Low-Dose Subcutaneous Anti-CD20 Treatment Depletes Disease Relevant B Cell Subsets and Attenuates Neuroinflammation.

To explore the B cell depleting capacity of a low-dose (20 µg) subcutaneous mouse anti-CD20 antibody treatment on disease-relevant B cell populations within lymph nodes and the spleen. B cell depleting capacity was explored in healthy female C57BL/6 and BALB/c mice; following immune activation in two different mouse models: trinitrophenylated lipopolysaccharide model (thymus-independent response) and dinitrophenyl-keyhole limpet hemocyanin model (thymus-dependent response); and in a chronic neuroinflammation experimental autoimmune encephalomyelitis model. CD20 protein expression on B cell subpopulations was also studied. The subcutaneous anti-CD20 regimen resulted in rapid depletion of B cells in blood, lymph nodes and spleen. Low-dose subcutaneous treatment did not reduce antigen-specific immunoglobulin M and immunoglobulin G titers in all subgroups, and relatively spared splenic marginal zone (MZ) B cells in both T cell dependent and T cell independent B cell immunization models. Analysis of immune compartments during anti-CD20-modulated autoimmune neuroinflammation showed that the maximal B cell depletion was achieved within 2 days of treatment and was highest in the lymph node. Regardless of the tissues analyzed, low-dose subcutaneous treatment was characterized by rapid B cell repletion following treatment cessation. CD20 protein expression was consistent on all B cell subsets in blood, and was more pronounced in germinal center B cells of lymph nodes and MZ B-cells of the spleen. Low-dose subcutaneous anti-CD20 therapy effectively depleted B cells within lymphatic tissues and reduced the severity of neuroinflammation. These data suggest that subcutaneous anti-CD20 therapies can effectively target disease-relevant B cell populations, have shorter repletion kinetics and maintain vaccination responses, thereby achieving autoimmune amelioration without severely impacting immune surveillance functions. Graphical Abstract *p < 0.05; **p < 0.01. CD, cluster of differentiation; DNP-KLH, dinitrophenyl-keyhole limpet hemocyanin; EC50, concentration of a drug that gives half-maximal response; Ig, immunoglobulin; MZ, marginal zone; s.c., subcutaneous; SEM, standard error of mean; TNP-LPS, trinitrophenylatedlipopolysaccharide.

Exacerbation of Murine Experimental Autoimmune Myositis by Toll-Like Receptor 7/8.

OBJECTIVE: Toll-like receptor 7 (TLR-7), TLR-8, and interferon (IFN)-induced genes are expressed in patients with idiopathic inflammatory myositis. This study was undertaken to investigate whether their activation influences the natural history of the disease. METHODS: Experimental autoimmune myositis was induced in mice by injection of the amino-terminal portion of the murine histidyl-transfer RNA synthetase (HisRS). Disease was compared in the presence or the absence of the TLR-7/8 agonist R-848 in wild-type mice and in mice that fail to express the IFNα/ß receptor (IFNα/ßR-null mice). RESULTS: Experimental autoimmune myositis induced by a single intramuscular immunization with HisRS spontaneously abated after 7-8 weeks. In contrast, levels of anti-HisRS autoantibodies, endomysial/perimysial leukocyte infiltration, and myofiber regeneration persisted at the end of the follow-up period (22 weeks after immunization) in mice immunized with HisRS in the presence of R-848. Myofiber major histocompatibility complex (MHC) class I molecules were detectable only in mice immunized with both HisRS and R-848. MHC up-regulation occurred early and in muscles that were not directly injected with HisRS. Muscle MHC expression paralleled with leukocyte infiltration. MHC class I molecules were selectively up-regulated in myotubes challenged with R-848 in vitro. Type I IFN was necessary for the prolonged autoantibody response and for the spreading of the autoimmune response, as demonstrated using IFNα/ßR-null mice. Muscle infiltration was maintained in the injected muscle up to the end of the follow-up period. CONCLUSION: TLR-7/8 activation is necessary to induce and maintain a systemic autoimmune response targeting the skeletal muscle. This experimental autoimmune myositis model reproduces many characteristics of human idiopathic inflammatory myopathies and may represent a tool for preclinical studies.

Autoimmune paraneoplastic syndromes associated to lung cancer: A systematic review of the literature: Part 3: Neurological paraneoplastic syndromes, involving the central nervous system.

The development of new immune treatment in oncology and particularly for lung cancer may induce new complications, particularly activation or reactivation of auto-immune diseases. In this context, a systematic review on the auto-immune paraneoplastic syndromes that can complicate lung cancer appears useful. This article is the third of a series of five and deals mainly with neurological paraneoplastic syndromes involving the central nervous system.

Development of an improved animal model of experimental autoimmune myositis.

Multiple animal models of experimental autoimmune myositis (EAM) have been developed. However, these models vary greatly in the severity of disease and reproducibility. The goal of this study was to test whether vaccination twice with increased dose of rat myosin and pertussis toxin (PT) could induce EAM with severer disease in mice. BALB/c mice were injected with 1 mg rat myosin in 50% complete Freund's adjuvant (CFA) weekly for four times and one time of PT (EAM) or twice with 1.5 mg myosin in CFA and PT (M-EAM). In comparison with that in the CFA and PT injected controls, vaccination with rat myosin and injection PT significantly reduced the muscle strength and EMG duration, elevated serum creatine kinase levels, promoted inflammatory infiltration in the muscle tissues, leading to pathological changes in the muscle tissues, demonstrating to induce EAM. Interestingly, we found that vaccination twice with the high dose of myosin and PT prevented EAM-related gain in body weights and caused significantly less muscle strength in mice. More importantly, all of the mice receiving high dose of myosin and PT survived while 3 out of 16 mice with four times of low dose of myosin died. Finally, vaccination with high dose of myosin promoted CD4(+) and CD8(+) T cell infiltration in the muscle tissues and up-regulated MHC-I expression in the muscle tissues of mice. Hence, the new model of EAM is a time-saving, efficient and easily replicable tool for studying autoimmune myositis.

Laminin-induced autoimmune myositis in rats.

The present study aimed to examine if immunization with laminin causes myositis in rats and whether the pathologic findings mirror human polymyositis and dermatomyositis. Rats were immunized with an emulsion of laminin and complete Freund's adjuvant. As a result, muscle fiber necrosis with infiltrating macrophages was frequently observed and mononuclear cells were observed in the endomysium. These mononuclear cells were composed of CD4+ cells, CD8+ T cells, and macrophages. CD4+ cells and CD8+ T cells were mainly located in the endomysium, whereas a large number of macrophages were located in the endomysium and infiltrating muscle fibers. A small number of B cells, detected by immunohistochemical staining, were mainly located in the perimysium. The nonnecrotic muscle fiber to which CD4+ T cells, CD8+ T cells, and perforin+ cells adhered was negative for antimerosin and antidystrophin antibodies. Muscle fiber necrosis in rats immunized with laminin may occur after denaturation of basement membrane proteins. In conclusion, the immunization with laminin induces moderate to severe myositis. We suggest that laminin may be an important antigen for connective tissue diseases such as polymyositis and dermatomyositis.

Immunization with a cannabinoid receptor type 1 peptide results in experimental allergic meningocerebellitis in the Lewis rat: a model for cell-mediated autoimmune neuropathology.

Neuronal elements are increasingly suggested as primary targets of an autoimmune attack in certain neurological and neuropsychiatric diseases. Type 1 cannabinoid receptors (CB1) were selected as autoimmune targets because they are predominantly expressed on neuronal surfaces in brain and display strikingly high protein levels in striatum, hippocampus, and cerebellum. Female Lewis rats were immunized with N-terminally acetylated peptides (50 or 400 microg per rat) of the extracellular domains of the rat CB1 and killed at various time points. Subsequent evaluation using immunohistochemistry and in situ hybridization showed dense infiltration of immune cells exclusively within the cerebellum, peaking 12-16 days after immunization with the CB1 peptide containing amino acids 9-25. The infiltrates clustered in meninges and perivascular locations in molecular and granular cell layers and were also scattered throughout the CB1-rich neuropil. They consisted primarily of CD4(+) and ED1(+) cells, suggestive of cell-mediated autoimmune pathology. There were no inflammatory infiltrates elsewhere in the brain or spinal cord. The results show that neuronal elements, such as neuronal cell-surface receptors, may be recognized as antigenic targets in a cell-mediated autoimmune attack and, therefore, support the hypothesis of cell-mediated antineuronal autoimmune pathology in certain brain disorders.

Pertussis toxin alters the innate and the adaptive immune responses in a pertussis-dependent model of autoimmunity.

Pertussis toxin (PTX) is used to promote development of autoimmune diseases. The mechanism(s) are still incompletely understood. We dissected the innate and adaptive immune responses in a PTX-dependent model of autoimmune retinal disease, experimental autoimmune uveoretinitis (EAU), a Th1-driven disease of the neural retina elicited in F344 rats with a peptide derived from the retinal antigen interphotoreceptor retinoid binding protein (IRBP). Our results showed that optimal doses of PTX led to strongly increased innate cytokine responses, followed by enhanced adaptive Th1 immunity and disease. At supraoptimal doses of PTX, EAU was suppressed, the animals exhibited persistent lymphocytosis and had an inhibited chemotactic response to chemokines. We suggest that the suppressive effect of PTX at supraoptimal doses is due to inhibition of lymphocyte emigration from the blood into the target tissue, secondary to inhibition of Gi-protein-coupled chemokine receptor signaling, that persists into the effector phase of disease.