Genetically encoded Runx3 and CD4+ intestinal epithelial lymphocyte deficiencies link SKG mouse and human predisposition to spondyloarthropathy.

Disturbances in immune regulation, intestinal dysbiosis and inflammation characterize ankylosing spondylitis (AS), which is associated with RUNX3 loss-of-function variants. ZAP70W163C mutant (SKG) mice have reduced ZAP70 signaling, spondyloarthritis and ileitis. In small intestine, Foxp3+ regulatory T cells (Treg) and CD4+CD8αα+TCRαß+ intraepithelial lymphocytes (CD4-IEL) control inflammation. TGF-ß and retinoic acid (RA)-producing dendritic cells and MHC-class II+ intestinal epithelial cells (IEC) are required for Treg and CD4-IEL differentiation from CD4+ conventional or Treg precursors, with upregulation of Runx3 and suppression of ThPOK. We show in SKG mouse ileum, that ZAP70W163C or ZAP70 inhibition prevented CD4-IEL but not Treg differentiation, dysregulating Runx3 and ThPOK. TGF-ß/RA-mediated CD4-IEL development, T-cell IFN-γ production, MHC class-II+ IEC, tissue-resident memory T-cell and Runx3-regulated genes were reduced. In AS intestine, CD4-IEL were decreased, while in AS blood CD4+CD8+ T cells were reduced and Treg increased. Thus, genetically-encoded TCR signaling dysfunction links intestinal T-cell immunodeficiency in mouse and human spondyloarthropathy.

Randomized phase I trial of antigen-specific tolerizing immunotherapy with peptide/calcitriol liposomes in ACPA+ rheumatoid arthritis.

BACKGROUNDAntigen-specific regulation of autoimmune disease is a major goal. In seropositive rheumatoid arthritis (RA), T cell help to autoreactive B cells matures the citrullinated (Cit) antigen-specific immune response, generating RA-specific V domain glycosylated anti-Cit protein antibodies (ACPA VDG) before arthritis onset. Low or escalating antigen administration under "sub-immunogenic" conditions favors tolerance. We explored safety, pharmacokinetics, and immunological and clinical effects of s.c. DEN-181, comprising liposomes encapsulating self-peptide collagen II259-273 (CII) and NF-κB inhibitor 1,25-dihydroxycholecalciferol.METHODSA double-blind, placebo-controlled, exploratory, single-ascending-dose, phase I trial assessed the impact of low, medium, and high DEN-181 doses on peripheral blood CII-specific and bystander Cit64vimentin59-71-specific (Cit-Vim-specific) autoreactive T cell responses, cytokines, and ACPA in 17 HLA-DRB1*04:01+ or *01:01+ ACPA+ RA patients on methotrexate.RESULTSDEN-181 was well tolerated. Relative to placebo and normalized to baseline values, Cit-Vim-specific T cells decreased in patients administered medium and high doses of DEN-181. Relative to placebo, percentage of CII-specific programmed cell death 1+ T cells increased within 28 days of DEN-181. Exploratory analysis in DEN-181-treated patients suggested improved RA disease activity was associated with expansion of CII-specific and Cit-Vim-specific T cells; reduction in ACPA VDG, memory B cells, and inflammatory myeloid populations; and enrichment in CCR7+ and naive T cells. Single-cell sequencing identified T cell transcripts associated with tolerogenic TCR signaling and exhaustion after low or medium doses of DEN-181.CONCLUSIONThe safety and immunomodulatory activity of low/medium DEN-181 doses provide rationale to further assess antigen-specific immunomodulatory therapy in ACPA+ RA.TRIAL REGISTRATIONAnzctr.org.au identifier ACTRN12617001482358, updated September 8, 2022.FUNDINGInnovative Medicines Initiative 2 Joint Undertaking (grant agreement 777357), supported by European Union's Horizon 2020 research and innovation programme and European Federation of Pharmaceutical Industries and Associations; Arthritis Queensland; National Health and Medical Research Council (NHMRC) Senior Research Fellowship; and NHMRC grant 2008287.

Evaluation of a fit-for-purpose assay to monitor antigen-specific functional CD4+ T-cell subpopulations in rheumatoid arthritis using flow cytometry-based peptide-MHC class-II tetramer staining.

Antigen-specific T cells can serve as a response biomarker in non-clinical or clinical immunotherapy studies in autoimmune disease. There are protocols with optimized multimer staining methods to detect peptide (p)MHCII+ CD4+ T cells, and some qualified and validated protocols for pMHCI+ CD8+ T cells. However, no protocol is fully or partially qualified to enumerate and characterize antigen-specific pMHCII+ CD4+ T cells from patient samples. Implementing such an assay requires a desired level of specificity and precision, in terms of assay repeatability and reproducibility. In transgenic type II collagen (CII)-immunized HLA-DR1/DR4 humanized mouse models of collagen-induced arthritis (CIA), CII259-273-specific T cells dominantly expand. Therefore antigen-specific T cells recognizing this epitope presented by rheumatoid arthritis (RA)-associated risk HLA-DR allomorphs are of interest to understand disease progression and responses to immunotherapy in RA patients. Using HLA-DRB1∗04:01 or ∗01:01-collagen type II (CII)259-273 tetramers, we evaluated parameters influencing precision and reproducibility of an optimized flow cytometry-based method for antigen-specific CD4+ T cells and eight specific subpopulations with and without tetramer positivity. We evaluated specificity, precision, and reproducibility for research environments and non-regulated laboratories. The assay has excellent overall precision with %CV<25% for intra-assay repeatability, inter-analyst precision, and inter-assay reproducibility. The precision of the assay correlated negatively with the cell viability after thawing, indicating that post-thaw viability is a critical parameter for reproducibility. This assay is suitable for longitudinal analysis of treatment response and disease activity outcome in RA patients, and adaptable for translational or immunotherapy clinical trial settings.

IL-6 receptor blockade does not slow ß cell loss in new-onset type 1 diabetes.

BackgroundIL-6 receptor (IL-6R) signaling drives development of T cell populations important to type 1 diabetes pathogenesis. We evaluated whether blockade of IL-6R with monoclonal antibody tocilizumab would slow loss of residual ß cell function in newly diagnosed type 1 diabetes patients.MethodsWe conducted a multicenter, randomized, placebo-controlled, double-blind trial with tocilizumab in new-onset type 1 diabetes. Participants were screened within 100 days of diagnosis. Eligible participants were randomized 2:1 to receive 7 monthly doses of tocilizumab or placebo. The primary outcome was the change from screening in the mean AUC of C-peptide collected during the first 2 hours of a mixed meal tolerance test at week 52 in pediatric participants (ages 6-17 years).ResultsThere was no statistical difference in the primary outcome between tocilizumab and placebo. Immunophenotyping showed reductions in downstream signaling of the IL-6R in T cells but no changes in CD4 memory subsets, Th17 cells, Tregs, or CD4+ T effector cell resistance to Treg suppression. A DC subset decreased during therapy but regressed to baseline once therapy stopped. Tocilizumab was well tolerated.ConclusionTocilizumab reduced T cell IL-6R signaling but did not modulate CD4+ T cell phenotypes or slow loss of residual ß cell function in newly diagnosed individuals with type 1 diabetes.Trial RegistrationClinicalTrials.gov NCT02293837.FundingNIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and National Institute of Allergy and Infectious Diseases (NIAID) UM1AI109565, UL1TR000004 from NIH/National Center for Research Resources (NCRR) Clinical and Translational Science Award (CTSA), NIH/NIDDK P30DK036836, NIH/NIDDK U01DK103266, NIH/NIDDK U01DK103266, 1UL1TR000064 from NIH/NCRR CTSA, NIH/National Center for Advancing Translational Sciences (NCATS) UL1TR001878, UL1TR002537 from NIH/CTSA; National Health and Medical Research Council Practitioner Fellowship (APP1136735), NIH/NIDDK U01-DK085476, NIH/CTSA UL1-TR002494, Indiana Clinical and Translational Science Institute Award UL1TR002529, Vanderbilt Institute for Clinical and Translational Research UL1TR000445. NIH/NCATS UL1TR003142, NIH/CTSA program UL1-TR002494, Veteran Affairs Administration, and 1R01AI132774.

Proinsulin-specific T-cell responses correlate with estimated c-peptide and predict partial remission duration in type 1 diabetes.

OBJECTIVE: Type 1 diabetes (T1D) is an autoimmune disorder in which autoreactive T cells destroy insulin-producing ß-cells. Interventions that preserve ß-cell function represent a fundamental therapeutic goal in T1D and biomarkers that predict and monitor ß-cell function, and changes in islet autoantigenic signatures are needed. As proinsulin and neoantigens derived from proinsulin peptides (hybrid insulin peptides, HIPs) are important T1D autoantigens, we analysed peripheral blood CD4+ T-cell autoantigen-specific proliferative responses and their relationship to estimated ß-cell function. METHODS: We recruited 72 people with and 42 without T1D, including 17 pre-diabetic islet antibody-positive and 9 antibody-negative first-degree relatives and 16 unrelated healthy controls with T1D-risk HLA types. We estimated C-peptide level at 3-month intervals for 2 years post-diagnosis and measured CD4+ T-cell proliferation to proinsulin epitopes and HIPs using an optimised bioassay. RESULTS: We show that CD4+ T-cell proliferation to any islet peptide and to multiple epitopes were significantly more frequent in pre-diabetic islet antibody-positive siblings and participants with T1D ≤ 3 months of duration, than in participants with T1D > 3 months or healthy controls. Among participants with T1D and first-degree relatives, CD4+ T-cell proliferation occurred most frequently in response to proinsulin33-63 (full-length C-peptide). Proinsulin33-63-specific responses were associated with HLA-DR3-DQ2 and/or HLA-DR4/DQ8. In children with T1D, proinsulin33-63-specific T-cell proliferation positively associated with concurrent estimated C-peptide and predicted survival in honeymoon. CONCLUSION: CD4+ T-cell proliferative responses to proinsulin-containing autoantigens are common before and immediately after diagnosis of T1D but decline thereafter. Proinsulin33-63-specific CD4+ T-cell response is a novel marker of estimated residual endogenous ß-cell function and predicts a better 2-year disease outcome.

Altered Repertoire Diversity and Disease-Associated Clonal Expansions Revealed by T Cell Receptor Immunosequencing in Ankylosing Spondylitis Patients.

OBJECTIVE: Ankylosing spondylitis (AS) is a common spondyloarthropathy primarily affecting the axial skeleton and strongly associated with HLA-B*27 carriage. Genetic evidence implicates both autoinflammatory processes and autoimmunity against an HLA-B*27-restricted autoantigen in immunopathology. In addition to articular symptoms, up to 70% of AS patients present with concurrent bowel inflammation, suggesting that adverse interactions between a genetically primed host immune system and the gut microbiome contribute to the disease. Accordingly, this study aimed to characterize adaptive immune responses to antigenic stimuli in AS. METHODS: The peripheral CD4 and CD8 T cell receptor (TCR) repertoire was profiled in AS patients (n = 47) and HLA-B*27-matched healthy controls (n = 38). Repertoire diversity was estimated using the Normalized Shannon Diversity Entropy (NSDE) index, and univariate and multivariate statistical analyses were performed to characterize AS-associated clonal signatures. Furthermore, T cell proliferation and cytokine production in response to immunogenic antigen exposure were investigated in vitro in peripheral blood mononuclear cells from AS patients (n = 19) and HLA-B*27-matched healthy controls (n = 14). RESULTS: Based on the NSDE measure of sample diversity across CD4 and CD8 T cell repertoires, AS patients showed increased TCR diversity compared to healthy controls (for CD4 T cells, P = 7.8 × 10-6 ; for CD8 T cells, P = 9.3 × 10-4 ), which was attributed to a significant reduction in the magnitude of peripheral T cell expansions globally. Upon in vitro stimulation, fewer T cells from AS patients than from healthy controls expressed interferon-γ (for CD8 T cells, P = 0.03) and tumor necrosis factor (for CD4 T cells, P = 0.01; for CD8 T cells, P = 0.002). In addition, the CD8 TCR signature was altered in HLA-B*27+ AS patients compared to healthy controls, with significantly expanded Epstein-Barr virus-specific clonotypes (P = 0.03) and cytomegalovirus-specific clonotypes (P = 0.02). HLA-B*27+ AS patients also showed an increased incidence of "public" CD8 TCRs, representing identical clonotypes emerging in response to common antigen encounters, including homologous clonotypes matching those previously isolated from individuals with bacterial-induced reactive arthritis. CONCLUSION: The dynamics of peripheral T cell responses in AS patients are altered, suggesting that differential antigen exposure and disrupted adaptive immunity are underlying features of the disease.

RelB suppresses type I Interferon signaling in dendritic cells.

Type I Interferon (IFN) signaling plays a critical role in dendritic cell (DC) development and functions. Inhibition of hyper type I IFN signaling promotes cDC2 subtype development. Relb is essential to development of cDC2 subtype and here we analyzed its effect on type I IFN signaling in DCs. We show that Relb suppresses the homeostatic type I IFN signaling in cDC2 cultures. TLR stimulation of FL-DCs led to RelB induction coinciding with fall in IFN signatures; conforming with the observation Relb expression reduced TLR stimulated IFN induction along with decrease in ISGs. Towards understanding mechanism, we show that effects of RelB are mediated by increased levels of IκBα. We demonstrate that RelB dampened antiviral responses by lowering ISG levels and the defect in cDC2 development in RelB null mice can be rescued in Ifnar1-/- background. Overall, we propose a novel role of RelB as a negative regulator of the type I IFN signaling pathway; fine tuning development of cDC2 subtype.

Optimization of a Method to Detect Autoantigen-Specific T-Cell Responses in Type 1 Diabetes.

The development of tolerizing therapies aiming to inactivate autoreactive effector T-cells is a promising therapeutic approach to control undesired autoimmune responses in human diseases such as Type 1 Diabetes (T1D). A critical issue is a lack of sensitive and reproducible methods to analyze antigen-specific T-cell responses, despite various attempts. We refined a proliferation assay using the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) to detect responding T-cells, highlighting the fundamental issues to be taken into consideration to monitor antigen-specific responses in patients with T1D. The critical elements that maximize detection of antigen-specific responses in T1D are reduction of blood storage time, standardization of gating parameters, titration of CFSE concentration, selecting the optimal CFSE staining duration and the duration of T-cell stimulation, and freezing in medium containing human serum. Optimization of these elements enables robust, reproducible application to longitudinal cohort studies or clinical trial samples in which antigen-specific T-cell responses are relevant, and adaptation to other autoimmune diseases.

Autoantigens in rheumatoid arthritis and the potential for antigen-specific tolerising immunotherapy.

Autoimmune diseases, including rheumatoid arthritis, develop and persist due to impaired immune self-tolerance, which has evolved to regulate inflammatory responses to injury or infection. After diagnosis, patients rarely achieve drug-free remission, and although at-risk individuals can be identified with genotyping, antibody tests, and symptoms, rheumatoid arthritis cannot yet be successfully prevented. Precision medicine is increasingly offering solutions to diseases that were previously considered to be incurable, and immunotherapy has begun to achieve this aim in cancer. Comparatively, modulating autoantigen-specific immune responses with immunotherapy for the cure of autoimmune diseases is at a relatively immature stage. Current treatments using non-specific immune or inflammatory suppression increase susceptibility to infection, and are rarely curative. However, early stage clinical trials suggesting that immunotherapy might allow extended duration of remission and even prevention of progression to disease suggest modulating tolerance in rheumatoid arthritis could be a promising opportunity for therapy.

PD-L1- and calcitriol-dependent liposomal antigen-specific regulation of systemic inflammatory autoimmune disease.

Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Airway cells from protracted bacterial bronchitis and bronchiectasis share similar gene expression profiles.

AIM: Protracted bacterial bronchitis (PBB) is a common cause of prolonged cough in young children, and may be a precursor of bronchiectasis. Bacteria are often present in the lower airways in both PBB and bronchiectasis and may cause persistent infections. However, there is a paucity of information available on the pathogenesis of PBB and the factors associated with persistent bacterial infection and progression to bronchiectasis. This study hypothesised that lung immune cells in recurrent PBB and bronchiectasis differentially express genes related to immune cell dysfunction compared to lung immune cells from control subjects. METHOD: Cells isolated from bronchoalveolar lavage (adult-control and PBB BAL cells) were stimulated with nontypeable Haemophilus influenzae (NTHi), and expression of genes involved in various inflammatory pathways was assessed. RESULT: NTHi induced production of large amounts of IL-1ß, IL-6, and IL-8 in adult-control BAL cells, however BAL cells from PBB airways appeared refractory to NTHi stimulation. BAL cells from PBB and bronchiectasis showed differential expression of several genes relative to control cells, including CCL20, MARCO, CCL24, IL-10, PPAR-γ, CD200R, TREM2, RelB. Expression of genes involved in resolution of inflammation and anti-inflammation response, such as CD200R and IL-10, was associated with the number of pathogenic bacteria found in the airways. CONCLUSION: In summary, we have shown that the expression of genes related to macrophage function and resolution of inflammation are similar in PBB and bronchiectasis. Lung immune cell dysfunction in PBB and bronchiectasis may contribute to poor bacterial clearance and prolonged resolution of inflammation.

Flow Cytometric Clinical Immunomonitoring Using Peptide-MHC Class II Tetramers: Optimization of Methods and Protocol Development.

With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerizing immunotherapy have become a reality. Besides safety, it will be essential to gather mechanistic data on responding CD4+ T cells to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide-MHC class II (pMHCII) multimers are an ideal tool for monitoring antigen-specific CD4+ T cell responses in unmanipulated cells directly ex vivo. Various protocols have been published but there are reagent and assay limitations across laboratories that could hinder their global application to immune monitoring. In this methodological analysis, we compare protocols and test available reagents to identify sources of variability and to determine the limitations of the tetramer binding assay. We describe a robust pMHCII flow cytometry-based assay to quantify and phenotype antigen-specific CD4+ T cells directly ex vivo from frozen peripheral blood mononuclear cell samples, which we suggest should be tested across various laboratories to standardize immune-monitoring results.

Dexamethasone and Monophosphoryl Lipid A Induce a Distinctive Profile on Monocyte-Derived Dendritic Cells through Transcriptional Modulation of Genes Associated With Essential Processes of the Immune Response.

There is growing interest in the use of tolerogenic dendritic cells (tolDCs) as a potential target for immunotherapy. However, the molecular bases that drive the differentiation of monocyte-derived DCs (moDCs) toward a tolerogenic state are still poorly understood. Here, we studied the transcriptional profile of moDCs from healthy subjects, modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), referred to as Dex-modulated and MPLA-activated DCs (DM-DCs), as an approach to identify molecular regulators and pathways associated with the induction of tolerogenic properties in tolDCs. We found that DM-DCs exhibit a distinctive transcriptional profile compared to untreated (DCs) and MPLA-matured DCs. Differentially expressed genes downregulated by DM included MMP12, CD1c, IL-1B, and FCER1A involved in DC maturation/inflammation and genes upregulated by DM included JAG1, MERTK, IL-10, and IDO1 involved in tolerance. Genes related to chemotactic responses, cell-to-cell signaling and interaction, fatty acid oxidation, metal homeostasis, and free radical scavenging were strongly enriched, predicting the activation of alternative metabolic processes than those driven by counterpart DCs. Furthermore, we identified a set of genes that were regulated exclusively by the combined action of Dex and MPLA, which are mainly involved in the control of zinc homeostasis and reactive oxygen species production. These data further support the important role of metabolic processes on the control of the DC-driven regulatory immune response. Thus, Dex and MPLA treatments modify gene expression in moDCs by inducing a particular transcriptional profile characterized by the activation of tolerance-associated genes and suppression of the expression of inflammatory genes, conferring the potential to exert regulatory functions and immune response modulation.

CD4+CD8ß+ double-positive T cells in skin-draining lymph nodes respond to inflammatory signals from the skin.

CD4+CD8+ double-positive (DP), mature, peripheral T cells are readily detectable in a variety of species and tissues. Despite a common association with autoimmune and malignant skin disorders, however, little is understood about their role or function. Herein, we show that DP T cells are readily detectable in the blood, spleen, and peripheral lymph nodes of naïve C57BL/6 mice. DP T cells were also present in Jα18-/- and CD1d-/- mice, indicating that these cells are not NK-T cells. After skin administration of CASAC adjuvant, but not Quil A adjuvant, both total DP T cells and skin-infiltrating DP T cells increased in number. We explored the possibility that DP T cells could represent aggregates between CD4+ and CD8+ single-positive T cells and found strong evidence that a large proportion of apparent DP T cells were indeed aggregates. However, the existence of true CD4+CD8+ DP T cells was confirmed by Amnis ImageStream (Millipore Sigma, Billerica, MA, USA) imaging. Multiple rounds of FACS sorting separated true DP cells from aggregates and indicated that conventional analyses may lead to ∼10-fold overestimation of DP T cell numbers. The high degree of aggregate contamination and overestimation of DP abundance using conventional analysis techniques may explain discrepancies reported in the literature for DP T cell origin, phenotype, and function.

Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features.

Tolerogenic dendritic cells (TolDCs) are promising tools for therapy of autoimmune diseases, such as rheumatoid arthritis (RA). Here, we characterize monocyte-derived TolDCs from RA patients modulated with dexamethasone and activated with monophosphoryl lipid A (MPLA), referred to as MPLA-tDCs, in terms of gene expression, phenotype, cytokine profile, migratory properties, and T cell-stimulatory capacity in order to explore their suitability for cellular therapy. MPLA-tDCs derived from RA patients displayed an anti-inflammatory profile with reduced expression of co-stimulatory molecules and high IL-10/IL-12 ratio, but were capable of migrating toward the lymphoid chemokines CXCL12 and CCL19. These MPLA-tDCs induced hyporesponsiveness of autologous CD4+ T cells specific for synovial antigens in vitro. Global transcriptome analysis confirmed a unique transcriptional profile of MPLA-tDCs and revealed that RA-associated genes, which were upregulated in untreated DCs from RA patients, returned to expression levels of healthy donor-derived DCs after treatment with dexamethasone and MPLA. Thus, monocyte-derived DCs from RA patients have the capacity to develop tolerogenic features at transcriptional as well as at translational level, when modulated with dexamethasone and MPLA, overcoming disease-related effects. Furthermore, the ability of MPLA-tDCs to impair T cell responses to synovial antigens validates their potential as cellular treatment for RA.

Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients.

In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritis in an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti-citrullinated peptide autoantibodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated with HLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCs modified with a nuclear factor κB (NF-κB) inhibitor exposed to four citrullinated peptide antigens, designated "Rheumavax," in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered once intradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype-positive RA patients with citrullinated peptide-specific autoimmunity. Sixteen RA patients served as controls. Rheumavax was well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a reduction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15 (IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin(447-455)-Cit450 relative to controls. Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreased within 1 month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety and biological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides, and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen immunomodulatory therapy in RA.

Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens.

BACKGROUND: The global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection. RESULTS: We demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice. CONCLUSION: Our data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

Participation of MyD88 and interleukin-33 as innate drivers of Th2 immunity to Trichinella spiralis.

Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses.