyte subpopulations were already at baseline significantly different in responder patients. The percentage of recent thymic emigrants (RTE) used to stratify fingolimod responder, and no responder patients was the best biomarker (4.0 ± 1.4% vs 7.4 ± 1.9%, respectively [P < 0.001]). CONCLUSION: The results support that immune-monitoring of lymphocyte subpopulations in peripheral blood is a promising tool to select RRMS candidate for fingolimod treatment.

Corrigendum: Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

[This corrects the article on p. 1870 in vol. 8, PMID: 29312346.].

Immunotheraphy in Allergic Diseases.

The prevalence of allergic diseases is increasing worldwide. It is estimated that more than 30% of the world population is now affected by one or more allergic conditions and a high proportion of this increase is in young people. The diagnosis of allergy is dependent on a history of symptoms on exposure to an allergen together with the detection of allergen-specific IgE. Accurate diagnosis of allergies opens up therapeutic options. Allergen specific immunotherapy is the only successful disease-modifying therapy for IgE-mediated allergic diseases. New therapeutic strategies have been developed or are currently under clinical trials. Besides new routes of administration, new types of allergens are being developed. The use of adjuvants may amplify the immune response towards tolerance to the antigens. In this review, we analyze different antigen-specific immunotherapies according to administration route, type of antigens and adjuvants, and we address the special case of food allergy.

Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

Adoptive therapy with regulatory T cells or tolerance-inducing antigen (Ag)-presenting cells is innovative and promising therapeutic approach to control undesired and harmful activation of the immune system, as observed in autoimmune diseases, solid organ and bone marrow transplantation. One of the critical issues to elucidate the mechanisms responsible for success or failure of these therapies and define the specificity of the therapy is the evaluation of the Ag-specific T-cell responses. Several efforts have been made to develop suitable and reproducible assays. Here, we focus on dye-based proliferation assays. We highlight with practical examples the fundamental issues to take into consideration for implementation of an effective and sensitive dye-based proliferation assay to monitor Ag-specific responses in patients. The most critical points were used to design a road map to set up and analyze the optimal assay to assess Ag-specific T-cell responses in patients undergoing different treatments. This is the first step to optimize monitoring of tolerance induction, allowing comparison of outcomes of different clinical studies. The road map can also be applied to other therapeutic interventions, not limited to tolerance induction therapies, in which Ag-specific T-cell responses are relevant such as vaccination approaches and cancer immunotherapy.

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.

Hurdles in therapy with regulatory T cells.

Improper activation of the immune system contributes to a variety of clinical conditions, including autoimmune and allergic diseases as well as solid organ and bone marrow transplantation. One approach to counteract this activation is through adoptive therapy with regulatory T cells (Tregs). Efforts to manufacture these cells have led to good maunfacturing practice-compliant protocols, and Treg products are entering early clinical trials. Here, we report the stance of the European Union Cooperation in Science and Technology Action BM1305, "Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies-A FACTT," which identifies hurdles hindering Treg clinical applications in Europe and provides possible solutions.