Rat Myelin Basic Protein Research Articles

Antigen-specific deletion of cloned T cells using peptide-toxin conjugate complexed with purified class II major histocompatibility complex antigen.

In a previous report, we showed that cloned T cells incubated with soluble, cognate major histocompatibility complex (MHC) II-peptide complex internalized the peptide moiety of the complex. Here, we report antigen-specific deletion of cloned T cells by treatment with soluble, cognate MHC II-(peptide-toxin) complexes. Toxin (doxorubicin or mycophenolic acid) was attached to synthetic AcMBP(1-14)Ala4 peptide, an analog of the natural acetylated NH2-terminal segment, AcMBP(1-14), of rat myelin basic protein (MBP). IAk-restricted, AcMBP(1-14)-Specific AJ1.2 and 4R3.9 cloned murine T cells were killed by IAk-(AcMBP(1-14)Ala4-toxin). No killing resulted from incubating AJ1.2 and 4R3.9 cells with irrelevant MHC II-(peptide-toxin) or treating IEk-restricted, pigeon cytochrome c-specific A.E7 cloned murine T cells with IAk-(AcMBP(1-14)Ala4-toxin). T cell receptor-mediated T cell uptake of the peptide-toxin moiety of relevant complex was blocked by anti-T cell receptor-alpha/beta antibody and by excess toxin-free complex. LD50 determinations revealed that cognate MHC II-(peptide-toxin) killed T cells much more effectively than did peptide-toxin conjugate alone. Finally, T cell uptake of peptide-toxin and intracellular release of toxin occurred after incubation with relevant MHC II-(peptide-toxin) containing radiolabeled toxin. These findings, which provide the first evidence that cloned T cells can be deleted with soluble, cognate MHC II-(peptide-toxin) complexes, may have significant clinical relevance for antigen-specific therapy of autoimmune or other T cell-mediated diseases.

The major histocompatibility complex influences myelin basic protein 63-88-induced T cell cytokine profile and experimental autoimmune encephalomyelitis.

Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP‐63–88. Analysis of MHC‐congenic LEWIS strains showed that RT1a, RT1c and RT1l haplotypes are permissive for disease induction, whereas RT1d and RT1u are resistant. All EAE responding strains showed peptide‐specific proliferation and interferon (IFN)‐γ secretion, but no early significant tendency to express interleukin (IL‐4) or transforming growth factor (TGF)‐β mRNA in lymphocytes in response to the MBP 63–88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide‐specific induction of IL‐4 and TGF‐β occurred in RT1l rats. Among the EAE non‐responders strains, only the RT1u rats showed an immune response to MBP 63‐88. This response, however, was qualitatively different from the immune response in the EAE‐susceptible strains. Thus, there was no proliferation and only moderate IFN‐γ production in response to peptide, but in contrast, a significant and early peptide‐induced IL‐4 and TGF‐β response was observed. The data suggest that the MHC‐associated susceptibility to EAE is partly related to the ability to mount a TH1‐like immune response while the MHC‐associated EAE resistance may either be related to MBP peptide non‐responsiveness or to peptide recognition and induction of a qualitatively different and disease down‐regulatory immune response.

Staphylococcal enterotoxin enhances the activation of rat encephalitogenic T cells by myelin basic protein

It is generally believed that the activation of autoreactive T cells is an essential step in the pathogenesis of autoimmune diseases; however, autologous antigens are often weak immunogens and their detectable levels in vivo are much lower than required for T-cell activation. In experimental autoimmune encephalomyelitis, encephalitogenic T cells specific for myelin basic protein (MBP) react weakly to autologous rat myelin basic protein (RBP) but strongly to guinea pig-derived basic protein, even though both ligands possess the essential epitope (MBP(68–88)). Here we demonstrate that RBP is converted to a strong immunogen in the presence of a small dose of bacterial superantigen, staphylococcal enterotoxin E (SEE). The enhancing effect of SEE on the rat protein was apparent with all encephalitogenic T-cell lines examined in this study, including those not responding to RBP alone. It depended, moreover, on the simultaneous presence of RBP and SEE; delaying the addition of SEE for 8–12 h greatly decreased the potency of RBP. None of a series of major cytokines was able to replace SEE as an enhancer. Our results indicate that two T ligands, one a bacterial superantigen, can interact to enhance the activation of autoreactive T cells. This observation has implications for the involvement of bacterial and viral infections in the pathogenesis of multiple sclerosis.

An autoantigenic T cell epitope forms unstable complexes with class II MHC: a novel route for escape from tolerance induction.

The peptide rAc1-11 represents the dominant T cell epitope of rat myelin basic protein (MBP) in mice of the H-2u haplotype. Residue 4 has been shown previously to govern binding of the peptide to the class II molecule, I-Au. We have constructed peptide analogues bearing amino acid substitutions at position 4 and have assessed their ability to stimulate an antigen-specific T cell hybridoma when presented by viable antigen presenting cells (APC). Complexes between I-Au and one such analogue, rAc1-11[4A], were rapidly lost from the surface of live APC displaying a half-life (t 1/2) of approximately 10 min. Neither shedding of intact complexes from the cell surface, nor their internalization and recycling through an acidic intracellular compartment were found to account for their loss. The possible dissociation of rAc1-11[4A] from the peptide binding cleft was therefore addressed by comparing the t 1/2 of complexes between I-Au and peptide analogues of higher affinity. The tyrosine-substituted analogue, rAc1-11[4Y], remained stably bound to I-Au for at least 4 h, thereby displaying a t 1/2 far in excess of that evident for rAc1-11[4A]. Significantly, the wild type peptide, rAc1-11, bound so transiently that functional complexes could not be detected on the surface of peptide-pulsed APC. The physiological relevance of these findings was confirmed by extending our studies to an analysis of the homologous epitope of murine MBP; evidence that this epitope likewise displays minimal affinity for I-Au suggests a novel strategy for the escape from tolerance induction by encephalitogenic T cells.

Cross-reactive antigen recognition by an encephalitogenic T cell receptor. Implications for T cell biology and autoimmunity.

The dominant immune response to rat myelin basic protein in H-2u mice is directed against the acetylated, N-terminal peptide Ac1-11 (AcASQKR-PSQRHG). This peptide causes encephalomyelitis on injection into mice of the H-2u haplotype. Only two residues of the peptide are required for ligation of the TCR from an Ac1-11-specific T cell hybridoma. Proline at position 6 could not be substituted by any other L-amino acid, whereas glutamine at position 3 could be replaced by phenylalanine, histidine, methionine, or tyrosine. Cross-reactive recognition of these residues appears to be specific, because increasing the affinity of each analogue for its MHC restriction element, by replacing lysine with tyrosine at position 4, did not alter the pattern of cross-reactivity. For the majority of substitutions at this position, a lack of stimulation could not be explained by failure to bind to I-Au. However, competition binding studies showed that introduction of proline at position 3 reduced the efficacy of binding to I-Au. Cross-reactive analogues of Ac1-11 were injected into H-2u mice to test the extent to which cross-reactive T cell activation might lead to autoimmune disease in this model. An analogue containing methionine at position 3 caused clinical experimental autoimmune encephalomyelitis in a small percentage of H-2u mice.

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. V. Hierarchy of suppression by myelin basic protein from different species

We have been investigating the suppression of experimental autoimmune encephalomyelitis (EAE) by oral tolerization to autoantigens. In the present study the tolerizing effect of orally administered myelin basic protein (MBP) from different species was examined in the Lewis rat, Hartley guinea pig, and SJL/J mouse model of EAE. Animals were fed guinea pig, rat, bovine, human or mouse-BMP and them immunized with the homologous species of MBP or myelin: Lewis rats were immunized with rat MBP, Hartley guinea pigs with guinea pig-MBP, and SJL/J mice with mouse myelin. Clinical expression of EAE and delayed-type hypersensitivity (DTH) responses to MBP were assessed. In each species, suppression of disease and DTH responses were most pronounced by tolerization with the homologous species of MBP. In addition, cross-species tolerization was observed in each species and in general was less suppressive than homologous MBP although in some instances MBP from a heterologous species was as effective as tolerization with the homologous species. We also studied guinea pig-MBP induced EAE in the Lewis rat because it is widely studied model of EAE and found that oral tolerization with guinea pig MBP was as suppressive as rat MBP. Of note is that oral tolerization with mouse MBP suppressed myelin-induced EAE in the SJL mouse in which autoimmunity to proteolipid protein appears to play a primary role, suggesting that antigen-driven bystander suppression following oral tolerization with autoantigens (Miller et al., 1991b) may be an important contributing mechanism for suppression of EAE following oral tolerization with MBP in this model. Taken together, these results suggest that epitopes of MBP distinct from the encephalitogenic region participate in triggering suppression following oral tolerization and have been important implications for treating human diseases by oral tolerization to autoantigens.

Antigen-specific inhibition of the adoptive transfer of experimental autoimmune enceophalomyelitis in Lewis rats

The efficacy of antigen-specific immunoregulation as a treatment for the efferent limb of an autoimmune disease was tested in a rat model of adoptive experimental autoimmune encephalomyelitis (EAE). Lewis rats receiving 4–5 × 10 7 guinea pig (GP) myelin basic protein (MBP)-activated lymph node T cell blasts from GPMBP/CFA sensitized donors routinely show clinical signs of disease 5–6 days post transfer. Intravenous injection of GPMBP coupled to syngeneic splenocytes using the chemical cross-linker carbodiimide was effective in completely abrogating the experssion of clinical EAA in rats that received MBP-specific T cells 2 days previously. Partial inhibition was also observed in rats injected as early as day 0 (the same day as MBP-specific T cell transfer) and as late as 1 day prior to the onset of clinicla signs (days 4–5 post trasfer). Unresponsiveness was shwon to be dose-dependent, dependent on the route of injection of the neuroantigen-coupled splenocytes, and was antigen-specific. Splenocytes coupled with GP or rat MBP (which are identical within the major encephalitogenic GP68–86 Lewis rat determinant with the exception of the residue at position 80) were equally efficient at eliminating disease expression in recipients of GPMBP-specific T cells. In contrast, splenocytes coupled with bovine or rabbit MBP (which differ significantly from GPMBP within the 68–86 region) had no inhibitory effects. The antigen specificity of the tolerance induction was also illustrated by the fact that splenocytes coupled with GP68–86, but not those coupled with the truncated GP68–84 peptide, induced profound unresponsiveness. Interestingly, de novo antigen processing by the antigen-coupled cells did not appear to be necessary as the inclusion of antigen processing inhibitors had no effect on inhibition of disease. However, the use of the carbodiimide couplign reagent was critical for the induction of unrespnosiveness as essentially equivalent amounts of 125I-labelled MBP were bound in its presence or absence, but only splenocytes incubated in the presence of both MBP and carbodiimide inhibited clinical expression of disease. Antigen-specific tolerance is thus an effective means of inhibiting expression of clinical disease in teh rat EAE model, and a powerful tool for determining the fine epitope specificity of encephalitogenic T cells.

Cellular immune crossreactivity between myelin basic protein and synapsin in rats with experimental allergic encephalomyelitis.

We previously demonstrated that antibodies against myelin basic protein (MBP) obtained from animals with experimental allergic encephalomyelitis (EAE), induced with MBP and purified by affinity chromatography, have the property to recognize a neuronal protein, synapsin Ia and Ib. To investigate whether this crossreactivity also occurs at the cellular level, we purified spleen and lymph node mononuclear cells from rats sensitized with MBP or synapsin using polystyrene plates coated with the respective antigen. We observed that animals injected with MBP have T lymphocytes that bind both antigens. Using the same system, each purified cell population was confronted again to the studied antigens. The anti-MBP cells recognized once more epitopes of MBP and about 40% of them also recognized synapsin. On the other hand, cells that first were attached to synapsin, in the second exposure to antigens bound to MBP and synapsin in similar amounts. Double immunofluorescent labeling of the mononuclear cells isolated from animals injected with bovine myelin or MBP showed that the same lymphocyte was able to recognize MBP as well as synapsin. In both experimental systems the quantitative results were similar indicating that in bovine myelin- or MBP-sensitized animals practically all the cells that recognize synapsin are anti-MBP cells, and of the total cells raised against MBP, around 40% of them show this crossreactivity. On the contrary, animals injected with synapsin have cells that bind to this protein but not to MBP indicating that the described crossreactivity, as observed at humoral level, is only in one way.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelin basic protein infused into cerebrospinal fluid suppresses experimental autoimmune encephalomyelitis

We have evaluated the antibody and the effector T-cell responses to a single cerebrospinal fluid (CSF) infusion of myelin basic protein (MBP) in Lewis rats by measuring serum anti-MBP antibodies and clinical signs of experimental autoimmune encephalomyelitis (EAE), respectively. Some rats developed anti-MBP antibodies, but none manifested EAE in response to the primary infusion. Antibody responses to an EAE challenge 3 weeks after CSF infusion were normal, but clinical symptoms of EAE were markedly suppressed. Brain trauma at the time of MBP pretreatment enhanced this suppression. The CSF route of MBP administration is more effective in inducing suppression of EAE than peripheral routes.

HgCl 2-induced perturbation of the T cell network in experimental allergic encephalomyelitis : I. In vitro characterization of T cells involved

Mercuric chloride (HgCl 2) induces in Lewis (LEW) rats a non-antigen-specific immuno-suppression and is able to down-modulate experimental allergic encephalomyelitis in about 70% of the rats. The aim of the present study was to determine the frequencies of lymph node cells involved in the proliferative response to myelin basic protein in rats injected with HgCl 2 and immunized with myelin by using limiting dilution analysis (LDA). Highly frequent CD8+ T suppressor cells and at least 10-fold less frequent protein basic-specific T helper cells were detected in these rats. A third cell type allowing the proliferative response of Th cells in spite of Ts cells was also demonstrated. These cells, which could act as contrasuppressor cells, were CD4+ and adhered to Vicia villosa lectin; their frequency was in the same range as that of T helper cells. These data illustrate the potential role of different levels of T cell immunoregulatory activity in autoimmunity and the major interest of LDA in their analysis.

Characterization of measles virus-induced cellular autoimmune reactions against myelin basic protein in Lewis rats

Subacute encephalomyelitis (SAME) in Lewis rats following infection with a neurotropic measles virus (MV) is associated with a cell-mediated autoimmune response (CMAI) to myelin badic protein (MBP). MBP-selected CD4 4+ T cell lines both from measles-infected animals as well as from rats challenged with guinea pig MBP (Gp-MBP) had a similar pattern of response in the presence of synthetic peptides to Gp-MBP and specifically responded in vitro only to the encephalitogenic and not the non-encephalitogenic or other control peptides. In primary splenic lymphocyte cultures from SAME animals, however, a low but significant T-cell response was obtained against the non-encephalitogenic peptide S67 (residues 69–81) of the Gp-MBP. Moreover, immunization of MV-infected rats with this peptide induced clinical and histological experimental allergic encephalomyelitis (EAE) in 38% of the animals. The results of the study show that the non-encephalitogenic peptide S67 can be rendered encephalitogenic in rats when an additional stimulus is given in the form of MV infection. The data indicate further that MV infection of the central nervous system (CNS) enhances the susceptibility of the CNS to autoimmune T cell aggression.

Transcriptional Regulation Studies of Myelin-Associated Genes in <i>Myelin-Deficient</i>Mutant Rats

To identify and assess the consequences of the mutation in myelin-deficient (md) rats, the myelin proteolipid protein (PLP) gene and its expression were studied in md rats. Southern blots of the PLP gene demonstrated that no major deletions or insertions have occurred in this gene. In addition, the mutation in this gene does not result in a splicing defect in the RNAs, since all exons are represented in md PLP RNAs. These data are consistent with results in another laboratory indicating that a point mutation in the PLP gene in md rats results in a single amino acid alteration in the protein. To elucidate the molecular mechanisms producing reduced levels of PLP, myelin basic protein (MBP) and glycerol phosphate dehydrogenase (GPDH) mRNAs, and their corresponding proteins in md rats, in vitro transcription assays were performed. Transcription of the PLP gene in nuclei isolated from 23-day-old md rat brains was dramatically reduced relative to normal tissue. Thus, the single amino acid alteration in this protein alters the regulation of transcription of this gene. In contrast, the transcriptional activities of the MBP and GPDH genes in md rats were indistinguishable from normal animals. Thus, the lower level of MBP and GPDH mRNA and protein in md rats relative to normal results from a posttranscriptional event.

Characteristics of in vitro cytotoxic effects of myelin basic protein-reactive T cell lines on syngeneic oligodendrocytes

We have previously found that Lewis rat myelin basic protein (MBP)-reactive lymphocytes (Lc) were cytotoxic in vitro to cultured syngeneic oligodendrocytes (oligos). We report here additional studies to characterize this reaction. The effector lymphocytes in the cytotoxic reaction are also encephalitogenic as evidenced by the capacity of other aliquots of these cells to transfer experimental allergic encephalomyelitis (EAE). We confirmed that the presence of both MBP and antigen-presenting cells (APC) are required for this in vitro cytotoxic effect. This reaction (measured by 51Cr release from labeled oligos) is dose-dependent on the effector/target ratio with marked 51Cr release at a 20/1 ratio. Effector/target cell contact is required since: (a) 51Cr release is not significantly increased when effector Lc and oligo are separated by a micropore membrane (28% vs. 24% spontaneous release); (b) no cytotoxic activity is present in the supernatant fluid of a toxic reaction. The adhesion of 51Cr-labeled effector Lc to unlabeled oligo is increased in the presence of both MBP and APC (21 ± 1.0% of cell adhering) as compared with effector Lc + APC (12 ± 2.7%), or effector Lc alone (14 ± 2.8%). Surface expression of class I major histocompatibility complex (MHC) antigens was expressed on the surface of the target oligos during this in vitro cytotoxic reaction. This may explain our previously observed MHC restriction in this reaction. The findings described here may explain some of the in vivo pathogenic events in EAE.

Clonotypic heterogeneity of lewis rat T cells specific for the encephalitogenic 68–86 region of myelin basic protein

Experimental autoimmune encephalomyelitis was induced in a Lewis rat by sensitization with synthetic peptide GP68-86, representing the 68–86 sequence of guinea pig myelin basic protein (GPMBP). To delineate T cell determinants of GP68-86, lymph node cells from this rat were activated in culture with GP68-86 and were fused with cells of the mouse thymoma BW5147. The resultant hybrids were cloned by limiting dilution and screened for GP68-86-evoked secretion of IL 2 in the presence of rat splenocytes. Twelve T cell hybrids derived in this manner were tested for reactivity to different heterologous species of MBP as well as to substituted or truncated analogs of GP68-86. The hybrids generally exhibited potent reactivity to GPMBP but differed markedly in their reactivity to autologous rat MBP (RMBP). A few exceptional hybrids exhibited crossreactivity with peptides in which native serine 75 or serine 80 residues of GPMBP were substituted with either alanine 75 (A 75) or proline 80 (P 80) residues. These cross-reactive hybrids also possessed high levels of anti-RMBP reactivity. The remaining hybrids were unresponsive to the A 75 and P 80 substituted peptides and, with one exception, had relatively low levels of anti-RMBP reactivity. Unique reactivity patterns were also revealed by hybrid responses to peptides having modified C-terminal 84–86 residues. In summary, the contrasting fine specificities of different hybrids indicated that several distinct clones of T cells mediate the immune response of Lewis rats against the 68–86 region of GPMBP. Furthermore, heterogeneity in the hybrid response to “self” RMBP may reflect substantial differences in encephalitogenic potency of the T cell clones from which these hybrids were derived.

Myelin protein expression in the myelin-deficient rat brain and cultured oligodendrocytes

The myelin-deficient (MD) rat does not express the major protein of CNS myelin, proteolipid protein (PLP). Here we further analyze whether this defect is reflected at the level of mRNA and whether the expression of other myelin proteins is affected in oligodendrocytes in vivo and in vitro. Both myelin basic protein (MBP) and PLP message levels were reduced in MD rats to 10-20% of the normal littermate controls, while the level of expression of an astrocyte-specific gene, glial fibrillary acidic protein (GFAP), was normal. Although MBP and PLP mRNAs were equally depressed, only MBP was detected with immunolabeling of corpus callosum, while PLP was absent in oligodendrocytes both in vivo and in vitro. A reduced number of MD rat oligodendrocytes express MBP in vitro compared to controls. The MD rat optic nerve contains normal numbers of 0-2A progenitors, but they tend to differentiate into GC-positive oligodendrocytes faster than oligodendrocytes from control littermates. In conclusion, the absence of PLP and reduced levels of MBP in the MD rats point to similarities with the jimpy mouse lesion. Moreover, the defect influences the expression of other myelin proteins and the oligodendrocyte developmental pathway.

Indomethacin augments in vitro proliferative responses of Lewis rat lymphocytes to myelin basic protein : Implications for experimental autoimmune encephalomyelitis

Indomethacin (IM), a specific inhibitor of prostaglandin (PG) synthesis, and PGE 2 were studied in terms of their ability to modulate in vitro immune responses associated with experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Lymphoid cells from either the spleens or the draining lymph nodes of myelin basic protein (MBP)-sensitized rats exhibited in vitro immune responses which were enhanced in the presence of IM. Specifically, IM enhanced (i) guinea pig MBP (GPMBP)- and rat MBP (RMBP)-stimulated lymphocyte proliferation, (ii) background proliferation, and (iii) interleukin 2 (IL-2)-stimulated proliferation. Conversely, PGE 2 inhibited both GPMBP- and IL-2-stimulated proliferation of MBP-sensitized lymphocytes. Together, these results indicate that PGs secreted by cultured lymphoid cells can directly mitigate MBP- or IL-2-stimulated lymphocyte proliferation. Furthermore, the observation that IM and PGE 2 modulate in vitro responses of MBP-specific lymphocytes may provide insight into how the in vivo administration of IM potentiates the severity of EAE (H. Ovadia and P. Y. Paterson, Clin. Exp. Immunol. 49, 386, 1982) and how PGs may be involved in the spontaneous remission of EAE in rats.

Peptide-specific prevention of experimental allergic encephalomyelitis. Neonatal tolerance induced to the dominant T cell determinant of myelin basic protein.

Experimental allergic encephalomyelitis (EAE) is a model of antigen-specific T cell-mediated autoimmune disease. The alpha-acetylated, NH2-terminal nine amino acids (1-9NAc) of myelin basic protein (MBP) represents the dominant T cell epitope for the induction of EAE in the B10.PL (H-2u) strain. We tolerized neonatal B10.PL mice to 1-9NAc and studied the proliferative responses to this peptide and to whole MBP. Mice exposed to 1-9NAc in the neonatal period were tolerant to subsequent challenge at the proliferative T cell level. Similarly, in the 1-9NAc-tolerant group, both the incidence and severity of 1-9NAc induced EAE were greatly reduced. The fact that we were able to tolerize mice normally responsive to MBP suggests that this self antigen is sequestered (within the central nervous system) and hence tolerance to it is not normally induced. No significant difference in disease incidence was seen in response to rat MBP between control animals and 1-9NAc-tolerized mice (50% in both groups), demonstrating the presence of at least one additional encephalitogenic determinant elsewhere on the molecule. We have successfully prevented disease induction by peptide-induced tolerization. Tolerance induction by peptides provides a new and specific strategy in the prevention of autoimmunity. However, it will be clearly necessary to fully define all epitopes potentially capable of inducing pathogenic T cells to ensure complete and effective therapy of T cell-mediated autoimmune disease.

Lewis rat lymphoid dendritic cells can efficiently present homologous myelin basic protein to encephalitogenic lymphocytes

Lymphocytes isolated from Lewis rats immunised with protein antigen in adjuvant were stimulated to proliferate in vitro by splenic dendritic cells (DC) which had been pulsed with purified homologous myelin basic protein (MBP). By contrast, in parallel experiments, lymphocytes did not respond to ovalbumin unless the protein was first processed by macrophages by a chloroquine-sensitive mechanism. DC, pulsed with rat MBP at concentrations as low as 6 μg/ml, activated lymphocytes for transfer of severe experimental autoimmune encephalomyelitis (EAE). MBP dissociating from myelin membranes in physiological medium, as well as MBP purified from highly acidic extracts of myelin, was effective for pulsing DC; preincubating myelin with macrophages led to a reduction rather than an enhancement in the severity of the EAE transferred. It is concluded that macrophage-mediated antigen processing is not required for immunogenic presentation of the determinants of MBP which cause EAE in Lewis rats. Furthermore, MBP-pulsed DC may prove useful in experiments requiring activation of encephalitogenic T cells.

Resistance to experimental autoimmune encephalomyelitis induced by neonatal tolerization to myelin basic protein: clonal elimination vs. regulation of autoaggressive lymphocytes

The target autoantigen of experimental autoimmune encephalomyelitis (EAE), myelin basic protein (MBP), appears late in ontogeny. In the rat MBP is expressed first on days 2-3 post partum, at a development stage, when self tolerance to most other autoantigens has already developed. To shed light on the cellular mechanisms that lead to immunological self tolerance to MBP, we treated neonatal rats with high doses of MBP before ontogenetic appearance of this autoantigen. We found that high doses are required to confer MBP-specific tolerance lasting until the adult life. Neonatally tolerized, adult rats are completely resistant to induction of EAE by injection of MBP in complete Freund's adjuvant (CFA). Upon MBP CFA challenge, these animals develop a limited humoral response to MBP, but are completely unreactive to MBP on the T cell level. The function of antigen-presenting cells is unchanged by neonatal tolerization, and there is no evidence for the induction of suppressive mechanisms. Transfers of large numbers of tolerized lymphocytes to normal hosts fails to interfere with EAE inducibility. Moreover, neonatally tolerized lymphocytes do not reduce MBP reactivity of primed lymph node cells or T line cells in vitro. Finally, neonatally tolerized rats are susceptible to EAE transferred by activated primed lymphocytes or by in vitro-activated MBP-specific T line cells. The apparent deletion of MBP-specific T lymphocytes in neonatally tolerized rats is in striking contrast to the physiological self tolerance to MBP, which is characterized by the presence of MBP-specific clones in the normal immune repertoire.

Lymphocyte proliferative response to glial cells in SJL/J mice immunized with rat whole spinal cord and rat myelin basic protein

Lymph node cells (LNC) from SJL/J mice immunized with either rat whole spinal cord (WSC) homogenate or rat myelin basic protein (MBP) in the presence of complete Freund's adjuvant were assayed for their in vitro proliferative response to MBP and whole or sonicated purified oligodendrocytes (OD), and astrocytes. WSC-immunized mice displayed a strong and persistent response to both syngeneic and rat OD, that disappeared after sonication (suggesting a preferential recognition of surface OD antigen(s)), and a much weaker response to rat MBP. LNC from MBP-immunized mice showed a small response to MBP, but no response to OD, indicating that OD are not able to present their endogenous MBP to lymphocytes. Conversely, rat astrocytes (whole or sonicated), isolated from cultures initially comprising OD, could trigger the proliferative response of MBP-sensitized LNC, suggesting that astrocytes may act as antigen-presenting cells for OD products.