Tolerance defects in New Zealand Black and New Zealand Black X New Zealand White F1 mice

The susceptibility of autoimmune NZB and (NZB X NZW)F1 mice to the induction of tolerance by monomeric BSA was compared with several normal mouse strains. Unresponsiveness in T and B lymphocyte compartments was probed by challenging with DNP8BSA and measuring anti-DNP and anti-BSA antibodies separately. Tolerance induced by monomeric BSA was carrier specific, and there was no evidence of epitope-specific suppression. Normal NZW, NFS, and B10.D2 mice were easily rendered tolerant with monomeric BSA and did not produce anti-DNP or anti-BSA antibodies after challenge with DNP8BSA. By contrast, the lack of anti-DNP antibody response in similarly treated NZB mice was dependent on the dose of monomeric BSA, indicating that the helper T cells were partially resistant to tolerance induction. NZB mice treated with a high dose of monomeric BSA produced anti-BSA, but not anti-DNP, antibodies after immunization. Thus, the anti-carrier B cells in NZB mice may have been primed by monomeric BSA. The presence of the xid gene on the NZB background rendered the mice susceptible to induction of tolerance, suggesting that the tolerance defect in NZB mice involves the B cell compartment. This abnormal antibody response was a dominant trait: (NZB X NFS)F1 and (NZB X B10.D2)F1 mice had the same characteristics as NZB mice. These F1 hybrids do not develop autoimmune disease, indicating that resistance to experimental tolerance induction expressed at a B cell level may not be sufficient for disease development. In contrast to NZB and other NZB F1 hybrids, (NZB X NZW)F1 hybrids treated with monomeric BSA and challenged with DNP8BSA responded to both DNP and BSA. The contribution of a B cell defect to the tolerance abnormality of (NZB X NZW)F1 mice was examined by analyzing the effect of the xid gene on the progeny of (NZB.xid X NZW)F1 mice. Unlike the effect of the xid gene on NZB mice, both phenotypically normal heterozygous female and phenotypically xid hemizygous male mice produced anti-DNP and anti-BSA antibodies after tolerance induction and immunization, demonstrating that a major helper T cell abnormality was present in (NZB X NZW)F1 mice. The (NZW X B10.D2)F1 hybrid was rendered tolerant by this procedure, indicating that the helper T cell defect (NZB X NZW)F1 mice may have resulted from gene complementation with the NZB mice contributing partial resistance of T helper cells to tolerance induction.(ABSTRACT TRUNCATED AT 400 WORDS)     

V region sequences of anti-DNA and anti-RNA autoantibodies from NZB/NZW F1 mice

The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft.     

IFN-alpha induces early lethal lupus in preautoimmune (New Zealand Black x New Zealand White) F1 but not in BALB/c mice

Recent studies indicate that IFN-alpha is involved in pathogenesis of systemic lupus erythematosus. However, direct proof that IFN-alpha is not only necessary, but also sufficient to induce lupus pathogenicity is lacking. In this study, we show that in vivo adenovector-mediated delivery of murine IFN-alpha results in preautoimmune (New Zealand Black (NZB) x New Zealand White (NZW))F(1), but not in normal, mice, in a rapid and severe disease with all characteristics of systemic lupus erythematosus. Anti-dsDNA Abs appeared as soon as day 10 after initiation of IFN-alpha treatment. Proteinuria and death caused by glomerulonephritis occurred in all treated mice within, respectively, approximately 9 and approximately 18 wk, at a time when all untreated (NZB x NZW)F(1) did not show any sign of disease. IFN-alpha in vivo induced an overexpression of B lymphocyte stimulator in circulation at similar levels in both the preautoimmune and the normal mouse strains. All effects elicited by IFN-alpha were dose dependent. (NZB x NZW)F(1) infused with purified murine IFN-alpha also showed acceleration of lupus. Thus, prolonged expression of IFN-alpha in vivo induces early lethal lupus in susceptible animals.     

Atorvastatin inhibits autoreactive B cell activation and delays lupus development in New Zealand black/white F1 mice

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by a wide range of immunological abnormalities that underlie the loss of tolerance. In this study we show that administration of atorvastatin to lupus-prone NZB/W F(1) mice resulted in a significant reduction in serum IgG anti-dsDNA Abs and decreased proteinuria. Histologically, the treatment was associated with reduced glomerular Ig deposition and less glomerular injury. Disease improvement was paralleled by decreased expression of MHC class II on monocytes and B lymphocytes and reduced expression of CD80 and CD86 on B lymphocytes. Consequent upon this inhibition of Ag presentation, T cell proliferation was strongly impaired by atorvastatin in vitro and in vivo. A significant decrease in MHC class II expression was also observed in the target organ of lupus disease (i.e., the glomerulus). Serum cholesterol in atorvastatin-treated lupus mice fell to the level found in young NZB/W mice before disease onset. This is the first demonstration that atorvastatin can delay the progression of a spontaneous autoimmune disease and may specifically benefit patients with systemic lupus erythematosus.     

Effects of gamma-interferon and FK506 on resting B cell proliferation of New Zealand black/white F1 mice.

We examined the effects of gamma-interferon (gamma-IFN) and the new immunosuppressant FK506 on resting B cell proliferation of New Zealand black/white F1 hybrid (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE). gamma-IFN and FK506 inhibited in a dose-dependent manner both B cell proliferation and autoantibody production of resting B cells respectively. There was a synergistic interaction between gamma-IFN and FK506 in their inhibition and they did not exhibit cell cytotoxicity. This in vitro synergism of gamma-IFN and FK506 may have clinical application in that low doses of gamma-IFN and FK506 combinations may be effective to correct polyclonal B cell activation of patients with SLE.     

Expansion and hyperactivity of CD1d-restricted NKT cells during the progression of systemic lupus erythematosus in (New Zealand Black x New Zealand White)F1 mice

CD1d-restricted NKT cells expressing invariant TCR alpha-chain rearrangements (iNKT cells) have been reported to be deficient in humans with a variety of autoimmune syndromes and in certain strains of autoimmune mice. In addition, injection of mice with alpha-galactosylceramide, a specific glycolipid agonist of iNKT cells, activates these T cells and ameliorates autoimmunity in several different disease models. Thus, deficiency and reduced function in iNKT cells are considered to be risk factors for the development of such diseases. In this study we report that the development of systemic lupus erythematosus in (New Zealand Black (NZB) x New Zealand White (NZW))F(1) mice was paradoxically associated with an expansion and activation of iNKT cells. Although young (NZB x NZW)F(1) mice had normal levels of iNKT cells, these expanded with age and became phenotypically and functionally hyperactive. Activation of iNKT cells in (NZB x NZW)F(1) mice in vivo or in vitro with alpha-galactosylceramide indicated that the immunoregulatory role of iNKT cells varied over time, revealing a marked increase in their potential to contribute to production of IFN-gamma with advancing age and disease progression. This evolution of iNKT cell function during the progression of autoimmunity may have important implications for the mechanism of disease in this model of systemic lupus erythematosus and for the development of therapies using iNKT cell agonists.     

Enlargement of Lyt-2-positive T cells is associated with functional impairment and autoimmune hemolytic anemia in New Zealand Black mice

We investigated the relationship between the increased cell diameter of Lyt-2+ T cells and the development of autoimmune disease in aging NZB and NZB X NZW F1 hybrid (BW) mice. Individual animals were analyzed for Lyt-2+ T cell size (by narrow-angle forward light scatter), anti-erythrocyte autoantibodies, anemia, proteinuria, and splenomegaly. The peak light scatter of the Lyt-2+ T cells correlated with the level of anti-erythrocyte autoantibodies and severity of hemolytic anemia, but not with proteinuria or splenomegaly. The cell size of this T cell subset did not increase in old BW or in NZB mice homozygous for the xid gene (NZB.xid). The in vivo administration of bacterial lipopolysaccharide to young NZB mice did not stimulate the enlargement of Lyt-2+ T cells. Ly-2+ T cells from old NZB mice could be stimulated by concanavalin A (Con A) to express interleukin 2 (IL 2) receptors and to synthesize DNA in vitro. However, in vivo administration of Con A to old NZB mice did not induce the expression of IL 2 receptors on Lyt-2+ T cells. Further, in vivo T suppressor function was impaired in old NZB mice with enlarged Lyt-2+ T cells. Thus, the enlargement of Lyt-2+ T cells in old NZB mice appears related to impaired T cell function in vivo and is associated with the development of anti-erythrocyte autoantibodies and autoimmune hemolytic anemia.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors

IntroductionAutoantibodies contribute significantly to the pathogenesis of systemic lupus erythematosus (SLE). Unfortunately, the long-lived plasma cells (LLPCs) secreting such autoantibodies are refractory to conventional immunosuppressive treatments. Although generated long before the disease becomes clinically apparent, it remains rather unclear whether LLPC generation continues in the established disease. Here, we analyzed the generation of LLPCs, including autoreactive LLPCs, in SLE-prone New Zealand Black/New Zealand White F1 (NZB/W F1) mice over their lifetime, and their regeneration after depletion.MethodsBromodeoxyuridine pulse-chase experiments in mice of different ages were performed in order to analyze the generation of LLPCs during the development of SLE. LLPCs were enumerated by flow cytometry and autoreactive anti-double-stranded DNA (anti-dsDNA) plasma cells by enzyme-linked immunospot (ELISPOT). For analyzing the regeneration of LLPCs after depletion, mice were treated with bortezomib alone or in combination with cyclophosphamide and plasma cells were enumerated 12 hours, 3, 7, 11 and 15 days after the end of the bortezomib cycle.ResultsAutoreactive LLPCs are established in the spleen and bone marrow of SLE-prone mice very early in ontogeny, before week 4 and before the onset of symptoms. The generation of LLPCs then continues throughout life. LLPC counts in the spleen plateau by week 10, but continue to increase in the bone marrow and inflamed kidney. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks. Persistent depletion of LLPCs was achieved only by combining a cycle of bortezomib with maintenance therapy, for example cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs.ConclusionsIn SLE-prone NZB/W F1 mice, autoreactive LLPCs are generated throughout life. Their sustained therapeutic elimination requires both the depletion of LLPCs and the inhibition of their regeneration.     

Autoimmune alterations induced by the New Zealand Black Lbw2 locus in BWF1 mice

The New Zealand Black (NZB) Lbw2 locus (lupus NZB x New Zealand White (NZW) 2 locus) was previously linked to mortality and glomerulonephritis, but not to IgG autoantibodies, suggesting that it played a role in a later disease stage. To define its contribution, (NZB x NZW)F1 hybrids (BWF1) containing two, one, or no copies of this locus were generated. Lack of the NZB Lbw2 indeed reduced mortality and glomerulonephritis, but not serum levels of total and anti-DNA IgG Abs. There were, however, significant reductions in the B cell response to LPS, total and anti-DNA IgM and IgG Ab-forming cells, IgM Ab levels, and glomerular Ig deposits. Furthermore, although serum IgG autoantibody levels correlated poorly with kidney IgG deposits, the number of spontaneous IgG Ab-forming cells had a significant correlation. Genome-wide mapping of IgM anti-chromatin levels identified only Lbw2, and analysis of subinterval congenics tentatively reduced Lbw2 to approximately 5 Mb. Because no known genes associated with B cell activation and lupus are in this interval, Lbw2 probably represents a novel B cell activation gene. These findings establish the importance of Lbw2 in the BWF1 hybrid and indicate that Lbw2, by enhancing B cell hyperactivity, promotes the early polyclonal activation of B cells and subsequent production of autoantibodies.     

Neuromuscular junction defects in mice with mutation of dynein heavy chain 1

Disruptions in axonal transport have been implicated in a wide range of neurodegenerative diseases. Cramping 1 (Cra1/+) and Legs at odd angles (Loa/+) mice, with hypomorphic mutations in the dynein heavy chain 1 gene, which encodes the ATPase of the retrograde motor protein dynein, were originally reported to exhibit late onset motor neuron disease. Subsequent, conflicting reports suggested that sensory neuron disease without motor neuron loss underlies the phenotypes of Cra1/+ and Loa/+ mice. Here, we present behavioral and anatomical analyses of Cra1/+ mice. We demonstrate that Cra1/+ mice exhibit early onset, stable behavioral deficits, including abnormal hindlimb posturing and decreased grip strength. These deficits do not progress through 24 months of age. No significant loss of primary motor neurons or dorsal root ganglia sensory neurons was observed at ages where the mice exhibited clear symptomatology. Instead, there is a decrease in complexity of neuromuscular junctions. These results indicate that disruption of dynein function in Cra1/+ mice results in abnormal morphology of neuromuscular junctions. The time course of behavioral deficits, as well as the nature of the morphological defects in neuromuscular junctions, suggests that disruption of dynein function in Cra1/+ mice causes a developmental defect in synapse assembly or stabilization.     

Ig-reactive CD4+CD25+ T cells from tolerized (New Zealand Black x New Zealand White)F1 mice suppress in vitro production of antibodies to DNA

We have recently shown that tolerogenic administration of an artificial peptide (pConsensus) that is based on sequences within the V(H) regions of several murine anti-dsDNA Ig delays appearance of autoantibodies in female (New Zealand Black (NZB) x New Zealand White (NZW))F(1) (NZB/W F(1)) mice and significantly prolongs their survival. The aim of this study was to characterize the T cell population(s) involved in pConsensus-induced down-regulation of autoimmune responses in tolerized NZB/W F(1) mice. Using MHC class II dimers loaded with tolerogenic peptide, we found that pCons favored expansion of peptide-reactive CD4(+)CD25(+) regulatory T cells (T(R)) that inhibited in vitro production of anti-dsDNA Ab-forming cells. Suppression by T(R) was abrogated by the presence in culture of Ab to glucocorticoid-induced TNFR family member 18 or to TGFbeta latency-associated protein. These findings suggest possible relevance of Ag specificity in the mechanism of T(R)-mediated immune tolerance to Ig-derived peptides in NZB/W F(1) mice.     

Blockade of programmed death-1 in young (New Zealand black x New Zealand white)F1 mice promotes the activity of suppressive CD8+ T cells that protect from lupus-like disease

The programmed death-1 (PD-1)/programmed death-1 ligand 1 (PD-L1) pathway regulates both stimulatory and inhibitory signals. In some conditions, PD-1/PD-L1 inhibits T and B cell activation, induces anergy, and reduces cytotoxicity in CD8(+) T cells. In other conditions, PD-l/PD-L1 has costimulatory effects on T cells. We recently showed that induction of suppressive CD8(+)Foxp3(+) T cells by immune tolerance of lupus-prone (New Zealand black × New Zealand white)F(1) (BWF(1)) mice with the anti-DNA Ig-based peptide pConsensus (pCons) is associated with significantly reduced PD-1 expression on those cells. In this study, we tested directly the role of PD-1 by administering in vivo neutralizing Ab to PD-1 to premorbid BWF(1) and healthy control mice. Anti-PD-1-treated mice were protected from the onset of lupus nephritis for 10 wk, with significantly improved survival. Although the numbers of T cells declined in aging control mice, they were maintained in anti-PD-1-treated mice, including CD8(+)Foxp3(+) T cells that suppressed syngeneic CD4(+)CD25(-) T cell proliferation and IFN-γ production, reduced production of IgG and anti-dsDNA IgG, induced apoptosis in syngeneic B cells, and increased IL-2 and TGF-β production. The administration of anti-PD-1 Ab to BWF(1) mice after induction of tolerance with pCons abrogated tolerance; mice developed autoantibodies and nephritis at the same time as control mice, being unable to induce CD8(+)Foxp3(+) T suppressor cells. These data suggest that tightly regulated PD-1 expression is essential for the maintenance of immune tolerance mediated by those CD8(+)Foxp3(+) T cells that suppress both T(h) cells and pathogenic B cells. PD-1 regulation could represent a target to preserve tolerance and prevent autoimmunity.     

Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells

An increasing number of studies indicate that a subset of CD4(+) T cells with regulatory capacity (regulatory T cells; T(regs)) can function to control organ-specific autoimmune disease. To determine whether abnormalities of thymic-derived T(regs) play a role in systemic lupus erythematosus, we evaluated T(reg) prevalence and function in (New Zealand Black x New Zealand White)F(1) (B/W) lupus-prone mice. To explore the potential of T(regs) to suppress disease, we evaluated the effect of adoptive transfer of purified, ex vivo expanded thymic-derived T(regs) on the progression of renal disease. We found that although the prevalence of T(regs) is reduced in regional lymph nodes and spleen of prediseased B/W mice compared with age-matched non-autoimmune mice, these cells increase in number in older diseased mice. In addition, the ability of these cells to proliferate in vitro was comparable to those purified from non-autoimmune control animals. Purified CD4(+)CD25(+)CD62L(high) B/W T(regs) were expanded ex vivo 80-fold, resulting in cells with a stable suppressor phenotype. Adoptive transfer of these exogenously expanded cells reduced the rate at which mice developed renal disease; a second transfer after treated animals had developed proteinuria further slowed the progression of renal disease and significantly improved survival. These studies indicate that thymic-derived T(regs) may have a significant role in the control of autoimmunity in lupus-prone B/W mice, and augmentation of these cells may constitute a novel therapeutic approach for systemic lupus erythematosus.     

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.     

Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice

Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCdelta. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCdelta were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCdelta in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.     

Ultraviolet cross-linking of helical oligonucleotides to two monoclonal MRL-1pr/1pr anti-DNA autoantibodies. Variations in H and L chain binding to DNA

Experiments were performed to determine whether both H and L chains of different anti-native DNA autoantibodies are uniformly involved in binding to DNA. Two purified monoclonal mouse (MRL-1pr/1pr) IgG autoantibodies, H241 and 2C10, were tested. They both bound synthetic helical oligonucleotides of 10 to 20 base pairs in a gel electrophoresis retardation assay but differed in their preferences for given base sequences. Exposure of antibody-radiolabeled oligonucleotide mixtures to UV light (254 nm) for 10 min led to specific covalent cross-linking of oligonucleotide to both the H and the L chains of H241 but only to the H chain of 2C10. Single labeling events were detected without higher aggregation. The oligonucleotides were not cross-linked to unrelated IgG, even after 2 h of irradiation. Cross-linked (radioactively labeled) H and L chains of H241 and 2C10 were isolated from denaturing electrophoresis gels and digested with lysyl endopeptidase and/or staphylococcal V8 protease. H241 and 2C10 H chains each yielded a major labeled peptide fragment, but the peptides from the two antibodies were different. These experiments measured only some of the antibody-DNA interactions, probably with bases in the major groove of the DNA. They indicated that two MRL-1pr/1pr IgG anti-native DNA antibodies differ in their H and L chain contacts with DNA and provide an approach to identifying affinity-labeled binding sites in the antibodies.     

Inhibition of experimental autoimmune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for IAs molecules.

Blocking of the Ag presenting function of MHC by peptides capable of high affinity binding to this molecule has been proposed as a potential immunotherapeutic intervention in MHC-associated diseases. Recent studies have used this strategy to prevent the induction of experimental allergic encephalomyelitis (EAE) in mice. However, because of the close structural relationship between the inhibitor and encephalitogenic peptides, the results of these previous studies have been difficult to interpret with regard to whether MHC blockade was the mechanism by which the inhibitory peptides functioned. In our study, we have determined the capacity of unrelated peptides capable of binding with high affinity to IAs in inhibiting the induction of EAE in SJL/J mice after immunization with the autoantigenic peptide PLP 139-151. Prevention of the disease was accomplished by two methods: 1) when inhibitor was administered together with the encephalitogenic peptide at the time of immunization, as in previous studies, and 2) when inhibitor was administered at a separate site from the autoantigen 1 day before the immunization with that Ag. Inhibition was due to binding of the inhibitor to IAs, as evidenced by the fact that a control peptide incapable of binding to this MHC had no effect on the course of the disease. The finding that inhibitor could also be efficacious when administered at a separate site has implications for potential use of such a strategy to reverse ongoing autoimmune diseases. The inhibitor had to be present during the time of Ag stimulation, and had no long term inhibitory effects, in that a secondary immune response to the encephalitogenic peptide was not inhibited in animals given the inhibitory peptide before the induction of a primary response. This is compatible with the conclusion that MHC blockade was, in fact, the mechanism of the inhibition, rather than as a result of any long term suppressive effects on immunoreactive T cells. Finally, not only did administration of the inhibitory peptide lead to a prevention of the induction of EAE, but it could also be shown to decrease the T cell proliferative response in vitro to the autoantigen.