Induction of antigen-specific tolerance for the treatment of ongoing, relapsing autoimmune encephalomyelitis: a comparison between oral and peripheral tolerance

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune demyelinating disease of the central nervous system that serves as an animal model for multiple sclerosis. Various forms of Ag-specific tolerance have been used prophylactically to prevent development of acute EAE. Here we compare the induction of Ag-specific tolerance using two regimens, proteolipid protein 139-151 (PLP139-151) peptide-coupled splenocytes and oral administration of PLP139-151, for efficacy in the reduction of established, chronic clinical EAE. PLP139-151-coupled splenocytes and not oral administration of PLP139-151 was able to down-regulate established EAE, including subsequent relapses. PLP139-151 peptide-coupled splenocytes were effective at reducing Ag-specific T cell proliferation and IL-2 and IFN-gamma production, while concomitantly increasing IL-4 production. Oral administration of PLP139-151 did not reduce IL-2 or IFN-gamma production and appeared to increase Ag-specific T cell proliferation. Neither multiple high nor low doses of PLP139-151 were effective at decreasing ongoing clinical EAE or PLP139-151-specific IL-2 and IFN-gamma production. These results suggest that PLP139-151 peptide-induced tolerance is an efficacious treatment for ongoing, R-EAE when the peptide is coupled to chemically fixed splenocytes and not when given orally.     

Presentation of a T cell receptor antagonist peptide by immunoglobulins ablates activation of T cells by a synthetic peptide or proteins requiring endocytic processing

T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell-mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (Ig-PLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR-specific T cells may be the operative mechanism for Ig-PLP-LR-mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell-mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.     

Lack of Th2 cytokine increase during spontaneous remission of experimental allergic encephalomyelitis

The mechanisms underlying spontaneous remission of autoimmune diseases are presently unknown, though regulatory T cells are believed to play a major role in this process. We tested the hypothesis that Th2 and/or other T cell regulatory cytokines cause the spontaneous remission of experimental allergic encephalomyelitis (EAE), a model of Th1-mediated autoimmunity. We analyzed the cytokine profile of lymph node and central nervous system-infiltrating cells in individual SJL mice at different stages of proteolipid protein (PLP) 139-151 peptide-induced EAE. We found that IFN-gamma slowly fades away after clinical recovery, whereas IL-4, IL-10 and transforming growth factor-beta remain low or undetectable. Our peptide-results therefore suggest that regulatory T cells producing anti-inflammatory cytokines are not involved in spontaneous remission of EAE and challenge the view that the Th1/Th2 balance has a key role in EAE regulation.     

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.     

IL-12 down-regulates autoantibody production in mercury-induced autoimmunity

In genetically susceptible H-2s mice, subtoxic doses of mercuric chloride (HgCl2) induce a complex autoimmune syndrome characterized by the production of anti-nucleolar IgG Abs, lymphoproliferation, increased serum levels of IgG1 and IgE Abs, and renal Ig deposits. Mercury-induced autoimmunity in H-2s mice provides a useful model for chemically related autoimmunity in humans. The increase in serum IgG1 and IgE, which are under IL-4 control, suggests a role for the Th2 subset in this syndrome. The IL-12 cytokine induces T cell proliferation and IFN-gamma production and is necessary for differentiation of naive T cells into the Th1 subset. To gain an understanding of T cell control in this syndrome and, in particular, Th1/Th2 regulation, we assessed the effect of IL-12 administration in mercury-induced autoimmunity. Groups of A.SW mice (H-2s) received HgCl2 plus IL-12, HgCl2 alone, or IL-12 alone. IL-12 treatment resulted in a dramatic reduction of the anti-nucleolar Ab titers. IL-12 also inhibited the HgCl2-induced serum IgG1 increase, but, in contrast, did not significantly affect IgE induction in this model. This observation may be related to our unexpected finding that IL-12 further potentiated the HgCl2-triggered IL-4 induction in this model. The levels of renal Ig deposits were similar in mice receiving HgCl2 alone or HgCl2 plus IL-12. Our results indicate that IL-12 can down-regulate the autoimmune component of this experimental syndrome and that the various manifestations of mercury-induced autoimmunity are independently regulated.     

IFN-gamma prevents Th2 cell-mediated pathology after neonatal injection of semiallogenic spleen cells in mice

BALB/c mice injected at birth with 10(8) (A/J X BALB/c)F1 hybrid spleen cells develop an autoimmune host-vs-graft (HVG) disease as a result of activation of donor B cells by host CD4+ cells. The antidonor CD4+ cells seem to be Th2-like cells, inasmuch as they are profoundly deficient in IL-2 and IFN-gamma production, but secrete high levels of IL-4 and IL-10. As IFN-gamma is known to inhibit the development of TH2 cells, we attempted to modulate HVG disease by injecting rIFN-gamma. First, we found that 10 micrograms of rIFN-gamma given on days 1 and 3 after birth reduced the serum hyper-IgE of HVG mice by 90% and the serum hyper-IgG1, by 70%. In addition, rIFN-gamma administration significantly decreased the anti-DNA IgG1 titers and prevented the occurrence of anti-glomerular basement membrane and anti-laminin IgG1 Abs as well as the formation of immune deposits in renal glomeruli. These effects were not caused by the abrogation of chimerism, as indicated by the persistence of donor-type B cells in lymph nodes and of Igs bearing donor allotype in serum. MLC experiments indicated that the major effect of early rIFN-gamma administration was to restore the production of IL-2 and IFN-gamma by donor-specific T cells while these cells still secreted significant amounts of IL-4 and IL-10. Unresponsiveness of antidonor cytolytic T cells was not influenced by rIFN-gamma. We conclude that rIFN-gamma prevents the TH2-type response induced by the neonatal injection of semiallogeneic spleen cells and the associated pathology.     

CD40 ligation prevents neonatal induction of transplantation tolerance

To investigate the consequences of CD40 engagement on the neonatal induction of transplantation tolerance, BALB/c mice were injected at birth with (A/J x BALB/c) F1 spleen cells together with activating anti-CD40 mAb and grafted 4 wk later with A/J skin. Whereas A/J allografts were accepted in mice neonatally injected with F1 cells and control Ab, they were acutely rejected in mice injected with F1 cells and anti-CD40 mAb. Neonatal administration of anti-CD40 mAb resulted in enhanced anti-A/J CTL activity, increased IFN-gamma, and decreased IL-4 production by donor-specific T cells in vitro. Experiments using anti-cytokine mAb and IFN-gamma-deficient mice demonstrated that CD40 ligation prevents neonatal allotolerance through an IFN-gamma- and IL-12-dependent pathway. Finally, we found that newborn T cells express less CD40L than adult T cells upon TCR engagement. Taken together these data indicate that insufficiency of CD40/CD40L interactions contribute to neonatal transplantation tolerance.     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J x BALB/c)F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-gamma in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

Neonatal exposure of TCR BV8S2 transgenic mice to recombinant TCR BV8S2 results in reduced T cell proliferation and elevated antibody response to BV8S2, and increased severity of EAE

Transgenic (Tg) mouse models are unique tools for investigating regulatory mechanisms of the immune system. Mice bearing a T cell receptor (TCR) BV8S2 transgene derived from an encephalitogenic T cell clone are highly susceptible to experimental autoimmune encephalomyelitis (EAE), a T cell-mediated neurological disorder. Although the pathogenesis of EAE is not yet fully understood, TCR-specific regulatory T cells seem to play a role in its remission and/or recovery process. In previous studies, we showed that immunization of BV8S2 Tg mice with recombinant BV8S2 protein induced TCR-specific T cells and protection against EAE, clearly indicating the persistence of a functional TCR regulatory network in spite of the highly skewed T cell repertoire. To further investigate the natural regulatory role of TCR-specific T cells, we evaluated the effect on EAE of inducing neonatal tolerance to heterologous (rat) and homologous BV8S2 proteins in Tg mice. Neonatal exposure to rat BV8S2 protein induced "split" tolerance, characterized by decreased T cell proliferation but increased antibody responses to both rat and mouse BV8S2 proteins that are known to be cross-reactive. When challenged as adults with an encephalitogenic emulsion, Tg mice tolerized with rat but not mouse BV8S2 protein developed more severe EAE compared to control mice. These results demonstrate that immunity to BV8S2 determinants in BV8S2 Tg mice is naturally induced and functions to limit the severity of EAE.     

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.     

Smoking status and relative weight by educational level in Finland, 1978-1995

Nasal administration of soluble antigens is an exciting means of specifically down-regulating pathogenic T-cell reactivities in autoimmune diseases. The mechanisms by which nasal administration of soluble antigens suppresses autoimmunity are poorly understood. To define further the principles of nasal tolerance induction, we studied the effects of nasal administration of myelin basic protein (MBP) on experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE is a CD4+ T-cell-mediated animal model for human multiple sclerosis. Nasal administration of guinea-pig (gp)-MBP at a dose as low as 30 micrograms/rat can completely prevent gp-MBP-induced EAE, whereas nasal administration of bovine (b)-MBP is not effective even at a much higher dosage. Cellular immune responses, as reflected by T-cell proliferation and interferon-gamma (IFN-gamma)-ELISPOT, were suppressed in rats receiving the two different doses (30 and 600 micrograms/rat) of gp-MBP, but not after administration of b-MBP. Rats tolerized with both doses of gp-MBP had also abrogated MBP-induced IFN-gamma mRNA expression in popliteal and inguinal lymph node mononuclear cells compared with rats receiving phosphate-buffered saline nasally. However, adoptive transfer revealed that only spleen mononuclear cells from rats pretreated with a low dose, but not from those pretreated with a high dose, of gp-MBP transferred protection to actively induced EAE. Low-dose (30 micrograms/rat) gp-MBP-tolerized rats also had high numbers of interleukin-4 (IL-4) mRNA-expressing lymph node cells, while high-dose (600 micrograms/rat) gp-MBP-tolerized rats had low numbers of IL-4 mRNA-expressing lymph node cells. Our data suggest an exquisite specificity of nasal tolerance. Dose-dependent mechanisms also relate to nasal tolerance induction and protection against EAE in the Lewis rat.     

Distribution of [35S]dATP alpha S binding sites in the adult rat neuraxis

IL-10 and IL-4 were studied with respect to their capacity to inhibit experimental allergic encephalomyelitis (EAE) induced in SJL/J mice by immunization with the proteolipid protein peptide PLP139-151. Treatment with 2 micrograms IL-10/day from day 0 until day 12 delayed onset of disease and inhibited the severity of EAE. By contrast, a daily dose of 0.5 microgram IL-4 was ineffective. Instead of acting in a synergistic fashion, IL-4 even abrogated the inhibitory effect of IL-10. The effects of IL-10 and IL-4 treatment were largely consistent with the (lack of) ability of these cytokines to down-regulate the inflammatory response in brain tissue. Although IL-4 was ineffective in the inhibition of EAE, lymph node cells from IL-4-treated mice displayed a strongly inhibited peptide-specific IFN-gamma production. By contrast, IL-10, which was effective in inhibiting EAE, showed no significant inhibition of IFN-gamma at this level. Neither cytokine treatment resulted in detectable levels of peptide-specific IL-4. Indirect evidence for the activity of Th2 cells in vivo came from the observation that IL-10 inhibited the primary PLP139-151-specific IgG2a and IgG3 response in favor of IgG1, whereas IL-4 inhibited the primary antibody response to the peptide, regardless of subclass. The combination of IL-4 and IL-10 did not affect the subclass composition. The observation that IL-10-treated mice remained sensitive to re-induction of EAE is not in support of an important role of Th2 cells in regulating disease activity in this model of actively induced EAE.     

Presentation of proteolipid protein epitopes and B7-1-dependent activation of encephalitogenic T cells by IFN-gamma-activated SJL/J astrocytes

There is controversy regarding the possible role of glial cells as APCs in the pathogenesis of central nervous system (CNS) demyelinating diseases such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Microglia have been clearly shown to present Ag in the CNS, and due to the proximity of activated astroglial cells to infiltrating T cells and macrophages in demyelinating lesions, it is also possible that astrocytes positively or negatively regulate disease initiation and/or progression. We examined the capacity of IFN-gamma-treated astrocytes from EAE-susceptible SJL/J mice to process and present myelin epitopes. IFN-gamma activation up-regulated ICAM-1, VCAM-1, MHC class II, invariant chain, H2-M, CD40, and B7-1 as determined by FACS and/or RT-PCR analyses. B7-2 expression was only marginally enhanced on SJL/J astrocytes. Consistent with the expression of these accessory molecules, IFN-gamma-treated SJL/J astrocytes induced the B7-1-dependent activation of Th1 lines and lymph node T cells specific for the immunodominant encephalitogenic proteolipid protein (PLP) epitope (PLP139-151) as assessed by proliferation and activation for the adoptive transfer of EAE. Interestingly, IFN-gamma-activated astrocytes efficiently processed and presented PLP139-151, but not the subdominant PLP178-191, PLP56-70, or PLP104-117 epitopes, from intact PLP and a recombinant variant fusion protein of PLP (MP4). The data are consistent with the hypothesis that astrocytes in the proinflammatory CNS environment have the capability of activating CNS-infiltrating encephalitogenic T cells specific for immunodominant epitopes on various myelin proteins that may be involved in either the initial or the relapsing stages of EAE.     

Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis

Specific oligonucleotide primers were used to identify and isolate IFN-gamma-inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as a model for multiple sclerosis. IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein that was used to induce anti-IGIF neutralizing Abs. These Abs significantly reduced the production of IFN-gamma by primed T cells proliferating in response to their target myelin basic protein 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 Abs significantly blocked the development of disease. Splenic T cells from protected rats were cultured with the encephalitogenic myelin basic protein epitope and evaluated for production of IL-4 and IFN-gamma. These cells, which proliferated, exhibited a profound increase in IL-4 production that was accompanied by a significant decrease in IFN-gamma and TNF-alpha production. Thus, we suggest that perturbation of the Th1/Th2 balance toward Th2 cells is the mechanism underlying EAE blockade by anti-IGIF immunotherapy.     

T score of trabecular and cortical bone in normal postmenopausal women

CD4+ T cells may be assigned a functional status (Th1 or Th2) according to the cytokines they produce including IL-2, IFN-gamma and IL-4. Th1 and Th2 CD4+ T cells deliver different isotype-switching signals to antigen-specific B cells which bias the serum Ig isotypes. The stimulation of Th1 or Th2 responses is influenced by adjuvants and administration of antigen in IFA results in Th1 unresponsiveness as evidenced by: (i) reduced T cell proliferation to antigen; (ii) reduced IFN-gamma production in response to antigen; and (iii) reduced IgG2a isotype antigen-specific antibodies following antigen/CFA challenge. The impact of established human gamma globulin (HGG) specific Th1 unresponsiveness on subsequent immunization with an unrelated antigen, human serum albumin (HSA) in Th1-inducing CFA was then examined. When subsequently challenged with a mixture of HSA and HGG in CFA the HGG-specific Th1 unresponsiveness was infectious and dominant, preventing the induction of a Th1 response to HSA. Reduced T cell proliferation, IFN-gamma production and IgG2a antibody were consequently observed in response to HSA. The HGG-specific Th1 unresponsiveness was not infectious when HGG/CFA and HSA/CFA were administered at separate sites. This demonstrates that antigen-specific Th1 unresponsiveness can be infectious for new, molecularly unrelated antigens and supports studies showing that Th1-mediated autoimmune diseases such as experimental allergic encephalomyelitis (EAE) and diabetes can be ameliorated using antigens molecularly distinct from the disease-inducing immunogen.     

The effect of gammadelta T cell depletion on cytokine gene expression in experimental allergic encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, we showed previously that depletion of gammadelta T cells using the mAb GL3 immediately before disease onset, or during the chronic phase, significantly ameliorated clinical severity. We now report on the effect of gammadelta T cell depletion on expression of five cytokine genes, IL-1, IL-6, TNF, lymphotoxin, and IFN-gamma in spinal cords of mice during the pre-onset, onset, height, and recovery phases of EAE, and on expression of type II nitric oxide synthase. In control animals, the mRNAs for IL-1 and IL-6 rose dramatically at disease onset and peaked before disease height, whereas the mRNAs for TNF, lymphotoxin, and IFN-gamma rose more slowly and peaked with peak of disease. In GL3-treated animals, a dramatic reduction in all five cytokines was noted at disease onset, but only IFN-gamma remained significantly reduced at a time point equivalent to height of disease in control animals. ELISA data confirmed the reduced levels of IL-1 and IL-6 at disease onset in GL3-treated animals, and pathologic analysis demonstrated a marked reduction in meningeal infiltrates at the same time point. Studies of type II NOS also demonstrated a significant reduction in both mRNA and protein expression at the height of disease in GL3-treated animals. These results suggest that gammadelta T cells contribute to the pathogenesis of EAE by regulating the influx of inflammatory cells into the spinal cord and by augmenting the proinflammatory cytokine profile of the inflammatory infiltrates.     

Certain norditerpenoid alkaloids and their cardiovascular action

Several investigators have reported experimental evidence of epitope spreading in experimental autoimmune encephalomyelitis (EAE). The role of epitope spreading in the pathogenesis of relapsing or chronic autoimmune disease is not established and the in vivo function of the T cells specific for new epitopes which appear during an autoimmune response is unclear. We recently demonstrated that mice which have recovered from an episode of EAE suffer a relapse shortly after reinjection with the original encephalitogen. The reinduced disease occurs in a reproducible fashion with an accelerated onset. This may be due to persistence of an expanded population of previously activated encephalitogenic cells which are rapidly reactivated when re-exposed to antigen. We reasoned that if epitope spread produces a significant number of encephalitogenic cells specific for a new epitope, then reinjection with that epitope should also cause the rapid onset of an episode of EAE. We tested this hypothesis using the known encephalitogenic epitopes in SJL mice. After recovery from EAE induced with the proteolipid protein peptide PLP139-151, five of 16 mice had an accelerated relapse of EAE when reinjected with a second encephalitogenic peptide, PLP178-191. All of the 10 mice reinjected with the original PLP139-151 peptide relapsed. We conclude that epitope spread may produce encephalitogenic cells specific for new epitopes, but that the response to new epitopes is minor compared to the response to the initial epitope.     

Inhibition of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68-86 and 87-99

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non-encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect na?ve rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.