Current status of immunological therapies for rheumatoid arthritis with a focus on antigen-specific therapeutic vaccines.

Rheumatoid arthritis (RA) is recognized as an autoimmune joint disease driven by T cell responses to self (or modified self or microbial mimic) antigens that trigger and aggravate the inflammatory condition. Newer treatments of RA employ monoclonal antibodies or recombinant receptors against cytokines or immune cell receptors as well as small-molecule Janus kinase (JAK) inhibitors to systemically ablate the cytokine or cellular responses that fuel inflammation. Unlike these treatments, a therapeutic vaccine, such as CEL-4000, helps balance adaptive immune homeostasis by promoting antigen-specific regulatory rather than inflammatory responses, and hence modulates the immunopathological course of RA. In this review, we discuss the current and proposed therapeutic products for RA, with an emphasis on antigen-specific therapeutic vaccine approaches to the treatment of the disease. As an example, we describe published results of the beneficial effects of CEL-4000 vaccine on animal models of RA. We also make a recommendation for the design of appropriate clinical studies for these newest therapeutic approaches, using the CEL-4000 vaccine as an example. Unlike vaccines that create or boost a new immune response, the clinical success of an immunomodulatory therapeutic vaccine for RA lies in its ability to redirect autoreactive pro-inflammatory memory T cells towards rebalancing the "runaway" immune/inflammatory responses that characterize the disease. Human trials of such a therapy will require alternative approaches in clinical trial design and implementation for determining safety, toxicity, and efficacy. These approaches include adaptive design (such as the Bayesian optimal design (BOIN), currently employed in oncological clinical studies), and the use of disease-related biomarkers as indicators of treatment success.

THEMIS increases TCR signaling in CD4+CD8+ thymocytes by inhibiting the activity of the tyrosine phosphatase SHP1.

The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4+CD8+ thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4+CD8+ thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis-/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis-/- developmental defect, whereas deletion of Ptpn6, Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4+CD8+ thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.

GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS-GRB2-SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis-/- thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands.

SHP-1 Protein Tyrosine Phosphatase Affects Early Postnatal Bone Development in Mice.

The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an intracellular tyrosine phosphatase that plays a negative regulatory role in immune cell signaling. Absent or diminished SHP-1 catalytic activity results in reduced bone mass with enhanced bone resorption. Here, we sought to investigate if Shp1 overexpression leads to increased bone mass and improved mechanical properties. Male and female wildtype (WT) and SHP1-transgenic (Tg) mice at 28, 56, and 84 days of age were compared. We applied microcomputed tomography to assess femoral cortical bone geometry and trabecular architecture and 3-point mechanical bending to assess mid-diaphyseal structural and estimated material properties. Serum OPG, RANKL, P1NP, and CTX-1 concentrations were measured by enzyme-linked immunoassay. The majority of transgene effects were restricted to the 28-day-old mice. Trabecular bone volume per total volume, trabecular number, and connectivity density were greater in 28-day-old female SHP1-Tg mice when compared to WTs. SHP1-Tg female mice showed increased total and medullary areas, with no difference in cortical area and thickness. Cortical tissue mineral density was strongly genotype-dependent. Failure load, yield load, ultimate stress, and yield stress were all lower in 28-day-old SHP1-Tg females. In 28-day-old SHP1-Tg females, circulating levels of OPG and P1NP were higher and RANKL levels were lower than WT controls. Our study demonstrates a role for SHP-1 in early postnatal bone development; SHP-1 overexpression negatively impacted whole bone strength and material properties in females.

Vaccination by Two DerG LEAPS Conjugates Incorporating Distinct Proteoglycan (PG, Aggrecan) Epitopes Provides Therapy by Different Immune Mechanisms in a Mouse Model of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) can be initiated and driven by immune responses to multiple antigenic epitopes including those in cartilage proteoglycan (PG, aggrecan) and type II collagen. RA is driven by T helper 1 (Th1) or Th17 pro-inflammatory T cell responses. LEAPS (Ligand Epitope Antigen Presentation System) DerG peptide conjugate vaccines were prepared using epitopes from PG that elicit immune responses in RA patients: epitope PG70 (DerG-PG70, also designated CEL-4000) and the citrullinated form of another epitope (PG275Cit). The LEAPS peptides were administered alone or together in Seppic ISA51vg adjuvant to mice with PG G1 domain-induced arthritis (GIA), a mouse model of RA. Each of these LEAPS peptides and the combination modulated the inflammatory response and stopped the progression of arthritis in the GIA mouse model. Despite having a therapeutic effect, the DerG-PG275Cit vaccine did not elicit significant antibody responses, whereas DerG-PG70 (alone or with DerG-PG275Cit) induced both therapy and antibodies. Spleen T cells from GIA mice, vaccinated with the DerG LEAPS peptides, preferentially produced anti-inflammatory (IL-4 and IL-10) rather than pro-inflammatory (IFN-γ or IL-17) cytokines in culture. Similarly, cytokines secreted by CD4+ cells of unvaccinated GIA mice, differentiated in vitro to Th2 cells and treated with either or both DerG vaccine peptides, exhibited an anti-inflammatory (IL-4, IL-10) profile. These results suggest that the two peptides elicit different therapeutic immune responses by the immunomodulation of disease-promoting pro-inflammatory responses and that the combination of the two LEAPS conjugates may provide broader epitope coverage and, in some cases, greater efficacy than either conjugate alone.

Restoring the Balance between Pro-Inflammatory and Anti-Inflammatory Cytokines in the Treatment of Rheumatoid Arthritis: New Insights from Animal Models.

Rheumatoid arthritis (RA) and other autoimmune inflammatory diseases are examples of imbalances within the immune system (disrupted homeostasis) that arise from the effects of an accumulation of environmental and habitual insults over a lifetime, combined with genetic predispositions. This review compares current immunotherapies-(1) disease-modifying anti-rheumatic drugs (DMARDs) and (2) Janus kinase (JAK) inhibitors (jakinibs)-to a newer approach-(3) therapeutic vaccines (using the LEAPS vaccine approach). The Ligand Epitope Antigen Presentation System (LEAPS) therapies are capable of inhibiting ongoing disease progression in animal models. Whereas DMARDs ablate or inhibit specific proinflammatory cytokines or cells and jakinibs inhibit the receptor activation cascade for expression of proinflammatory cytokines, the LEAPS therapeutic vaccines specifically modulate the ongoing antigen-specific, disease-driving, proinflammatory T memory cell responses. This decreases disease presentation and changes the cytokine conversation to decrease the expression of inflammatory cytokines (IL-17, IL-1(α or ß), IL-6, IFN-γ, TNF-α) while increasing the expression of regulatory cytokines (IL-4, IL-10, TGF-ß). This review refocuses the purpose of therapy for RA towards rebalancing the immune system rather than compromising specific components to stop disease. This review is intended to be thought provoking and look forward towards new therapeutic modalities rather than present a final definitive report.

Regulation of autoimmune arthritis by the SHP-1 tyrosine phosphatase.

BACKGROUND: The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to exert negative regulatory effects on immune cell signaling. Mice with mutations in the Shp1 gene develop inflammatory skin disease and autoimmunity, but no arthritis. We sought to explore the role of SHP-1 in arthritis using an autoimmune mouse model of rheumatoid arthritis. We generated Shp1 transgenic (Shp1-Tg) mice to study the impact of SHP-1 overexpression on arthritis susceptibility and adaptive immune responses. METHODS: SHP-1 gene and protein expression as well as tyrosine phosphatase activity were evaluated in spleen cells of transgenic and wild type (WT) mice. WT and Shp1-Tg (homozygous or heterozygous for the transgene) mice were immunized with human cartilage proteoglycan (PG) in adjuvant, and arthritis symptoms were monitored. Protein tyrosine phosphorylation level, net cytokine secretion, and serum anti-human PG antibody titers were measured in immune cells from WT and Shp1-Tg mice. WT mice were treated with regorafenib orally to activate SHP-1 either before PG-induced arthritis (PGIA) symptoms developed (preventive treatment) or starting at an early stage of disease (therapeutic treatment). Data were statistically analyzed and graphs created using GraphPad Prism 8.0.2 software. RESULTS: SHP-1 expression and tyrosine phosphatase activity were elevated in both transgenic lines compared to WT mice. While all WT mice developed arthritis after immunization, none of the homozygous Shp1-Tg mice developed the disease. Heterozygous transgenic mice, which showed intermediate PGIA incidence, were selected for further investigation. We observed differences in interleukin-4 and interleukin-10 production in vitro, but serum anti-PG antibody levels were not different between the genotypes. We also found decreased tyrosine phosphorylation of several proteins of the JAK/STAT pathway in T cells from PG-immunized Shp1-Tg mice. Regorafenib administration to WT mice prevented the development of severe PGIA or reduced disease severity when started after disease onset. CONCLUSIONS: Resistance to arthritis in the presence of SHP-1 overexpression likely results from the impairment of tyrosine phosphorylation (deactivation) of key immune cell signaling proteins in the JAK/STAT pathway, due to the overwhelming tyrosine phosphatase activity of the enzyme in Shp1-Tg mice. Our study is the first to investigate the role of SHP-1 in autoimmune arthritis using animals overexpressing this phosphatase. Pharmacological activation of SHP-1 might be considered as a new approach to the treatment of autoimmune arthritis.

Amelioration of Autoimmune Arthritis in Mice Treated With the DNA Methyltransferase Inhibitor 5'-Azacytidine.

OBJECTIVE: Disease-associated, differentially hypermethylated regions have been reported in rheumatoid arthritis (RA), but no DNA methyltransferase inhibitors have been evaluated in either RA or any animal models of RA. The present study was conducted to evaluate the therapeutic potential of 5'-azacytidine (5'-azaC), a DNA methyltransferase inhibitor, and explore the cellular and gene regulatory networks involved in the context of autoimmune arthritis. METHODS: A disease-associated genome-wide DNA methylation profile was explored by methylated CpG island recovery assay-chromatin immunoprecipitation (ChIP) in arthritic B cells. Mice with proteoglycan-induced arthritis (PGIA) were treated with 5'-azaC. The effect of 5'-azaC on the pathogenesis of PGIA was explored by measuring serum IgM and IgG1 antibody levels using enzyme-linked immunosorbent assay, investigating the efficiency of class-switch recombination (CSR) and Aicda gene expression using real-time quantitative polymerase chain reaction, monitoring germinal center (GC) formation by immunohistochemistry, and determining alterations in B cell subpopulations by flow cytometry. The 5'-azaC-induced regulation of the Aicda gene was explored using RNA interference, ChIP, and luciferase assays. RESULTS: We explored arthritis-associated hypermethylated regions in mouse B cells and demonstrated that DNA demethylation had a beneficial effect on autoimmune arthritis. The 5'-azaC-mediated demethylation of the epigenetically inactivated Ahr gene resulted in suppressed expression of the Aicda gene, reduced CSR, and compromised GC formation. Ultimately, this process led to diminished IgG1 antibody production and amelioration of autoimmune arthritis in mice. CONCLUSION: DNA hypermethylation plays a leading role in the pathogenesis of autoimmune arthritis and its targeted inhibition has therapeutic potential in arthritis management.

Citrullinated Aggrecan Epitopes as Targets of Autoreactive CD4+ T Cells in Patients With Rheumatoid Arthritis.

OBJECTIVE: Recognition of citrullinated antigens such as vimentin, fibrinogen, and α-enolase is associated with rheumatoid arthritis (RA). Emerging data suggest that the matrix protein aggrecan is also recognized as a citrullinated antigen. This study was undertaken to directly visualize Cit-aggrecan-specific T cells and characterize them in patients with RA. METHODS: Citrullinated aggrecan peptides with likely DRB1*04:01 binding motifs were predicted using a previously published scanning algorithm. Peptides with detectable binding were assessed for immunogenicity by HLA tetramer staining, followed by single cell cloning. Selectivity for citrullinated peptide was assessed by tetramer staining and proliferation assays. Ex vivo tetramer staining was then performed to assess frequencies of aggrecan-specific T cells in peripheral blood. Finally, disease association was assessed by comparing T cell frequencies in RA patients and controls and correlating aggrecan-specific T cells with levels of aggrecan-specific antibodies. RESULTS: We identified 6 immunogenic peptides, 2 of which were the predominant T cell targets in peripheral blood. These 2 epitopes were citrullinated at HLA binding residues and shared homologous sequences. RA patients had significantly higher frequencies of Cit-aggrecan-specific T cells than healthy subjects. Furthermore, T cell frequencies were significantly correlated with antibodies against citrullinated aggrecan. CONCLUSION: Our findings indicate that T cells that recognize citrullinated aggrecan are present in patients with RA and correlate with antibodies that target this same antigen. Consequently, aggrecan-specific T cells and antibodies are potentially relevant markers that could be used to monitor patients with RA or at-risk subjects.

An epitope-specific DerG-PG70 LEAPS vaccine modulates T cell responses and suppresses arthritis progression in two related murine models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune joint disease maintained by aberrant immune responses involving CD4+ T helper (Th)1 and Th17 cells. In this study, we tested the therapeutic efficacy of Ligand Epitope Antigen Presentation System (LEAPS™) vaccines in two Th1 cell-driven mouse models of RA, cartilage proteoglycan (PG)-induced arthritis (PGIA) and PG G1-domain-induced arthritis (GIA). The immunodominant PG peptide PG70 was attached to a DerG or J immune cell binding peptide, and the DerG-PG70 and J-PG70 LEAPS vaccines were administered to the mice after the onset of PGIA or GIA symptoms. As indicated by significant decreases in visual and histopathological scores of arthritis, the DerG-PG70 vaccine inhibited disease progression in both PGIA and GIA, while the J-PG70 vaccine was ineffective. Splenic CD4+ cells from DerG-PG70-treated mice were diminished in Th1 and Th17 populations but enriched in Th2 and regulatory T (Treg) cells. In vitro spleen cell-secreted and serum cytokines from DerG-PG70-treated mice demonstrated a shift from a pro-inflammatory to an anti-inflammatory/regulatory profile. DerG-PG70 peptide tetramers preferentially bound to CD4+ T-cells of GIA spleen cells. We conclude that the DerG-PG70 vaccine (now designated CEL-4000) exerts its therapeutic effect by interacting with CD4+ cells, which results in an antigen-specific down-modulation of pathogenic T-cell responses in both the PGIA and GIA models of RA. Future studies will need to determine the potential of LEAPS vaccination to provide disease suppression in patients with RA.

Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA) are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients. METHODS: We used mice with PG-induced arthritis (PGIA) as a screening tool to select citrulline (Cit)-containing PG peptides that were more immunogenic than the arginine (R)-containing counterparts. The selected peptide pairs were tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC) cultures using ELISA and flow cytometry. Anti-Cit and anti-R peptide antibodies were detected by ELISA. RESULTS: Splenocytes from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49) and anti-P49 antibodies were found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma. CONCLUSIONS: We identified a novel citrullinated PG epitope (Cit49) that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.

FTY720 (Gilenya) treatment prevents spontaneous autoimmune myocarditis and dilated cardiomyopathy in transgenic HLA-DQ8-BALB/c mice.

Although dilated cardiomyopathy (DCM) is often caused by viral infections, it frequently involves autoimmune mechanisms associated with particular HLA-DR and DQ alleles. Our homozygous HLA-DQ8Ab(0) transgenic mice in the BALB/c background (HLA-DQ8(BALB/c)-Tg) developed early and progressive fatal heart failure from 4 to 5 weeks of age. Clinical signs of the disease included cyanotic eyes, tachycardia with dyspnea (from pale to cyanotic limbs), and terminal whole body edema. Sick mice had extremely dilated hearts, enlarged liver and spleen, and pleural/peritoneal effusion. Histology of the heart showed extensive heart muscle destruction with signs of fibrosis. The autoimmune nature of the disease was shown by high titers of antimyosin antibodies in the sera and IgG deposits in sick heart muscles, as well as focal neutrophil, T cell, and macrophage infiltration of the heart muscle. The sera of the sick mice showed a granular staining pattern on sections of healthy heart muscle. Quantitative analyses of DCM-specific gene expression studies revealed that sets of genes are involved in inflammation, hypoxia, and fibrosis. Treatment with FTY720 (Fingolimod/Gilenya) protected animals from the development of cardiomyopathy. HLA-DQ8(BALB/c)-Tg mice represent a spontaneous autoimmune myocarditis model that may provide a useful tool for studying the autoimmune mechanism of DCM and testing immunosuppressive drugs.

Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA. METHODS: CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry. FINDINGS: Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage. CONCLUSIONS: CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.

Enigma of IL-17 and Th17 Cells in Rheumatoid Arthritis and in Autoimmune Animal Models of Arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders characterized by the chronic and progressive inflammation of various organs, most notably the synovia of joints leading to joint destruction, a shorter life expectancy, and reduced quality of life. Although we have substantial information about the pathophysiology of the disease with various groups of immune cells and soluble mediators identified to participate in the pathogenesis, several aspects of the altered immune functions and regulation in RA remain controversial. Animal models are especially useful in such scenarios. Recently research focused on IL-17 and IL-17 producing cells in various inflammatory diseases such as in RA and in different rodent models of RA. These studies provided occasionally contradictory results with IL-17 being more prominent in some of the models than in others; the findings of such experimental setups were sometimes inconclusive compared to the human data. The aim of this review is to summarize briefly the recent advancements on the role of IL-17, particularly in the different rodent models of RA.

Rheumatoid arthritis vaccine therapies: perspectives and lessons from therapeutic ligand epitope antigen presentation system vaccines for models of rheumatoid arthritis.

The current status of therapeutic vaccines for autoimmune diseases is reviewed with rheumatoid arthritis as the focus. Therapeutic vaccines for autoimmune diseases must regulate or subdue responses to common self-antigens. Ideally, such a vaccine would initiate an antigen-specific modulation of the T-cell immune response that drives the inflammatory disease. Appropriate animal models and types of T helper cells and signature cytokine responses that drive autoimmune disease are also discussed. Interpretation of these animal models must be done cautiously because the means of initiation, autoantigens, and even the signature cytokine and T helper cell (Th1 or Th17) responses that are involved in the disease may differ significantly from those in humans. We describe ligand epitope antigen presentation system vaccine modulation of T-cell autoimmune responses as a strategy for the design of therapeutic vaccines for rheumatoid arthritis, which may also be effective in other autoimmune conditions.

Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA. METHODS: Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined. RESULTS: BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels. CONCLUSIONS: Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

Identification of myeloid-derived suppressor cells in the synovial fluid of patients with rheumatoid arthritis: a pilot study.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of innate immune cells with a granulocyte-like or monocyte-like phenotype and a unique ability to suppress T-cell responses. MDSCs have been shown to accumulate in cancer patients, but recent studies suggest that these cells are also present in humans and animals suffering from autoimmune diseases. We previously identified MDSCs in the synovial fluid (SF) of mice with experimental autoimmune arthritis. The goal of the present study was to identify MDSCs in the SF of patients with rheumatoid arthritis (RA). METHODS: RA SF cells were studied by flow cytometry using antibodies to MDSC cell surface markers as well as by analysis of cell morphology. The suppressor activity of RA SF cells toward autologous peripheral blood T cells was determined ex vivo. We employed both antigen-nonspecific (anti-CD3/CD28 antibodies) and antigen-specific (allogeneic cells) induction systems to test the effects of RA SF cells on the proliferation of autologous T cells. RESULTS: SF from RA patients contained MDSC-like cells, the majority of which showed granulocyte (neutrophil)-like phenotype and morphology. RA SF cells significantly suppressed the proliferation of anti-CD3/CD28-stimulated autologous T cells upon co-culture. When compared side by side, RA SF cells had a more profound inhibitory effect on the alloantigen-induced than the anti-CD3/CD28-induced proliferation of autologous T cells. CONCLUSION: MDSCs are present among RA SF cells that are commonly regarded as inflammatory neutrophils. Our results suggest that the presence of neutrophil-like MDSCs in the SF is likely beneficial, as these cells have the ability to limit the expansion of joint-infiltrating T cells in RA.

Epigenetics in the pathogenesis of rheumatoid arthritis.

An increasing number of studies show that besides the inherited genetic architecture (that is, genomic DNA), various environmental factors significantly contribute to the etiology of rheumatoid arthritis. Epigenetic factors react to external stimuli and form bridges between the environment and the genetic information-harboring DNA. Epigenetic mechanisms are implicated in the final interpretation of the encoded genetic information by regulating gene expression, and alterations in their profile influence the activity of the immune system. Overall, epigenetic mechanisms further increase the well-known complexity of rheumatoid arthritis by providing additional subtle contributions to rheumatoid arthritis susceptibility. Although there are controversies regarding the involvement of epigenetic and genetic factors in rheumatoid arthritis etiology, it is becoming obvious that the two systems (genetic and epigenetic) interact with each other and are ultimately responsible for rheumatoid arthritis development. Here, epigenetic factors and mechanisms involved in rheumatoid arthritis are reviewed and new, potential therapeutic targets are discussed.