Murine Models Of Autoimmunity Research Articles

Development of anti-CXCL13 monoclonal antibody for the treatment of autoimmune and immuno-oncological disorders (THER2P.950)

Abstract The chemokine CXCL13 is expressed in secondary lymphoid organs by follicular dendritic cells and macrophages and is also expressed by TH17 cells. It is the only known ligand for the CXCR5 receptor expressed on mature B cells, multiple subsets of T cells, macrophages and endothelial cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune and cancer-promoting inflammatory disorders. Over-expression of CXCR5 receptor in certain cancers has been reported to induce CXCL13-dependent cell proliferation and metastasis. We developed a human IgG1 monoclonal antibody that specifically binds to human, rodent and primate CXCL13 and is capable of neutralizing CXCL13 function of these various species in in vitro functional assays. For preclinical in vivo studies we have engineered a chimeric antibody that contains the same human heavy and light chain variable genes as the human antibody along with mouse constant regions. This antibody has demonstrated efficacy in murine models of autoimmunity (Experimental Autoimmune Encephalomyelitis, Collagen-Induced Arthritis) and immuno-oncology (MALT lymphoma). In adoptive transfer and immunization studies, treatment with this antibody reduced B cell trafficking to the spleen and lymph nodes and interfered with the formation and expansion of germinal centers. The human antibody is currently undergoing pre-clinical development to support a future FDA Investigational New Drug application.

Glucocorticoid-resistant Th17 cells are selectively attenuated by cyclosporine A

Glucocorticoids remain the cornerstone of treatment for inflammatory conditions, but their utility is limited by a plethora of side effects. One of the key goals of immunotherapy across medical disciplines is to minimize patients' glucocorticoid use. Increasing evidence suggests that variations in the adaptive immune response play a critical role in defining the dose of glucocorticoids required to control an individual's disease, and Th17 cells are strong candidate drivers for nonresponsiveness [also called steroid resistance (SR)]. Here we use gene-expression profiling to further characterize the SR phenotype in T cells and show that Th17 cells generated from both SR and steroid-sensitive individuals exhibit restricted genome-wide responses to glucocorticoids in vitro, and that this is independent of glucocorticoid receptor translocation or isoform expression. In addition, we demonstrate, both in transgenic murine T cells in vitro and in an in vivo murine model of autoimmunity, that Th17 cells are reciprocally sensitive to suppression with the calcineurin inhibitor, cyclosporine A. This result was replicated in human Th17 cells in vitro, which were found to have a conversely large genome-wide shift in response to cyclosporine A. These observations suggest that the clinical efficacy of cyclosporine A in the treatment of SR diseases may be because of its selective attenuation of Th17 cells, and also that novel therapeutics, which target either Th17 cells themselves or the effector memory T-helper cell population from which they are derived, would be strong candidates for drug development in the context of SR inflammation.

IL-35 and Autoimmunity: a Comprehensive Perspective.

Interleukin 35 (IL-35) is the most recently identified member of the IL-12 family of cytokines and offers the potential to be a target for new therapies for autoimmune, inflammatory, and infectious diseases. Similar to other members of the IL-12 family including IL-12, IL-23, and IL-27, IL-35 is composed of a heterodimer of α and β chains, which in the case of IL-35 are the p35 and Epstein-Barr virus-induced gene 3 (EBI3) proteins. However, unlike its proinflammatory relatives, IL-35 has immunosuppressive effects that are mediated through regulatory T and B cells. Although there are limited data available regarding the role of IL-35 in human autoimmunity, several murine models of autoimmunity suggest that IL-35 may have potent effects in regulating immunoreactivity via IL-10-dependent mechanisms. We suggest that similar effects are operational in human disease and IL-35-directed therapies hold significant promise. In particular, we emphasize that IL-35 has immunosuppressive ability that are mediated via regulatory T and B cells that are IL-10 dependent. Further, although deletion of IL-35 does not result in spontaneous breach of tolerance, recombinant IL-35 can improve autoimmune responses in several experimental models.

Tolerogenic dendritic cells: role and therapeutic implications in systemic lupus erythematosus.

Dendritic cells (DCs) are antigen presenting cells that activate T cells and determine the outcome of immune response. In addition to their important function in defense against pathogens, DCs are increasingly recognized as playing a crucial role in the regulation of immune tolerance. Plasticity of DCs with different maturity status and functions enable them to be exploited as potential cell-based therapy to restore immune tolerance in autoimmune diseases. Various ex vivo methods have been developed to generate stable tolerogenic DCs that are able to induce and maintain regulatory T cell homeostasis. The beneficial effect of tolerogenic DCs have been studied in murine autoimmune models with promising results. Systemic lupus erythematosus (SLE) is a prototypic multi-systemic autoimmune disease characterized by autoantibody production and deposition of immune complexes in organs. There are evidences that dysregulated DCs play a pivotal role in the initiation and perpetuation of lupus disease. Peripheral blood monocytes in SLE patients were found to have active phenotype with accelerated differentiation into DCs efficient in antigen presentation. Plasmacytoid DCs in SLE patients produce high levels of interferon-alpha, the signature cytokine of this disease, that cause a positive feedback loop in the amplification of activation of innate and adaptive immunity. Furthermore, manipulation of DCs via toll-like receptor knockout in a murine lupus model leads to alteration in disease severity and survival. Thus, tolerogenic DCs may appear as a potential cell-based therapeutic option in SLE.

UVB light attenuates the systemic immune response in CNS autoimmunity

Environmental conditions are critical in the susceptibility and severity of autoimmune disorders. Multiple Sclerosis (MS) is characterized by a positive latitude gradient (the further away from the equator, the higher the disease incidence). Here, we investigated the mechanisms underlying the beneficial effects of immune regulatory processes induced in the skin by moderate ultraviolet B (UVB) radiation on central nervous system (CNS) autoimmunity. Effects of UVB light were analyzed in a murine model of CNS autoimmunity (experimental autoimmune encephalomyelitis (EAE)).

B cells with immune-regulating function in transplantation.

In transplantation, the contribution of B cells to the rejection or acceptance of the allograft is a topic of major interest. The presence of donor-specific antibodies in transplant recipients is often associated with decreased graft function and rejection, clearly indicating a pathogenetic role of B cells in transplantation. However, data from studies in humans and rodents suggest that under certain conditions, B cells have the capacity to control or regulate the immune response to a transplanted organ. Although a great deal of attention has been focused on B cells in human and murine models of autoimmunity, our understanding of the role of these cells in transplantation is limited at present. Indeed, results in this setting are controversial and seem to depend on the model system used or the clinical situation studied. Here, we review the current understanding of the various phenotypes and roles that have been associated with immune-regulating B cells. We also discuss the mechanisms employed by subsets of these regulatory B cells to control the immune response in transplant recipients and in animal models of transplantation.

Ultraviolet B light attenuates the systemic immune response in central nervous system autoimmunity.

Environmental conditions (eg, latitude) play a critical role in the susceptibility and severity of many autoimmune disorders, including multiple sclerosis (MS). Here, we investigated the mechanisms underlying the beneficial effects of immune regulatory processes induced in the skin by moderate ultraviolet B (UVB) radiation on central nervous system (CNS) autoimmunity. Effects of UVB light were analyzed in a murine model of CNS autoimmunity (experimental autoimmune encephalomyelitis). Additionally, patients with relapsing-remitting MS were treated with narrowband UVB phototherapy. Immunomodulatory effects were examined in skin biopsies, serum samples, and immune cells of the peripheral blood. Regulatory T cells (Tregs), which are induced locally in the skin-draining lymph nodes in response to UVB exposure, connect the cutaneous immune response to CNS immunity by migration to the sites of inflammation (blood, spleen, CNS). Here, they attenuate the inflammatory response and ameliorate disease symptoms. Treg-inducing tolerogenic dendritic cells (DCs) were further necessary for induction of this systemic immune regulation by UVB radiation, because ablation of Langerhans cells abolished the UVB-induced phenotype. MS patients treated with UVB phototherapy showed an increase in induced Tregs and tolerogenic DCs accompanied by the downregulation of the T-cell effector cytokine interleukin 21. The treatment further induced elevated serum levels of vitamin D. Local UVB radiation of the skin influences systemic immune reactions and attenuates systemic autoimmunity via the induction of skin-derived tolerogenic DCs and Tregs. Our data could have implications for the understanding or therapeutic modulation of environmental factors that influence immune tolerance.

PReS-FINAL-2113: The association of the NFKB1 gene polymorphism with systemic onset juvenile idiopathic arthritis in Russia

Juvenile idiopathic arthritis (JIA) is a childhood onset autoimmune disorder characterized by inflammation of joints and other tissues. The basis for susceptibility to common autoimmune and inflammatory diseases, including JIA, is a complex interplay between multiple genetic and environmental risk factors. NF-κb appears to be a central player in several autoimmune diseases, according to recent studies of genetic defects in autoreactive lymphoid cells in both murine models of autoimmunity and humans with diverse forms of autoimmunity. Meta-analysis suggested a possible association between NFKB1 -94 ins/delattg promoter polymorphism and certain autoimmune and inflammatory diseases in the Asian population, but not in the Caucasian population.

The progression in the role of regulatory B cells

Background B cells are typically characterized by the ability to produce antibodies and cytokines,also possess the ability to present agents.Regulatory B cells,as the B cell functionally distinct subset,performing both pathogenic and regulatory role in several murine models of autoimmunity and inflammation.Objective To discuss the most recently progress in the role of regulatory B cells.Content This paper sums up the characters of regulatory B cells,and the functions of regulatory B cells in the autoimmune diseases and tumor.Trend Currently,the exact identity and origin of the regulatory B cells remains elusive.The complex cellular network may differ for each pathology.Control of regulatory activity would be an alternative therapy for autoimmune diseases and tumors. Key words: B10; Interleukin-10; Autoimmunity; Tumor

Close Relations between Podocyte Injuries and Membranous Proliferative Glomerulonephritis in Autoimmune Murine Models

Background: Membranous proliferative glomerulonephritis (MPGN) is a major primary cause of chronic kidney disease (CKD). Podocyte injury is crucial in the pathogenesis of glomerular disease with proteinuria, leading to CKD. To assess podocyte injuries in MPGN, the pathological features of spontaneous murine models were analyzed. Methods: The autoimmune-prone mice strains BXSB/MpJ-Yaa and B6.MRL-(D1Mit202-D1Mit403) were used as the MPGN models, and BXSB/MpJ-Yaa⁺ and C57BL/6 were used as the respective controls. In addition to clinical parameters and glomerular histopathology, the protein and mRNA levels of podocyte functional markers were evaluated as indices for podocyte injuries. The relation between MPGN pathology and podocyte injuries was analyzed by statistical correlation. Results: Both models developed MPGN with albuminuria and elevated serum anti-double-strand DNA (dsDNA) antibody levels. BXSB/MpJ-Yaa and B6.MRL showed severe proliferative lesions with T and B cell infiltrations and membranous lesions with T cell infiltrations, respectively. Foot process effacement and microvillus-like structure formation were observed ultrastructurally in the podocytes of both MPGN models. Furthermore, both MPGN models showed a decrease in immune-positive areas of nephrin, podocin and synaptopodin in the glomerulus, and in the mRNA expression of Nphs1, Nphs2, Synpo, Actn4, Cd2ap, and Podxl in the isolated glomerulus. Significant negative correlations were detected between serum anti-dsDNA antibody levels and glomerular Nphs1 expression, and between urinary albumin-to-creatinine ratio and glomerular expression of Nphs1, Synpo, Actn4, Cd2ap, or Podxl.Conclusion: MPGN models clearly developed podocyte injuries characterized by the decreased expression of podocyte functional markers with altered morphology. These data emphasized the importance of regulation of podocyte injuries in MPGN.

Galectin-9 Activates and Expands Human T-Helper 1 Cells

Galectin-9 (Gal-9) is known for induction of apoptosis in IFN-γ and IL-17 producing T-cells and amelioration of autoimmunity in murine models. On the other hand, Gal-9 induced IFN-γ positive T-cells in a sarcoma mouse model and in food allergy, suggesting that Gal-9 can have diametric effects on T-cell immunity. Here, we aimed to delineate the immunomodulatory effect of Gal-9 on human resting and ex vivo activated peripheral blood lymphocytes. Treatment of resting lymphocytes with low concentrations of Gal-9 (5–30 nM) induced apoptosis in ∼60% of T-cells after 1 day, but activated the surviving T-cells. These viable T-cells started to expand after 4 days with up to 6 cell divisions by day 7 and an associated shift from naïve towards central memory and IFN-γ producing phenotype. In the presence of T-cell activation signals (anti-CD3/IL-2) Gal-9 did not induce T-cell expansion, but shifted the CD4/CD8 balance towards a CD4-dominated T-cell response. Thus, Gal-9 activates resting T-cells in the absence of typical T-cell activating signals and promotes their transition to a TH1/C1 phenotype. In the presence of T-cell activating signals T-cell immunity is directed towards a CD4-driven response by Gal-9. Thus, Gal-9 may specifically enhance reactive immunological memory.

A disease-associated PTPN22 variant promotes systemic autoimmunity in murine models

Multiple autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus, are associated with an allelic variant of protein tyrosine phosphatase nonreceptor 22 (PTPN22), which encodes the protein LYP. To model the human disease-linked variant LYP-R620W, we generated knockin mice expressing the analogous mutation, R619W, in the murine ortholog PEST domain phosphatase (PEP). In contrast with a previous report, we found that this variant exhibits normal protein stability, but significantly alters lymphocyte function. Aged knockin mice exhibited effector T cell expansion and transitional, germinal center, and age-related B cell expansion as well as the development of autoantibodies and systemic autoimmunity. Further, PEP-R619W affected B cell selection and B lineage-restricted variant expression and was sufficient to promote autoimmunity. Consistent with these features, PEP-R619W lymphocytes were hyperresponsive to antigen-receptor engagement with a distinct profile of tyrosine-phosphorylated substrates. Thus, PEP-R619W uniquely modulates T and B cell homeostasis, leading to a loss in tolerance and autoimmunity.

Induction of atypical EAE mediated by transgenic production of IL‐6 in astrocytes in the absence of systemic IL‐6

Interleukin (IL)-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. While IL-6-deficient mice (IL-6 KO) are resistant to EAE, we showed previously that in transgenic mice with astrocyte-targeted production of IL-6-restricted to the cerebellum (GFAP-IL6), EAE induced with MOG(35-55) was redirected away from the spinal cord to the cerebellum. To further establish the importance of IL-6 produced in the central nervous system, we have generated mice producing IL-6 essentially only in the brain by crossing the GFAP-IL6 mice with IL-6 KO mice. Interestingly, GFAP-IL6-IL-6 KO mice showed a milder but almost identical phenotype as the GFAP-IL6 mice, which correlated with a lower load of inflammatory cells and decreased microglial reactivity. These results indicate that not only is cerebellar IL-6 production and eventual leakage into the peripheral compartment the dominating factor controlling this type of EAE but that it can also facilitate induction of autoimmunity in the absence of normal systemic IL-6 production.

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

Autoimmune mechanisms in antitumor immunity: tumor as an immunoprivileged self (127.20)

Abstract Blocking cytotoxic T lymphocyte antigen 4 (CTLA4), a prototypical inhibitory molecule of antitumor immunity, is emerging as a promising immunotherapy against cancers. Its clinical efficacy, however, is intricately associated with autoimmune toxicity. This association presents a challenge to selective immunosurveillance against tumors versus normal tissues, and suggests a paradigm change whereto autoimmunity is exploited as an effector mechanism for the destruction of tumor cells. Aided with well-defined murine models of autoimmunity, this study demonstrated that autoantigen-specific effector T (Teff) cell clones were indeed effective in eradicating tumor cells. However, a combination of the tumor microenvironment and regulatory T (Treg) cells presented a formidable barrier to the autoimmune effectors. CTLA4 critically regulated autoimmunity-mediated tumor destruction, such that a subtle reduction in CTLA4, engineered by shRNA transgenesis to mimic human CTLA4 genetic variations, substantially enhanced antitumor immunity. Surprisingly, this enhancement was not due to an extrinsic mechanism of crippling Treg-cell suppression, but an intrinsic promotion of Teff cell function. Overall, this study suggests that the tumor microenvironment may represent an “immunoprivileged self” status, wherein physiological mechanisms of immunoregulation evolved to protect nonmalignant “self” could be exploited to an increased potency within a solid tumor.

Therapeutic effect of Bushen Huoxue Recipe (补肾活血方) on autoimmune premature ovarian failure mice established by immunization with recombinant porcine zona pellucida 4 antigen

To investigate the efficacy and mechanism of Bushen Huoxue Recipe (, BHR) in the treatment of murine autoimmune premature ovarian failure (POF). The recombinant porcine zona pellucida 4 (pZP4) was expressed in E. coli BL21 (DE3) strain within prokaryotic plasmid pET28a (+), purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low (3.75 mg/kg), moderate (7.5 mg/kg), and high dose (15.0 mg/kg) of BHR by gastrogavage once daily for 20 days, with 17-β-estradiol (0.13 mg/kg) and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol (E) levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [(3)H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope. The purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice (P <0.05). However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E levels (P <0.01), and decreased the serum anti-pZP4 antibody titers of model mice P<0.05). The recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E levels and protect ovarian functions from the autoimmune injury in murine POF model.

Double-negative T cells during HIV/SIV infections: potential pinch hitters in the T-cell lineup.

This review summarizes the role of CD3+CD4-CD8- double-negative T cells, which have both regulatory and helper T-cell functions and may have the potential to compensate for the reduced levels of CD4 T cells during SIV/HIV infection. Double-negative T cells have been characterized in several human diseases and in murine models of autoimmunity and transplantation, where they exhibit both immunoregulatory and helper T-cell-like function. During the natural nonpathogenic SIV infection of African nonhuman primates, the lack of clinical disease progression is associated with the presence of double-negative T cells that maintain helper T-cell functions while remaining refractory to viral infection. Moreover, DN T cells may compensate for very low levels of CD4+ T cells observed in a cohort of SIV-infected sooty mangabeys that have remained free of clinical AIDS for over 10 years. These studies identify a potential for double-negative T cells to provide critical helper function during HIV infection. Double-negative T cells with some CD4+ T-cell functions are associated with a nonpathogenic outcome during SIV infection and represent a potential immune therapeutic target in HIV-infected patients.

Soluble Peptide Treatment Reverses CD8 T-Cell-Induced Disease in a Mouse Model of Spontaneous Tissue-Selective Autoimmunity

Transgenic (Tg) mouse models of autoimmunity have been used to express model antigens that can be recognized by T cells or by autoantibodies. To identify mechanisms of CD8-mediated tissue-specific autoimmune reactions and to identify potential treatments, we generated a double-transgenic (DTg) murine model of autoimmunity by crossing keratin-14 (K14)-soluble chicken ovalbumin (sOVA) mice, which express sOVA predominantly in external ear skin, with OT-I mice whose CD8 T cells express Vα2/Vβ5 regions of the TCR and are specific for SIINFEKL peptide (chicken ovalbumin (OVA) peptide 257-264) in association with class I major histocompatibility complex. The K14-sOVA/OT-I DTg mice develop a destructive process selectively targeting the external ear pinnae in the first 6 days of life. The ear bud area develops an intense inflammatory infiltrate of OT-I cells. Administration of the SIINFEKL peptide intravenously to pregnant F1 (filial 1, first filial generation of animal offspring from cross-mating two parental types) mice and subsequently intraperitoneally to newborn pups resulted in normal external ear development. Treatment with this self-peptide markedly reduced OT-I cell numbers, as well as downregulated the CD8 co-receptor. This model can be useful in studying localized, tissue-specific, immune-mediated skin disease, and provide information about potential therapies for autoimmune diseases in which specific molecular targets are known.

Epidermal Cadm1 Expression Promotes Autoimmune Alopecia via Enhanced T Cell Adhesion and Cytotoxicity

Autoimmune alopecia is characterized by an extensive epidermal T cell infiltrate that mediates hair follicle destruction. We have investigated the role of cell adhesion molecule 1 (Cadm1; Necl2) in this disease. Cadm1 is expressed by epidermal cells and mediates heterotypic adhesion to lymphocytes expressing class 1-restricted T cell-associated molecule (CRTAM). Using a murine autoimmune alopecia model, we observed an increase in early-activated cytotoxic (CD8-restricted, CRTAM-expressing) T cells, which preferentially associated with hair follicle keratinocytes expressing Cadm1. Coculture with Cadm1-transduced MHC-matched APCs stimulated alopecic lymph node cells to release IL-2 and IFN-γ. Overexpression of Cadm1 in cultured human keratinocytes did not promote cytokine secretion, but led to increased adhesion of alopecic cytotoxic T cells and enhanced T cell cytotoxicity in an MHC-independent manner. Epidermal overexpression of Cadm1 in transgenic mice led to increased autoimmune alopecia susceptibility relative to nontransgenic littermate controls. Our findings reveal that Cadm1 expression in the hair follicle plays a role in autoimmune alopecia.

Tracking Interferon in Autoimmunity

Type I interferon is a family of antiviral cytokines linked to human autoimmune diseases. In this issue of Immunity, Gall et al. (2012) characterize, in a murine model of autoimmunity, the origin and progression of the type I interferon response leading to disease.