Gene therapy delivery of myelin oligodendrocyte glycoprotein (MOG) via hematopoietic stem cell transfer induces MOG-specific B cell deletion.

The various mechanisms that have been described for immune tolerance govern our ability to control self-reactivity and minimize autoimmunity. However, the capacity to genetically manipulate the immune system provides a powerful avenue to supplement this natural tolerance in an Ag-specific manner. We have previously shown in the mouse model of experimental autoimmune encephalomyelitis that transfer of bone marrow (BM) transduced with retrovirus encoding myelin oligodendrocyte glycoprotein (MOG) promotes disease resistance and CD4(+) T cell deletion within the thymus. However, the consequence of this strategy on B cell tolerance is not known. Using BM from IgH(MOG) mice that develop MOG-specific B cell receptors, we generated mixed chimeras together with BM-encoding MOG. In these animals, the development of MOG-specific B cells was abrogated, resulting in a lack of MOG-specific B cells in all B cell compartments examined. This finding adds a further dimension to our understanding of the mechanisms of tolerance that are associated with this gene therapy approach to treating autoimmunity and may have important implications for Ab-mediated autoimmune disorders.

Thymic gene transfer of myelin oligodendrocyte glycoprotein ameliorates the onset but not the progression of autoimmune demyelination.

Tolerance induction, and thus prevention of autoimmunity, is linked with the amount of self-antigen presented on thymic stroma. We describe that intrathymic (i.t.) delivery of the autoantigen, myelin oligodendrocyte glycoprotein (MOG), via a lentiviral vector (LV), led to tolerance induction and prevented mice from developing fulminant experimental autoimmune encephalomyelitis (EAE). This protective effect was associated with the long-term expression of antigen in transduced stromal cells, which resulted in the negative selection of MOG-specific T cells and the generation of regulatory T cells (Tregs). These selection events were effective at decreasing T-cell proliferative responses and reduced Th1 and Th17 cytokines. In vivo, this translated to a reduction in inflammation and demyelination with minimal, or no axonal loss in the spinal cords of treated animals. Significantly intrathymic delivery of MOG to mice during the priming phase of the disease failed to suppress clinical symptoms despite mice being previously treated with a clearing anti-CD4 antibody. These results indicate that targeting autoantigens to the thymic stroma might offer an alternative means to induce the de novo production of tolerant, antigen-specific T cells; however, methods that control the number and or the activation of residual autoreactive cells in the periphery are required to successfully treat autoimmune neuroinflammation.

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis.

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development.

Hematopoietic stem cell gene therapy as a treatment for autoimmune diseases.

A key function of the immune system is to protect us from foreign pathogens such as viruses, bacteria, fungi and multicellular parasites. However, it is also important in many other aspects of human health such as cancer surveillance, tissue transplantation, allergy and autoimmune disease. Autoimmunity can be defined as a chronic immune response that targets self-antigens leading to tissue pathology and clinical disease. Autoimmune diseases, as a group of diseases that include type 1 diabetes, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, have no effective cures, and treatment is often based on long-term broad-spectrum immunosuppressive regimes. While a number of strategies aimed at providing disease specific treatments are being explored, one avenue of study involves the use of hematopoietic stem cells to promote tolerance. In this manuscript, we will review the literature in this area but in particular examine the relatively new experimental field of gene therapy and hematopoietic stem cell transplantation as a molecular therapeutic strategy to combat autoimmune disease.

B-cells expressing NgR1 and NgR3 are localized to EAE-induced inflammatory infiltrates and are stimulated by BAFF.

We have previously reported evidence that Nogo-A activation of Nogo-receptor 1 (NgR1) can drive axonal dystrophy during the neurological progression of experimental autoimmune encephalomyelitis (EAE). However, the B-cell activating factor (BAFF/BlyS) may also be an important ligand of NgR during neuroinflammation. In the current study we define that NgR1 and its homologs may contribute to immune cell signaling during EAE. Meningeal B-cells expressing NgR1 and NgR3 were identified within the lumbosacral spinal cords of ngr1+/+ EAE-induced mice at clinical score 1. Furthermore, increased secretion of immunoglobulins that bound to central nervous system myelin were shown to be generated from isolated NgR1- and NgR3-expressing B-cells of ngr1+/+ EAE-induced mice. In vitro BAFF stimulation of NgR1- and NgR3-expressing B cells, directed them into the cell cycle DNA synthesis phase. However, when we antagonized BAFF signaling by co-incubation with recombinant BAFF-R, NgR1-Fc, or NgR3 peptides, the B cells remained in the G0/G1 phase. The data suggest that B cells express NgR1 and NgR3 during EAE, being localized to infiltrates of the meninges and that their regulation is governed by BAFF signaling.

Transplantation of bone marrow transduced to express self-antigen establishes deletional tolerance and permanently remits autoimmune disease.

Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-gamma, and GM-CSF in response to MOG(35-55) peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4(+) MOG(35-55)-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.

Tetraspanin CD53 Promotes Lymphocyte Recirculation by Stabilizing L-Selectin Surface Expression.

Tetraspanins regulate key processes in immune cells; however, the function of the leukocyte-restricted tetraspanin CD53 is unknown. Here we show that CD53 is essential for lymphocyte recirculation. Lymph nodes of Cd53-/- mice were smaller than those of wild-type mice due to a marked reduction in B cells and a 50% decrease in T cells. This reduced cellularity reflected an inability of Cd53-/- B and T cells to efficiently home to lymph nodes, due to the near absence of L-selectin from Cd53-/- B cells and reduced stability of L-selectin on Cd53-/- T cells. Further analyses, including on human lymphocytes, showed that CD53 stabilizes L-selectin surface expression and may restrain L-selectin shedding via both ADAM17-dependent and ADAM17-independent mechanisms. The disruption in lymphocyte recirculation in Cd53-/- mice led to impaired immune responses dependent on antigen delivery to lymph nodes. Together these findings demonstrate an essential role for CD53 in lymphocyte trafficking and immunity.

Activated CD8+ T Cells Cause Long-Term Neurological Impairment after Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) leaves many survivors with long-term disabilities. A prolonged immune response in the brain may cause neurodegeneration, resulting in chronic neurological disturbances. In this study, using a TBI mouse model, we correlate changes in the local immune response with neurodegeneration/neurological dysfunction over an 8-month period. Flow cytometric analysis reveals a protracted increase in effector/memory CD8+ T cells (expressing granzyme B) in the injured brain. This precedes interleukin-17+CD4+ T cell infiltration and is associated with progressive neurological/motor impairment, increased circulating brain-specific autoantibodies, and myelin-related pathology. Genetic deficiency or pharmacological depletion of CD8+ T cells, but not depletion of CD4+ T cells, improves neurological outcomes and produces a neuroprotective Th2/Th17 immunological shift, indicating a persistent detrimental role for cytotoxic T cells post-TBI. B cell deficiency results in severe neurological dysfunction and a heightened immune reaction. Targeting these adaptive immune cells offers a promising approach to improve recovery following TBI.

Gene therapy strategies towards immune tolerance to treat the autoimmune diseases.

Autoimmune diseases such as type 1 diabetes and multiple sclerosis pose a significant health burden on our society. As a whole, autoimmune diseases affect approximately 6% of the population and are the third largest disease burden after heart disease and cancer. Such pathologic manifestations arise by way of damaging reactions of B-cell derived antibodies and/or T-cells to self-antigens and are triggered by genetic and environmental factors. Currently there is no known cure, with treatment restricted to toxic, long-term immunosuppressive regimes, replacement therapy and in intractable cases, transplantation of autologous or allogeneic haematopoietic stem cells. In experimental models of autoimmunity, gene therapeutic approaches have demonstrated promise in treating the autoimmune diseases. These include delivery of anti-inflammatory cytokines and exploitation of regulatory T cells. However, none of these approaches provide lasting, long-term benefit. We hypothesise that therapeutically transduced haematopoietic stem cells followed by transplantation is an alternative strategy to establish permanent immune tolerance that can not only prevent autoimmunity but also cure these diseases. Our approach is focused on directing autoimmune disease-specific autoantigen expression in the thymus by genetic manipulation of haematopoietic stem cells to establish molecular chimeras. Our hypothesis originates from experimental studies with a mouse model of experimental autoimmune gastritis (EAG) and more recently with the non-obese diabetic (NOD) mouse model for type 1 diabetes (T1D).

The Influence of Differentially Expressed Tissue-Type Plasminogen Activator in Experimental Autoimmune Encephalomyelitis: Implications for Multiple Sclerosis.

Tissue type plasminogen activator (t-PA) has been implicated in the development of multiple sclerosis (MS) and in rodent models of experimental autoimmune encephalomyelitis (EAE). We show that levels of t-PA mRNA and activity are increased ~4 fold in the spinal cords of wild-type mice that are mice subjected to EAE. This was also accompanied with a significant increase in the levels of pro-matrix metalloproteinase 9 (pro-MMP-9) and an influx of fibrinogen. We next compared EAE severity in wild-type mice, t-PA-/- mice and T4+ transgenic mice that selectively over-express (~14-fold) mouse t-PA in neurons of the central nervous system. Our results confirm that t-PA deficient mice have an earlier onset and more severe form of EAE. T4+ mice, despite expressing higher levels of endogenous t-PA, manifested a similar rate of onset and neurological severity of EAE. Levels of proMMP-9, and extravasated fibrinogen in spinal cord extracts were increased in mice following EAE onset regardless of the absence or over-expression of t-PA wild-type. Interestingly, MMP-2 levels also increased in spinal cord extracts of T4+ mice following EAE, but not in the other genotypes. Hence, while the absence of t-PA confers a more deleterious form of EAE, neuronal over-expression of t-PA does not overtly protect against this condition with regards to symptom onset or severity of EAE.

Mechanisms of gastric mucosal cell loss in autoimmune gastritis.

The causes of target cell death in organ-specific autoimmune diseases are not precisely known. In the case of EAG, parietal cell death depends on Th1 CD4 T cells and Fas/Fas-ligand, either through interaction between infiltrating CD4 T cells with gastric parietal cells that have upregulated Fas expression or through homotypic interactions between the parietal cells. TNF-alpha does not appear to have a role in this process. The accompanying loss of zymogenic cells is likely a consequence of the interruption of the normal developmental pathway in the gastric mucosa that follows the destruction of parietal cells in the gastric mucosa.

Reversing the autoimmune condition: experience with experimental autoimmune gastritis.

Autoimmune diseases remain a significant health problem in our society, despite the best efforts to understand and treat these conditions. Current clinical treatments are aimed at alleviating the consequences of these diseases, with limited prospects for cure. Our studies with the experimental model of autoimmune gastritis have led us to explore potential curative strategies that can reverse the autoimmune condition. Using mouse models, we have shown that expression of the known gastric autoantigen in the thymus results in immunological tolerance and resistance to the induction of autoimmune gastritis. Also, induced tolerance in donor mice can be transferred to syngeneic recipient mice by bone marrow cells. Strategies based on these observations could lead to reversal of established disease. Transfer of ensuing knowledge to the cure of serious human autoimmune diseases is our ultimate goal.

Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals.

Organ-specific autoimmunity in granulocyte macrophage-colony stimulating factor (GM-CSF) deficient mice.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a pleiotrophic proinflammatory cytokine that augments adaptive immunity by linking it to innate immunity. Experimental autoimmune gastritis is an animal model of human autoimmune gastritis and pernicious anaemia. We have previously shown that GM-CSF is expressed in the gastric mucosa of mice with gastritis initiated by neonatal thymectomy (Gastroenterology 110 (1996) 1791) and that transgenic expression of GM-CSF in the stomach induces autoimmune gastritis in mice (J. Immunol. 166 (2001) 2090). Here we have examined the development of autoimmune gastritis initiated by immunisation or by neonatal thymectomy in GM-CSF deficient mice. We found that gastritis develops in GM-CSF deficient mice initiated by neonatal thymectomy but not by immunisation with gastric antigen. These observations suggest that GM-CSF is not absolutely required for the initiation of autoimmunity and highlights the different conclusions that can be drawn using different disease models.

Autoantibodies in neuropsychiatric lupus.

The American College of Rheumatology presented a consensus document in 1999 proposing the classification of 19 different syndromes defined by neurological and psychiatric manifestations of systemic lupus erythematosus (SLE). The detection of autoantibodies in patient's serum or cerebrospinal fluid has not been used as diagnostic markers for the proposed neuropsychiatric lupus classifications as their disease associations remain highly contentious. Autoantibodies detected in the serum and/or cerebrospinal fluid, that have been reported to segregate with patients presenting with neuropsychiatric lupus include: (1) anti-neuronal antibodies, (2) brain-lymphocyte cross-reactive antibodies, (3) anti-ribosomal P antibodies, (4) anti-phospholipid antibodies and (5) anti-ganglioside antibodies. Tests for anti-neuronal, anti-brain-lymphocyte cross-reactive and anti-ganglioside antibodies remain highly specialized whereas tests for ribosomal P antibodies and for antiphospholipid antibodies are currently routinely available in most diagnostic laboratories. Anti-ribosomal antibodies segregate with SLE. Antiphospholipid P antibodies are markers for the antiphospholipid syndrome. This syndrome may be associated with another disease, commonly SLE. In this setting, neuropsychiatric manifestations in SLE may arise as a consequence of thrombotic episodes involving the cerebral vasculature. There is a pressing need for antibodies to ribosomal P and to phospholipids to be standardized for routine diagnostic application. We conclude that the search for specific antibody marker(s) that can be applied for the routine laboratory diagnosis for neuropsychiatric lupus remains elusive.

Tackling autoimmunity with gene therapy.

Autoimmune diseases result from an aberrant response of the immune system that target self-tissues. Our understanding of normal immune development has been used to subvert this self-reactivity and involves exposing self-antigen to the developing immune system. This can be achieved through bone marrow derived cells, thus introducing potential clinical application. We have used the mouse model of multiple sclerosis to demonstrate that the transfer of bone marrow encoding a target autoantigen can be used to promote immune tolerance. The process of preconditioning recipients for hematopoietic stem cell transfer is critical for potential human translation. Thus, we have directly addressed if our model can also be applied in non-myeloablative and less toxic conditioning to promote tolerance and reverse established disease. Our studies to date indicate that this can indeed be achieved and that only low levels of chimerism are required to achieve tolerance.

Cutting edge issues in autoimmune gastritis.

Autoimmune gastritis is the outcome of a pathological CD4 T cell-mediated autoimmune response directed against the gastric H/K-ATPase. Silent initially, the gastric lesion becomes manifest in humans by the development of megaloblastic pernicious anemia arising from vitamin B12 deficiency. Cutting edge issues in this disease relate to its epidemiology, immunogenetics, a role for Helicobacter pylori as an infective trigger through molecular mimicry, its immunopathogenesis, associated organ-specific autoimmune diseases, laboratory diagnosis, and approaches to curative therapy.

Transplantation of genetically modified haematopoietic stem cells to induce antigen-specific tolerance as a cure for autoimmune diseases.

Autoimmune diseases are incurable and are managed using therapeutic agents. Bone marrow transplantation is being trialled as a treatment for these diseases. While allogeneic bone marrow transplantation shows impressive benefit, its application is hindered by GVHD and high mortality. On the other hand, autologous bone marrow transplantation has lower mortality rate and no GVHD but is associated with higher relapse rates. Given that autoimmune diseases are a result of a failure of immune tolerance and that bone marrow-derived dendritic cells play an important role in establishing immune tolerance, the transplantation of genetically modified haematopoietic stem cells to generate molecular chimerism to induce antigen-specific tolerance offers the potential for developing a cure for autoimmune diseases. In this review, we will discuss key findings from clinical data and animal studies to provide evidence to support the above concept.

Targeting MOG expression to dendritic cells delays onset of experimental autoimmune disease.

Haematopoietic stem cell (HSC) transfer coupled with gene therapy is a powerful approach to treating fatal diseases such as X-linked severe combined immunodeficiency. This ability to isolate and genetically manipulate HSCs also offers a strategy for inducing immune tolerance through ectopic expression of autoantigens. We have previously shown that retroviral transduction of bone marrow (BM) with vectors encoding the autoantigen, myelin oligodendrocyte glycoprotein (MOG), can prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, ubiquitous cellular expression of autoantigen driven by retroviral promoters may not be the best approach for clinical translation and a targeted expression approach may be more acceptable. As BM-derived dendritic cells (DCs) play a major role in tolerance induction, we asked whether targeted expression of MOG, a target autoantigen in EAE, to DCs can promote tolerance induction and influence the development of EAE. Self-inactivating retroviral vectors incorporating the mouse CD11c promoter were generated and used to transduce mouse BM cells. Transplantation of gene-modified cells into irradiated recipients resulted in the generation of chimeric mice with transgene expression limited to DCs. Notably, chimeric mice transplanted with MOG-expressing BM cells manifest a significant delay in the development of EAE suggesting that targeted antigen expression to tolerogenic cell types may be a feasible approach to inducing antigen-specific tolerance.

Transplantation of retrovirally transduced bone marrow prevents autoimmune disease in aged mice by peripheral tolerance mechanisms.

Transplantation of bone marrow (BM) engineered to express self-antigen has been shown to protect 100% of young mice from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), with thymic clonal deletion as a tolerance mechanism. Here, we asked whether aged mice can also be tolerised following transplantation with self-antigen-engineered BM and whether castration-induced thymus regrowth can enhance this outcomes. Then, 50% of aged mice were protected from EAE regardless of castration-induced thymus regrowth. EAE-free and diseased mice demonstrated MOG-specific lymphocyte proliferation and antibody production regardless of castration-induced thymus regrowth, consistent with lack of intrathymic deletion of self-antigen-reactive T cells. Although low chimerism levels ( < 4%) were observed, EAE-free mice showed significantly higher chimerism levels in lymphocytes in peripheral lymphoid organs compared with thymus. CD4(+)CD25(+) regulatory T cells were elevated in lymph nodes of EAE-free mice. We conclude that transplantation of self-antigen expressing BM protects 50% of aged mice and castration-induced thymic regrowth had no effect on outcomes. Peripheral tolerance mechanisms are implicated since protection is associated with higher chimerism levels in peripheral T and B lymphocytes and with elevated regulatory T cells.