Amelioration of Murine Passive Immune Thrombocytopenia by IVIg and a Therapeutic Monoclonal CD44 Antibody Does Not Require the Myd88 Signaling Pathway

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a low platelet count and the production of anti-platelet antibodies. The majority of ITP patients have antibodies to platelet integrin α_IIbβ₃ (GPIIbIIIa) which can direct platelet phagocytosis by macrophages. One effective treatment for patients with ITP is intravenous immunoglobulin (IVIg) which rapidly reverses thrombocytopenia. The exact mechanism of IVIg action in human patients is unclear, although in mouse models of passive ITP, IVIg can rapidly increase platelet counts in the absence of adaptive immunity. Another antibody therapeutic that can similarly increase platelet counts independent of adaptive immunity are CD44 antibodies. Toll-like receptors (TLRs) are pattern recognition receptors which play a central role in helping direct the innate immune system. Dendritic cells, which are notable for their expression of TLRs, have been directly implicated in IVIg function as an initiator cell, while CD44 can associate with TLR2 and TLR4. We therefore questioned whether IVIg, or the therapeutic CD44 antibody KM114, mediate their ameliorative effects in a manner dependent upon normal TLR function. Here, we demonstrate that the TLR4 agonist LPS does not inhibit IVIg or KM114 amelioration of antibody-induced thrombocytopenia, and that these therapeutics do not ameliorate LPS-induced thrombocytopenia. IVIg was able to significantly ameliorate murine ITP in C3H/HeJ mice which have defective TLR4. All known murine TLRs except TLR3 utilize the Myd88 adapter protein to drive TLR signaling. Employing Myd88 deficient mice, we found that both IVIg and KM114 ameliorate murine ITP in Myd88 deficient mice to the same extent as normal mice. Thus both IVIg and anti-CD44 antibody can mediate their ameliorative effects in murine passive ITP independent of the Myd88 signaling pathway. These data help shed light on the mechanism of action of IVIg and KM114 in the amelioration of murine ITP.     

Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation

The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.     

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.     

Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease

Humans and mice deficient in Fas, a tumor necrosis factor (TNF)-receptor family member, cannot induce apoptosis of autoreactive cells, and consequently develop progressive lymphoproliferative disorders and lupus-like autoimmune diseases. Previous studies have shown that short-term administrations of agonistic monoclonal antibodies against CD137, another TNF-receptor family member, activate T cells and induce rejection of allografts and established tumors. Here we report that treatment with an agonistic monoclonal antibody to CD137 (2A) blocks lymphadenopathy and spontaneous autoimmune diseases in Fas-deficient MRL/lpr mice, ultimately leading to their prolonged survival. Notably, 2A treatment rapidly augments IFN-gamma production, and induces the depletion of autoreactive B cells and abnormal double-negative T cells, possibly by increasing their apoptosis through Fas- and TNF receptor-independent mechanisms. This study demonstrates that agonistic monoclonal antibodies specific for costimulatory molecules can be used as novel therapeutic agents to delete autoreactive lymphocytes and block autoimmune disease progression.     

CD3-specific antibody-induced active tolerance: from bench to bedside

Although they were used initially as non-specific immunosuppressants in transplantation, CD3-specific monoclonal antibodies have elicited renewed interest owing to their capacity to induce immune tolerance. In mouse models of autoimmune diabetes, CD3-specific antibodies induce stable disease remission by restoring tolerance to pancreatic beta-cells. This phenomenon was extended recently to the clinic--preservation of beta-cell function in recently diagnosed patients with diabetes was achieved by short-term administration of a CD3-specific antibody. CD3-specific antibodies arrest ongoing disease by rapidly clearing pathogenic T cells from the target. Subsequently, they promote long-term T-cell-mediated active tolerance. Recent data indicate that transforming growth factor-beta-dependent CD4+CD25+ regulatory T cells might have a central role in this effect.     

CD3-specific antibody-induced immune tolerance involves transforming growth factor-beta from phagocytes digesting apoptotic T cells

Intact CD3-specific antibody transiently depletes large numbers of T cells and subsequently induces long-term immune tolerance. The underlying mechanisms for the systemic tolerance, however, remain unclear. We show here that treatment of normal mice with intact antibody to CD3 increases systemic transforming growth factor-beta (TGF-beta) produced by phagocytes exposed to apoptotic T cells. Among the phagocytes, macrophages and immature dendritic cells (iDCs) secrete TGF-beta upon ingestion of apoptotic T cells, which induces CD4+Foxp3+ regulatory T cells in culture and contributes to immune tolerance mediated by CD3-specific antibody in vivo. In accordance with these results, depletion of macrophages and iDCs not only abrogates CD3-specific antibody-mediated prevention of myelin oligodendrocyte glycoprotein-induced acute experimental autoimmune encephalomyelitis (EAE), but also reverses the therapeutic effects of antibody to CD3 on established disease in a model of relapsing-remitting EAE. Thus, CD3-specific antibody-induced immune tolerance is associated with TGF-beta production in phagocytes involved in clearing apoptotic T cells, which suggests that apoptosis is linked to active suppression in immune tolerance.     

Preventing autoimmune arthritis using antigen-specific immature dendritic cells: a novel tolerogenic vaccine

Conventional treatments for autoimmune diseases have relied heavily on nonspecific immune suppressants, which possess a variety of adverse effects without inhibiting the autoimmune process in a specific manner. In the present study we demonstrate the effectiveness of antigen-specific, maturation-resistant, tolerogenic dendritic cells (DC) in suppressing collagen-induced arthritis, a murine model of rheumatoid arthritis. Treatment of DC progenitors with the NF-κB inhibiting agent LF 15-0195 (LF) resulted in a population of tolerogenic DC that are characterized by low expression of MHC class II, CD40, and CD86 molecules, as well as by poor allostimulatory capacity in a mixed leukocyte reaction. Administering LF-treated DC pulsed with keyhole limpet hemocyanin antigen to naïve mice resulted hyporesponsiveness specific for this antigen. Furthermore, administration of LF-treated DC to mice with collagen-induced arthritis resulted in an improved clinical score, in an inhibited antigen-specific T-cell response, and in reduced antibody response to the collagen. The efficacy of LF-treated DC in preventing arthritis was substantiated by histological examination, which revealed a significant decrease in inflammatory cell infiltration in the joints. In conclusion, we demonstrate that in vitro-generated antigen-specific immature DC may have important potential as a tolerogenic vaccine for the treatment of autoimmune arthritis.     

Inhibition of experimental autoimmune thyroiditis in mice by anti-I-A antibodies

Experimental autoimmune thyroiditis is induced in mice by immunization with thyroglobulin emulsified in Freund's complete adjuvant. The disease is characterized both by thyroid infiltration with mononuclear cells and by circulating thyroglobulin antibodies. The magnitude of the thyroid infiltration and the titer of thyroglobulin antibodies are controlled by genes in the I-A subregion of the major histocompatibility complex (H-2). We investigated the in vivo effect of monoclonal anti-Ia antibodies on experimental autoimmune thyroiditis in susceptible mice. Antibodies were given around the time of immunization, later after immunization, and to mice with established disease. Monoclonal antibody produced by the hybridoma line 10-3.6 (anti-I-Ak, s, u, v, z, f) completely prevented both production of thyroglobulin antibodies and thyroid infiltrates, when given shortly before or at the time of antigen administration. This effect was dose-dependent and this monoclonal antibody decreased the severity of the disease when given after the antigen challenge but did not fully suppress established thyroiditis. The same antibody markedly decreased the number of B lymphocytes in the spleen and decreased the thyroglobulin-induced spleen cell proliferation when either given in vivo or added in vitro to cell cultures. Antibodies produced by the hybridoma line 11.2.12 (anti-I-Ak) did not show an inhibitory effect on the disease. These experiments suggest that in this model of murine thyroiditis anti-Ia antibodies act on antigen-presenting cells. Furthermore, only one monoclonal antibody, anti-Ia, suppressed the immune response to thyroglobulin, suggesting a possible role for the isotype and specificity of anti-Ia antibody.     

Immunological evaluation of rheumatoid arthritis patients treated with itolizumab

Rheumatoid arthritis is an autoimmune disease characterized by joint inflammation that affects approximately 1% of the general population. Itolizumab, a monoclonal antibody specific for the human CD6 molecule mainly expressed on T lymphocytes, has been shown to inhibit proliferation of T cells and proinflammatory cytokine production in psoriasis patients. We have now assessed the immunological effect of itolizumab in combination with methotrexate in rheumatoid arthritis by analyzing clinical samples taken from 30 patients enrolled in a clinical trial. T and B cell subpopulations were measured at different time points of the study. Plasma cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. The combined treatment of itolizumab and methotrexate led to a reduction in the frequency of T cell subpopulations, and plasma levels of proinflammatory cytokines showed a significant decrease up to at least 12 weeks after treatment ended. No anti-idiotypic antibody response was detected. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.     

Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

Therapeutic activity of agonistic monoclonal antibodies against CD40 in a chronic autoimmune inflammatory process

The use of agonistic monoclonal antibody against CD40 has emerged as one the most effective ways to boost immune responses against infectious agents or to fight cancer. Here, we report that the same monoclonal antibodies against CD40 (FGK45 and 3/23) previously used to elicit protective immune responses treated the autoimmune inflammatory process of chronic collagen-induced arthritis in DBA/1-TCR-beta transgenic mice, as well as collagen-induced arthritis in DBA/1 mice, both animal models of rheumatoid arthritis. This study indicates that agonistic monoclonal antibody against CD40 can potentially be used to treat chronic autoimmune inflammatory processes.     

Therapy of rat autoimmune disease by a monoclonal antibody specific for T lymphoblasts

The central role of T lymphocytes in the initiation, regulation and propagation of autoimmune diseases defines them as most suitable targets for selective immunotherapy. The recent advance in culturing human and animal T cell lines allows us to select monoclonal antibodies specific for differentiation antigens expressed by activated T lymphocytes. We selected a monoclonal antibody cytotoxic for a subpopulation of activated rat T cells. In vivo, this antibody effectively blocks immune responses to foreign antigens or autoantigen and prevents development of autoimmune diseases like experimental allergic encephalomyelitis and adjuvant arthritis. Even already established disease can be blocked by a single injection of antibody. Furthermore, this monoclonal antibody can be used to monitor the course of autoimmune disease progression from peripheral blood samples.     

Murine IL-10 gene transfer inhibits established collagen-induced arthritis and reduces adenovirus-mediated inflammatory responses in mouse liver

The effects of homologous IL-10 administration during an established autoimmune disease are controversial, given its reported immunostimulatory and immunosuppressive properties. Studies of collagen-induced arthritis have shown efficacy with repeated administrations of IL-10; however, when the EBV IL-10 homologue was administered via adenovirus gene transfer technology the results were equivocal. Therefore, the present study was undertaken to elucidate the effects of prolonged homologous IL-10 administration via adenovirus-mediated gene delivery on the progression of established arthritis. Collagen type II (CII)-immunized mice received i.v. injections of 10(7) or 10(8) PFU of an E1-deleted adenoviral vector containing the murine IL-10 gene (AdIL-10), after arthritis onset. Mice were monitored for 3 wk for disease progression, and gene transduction was assessed by quantification of serum mIL-10. CII-specific cell-mediated and humoral immune responses were analyzed by lymph node cell proliferation, cytokine production, and anti-CII Ab responses. Furthermore, because adenoviral vectors have been reported to induce organ dysfunction due to cell-mediated immune responses to the viral Ags, we have also evaluated delayed-type hypersensitivity responses and reactive hepatitis to the systemically delivered adenovirus and whether the IL-10 produced could influence those responses. Sustained suppression of autoimmune arthritis and elevated serum levels of IL-10 were achieved in our study. AdIL-10 treatment reduced cell-mediated immune reactivity, but did not affect humoral responses. Furthermore, IL-10 was able to reduce, but not totally abrogate, adenovirus-induced hepatic inflammation. These findings provide further insights into the diverse interplay of immune processes involved in autoimmune inflammation and the mechanism of cytokine immunotherapy.     

Immunotherapy for myasthenia gravis: a murine model

In vivo therapy with monoclonal antibody (mAb) GK1.5, which recognizes a glycoprotein antigen designated L3T4 on murine helper T lymphocytes, either prevented or suppressed the development of murine lupus, autoimmune encephalomyelitis, and collagen arthritis. The L3T4 antigen in the mouse is analogous to the human Leu-3/T4 antigen expressed on helper T lymphocytes, because they both participate in the T cell response to class II major histocompatibility complex (MHC) antigens. Class II MHC genes and I-A antigens mediate murine experimental autoimmune myasthenia gravis (EAMG) induced by acetylcholine receptor (AChR) autoimmunity. We studied the efficacy of mAb GK1.5 as an immunotherapeutic agent for murine EAMG. Therapy with mAb GK1.5 not only suppressed established autoimmunity to AChR but also prevented loss of muscle AChR in mice with EAMG. Moreover, permanent remission of clinical muscle weakness was induced if mAb GK1.5 therapy was initiated after the onset of clinical disease. Because the function of the Leu-3/T4 determinant on human helper T lymphocytes is analogous to the murine L3T4 determinant, use of antibody to the Leu-3/T4 determinant as an immunotherapeutic agent may provide a way to control the progression of human MG.     

Antibody-induced arthritis: disease mechanisms and genes involved at the effector phase of arthritis

During the development of rheumatoid arthritis (RA) autoantibodies to IgG-Fc, citrullinated proteins, collagen type II (CII), glucose 6 phosphoisomerase (G6PI) and some other self-antigens appear. Of these, a pathogenic effect of the anti-CII and anti-G6PI antibodies is well demonstrated using animal models. These new antibody mediated arthritis models have proven to be very useful for studies involved in understanding the molecular pathways of the induction of arthritis in joints. Both the complement and FcgammaR systems have been found to play essential roles. Neutrophils and macrophages are important inflammatory cells and the secretion of tumour necrosis factor-alpha and IL-1beta is pathogenic. The identification of the genetic polymorphisms predisposing to arthritis is important for understanding the complexity of arthritis. Disease mechanisms and gene regions studied using the two antibody-induced arthritis mouse models (collagen antibody-induced arthritis and serum transfer-induced arthritis) are compared and discussed for their relevance in RA pathogenesis.     

Suppression of collagen arthritis with antibodies to an arthritogenic, oligoclonal T cell line.

Rats immunized with type II collagen (CII) develop an immunologically mediated polyarthritis. T cells have been implicated in the pathogenesis of this model since they can adoptively transfer the disease. A CII-specific T cell line (VA), consisting of three distinct clones by Southern blot analysis, has been shown to be arthritogenic. Antibodies specific for this line were generated by immunizing rabbits. In an attempt to prevent collagen-induced arthritis (CIA), Louvain rats were injected with 1 ml of anti-VA ip on Days -1, +1, +3 and 0.5 ml on Day +5 (early treatment). To evaluate its effect on existing disease, rats received anti-VA on the day of arthritis onset and subsequently on 4 successive alternate days using the same dosage protocol (late treatment). Control rats received no therapeutic injections or were administered normal rabbit serum. All rats were immunized with CII on Day 0 to induce CIA. Rats administered antibodies using the early anti-VA treatment protocol had a significantly diminished incidence of arthritis compared to controls. Established arthritis was significantly diminished compared to controls in rats given the late anti-VA treatment. In both protocols, radiographic evidence of joint destruction was significantly reduced compared to controls. T cell phenotyping using flow cytometry analysis demonstrated that the anti-VA antibody therapy selectively eliminated a small subset of T cells since there was little difference in total T cell counts in the experimental versus control groups. Delayed type hypersensitivity and IgG antibody titers to CII were minimally decreased in the experimental versus control group. These results suggest that antibodies raised to an oligoclonal arthritogenic T cell line can suppress collagen arthritis. This may have implications with respect to 1) the size of the T cell receptor repertoire involved in the pathogenesis of collagen arthritis and 2) immunospecific protocols for CIA and other autoimmune diseases.     

Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis

We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular administration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune diseases.     

CDP7657, an anti-CD40L antibody lacking an Fc domain,inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study

IntroductionCD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG₁ antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab'' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8.MethodsThe potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE.ResultsCDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab'' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria.ConclusionsThese findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0757-4) contains supplementary material, which is available to authorized users.     

IL-23 Dependent and Independent Stages of Experimental Arthritis: No Clinical Effect of Therapeutic IL-23p19 Inhibition in Collagen-induced Arthritis

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.