Inducible costimulator (ICOS) and its ligand (ICOSL) regulate T and B cell responses. Glucose-6-phosphate isomerase (G6PI)-induced arthritis requires T and B lymphocytes. It was hypothesised that blocking ICOS/ICOSL interactions ameliorates G6PI-induced arthritis and reduces G6PI-specific B and T lymphocyte responses.
Methods
DBA/1 mice were injected with a blocking, non-depleting anti-ICOSL monoclonal antibodies (mAbs) during the induction or effector phase of G6PI-induced arthritis. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry.
Results
Transient blockade of ICOS/ICOSL interactions profoundly reduced the severity of G6PI-induced arthritis. ELISA and proliferation assays showed no clear ex vivo correlates of protection. Polychromatic flow cytometry revealed two major findings: the absolute number of G6PI-specific Th cells was markedly diminished in secondary lymphatic organs from mice with blocked ICOS/ICOSL interactions. Within the pool of G6PI-specific Th cells the frequency of interleukin 17 (IL17), interferon γ or tumour necrosis factor α producers or polyfunctional Th cells (expressing two or more of these cytokines) was higher in treated than in control mice.
Conclusions
ICOS costimulation is not mandatory for the differentiation of Th1 or Th17 cells. Instead, the lack of ICOS costimulation results in reduced survival of G6PI-specific Th cells irrespective of their functional differentiation. This study demonstrates that a thorough examination of the quantity and the quality of antigen-specific immune responses is useful to determine ex vivo correlates of efficacy for immunomodulating treatments.
The therapeutic benefit of B cell depletion in patients with rheumatoid arthritis has provided proof of concept that B cells are relevant for the pathogenesis of arthritis. It remains unknown which B cell effector functions contribute to the induction or chronification of arthritis. We studied the clinical and immunological effects of B cell depletion in glucose-6-phosphate isomerase-induced arthritis. We targeted CD22 to deplete B cells. Mice were depleted of B cells before or after immunization with glucose-6-phosphate isomerase (G6PI). The clinical and histological effects were studied. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. B cell depletion prior to G6PI-immunization prevented arthritis. B cell depletion after immunization ameliorated arthritis, whereas B cell depletion in arthritic mice was ineffective. Transfer of antibodies from arthritic mice into B cell depleted recipients did not reconstitute arthritis. B cell depleted mice harbored much fewer G6PI-specific Th cells than control animals. B cell depletion prevents but does not cure G6PI-induced arthritis. Arthritis prevention upon B cell depletion is associated with a drastic reduction in the number of G6PI-specific effector Th cells.
SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.
Abstract Because of its role in mediating both B cell and Fc receptor signaling, Bruton’s tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor–mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell–driven autoimmune disorders.
T-helper (Th) lymphocytes are critically required for the pathogenesis of glucose-6-phosphate isomerase (G6PI)-induced arthritis, but neither the G6PI epitopes recognized by arthritogenic T cells nor their pathogenic effector functions have been fully elucidated to date. We aimed at identifying arthritogenic G6PI peptides.We used a library of overlapping peptides spanning the entire G6PI sequence to identify the epitopes recognized by G6PI-specific Th cells. Immunodominant peptides were then used to immunize mice. Arthritis development was evaluated clinically and histologically. The humoral and cellular immune responses upon peptide immunization were analyzed by ELISA and multiparameter flow cytometry, respectively.We identified six immunodominant T-cell epitopes in DBA/1 mice, of which three are arthritogenic. One of these peptides (G6PI469-483) is identical in man and mice. Immunization with this peptide induces arthritis, which is less severe and of shorter duration than arthritis induced by immunization with full-length G6PI. Upon immunization with either G6PI or peptide, the antigen-specific Th cells produce IL-17, RANKL, IFNgamma and TNFalpha.We identified immunodominant and arthritogenic epitopes of G6PI. Not all immunodominant peptides are arthritogenic. This is the first description of arthritis induced by immunization with a self-peptide in mice.
Despite several side effects, glucocorticoids (GCs) have been widely used for 60 y to treat rheumatoid arthritis on the basis of their antiinflammatory effects. However, the cells targeted by GCs and the transcriptional mechanisms underlying their actions through the glucocorticoid receptor (GR) in steroid therapy remain poorly defined. Using cell type-specific GR-deficient mice subjected to antigen-induced arthritis (AIA) as a model of human rheumatoid arthritis, we show that GC action on T cells but not myeloid cells is critical for therapeutic intervention in AIA. Furthermore, the resistance of mice expressing a DNA binding-defective GR (GR dim ) to GC treatment reveals that dimerization of the GR is indispensable for the antiinflammatory effects. In these mice, the GC-induced suppression of T H 1 and T H 17 cell-derived proinflammatory cytokines is impaired. Our finding that IL-17A −/− mice are resistant to GC therapy, whereas IFN-γ −/− mice respond as efficiently as WT mice implies that IL-17–producing T cells and not IFN-γ–producing T cells are the most important targets for an efficient GC therapy. The present study's identification of the critical cell type and the mode of GR action in steroid therapy of AIA significantly advances our understanding of steroid therapy and should lead to therapies with greater efficiency and fewer side effects.
Trotz intensiver Forschung sind die Atiologie und die pathologischen Mechanismen der Rheumatoiden Arthritis (RA) bisher unvollstandig geklart. Tiermodelle der RA, wie die Glukose-6-phosphat- Isomerase-induzierte Arthritis, bieten die Moglichkeit pathologisch-relevante Mechanismen vor allen Dingen in der initialen Phase von Arthritiden zu untersuchen und mogliche Ansatzpunkte fur neue Therapien zu definieren; daruber hinaus ermoglichen sie, den Erfolg von Therapien zu testen.
Im Rahmen dieser Arbeit wird die Relevanz von Th17-Zellen in der Pathogenese der Glukose-6-phosphat-Isomerase-induzierten Arthritis untersucht. Zusatzlich beschreibt die vorliegende Arbeit die Identifizierung von zwei immunodominanten Peptiden (G6PI85-99 und G6PI469-483), abgeleitet von der Glukose-6-phosphat-Isomerase, im Kontext von I-Aq restringierten Th-Zellantworten. Die Immunisierung von DBA/1 Mausen mit einem dieser Peptide fuhrt zur Entwicklung einer Arthritis. Somit ist die G6PI-induzierte Arthritis nach dem bisherigen Kenntnisstand das einzige Mausmodell, in dem eine Arthritis durch die Immunisierung mit einem autologen Peptid („self-peptide“) hervorgerufen werden kann, das von einem Autoantigen abgeleitet wurde. Die Identifizierung immunodominanter arthritogener Peptide ermoglicht in der Zukunft die detaillierte Analyse pathogener G6PI-spezifischer T-Zellen unabhangig von ihrem Aktivierungsstatus durch Tetramere. Zusatzlich eroffnet sie neue therapeutische Ansatze im Hinblick auf Antigen-spezifische Therapien zur Toleranzinduktion.
SJL-Mause wurden als weiterer suszeptibler Maussstamm fur die G6PI-induzierte Arthritis identifiziert und naher charakterisiert. Zusatzlich liefert diese Arbeit Hinweise darauf, dass B-Zellen als Antigen-prasentierende Zellen fur die Pathogenese der Arthritis relevant sind und ruckt diese erneut als interessantes therapeutisches Target in den Mittelpunkt des Interesses.
Bruton9s tyrosine kinase (BTK) is a clinically proven target in several hematological indications. Due to its role in mediating the signaling of both B cell receptors (BCR) and Fc receptors (FcR), BTK is also a potential target for the treatment of autoimmune disease such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), where B cell and innate immune cell activation are key drivers of pathology.
Objectives
We are developing M2951, a novel,highly selective BTK inhibitor that is suitable for the treatment of chronic diseases. This work aimed at characterizing its efficacy in various cellular assays in vitro as well as in disease models for RA and SLE. Based on in vivo efficacy and target occupancy data, a PKPD model describing the correlation of BTK inhibition and disease severity reduction was built.
Methods
The BTK inhibitor M2951 was characterized in various biochemical and cellular assays to demonstrate its potency and specificity. To determine disease-modifying activity, M2951 was tested in NZB/W F1 mice which had been infected with e replication-deficient adenovirus expressing IFN-alpha in order to synchronize disease onset. Efficacy was determined by measuring proteinuria and histological kidney damage. Furthermore, M2951 was tested in a collagen-induced arthritis model (CIA) in the mouse. Paw scores were used to measure efficacy. A biochemical assay was developed that allowed to measure the degree of BTK occupancy in blood cells and splenocytes was developed. This assay was used to build a translational PKPD model linking target occupancy to efficacy.
Results
M2951 potently inhibits BCR- and FcR-mediated signaling and subsequent activation and function of B cells and certain myeloid cells. In mouse models of RA and SLE, M2951 displayed robust efficacy as demonstrated by a marked reduction of disease severity. In the NZB/W F1 IFN-α-accelerated SLE model, efficacy correlated with B cell inhibition, reduction of autoantibodies, and decreased circulating memory B and T cells. In addition to SLE, RA-like symptoms were inhibited in a collagen-induced arthritis model. In order to translate preclinical efficacious doses to humans, we determined the degree of target occupancy necessary to achieve disease reduction. Pharmacodynamic modeling showed that BTK occupancy of 60 and 80% was linked to 80% and near complete disease inhibition, respectively, in both the RA and SLE models.
Conclusions
In summary, these results demonstrate the potential of M2951 to treat autoimmune disease and may inform rational dose decisions as M2951 is advanced for development in rheumatologic diseases.
Disclosure of Interest
P. Haselmayer Employee of: Merck KGaA, M. Camps Employee of: Merck Serono, L. Liu-Bujalski Employee of: EMD Serono, F. Morandi Employee of: EMD Serono, J. Head Employee of: EMD Serono, S. Zimmerli Employee of: EMD Serono, L. Bruns Employee of: Merck KGaA, A. Bender Employee of: EMD Serono, P. Schroeder Employee of: EMD Serono, R. Grenningloh Employee of: EMD Serono
Background: we have established a new arthritis model, induced by immunization of normal mice with the glycolytic enzyme glucose‐6‐phosphate isomerase (G6PI) (Schubert et al. J. Immunol 2004;172:4503). Arthritis occurs in > 90% of the animals from d9 after the immunization and is usually self‐limiting within 30–40 days. We examined if regulatory T cells (Treg) were involved in the resolution of arthritis. Results: Injection of anti‐CD25 antibody on d‐11 and –8 before immunization resulted in a massive yet transient reduction of CD25 + FoxP3 + Treg. The percentage of Treg was normal at d12 after immunization i.e. early during the course of arthritis. This transient depletion affected the disease course profoundly, resulting in chronic‐destructive arthritis with histologically active inflammation over the whole follow‐up period (80d). Exacerbated arthritis was accompanied by increased T‐cell proliferation and increased numbers of IL‐17 producing T cells. Conclusions: CD25 + FoxP3 + Treg may not be effective in the resolution of ongoing arthritis. Rather, they seem to act in the induction phase of the disease by controlling the extent of T cell activation and differentiation.
Background IL-21 is secreted by activated T cells and modulates immune cell functions with both proinflammatory and anti-inflammatory effects.IL-21 receptor (IL-21R), homologous to IL-2Rβ and IL-4Rα, associates with the gamma common chain upon ligand binding.It was recently described that IL-21R is overexpressed in the inflamed synovial membrane and on leucocytes of rheumatoid arthritis patients.Objective Previously we have shown that blockade of the IL-21 pathway with soluble IL-21R-Fc resulted in a reduction of clinical signs of arthritis in rodent models.To understand potential mechanisms of IL-21 regulation in arthritis, we analyzed serum immunoglobulin levels, and cytokine expression in the paws, serum, and collagen-restimulated splenocytes, in response to IL-21 pathway blockade.Methods Arthritis was induced in DBA/1 male mice with bovine type II collagen.Animals were treated with either soluble mIL-21R-Fc, which neutralizes murine IL-21 bioactivity, with TNFRII-Fc or with control IgG.Spleens from each group of treated mice were cultured in vitro with collagen and assayed for cytokine secretion.Cytokines and anticollagen-specific IgG levels were also measured in the serum by ELISA.Cytokine mRNA levels in the paws were evaluated by quantitative PCR analysis.Results Treatment of mice with IL-21R-Fc or TNFRII-Fc reduced clinical and histological signs of collagen-induced arthritis.IL-6 mRNA in paws and serum IL-6 levels were decreased after IL-21R-Fc treatment.IFNγ mRNA was increased in paws of IL-21R-Fctreated mice.Collagen-specific spleen cell responses from IL-21R-Fc-treated mice exhibited increased IFNγ and IL-2, and reduced IL-6 and IL-17 levels.Serum levels of total IgG 1 were also reduced in response to IL-21R-Fc treatment.Conclusion These data demonstrate a role for IL-21 in the modulation of collagen-specific T-cell responses and the pathology of arthritis, supporting a rationale for blockade of the IL-21 pathway in rheumatoid arthritis.
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