Conceptually, gene therapy has been used as an efficient methodology to circumvent genetic deficiency by transfection of cDNA encoding the appropriate functional gene product. It is therefore conceivable that the best candidates for this form of therapy would be genetic diseases associated with a single gene mutation, such as X-linked agammaglobulinemia (XLA) or cystic fibrosis (CF). Paradoxically, it appears that gene therapy needs to confront similar levels of technological challenges when encountering genetic disorders, such as XLA or CF, to those a involved in a successful intervention in multifactorial diseases. Yet, while genetic disorders that involve a mutation in a single gene are rare, multifactorial diseases comprise a major cause of illness and death in the developed countries. This has motivated scientists to explore gene therapy strategies in multifactorial disorders. This chapter discusses the use of a modification of gene therapy named DNA vaccination to provide novel ways of interfering in the regulation of the inflammatory process in T-cell-mediated autoimmune diseases, such as mutiple sclerosis (MS), and thus provide protective immunity against these multi-factorial diseases. One approach to applying gene therapy in T-cell-mediated autoimmunity involves in vitro transfection of antigen-specific autoimmune T-cells with a regulatory gene of interest, such as inter-leukin-4 (IL-4) or IL-10.1,2 Upon injection of the manipulated T-cells into the circulation, the cells are expected to home to the target autoimmune organ and propagate the response to the specific autoimmune determinate.1,2 While so doing, they produce and secrete the desired regulatory gene product at this site and restrain the relevant autoimmune disease.1,2 My group has utilized another modification of gene therapy, by which the immune system could be 're-educated' to restrain its harmful activities.
Experimental autoimmune encephalomyelitis (EAE) is a paralytic autoimmune disease of the central nervous system that serves as an animal model for MS. In both diseases, circulating leukocytes enter the brain blood to interact with their target antigens, resulting in impaired nerve conduction and paralysis. The disease can be induced in various susceptible strains of animals, one of which is the Lewis strain of rats. Upon a single immunization with myelin basic protein (MBP) emulsified in an appropriate adjuvant, these rats develop active EAE.3 MBP-specific CD4+ T-cell clones and lines selected from EAE rats are capable of transferring the disease to naive recipients.3 When being attenuated to become non-encephalitogenic, or if administered at a sub-pathogenic dose, these cells can endow recipients with a high state of resistance to any further attempt to induce the disease.4-6 The suggested mechanism includes elicitation of a self-specific anti-idiotypic response, which includes CD4+6 and CD8+7,8 regulatory T-cells. These studies suggest that some cells of the specific arm of the immune system not only do not participate in the induction of antigen-specific effector function, but also regulate the harmful function of autoimmune effector T-cells. As discussed below, my group has recently defined another mechanism by which immune cells mount an immune response against self-antigens (pro-inflammatory cytokines and chemokines) to restrain the harmful activity of autoimmune T-cells.
The role of cytokines and chemokines in the regulation of T-cell-mediated autoimmunity has been extensively studied. Based on their cytokine profile, CD4+ T-cells can be divided into: Th1 cells that produce large amounts of interferon gamma (IFN-7) and tumor necrosis factor alpha (TNF-a), and, to a much lesser extent, IL-4 and IL-10; and Th2 cells that produce IL-4, IL-10, and IL-13, and, to a much lesser extent, IFN-7 and TNF-a.9-18 More recently defined subsets comprise the Th3 cells that produce significant amounts of transforming growth factor beta (TGF-p) and have been associated with oral tolerance,19 and the high-IL-10- low-IL-4-producing regulatory T-cells that have been implicated in colitis.20 Th1 cells selected in response to various autoantigens transfer T-cell-mediated autoimmune diseases, whereas IL-4-secreting Th2 cells, selected in response to these same antigens, either inhibit or exert no profound effect on the inflammatory process.13,21-33 High levels of IFN-7 and low levels of IL-4 positively select for Th1 cells, whereas low levels of IFN-7 together with high levels of IL-4 mediate Th2 selection.9-14 In a recent study, Wildbaum and Youssef isolated mRNA encoding rat IFN-7 inducing factor (IGIF, IL-18) from the EAE brain, generated neutralizing antibodies against its gene product, and used them to explore the role of IGIF in T-cell deviation and function.34 These antibodies significantly reduced the production of IFN-7 by primed T-cells proliferating in response to their target MBP epitope and by Con A-activated T-cells from naive donors. When administered to rats during the development of either active or transferred EAE, these antibodies significantly blocked the development of disease. Splenic T-cells from protected rats were cultured with the encephalitogenic MBP epitope and evaluated for production of IL-4 and IFN-7. These cells, which proliferated, exhibited a profound increase in IL-4 production, accompanied by a significant decrease in IFN-7 and TNF-a production.34 This study demonstrates, again, that by simple means one may interfere in a natural mechanism by which self-tolerance is maintained by the peripheral immune system to keep autoreactive lymphocytes under control.34 As an alternative approach to treat T-cell-mediated autoimmune diseases, one may use antibodies to key adhesion molecules that mediate leukocyte migration to their target organ. Eight years ago, we defined the a4 p1 integrin (VLA-4) as the key adhesion molecule that mediates T-cell and monocyte transmigration to the target autoimmune site in EAE, and demonstrated that VLA-4-specific antibodies can inhibit the development and progression of disease.35 The underlying mechanism includes blockade of the secondary influx of leukocytes that is required for the development and progression of disease.36 Once again, this form of therapy is not disease, or organ, specific and is dependent on continuing exposure to these antibodies.
In an attempt to develop a highly specific therapeutic strategy, we have explored the abilities of dominant epitopes of MBP, or even soluble altered analogs of these epitopes, to inhibit an ongoing disease.37-39 In a recent study, we demonstrated that an engagement of the antigenic determinant to one major histocompatibility complex (MHC) anchor and to one TCR binding site of an MBP determinant could be sufficient for the generation of antigen-specific T-cell tolerances,37 whereas five-seven simultaneous engagements of an MBP determinant to MHC and TCR are required for mounting an encephalitogenic response in self-reactive T-cells.37 Perhaps during the evolution of self-non-self recognition, the immune system has evolved to induce self-specific unresponsiveness more readily than self-specific pro-inflammatory responses. The major advantage of using such a non-pathogenic altered antigen analog for therapy resides in its specificity. Yet, as with previous means of therapy discussed above, it requires continuing administration, which is impractical for the treatment of long-lasting chronic diseases.
In an attempt to overcome the above disadvantage, we have recently explored two ways by which the immune system could be 're-educated' to restrain the aggressiveness of autoimmune T-cells EAE is attenuated. The first approach interferes in the polarization of MBP-specific T-cells during the neonatal period of life. The other approach uses naked DNA vaccination to generate self-specific immunity cytokines/chemokines capable of neutralizing the harmful effect of autoimmune T-cells and macrophages. The autoimmune response of T-cells to components of the central nervous system (CNS) begins with recognition of a single or limited number of self-determinants, and then expands into a reaction to several self-determinants on the same molecule, termed intramolecular epitope spreading, or to other molecules within the nervous system, termed intermolecular epitope spreading.40-43 The first approach explores determinate spread following neonatal exposure to encephalitogenic determinants. The embryonic and neonatal periods have been thought of as a window in ontogeny during which the developing immune system is particularly susceptible to tolerization. Thus, antigenic challenge in neonatal life may result in specific T-cell unresponsiveness in the adult.44-48 Antigen-specific T-cell deletion was suggested to be a pivotal mechanism by which central tolerance, including tolerance to neonatally administered antigens, is induced and maintained.49-50 Our observations clearly show that neonatal exposure to MBP epitopes results not only in antigen-specific T-cell deletion, but also in altered deviation of some of the escaping CD4+ antigen-specific T-cells. In adult life, these cells can endow disease resistance that spreads in an intramolecular manner.51,52 As with soluble peptide therapy,38 here again the tolerant state is IL-4 dependent.51,52 Most importantly, our observations indirectly suggest that tolerizing T-cells selected during the neonatal period of life, to maintain central tolerance, become effective when they encounter their target antigen in the periphery during adult life.51,52
The second approach which we have recently proposed suggests that some of the autoreactive lymphocytes that escape central selection elicit the production of neutralizing antibodies to pro-inflammatory mediators of the immune system, such as TNF-a and pro-inflammatory chemokines.53-55 As this response in not sufficient to prevent the development and progression of an autoimmune condition, we have looked for ways in which this response could be amplified in accordance with the development and progression of the autoimmune condition. For this purpose, we have selected a modification of gene therapy named naked DNA vaccination. A major current use of this technology is to increase the cell-mediated antigen-specific immune response against infectious agents such as tuberculosis and HIV, and allergens such as mite proteins.56-62 The interesting work of Waisman et al paved the way for applying this powerful technology to elicit protective immunity to experimental autoimmune diseases.63 In their study, Waisman et al inhibited EAE by immunizing mice with cDNA encoding the T-cell receptor V genes.63 In our studies, we tried to use naked DNA vaccination to break down tolerance to pro-inflammatory mediators of the autoimmune process, thus generating immunological memory against these pro-inflammatory factors.53,54 Thus each gene of interest was cloned into a mammalian vector with a strong viral promoter (cytomegalo-virus (CMV)) and a repeated immunostimulatory sequence (ISS).60,61 We have demonstrated that upon repeated administrations of each vaccine, tolerance to its gene product was broken and immunological memory was established.53,54 In these experiments, rats were immunized with MBP/CFA to induce active EAE 2 months after the last administration of each vaccine. At this time, the self-specific antibody titer to each gene product regressed to background levels. Interestingly, immunization with MBP/CFA to induce active EAE, and not with the CFA alone, to elicit a local inflammatory process, elicited the rapid production of self-specific antibodies to the product of each given vaccine.53,54 Thus, rats that were previously subjected to naked DNA vaccines encoding MIP-1a, MCP-1 or TNF-a were EAE resistant, and at that time exhibited a marked antibody titer against the gene product of each vaccine. Each titer accelerated in accordance with the progression of disease in control EAE rats and regressed background levels upon recovery. MCP-1-, MIP-1a-and TNF-a-specific antibodies generated in EAE-resistant rats were neutralizing in vitro, and could transfer EAE resistance in adoptive transfer experi-ments.53,54 Thus by applying pro-inflammatory cytokine/chemokine-based naked DNA vaccination, one may re-educate the immune system to use self-specific immunity to restrain its own harmful activities.53,54
The biological significance of the association between the elevated levels of TNF-a or C-C chemokine mRNA at a privileged autoimmune site (CNS) and the enhancement in anti-self-response against pro-inflammatory cytokines and chemokines is apparent. After all, an ideal immune system would be selected in evolution to centralize its destructive competence against invading microbes rather than against the self-tissues it was designed to protect.64-66 The underlying mechanism by which the immune system distinguishes a gene product transcribed at a privileged autoimmune site from the same gene product transcribed at a local site of inflammation is, however, still elusive. A partial explanation for these intriguing observations was previously suggested by Cyster et al.67 This group provided compelling evidence to suggest that peripheral clonal exclusion of self-reactive B-cells occurs in germinal centers of lymph nodes that drain tissues lacking immune surveillance (i.e. immune privileged areas), where competition for follicular niches does not exclude self-reactive cells from the recirculating B-cell repertoire.67 This may suggest that the expression and production of pro-inflammatory cytokines/chemokines will lead to the exclusion of self-specific B-cells, capable of generating an immune response to these self-gene products, unless they are transcribed and produced at an autoimmune site, such as the CNS.67 Pro-inflammatory chemokine/cytokine-based DNA vaccination probably amplifies this process of tolerance breakdown.
From the basic science perspective, the above observations may provide a new perspective for understanding the role of T-cell and B-cell selection in induction and maintenance of tolerance to self. In the process of negative selection, self-reactive T-cells die when they encounter self-antigen in the thymus.49,50 Similarly, self-specific pre-B-cells either die or undergo receptor editing in the bone marrow.68 It is believed that those cells escaping central tolerance are subjected to various mechanisms acting outside the thymus or the bone marrow to keep them under control. This type of control has been termed peripheral tolerance. T-cell anergy,69 active suppression,30,69-71 T-cell deletion72,73 and generation of anti-idiotypic immunity74 have been described as key mechanisms that contribute to the maintenance of peripheral tolerance. The current study suggests for the first time that self-specific T- and B-cells, capable of mounting self-specific immunity against pro-inflammatory mediators, escape central tolerance to provide the immune system with a powerful tool with which to keep its dangerous anti-self activity under control and thus maintain tolerance to self in the periphery. Moreover, as microbes and self-components are constructed from similar 'building blocks' and as central selection manifests its own limitations, antiself immunity cannot be avoided, but rather has to be restrained by peripheral mechanisms. Moreover, it could well be that a substantial increase in the competence of the immune system to effectively limit its T- and B-cell repertoire would result in a constrained ability to effectively confront infectious diseases. The case of natural immunity to TNF-a, evoked during the course of a T-cell-mediated autoimmune disease, demonstrates how the immune system has evolved to benefit from its own limited competence to effectively select against self-reactivity.
From a clinical perspective, the advantage of interfering in the autoimmune process with cytokine and chemokine DNA vaccines is apparent. A major disadvantage in treating chronic diseases with xenogenic neutralizing antibodies lies in their immunogenicity. This has motivated investigators to develop chimeric humanized antibodies (review: Riethmuller et al75) and monoclonal antibodies engineered with human Ig heavy and light chain yeast artificial chromosome (YAC).76 However, following repeated immunization, these engineered antibodies do trigger an apparently allotypic response. The therapeutic strategy suggested by our studies has an advantage over the above methods, since it resulted in the generation of immunity to autologous antigen only during the course of disease at the time when mRNA encoding the pro-inflammatory cytokine is profoundly evated at the site of inflammation. Yet another major disadvantage of applying anti-chemokine/cytokine immunotherapy in T-cell-mediated autoimmunity is that the treatment is not disease specific and may lead to suppression/alteration of other immuno-logical functions. Ultimately, an ideal DNA vaccine would exert a maximal effect on the clinical manifestation of an autoimmune condition with a minimal effect on other immunological functions. We believe that the next breakthrough in the development of genetic vaccines for T-cell-mediated autoimmunity will depend on defining disease-specific chemokine/cytokine encoding DNA vaccines. This goal is still dependent on future characterization of organ-specific/disease-specific pro-inflammatory factors.
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