Masha Fridkis-Hareli

Copolymer 1 inhibits chronic relapsing experimental allergic encephalomyelitis induced by proteolipid protein (PLP) peptides in mice and interferes with PLP-specific T cell responses

Copolymer 1 (Cop 1) is a synthetic amino acid copolymer effective in suppression of experimental allergic encephalomyelitis (EAE) and developed as a candidate drug for multiple sclerosis (MS). In the present study, we induced chronic relapsing (CR)-EAE in (SJL/J X BALB/c)F1 mice by either whole spinal cord homogenate or two synthetic peptides of proteolipid protein (PLP), p139-151 and p178-191. When Cop 1 was added to the encephalitogenic inoculum, mice were almost completely resistant to disease induction. T cell lines to p139-151 and p178-191 were specific to these peptides. Their antigen-specific responses were inhibited by Cop 1 in a dose-dependent manner, while their polyclonal response to the superantigen staphylococcal enterotoxin A (SEA) was not affected by Cop 1. Using biotinylated PLP derivatives, we demonstrated that the two PLP peptides bound to I-A(s) molecules, and that their binding was completely inhibited by unlabelled Cop 1. Furthermore, Cop 1 could displace the PLP peptides from the MHC binding site. These results support the potential of Cop 1 as a broad-spectrum drug for MS.

The autoimmune reactivity to myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is potentially pathogenic: effect of copolymer 1 on MOG-induced disease.

Multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) characterized by primary demyelination, is believed to result from an autoimmune attack against myelin components. In view of their ability to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS, the quantitatively major myelin proteins — myelin basic protein (MBP) and proteolipid protein (PLP) — have been extensively studied as the relevant primary antigens in MS, and therapeutic approaches have been targeted to counteract autoimmune reactivity to MBP and PLP. Accordingly, copolymer 1, a random synthetic amino acid copolymer cross-reactive with MBP and highly protective against the induction of EAE with MBP or PLP, is now being extensively tested in clinical studies as a therapeutic agent for MS. However, increasing evidence suggests that autoimmune reactivity against other CNS-specific myelin proteins could also be involved in the pathogenesis of MS. In this context, we have demonstrated that peripheral blood lymphocytes from patients with MS respond predominantly to myelin oligodendrocyte glycoprotein (MOG) rather than to MBP or PLP, suggesting an important role for cell reactivity against MOG in the pathogenesis of MS. We have demonstrated that T-cell reactivity to MOG can also be pathogenic by inducing neurological disease in H-2u and H-2b mice with the same peptide of MOG, pMOG 35–55. Most interestingly, the expression of the disease differed with the different MHC backgrounds. Induction of a differentially expressed disease in different strains of mice with the same myelin antigen makes this new model particularly relevant to MS, where different expression of the disease is seen in different patients. Therefore, notwithstanding the importance of the autoimmune reactivity to MBP and PLP in MS, the potentially pathogenic autoimmune reactivity to MOG must now also be taken into consideration in therapeutic approaches to MS. In this context, we have investigated the possible effect of copolymer 1 treatment on autoimmune reactivity to MOG and on the development of EAE induced by MOG. Copolymer 1 was found to inhibit the binding of MOG peptides to MHC molecules, as well as the proliferation of MOG-reactive T cells, in a dose-dependent manner. In parallel, injection of copolymer 1 concomitantly with the encephalitogenic MOG peptide exerted a strong protective effect against the development of EAE. These preliminary data on the effect of copolymer 1 on the autoimmune response to MOG in mice indicate that copolymer 1 may also be effective in cases of MS where the autoimmune response to MOG prevails, and should therefore be further investigated in this context.

Novel synthetic amino acid copolymers that inhibit autoantigen-specific T cell responses and suppress experimental autoimmune encephalomyelitis

Copolymer 1 (Cop 1, Copaxone [Teva Marion Partners, Kansas City, Missouri, USA]), a random amino acid copolymer of tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K), reduces the frequency of relapses by 30% in relapsing-remitting multiple sclerosis (MS) patients. In the present study, novel random four-amino acid copolymers, whose design was based on the nature of the anchor residues of the immunodominant epitope of myelin basic protein (MBP) 85-99 and of the binding pockets of MS-associated HLA-DR2 (DRB1*1501), have been synthesized by solid-phase chemistry. Poly (Y, F, A, K) (YFAK) inhibited binding of the biotinylated MBP 86-100 epitope to HLA-DR2 molecules more efficiently than did either unlabeled MBP 85-99 or any other copolymer including Cop 1. Moreover, YFAK and poly (F, A, K) (FAK) were much more effective than Cop 1 in inhibition of MBP 85-99-specific HLA-DR2-restricted T cell clones. Most importantly, these novel copolymers suppressed experimental autoimmune encephalomyelitis, induced in the susceptible SJL/J (H-2(s)) strain of mice with the encephalitogenic epitope PLP 139-151, more efficiently than did Cop 1. Thus, random synthetic copolymers designed according to the binding motif of the human immunodominant epitope MBP 85-99 and the binding pockets of HLA-DR2 might be more beneficial than Cop 1 in treatment of MS.

Feedback regulation of T cell development: manifestations in aging

Recent findings have indicated that mature T cells may regulate thymocytopoiesis in an age-related differential manner. The studies were based on T lymphocyte development in mouse fetal thymus stroma colonized with immature thymocytes and CD4+ T cells from young or old donors. In the present study, we used mathematical modeling and computer simulations in order to identify the thymocyte subsets that are targets for this type of regulation, and the processes affected by it. Our results suggest that thymocyte development is subject to regulation through 2 feedback loops: mature CD4+ cells exert a negative feedback on the double-negative to double-positive transition and on double-positive subset growth, and a positive feedback on the double-positive to CD4 single-positive transition. These effects may operate, in young mice, through a reduction in the rate of death of CD4+8- thymocytes, and a faster maturation of double-positive cells. In old mice, our simulations suggest that there may additionally be a reduction in double-positive proliferation rate. In some, but not all, of the simulations of old donor- derived thymocytes, we also had to assume a reduction in double-negative to double-positive differentiation, an increase in double-positive death rates, an increase of CD4+8- cell division rate, and a decrease of differentiation to the CD8 lineage.

Cytokine profiles in pemphigus vulgaris patients treated with intravenous immunoglobulins as compared to conventional immunosuppressive therapy

Pemphigus vulgaris (PV) is a potentially fatal blistering disease of the skin and mucous membranes, characterized by the presence of autoantibodies against adhesion molecules (desmoglein, Dsg3) present on the surface of keratinocytes, which lead to the loss of cellular adhesion or acantholysis. The mainstay of treatment is conventional immunosuppressive therapy (CIST), i.e. high dose, long-term systemic corticosteroids with or without immunosuppressive drugs. Intravenous immunoglobulin (IVIg) has been used in patients refractory to CIST, and its use has resulted in long-term clinical remission. Since cytokines play an important role in the immunopathogenesis of PV, it would be useful to compare how both IVIg and CIST therapies affect cytokine levels in the serum of PV patients. Thus, the goal of this study was to conduct a comparative analysis of levels of various cytokines, during an 18 month consecutive period, after the initiation of CIST or IVIg treatment in PV patients, with similar extent and severity of disease in the two study groups, with 11 patients in each group. The cytokines measured were IL-1beta, IL-6, IL-8, IFN-gamma, IL-4 and IL-10. The levels of most of these cytokines were higher in the sera of untreated patients in both groups, compared to normal controls. The cumulative data collected over an 18 month period of treatment demonstrates that there is a gradual reduction in the levels of these cytokines, until they are at levels observed in normal individuals. The conclusions from this limited number of patients, prospectively studied, would suggest that both CIST and IVIg therapies are similar in their ability to influence a panel of cytokines in patients with pemphigus vulgaris.

Developmental interactions of CD4 T cells and thymocytes: age-related differential effects

The study was designed to determine whether the developmental potential of immature thymocytes in the thymus is altered in aging, and whether concomitantly present mature T cells have any feedback effect. The strategy was to seed sorted double negative, CD4-CD8-(DN) thymocytes on their own, or in the presence of mature T cells, onto lymphoid depleted fetal thymus (FT) explants, and to examine the resulting T cell subsets. Thymocyte donors were young (2-3 months) and old (24 months) C57BL/6J, Thy1.2 mice and splenocytes were from C57BL/Ka, Thy1.1 mice. The DN cells of the old gave rise to lower values of double positive CD4+CD8+ (DP) cells than those of the young. Cocultures containing a mixture of DN thymocytes and CD4+CD8- splenocytes showed higher CD4+CD8- and DN, and lower DP and CD4-CD8+ levels in the old-donor derived cells, as compared with the young ones. Similar results were obtained with CD4+CD8- thymocytes. In contrast, the presence of CD4-CD8+ splenocytes had no effect on the pattern of DN cell development. Our data indicate that differentiation of CD4/CD8 thymocyte phenotypes is affected by CD4+ cells, in an age-associated differential manner.

Immunogenetic mechanisms for the coexistence of organ-specific and systemic autoimmune diseases.

BackgroundOrgan-specific autoimmune diseases affect particular targets in the body, whereas systemic diseases engage multiple organs. Both types of autoimmune diseases may coexist in the same patient, either sequentially or concurrently, sustained by the presence of autoantibodies directed against the corresponding autoantigens. Multiple factors, including those of immunological, genetic, endocrine and environmental origin, contribute to the above condition. Due to association of certain autoimmune disorders with HLA alleles, it has been intriguing to examine the immunogenetic basis for autoantigen presentation leading to the production of two or more autoantibodies, each distinctive of an organ-specific or systemic disease. This communication offers the explanation for shared autoimmunity as illustrated by organ-specific blistering diseases and the connective tissue disorders of systemic nature.Presentation of the hypothesisSeveral hypothetical mechanisms implicating HLA determinants, autoantigenic peptides, T cells, and B cells have been proposed to elucidate the process by which two autoimmune diseases are induced in the same individual. One of these scenarios, based on the assumption that the patient carries two disease-susceptible HLA genes, arises when a single T cell epitope of each autoantigen recognizes its HLA protein, leading to the generation of two types of autoreactive B cells, which produce autoantibodies. Another mechanism functioning whilst an epitope derived from either autoantigen binds each of the HLA determinants, resulting in the induction of both diseases by cross-presentation. Finally, two discrete epitopes originating from the same autoantigen may interact with each of the HLA specificities, eliciting the production of both types of autoantibodies.Testing the hypothesisDespite the lack of immediate or unequivocal experimental evidence supporting the present hypothesis, several approaches may secure a better understanding of shared autoimmunity. Among these are animal models expressing the transgenes of human disease-associated HLA determinants and T or B cell receptors, as well as in vitro binding studies employing purified HLA proteins, synthetic peptides, and cellular assays with antigen-presenting cells and patients lymphocytes. Indisputably, a bioinformatics-based search for peptide motifs and the modeling of the conformation of bound autoantigenic peptides associated with their respective HLA alleles will reveal some of these important processes.Implications of the hypothesisThe elucidation of HLA-restricted immune recognition mechanisms prompting the production of two or more disease-specific autoantibodies holds significant clinical ramifications and implications for the development of more effective treatment protocols.

Synthetic peptides that inhibit binding of the myelin basic protein 85-99 epitope to multiple sclerosis-associated HLA-DR2 molecules and MBP-specific T-cell responses.

Copolymer 1 (Cop 1, poly [Y, E, A, K]) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS), a disease that is linked to HLA-DR2 (DRB1*1501). In the present study various peptides, synthesized according to the binding motifs for both the immunodominant epitope of myelin basic protein (MBP) 85-99, a candidate autoantigen in MS, and Cop 1, differentially inhibited binding of these antigens to disease-associated HLA-DR2 (DRB1*1501) molecules. In particular, two peptides with residue K at position P-1, as referred to MBP 85-99, inhibited effectively the binding of both biotinylated MBP 85-99 and Cop 1 to HLA-DR2 molecules as well as IL-2 production by two MBP-specific HLA-DR2-restricted T-cell clones. These findings suggest the possible utility of these compounds or their more stable derivatives in treatment of MS.

Aging in the T Lymphocyte Compartment

A decline in the capacity of bone marrow cells to differentiate to T lymphocytes was found when cells from young and old donors were seeded onto an alymphoid fetal thymus. A step-by-step analysis of cell-cell interactions of the lymphohemopoietic cells and the thymic stroma indicated an effect of age on a variety of cell differentiation parameters. These included a decrease in the affinity of bone marrow cells to the stroma, and in their capacity to compete with the thymic lymphoid resident cells on colonization of the thymus. There was a significant decrease in the ability of cells of old donors to replicate sequentially within the thymic microenvironment. There was a reduced capacity of bone marrow cells from aging mice to express a developmental preference after seeding onto a syngeneic fetal thymus in a mixture with cells from allogeneic donors. We addressed the question whether the aging thymus contains increased levels of immature cells that fail to differentiate in the involuted thymic microenvironment by seeding thymocytes from young and old donors onto the fetal thymic stroma. The values of T cells that developed from the old donor inoculum were lower under these conditions. Our studies suggest that at least some of the manifestations of aging in the T cell compartment are related to developmentally programmed events in the lymphohemopoietic cell compartment.

Differential expression of membrane polypeptides on fetal thymic stroma co-cultured with bone marrow cells from young and old mice

Cell membrane polypeptides from co-cultures of lymphocyte depleted fetal thymuses (C57BL/Ka, H-2b, Thy 1.1) and bone marrow (BM) cells (C57BL/6J, H-2b, Thy 1.2) were analyzed by two-dimensional gel electrophoresis (2D-PAGE). Donors of BM cells were young (3 months) and old (24 months) mice. Two polypeptides, not detected in either the alymphoid thymus itself or the BM cells prior to seeding, were expressed in the co-cultures, and their levels in the presence of old BM cells were higher than in the presence of young BM cells. Two other polypeptides, originally observed in the thymic stroma, exhibited reduced levels by day 3 in cocultures containing BM cells from young donors, but not from old donors. It is concluded that distinct changes in membrane proteins are manifested in the thymic stroma, as well as in the colonizing BM cells.