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-MBP and then 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 a 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of timing of intravenous administration of myelin basic protein on the induction of tolerance in experimental allergic encephalomyelitis

Immunological tolerance and suppression of clinical and histological experimental allergic encaphalomyelitis (EAE) can be induced by the intravenous (i.v.) administration of myelin basic protein (MBP). In this report we have characterized the effect of the time of i.v. administration of MBP on the course of EAE in Lewis rats. Rats were treated with the i.v. administration of one or two 500 micrograms doses of MBP either before or after active immunization. Results indicated that i.v. administration of MBP in rats before active immunization with MBP/CFA (na?ve rats) was most effective when given 14 days before active immunization, but treatment of rats actively immunized with MBP (immunized rats) was most effective at the onset of disease. Treatment at other times was less effective. The i.v. administration of the peptide MBP 68-88 (p68-88) containing the dominant encephalitogenic epitope could also suppress MBP-induced EAE in a dose dependent manner. Intravenous administration of two injections of p68-88 to na?ve rats on days 10 and 3 before, or on days 0 and 7 after, active immunization with MBP suppressed the development of EAE in a dose dependent manner. Treatment of rats with i.v. MBP after, but not before, the transfer of MBP-reactive EAE effector cells suppressed the development of EAE in the recipient rats. Transfer of lymphoid cells from tolerized na?ve rats failed to protect recipient rats against development of active or passive EAE. These results indicate the importance of timing and dose of the antigen on the induction of tolerance and suggests different mechanisms of tolerance induction by intravenous MBP in immunized and na?ve rats. They also emphasize the importance of timing in designing efficient treatment strategies when i.v. tolerance is contemplated in EAE and possibly multiple sclerosis.     

经腹腔诱导免疫耐受抑制EAE的实验研究

目的 通过建立实验性自身免疫性脑脊髓炎（EAE）动物模型。研究经腹腔诱导免疫耐受的方法及机制。方法 腹腔内注射可溶性髓鞘碱性蛋白（MBP）或转输可溶性MBP致敏的腹腔抗原提呈细胞（MBP-APC），观察各组动物EAE的发病情况，检测各组动物迟发型超敏反应（DTH）和淋巴细胞体外增殖应答。结果 腹腔内注射MBP及MBP-APC的动物发病率明显低于EAE组。临床症状减轻，由MBP引起的DTH和体外特异的MBP淋巴细胞增殖反应也明显降低。结论 腹腔注射MBP或MBP-APC可引起黏膜耐受，抑制EAE的发生。     

Adoptive transfer of experimental allergic encephalomyelitis and localization of the encephalitogenic epitope in the SWR mouse.

Adoptively-transferred experimental allergic encephalomyelitis (EAE) was induced in the SWR (H-2q) strain of mouse using lymph node cells, spleen cells, or cell lines sensitized to whole myelin basic protein (MBP). With the aid of synthetic peptides, the minimal encephalitogenic epitope of MBP for the SWR strain was localized to amino acids 87-99 of the MBP molecule. The 87-99 sequence is also encephalitogenic for the SJL strain of mouse and the Lewis rat. EAE was induced with a protocol similar to that for the induction of EAE in the SJL strain with the exception that sublethal irradiation of recipients was necessary. Mice developed typical clinical and pathological EAE 6-14 days post-transfer of cells sensitized to either whole MBP or peptide 87-99, after which they remitted. No relapses were observed. Thus, adoptively transferred EAE can be induced in irradiated H-2q mice for which the encephalitogenic epitope is one of the nested encephalitogenic epitopes for SJL (H-2s) mice, namely residues 87-99.     

Cloning of myelin basic protein-reactive T cells from the experimental allergic encephalomyelitis-resistant rat strain, LER

Rats of the LER inbred strain are resistant to the active induction of experimental allergic encephalomyelitis (EAE), although they are susceptible to adoptively transferred EAE when they are injected with encephalitogenic T cells from EAE-susceptible Lewis rats. The mechanism of resistance remains to be elucidated. We report here that myelin basic protein (MBP)-specific T cells can be cloned from LER rats immunized with MBP, that these CD4⁺ LER T cells can recognize the encephalitogenic peptide (MBP-EP) and will divide vigorously when it is presented to them, and that these T cells bear Vβ8 + TCR chains. Nevertheless, in contrast to Lewis T cells with the same specificity and TCR β chains, LER T cells from MBP-EP-specific clones cannot induce EAE when adoptively transferred into naive rats of either strain. Thus, LER T cells can assemble and use a TCR with the canonical encephalitogenic Vβ8.2-Dβ-Jβ region in response to immunization with MBP, yet they continue to display resistance to EAE.     

Synergy between encephalitogenic T cells and myelin basic protein-specific antibodies in the induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an experimentally induced demyelinating disease mediated by CD4+ T cells specific for various myelin proteins including myelin basic protein (MBP) and myelin proteolipid protein (PLP). Although myelin- and other CNS-specific antibodies are produced in EAE, B cells and antibodies are thought by most not to play a decisive role in the induction of EAE. In this report we show that B cells serve as the major antigen-presenting cells (APC) during the T cell activation stage in lymph nodes, and that MBP-specific antibodies can greatly enhance the induction of EAE. The role of B cells as APC is demonstrated in B cell-depleted mice. EAE cannot be induced by antigen/complete Freund's adjuvant immunization unless these mice are locally reconstituted with B cells prior to immunization. The enhancing effect of antibodies is demonstrated in experiments in which EAE is induced by the adoptive transfer of encephalitogenic T cells. The adoptive transfer of large numbers of encephalitogenic T cells induces EAE in 90% of normal recipient mice, but only 33% of B cell-depleted mice get EAE at the same cell dose. The efficiency of EAE induction in B cell-depleted mice can be enhanced if MBP-specific antibodies are simultaneously administered. A similar enhancement is also seen in normal mice when the number of adoptively transferred T cells is limiting. We propose that MBP-specific antibodies enhance the presentation of myelin-derived antigens by APC in the CNS to the adoptively transferred encephalitogenic T cells.     

Suppression of experimental autoimmune myasthenia gravis and experimental allergic encephalomyelitis by oral administration of acetylcholine receptor and myelin basic protein: double tolerance

Oral administration of acetylcholine receptor (AChR) or myelin basic protein (MBP) to Lewis rat prior to immunization with AChR or MBP and complete Freund's adjuvant (CFA) has previously been shown to prevent or delay the onset of experimental autoimmune myasthenia gravis (EAMG) or experimental allergic encephalomyelitis (EAE), which represent animal models of myasthenia gravis and multiple sclerosis, respectively. Here we show that Lewis rats immunized with AChR + MBP + CFA developed both signs of muscular weakness seen in EAMG and paresis characteristic for EAE. This disease was associated with high levels of anti-AChR and anti-MBP antibody secreting cells and of AChR- and MBP-reactive INF-γ secreting Th1-like cells in lymph nodes. The diseased rats also showed upregulation of AChR- and MBP-induced mRNA expression of IFN-γ in lymph node cells. Oral tolerization with AChR and MBP in combination prior to immunization with AChR + MBP + CFA alleviated clinical disease as well as AChR- and MBP-specific B cell responses and autoantigen-induced IFN-γ secretion and production, but upregulated antigen-induced TGF-β mRNA expression in lymph node cells. The results implicate that oral tolerization simultaneously to more than one autoimmune disease-related autoantigen is feasible, and that suppression of autoantigen-induced IFN-γ and augmentation of TGF-β are pivotal in tolerance induction.     

T-cell vaccination prevents EAE effector cell development but does not inhibit priming of MBP responsive cells

Cell recipients which have recovered from adoptively transferred Experimental Allergic Encephalomyelitis (EAE) mediated by encephalitogenic T-cell lines do not develop clinical disease following subsequent challenge with myelin basic protein (MBP) emulsified in CFA (MBP-CFA), a recipient response termed vaccination. The immune mechanism(s), which accounts for the vaccination-induced resistance response, is not known. We have used an adoptive transfer system to investigate the point(s) of control within the pathway of EAE effector cell development from MBP-specific naive precursors that prevents clinical disease in T-cell line vaccinated, MBP-CFA challenged Lewis rats. Although EAE effector cells do not develop in T-cell line vaccinated recipients, our data shows that MBP precursor cells are primed in T-cell line vaccinated MBP-CFA challenged animals, and these MBP-specific precursor cells can be stimulated in culture to the EAE effector cell level. MBP-memory cells also arise in T-cell line vaccinated MBP-CFA challenged donors, as demonstrated by the early and rapid onset of EAE in MBP-CFA challenged recipients of lymphnode cells from T-cell line vaccinated MBP-CFA challenged donors. We also found that it was possible to adoptively transfer resistance to MBP-CFA challenge using spleen cells from donors previously vaccinated with encephalitogenic T-cells. These results show that although EAE effector cells do not develop in T-cell line vaccinated animals, T-cell vaccination does not inhibit the initial MBP precursor cell response and does not prevent the development of MBP memory cells. © 1996 Wiley-Liss, Inc.     

Oral tolerance in experimental autoimmune encephalomyelitis: serum and salivary antibody responses.

We have recently reported that the oral administration of myelin basic protein (MBP) prior to encephalitogenic challenge results in suppression of experimental autoimmune encephalomyelitis (EAE). We examined the serum and salivary antibody responses to MBP in orally tolerant rats using an avidin-biotin enzyme-linked immunosorbent assay. Serum anti-MBP IgA and IgG, but not IgM levels are suppressed in orally tolerant versus control rats. This suppression is time dependent and is confined to the period when animals would otherwise be manifesting EAE clinical signs. In contrast, there is an increase in salivary anti-MBP IgA levels in MBP-fed rats relative to vehicle-fed controls. Thus, MBP-induced unresponsiveness is demonstrable at the humoral level, and moreover, a discrete compartmentalization between the serum and salivary anti-MBP responses exists.     

Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens

Experimental autoimmune neuritis (EAN) in Lewis rats is a T cell-mediated disease and serves as an animal model of human inflammatory demyelinating neuropathies. EAN can be induced by immunization with complete bovine peripheral nerve myelin (BPM), the myelin protein P2 or its neuritogenic peptide, each emulsified in complete Freund's adjuvant (CFA). The present study evaluates the effect of oral tolerization with BPM or P2 protein on the development of actively induced EAN. Oral administration of BPM strongly suppressed clinical and histological signs of EAN subsequently induced by BPM/CFA, but feeding of P2 protein alone did not affect its course. In contrast, feeding of BPM did not mitigate the course of EAN subsequently induced by immunization with neuritogenic P2 peptide/CFA. Oral therapy with BPM after onset of myelin-induced EAN only slightly ameliorated the further course of disease, but significantly reduced lethality of this severe form of disease. The findings suggest that immunogenicity of the antigens fed determine strength of tolerance, that downregulation of EAN occurs at the site of immunization and not in the nerve, and that active suppression rather than specific anergization is operative in mediating resistance to EAN. However, only partial tolerance to myelin-induced EAN was achieved in naive animals by transfer of spleen/LN cells from rats orally tolerized with BPM. Although methodic factors may have limited the effect of the cells, the result is suggestive of some contribution of anergy to oral tolerance in the present model. Cholera toxin and LPS were identified as oral adjuvants for BPM and prolonged the state of tolerance. However, LPS exhibited proinflammatory properties if EAN was induced early after BPM/LPS-feeding. Thus, oral application of a mixture of myelin components in combination with cholera toxin may be a useful treatment for chronic inflammatory neuropathies considered autoimmune in nature.     

Gender differences in protection from EAE induced by oral tolerance with a peptide analogue of MBP-Ac1-11

Mechanisms that contribute to increased female susceptibility to multiple sclerosis can be studied in the murine model of experimental autoimmune encephalomyelitis (EAE). In this report, we compared oral tolerance induction in male and female B10.PL mice using fed myelin basic protein (MBP) Ac1-11 peptide or a high-affinity analogue, Ac1-11[4Y]. We found that fed Ac1-11[4Y] peptide, but not native Ac1-11, could limit cellular infiltration into the central nervous system (CNS) and protect male mice from EAE, an effect that was completely obviated by castration. In contrast, female mice could not be orally tolerized or protected from EAE with either peptide. Tolerance induction in males was reflected by the appearance of Ac1-11[4Y]-reactive splenocytes that produced a sharply increased ratio of transforming growth factor (TGF)-beta:interleukin (IL)-2 and induced bystander suppression. These data directly demonstrate gender differences in regulatory T cells, and support the concept that androgens are involved in governing oral tolerance to EAE.     

Oral administration of human or murine interferon alpha suppresses relapses and modifies adoptive transfer in experimental autoimmune encephalomyelitis

Chronic relapsing experimental autoimmune encephalitis (CR-EAE) is an inflammatory process of the central nervous system (CNS) that closely resembles the human disease multiple sclerosis (MS). EAE was induced in SJL/J mice and following recovery from the initial attack, animals were fed varying doses of human or murine interferon alpha (IFN-α), or mock IFN three times per week. After relapse, concanavalin A-activated spleen cells were transferred adoptively from orally fed animals into recipient animals. Oral administration of human or murine IFN-α suppressed relapse in actively immunized animals, modified adoptive transfer of EAE, and decreased mitogen/antigen proliferation and IFN-γ secretion in both donors and recipients. IFN-α acts orally by modifying the encephalitogenicity of donor spleen T cells.     

Effects of oral tolerance induction by myelin basic protein on Vβ8+ lewis rat T cells

Encephalitogenic T cells from Lewis rats use a restricted T cell receptor (TCR) gene combination, Vβ8.2 and Vα2. The oral administration of myelin basic protein (MBP) to Lewis rats prior to encephalito-genic challenge results in a marked inhibition of clinical neurologic signs of encephalitis, reduced central nervous system pathology, suppressed T cell reactivity to MBP, and decreased serum anti-MBP antibody responses. The present study determined the TCR Vβ8 gene usage in rats rendered orally tolerant to MBP as compared with vehicle-fed or unfed controls. Total RNA was extracted from lymph node cells (LNC), Northern blots run, and hybridizations performed using a rat β chain V region probe positive for Vβ8.2. The results indicate that feeding MBP results in a decrease in Vβ8+ TCR RNA expression in lymph nodes draining the site of encephalitogenic challenge. T cell proliferation was reduced in LNC of tolerized rats relative to control rats. No change in the Vβ8+ TCR RNA expression or MBP reactivity was observed in the mesenteric lymph nodes (MLN) of vehicle-fed or MBP-fed rats, although an increase in cell number was found in the MLN of both groups. These results suggest that the mechanisms of orally induced tolerance involve local clonal deletion or migration of Vβ8+ T cells, of which MBP-specific T cells are a part.     

Encephalitogenic T cells are present in Lewis rats protected from autoimmune encephalomyelitis by coimmunization with MBP73-84 and its analog

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that is mediated by T helper 1 (Th1) CD4⁺ T cells. Lewis rats can be protected from actively induced EAE by coimmunization with the encephalitogenic myelin basic protein (MBP) epitope 73–84 and its single alanine-substituted analog 1028. Although analog 1028 cannot induce either active or passive EAE, it does elicit a Th1-like response that is cross-reactive with MBP73–84. Analog 1028 can effectively inhibit clinical EAE in a dose-dependent manner when rats are coimmunized with the encephalitogenic peptide MBP73–84 and 1028 in complete Freund adjuvant (CFA). Stimulation of cells from MBP73–84:1028—coimmunized protected rats proliferate and secrete interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in vitro in response to MBP73–84. Furthermore, coimmunized protected rats harbor a population of MBP73–84—reactive potentially encephalitogenic T cells, because splenocytes from these rats can be stimulated to transfer passive EAE to naive recipients. Thus, the protection of coimmunized rats by analog 1028 is not due to the inhibition of priming of MBP73–84—reactive T cells or alteration of the cytokine secretion profile of the MBP73–84—reactive cell population. Rather, MBP73–84—reactive potentially encephalitogenic T cells are primed in these protected animals. © 1996 Wiley-Liss, Inc.     

Suppressive activity of long-term myelin basic protein-specific SJL T cell lines

Myelin basic protein (MBP)-specific T cell lines derived from SJL mice lose the ability to transfer adoptively experimental allergic encephalomyelitis (EAE) after 5-6 restimulations with antigen in vitro. In order to test whether such lines were suppressive, non-encephalitogenic T cell lines were co-cultured with a freshly derived encephalitogenic T cell line. Following co-culture in the presence of MBP and irradiated syngeneic spleen cells the mixture was transferred adoptively to syngeneic recipients. Severe EAE was observed in recipients of the encephalitogenic cell line alone but not in animals which received the co-culture. A co-culture period was required as mixing the encephalitogenic and non-encephalitogenic T cell lines just prior to transfer was without effect. Not all non-encephalitogenic cell lines were found to be suppressive. Culture fluids from the suppressive, but not the non-suppressive lines were found to inhibit MBP-driven proliferation of T cell clones and encephalitogenic lines in vitro. Nineteen of 55 MBP-specific T cell clones derived from suppressive lines were found to elaborate the suppressive supernatant activity. The suppressive effect was not antigen-specific since the same culture supernatants inhibited proliferation of an ovalbumin-specific SJL T cell clone. The suppressive effect became apparent only after T cell lines had lost encephalitogenicity and was not mediated by tumor necrosis factor, lymphotoxin or prostaglandin.     

Suppression of experimental allergic encephalomyelitis by the encephalitogenic peptide, in solution or bound to liposomes

We have investigated the ability of liposome-bound encephalitogenic peptide to suppress experimental allergic encephalomyelitis (EAE) in the guinea pig. EAE was induced by challenge with the encephalitogenic peptide, residues 113-122 of human myelin basic protein (MBP) in complete Freund's adjuvant. The peptide was acylated with stearic acid in order to anchor it to the lipid bilayer. The liposomal-bound peptide effectively suppressed clinical signs of EAE at relatively low doses, when given subcutaneously or intraperitoneally without incomplete Freund's adjuvant, several days after challenge. In vitro proliferation of lymphocytes from treated, protected animals in response to the peptide was greatly decreased but that to the purified protein derivative of tuberculin antigen was not, indicating an antigen-specific effect. However, histological signs of EAE were not reduced. The free peptide in solution was somewhat less effective when given intraperitoneally but was as or nearly as effective as liposome-bound peptide when given subcutaneously. Binding to liposomes may decrease the rate of clearance or degradation of the peptide when given intraperitoneally.     

Localization of an encephalitogenic epitope for the SJL mouse in the N-terminal region of myelin basic protein.

T cells from SJL mice reactive with myelin basic protein peptide 1-38 have been reported to be encephalitogenic when adoptively transferred into naive syngeneic recipients. To determine whether the encephalitogenic epitope recognized by peptide 1-38-specific SJL T cells was different from those recognized by H-2u-restricted MBP peptide 1-38-specific T cells, peptide 1-38-specific SJL T cell lines were developed following immunization with guinea pig MBP peptide 1-38. Following a period of in vitro selection in the presence of peptide 1-38 and syngeneic antigen-presenting cells, one of two T cell lines transferred severe clinical disease adoptively. The second line was not encephalitogenic. When the fine specificity for antigen of the two T cell lines was determined by the use of overlapping synthetic peptides, the encephalitogenic epitope recognized by the encephalitogenic line was localized to residues 17-27. This epitope is clearly distinct from that recognized by H-2u mice. The non-encephalitogenic line was found to react only with peptide 1-38, and did not react with mouse MBP.     

Adoptive transfer of murine chronic-relapsing autoimmune encephalomyelitis: Analysis of basic protein-reactive cells in lymphoid organs and nervous system of donor and recipient animals

Frequency analysis of myelin basic protein (MBP)-reactive lymphocytes was performed in the chronic relapsing murine experimental allergic encephalomyelitis (EAE) model induced by the adoptive transfer of myelin basic protein (MBP)-primed lymphocytes to naive recipients. During the first attack, MBP-reactive cell frequencies were: 1/41,700 in spleen, 1/328,000 in lymph nodes, 1/64,500 in the peripheral blood. After recovery from a second attack, the frequencies were: 1/11,000 in spleen, 1/46,000 in lymph node, and 1/195,000 in the blood. In addition, lymph node cells obtained from animals following a second attack had increased encephalitogenic properties. CNS-derived lymphocytes analyzed during the first attack were 50% Lyt 1.2+ and 16% Lyt 2.2+. After recovery from the second attack, phenotypes were 20% Lyt 1.2+ and 49% Lyt 2.2+. There were only minimal responses to MBP in CNS-derived lymphocytes. Susceptibility to adoptively transferred EAE was in general predicted by whether a proliferative response to MBP occurred following immunization and was not solely H-2 linked. These studies demonstrate an accumulation of autoreactive cells in the spleen and lymph nodes and a shift of the phenotype of cells in the target organ as EAE becomes chronic and suggest there are dynamic immunologic processes, both in the peripheral immune system and target organ associated with relapsing EAE.     

Chronic relapsing experimental allergic encephalomyelitis in the Lewis rat: studies on immune regulation

To study immunoregulation of chronic relapsing experimental allergic encephalomyelitis (CR-EAE) in Lewis rats, we adoptively transferred concanavalin A-activated lymph node cells (LNC) or splenocytes, from hind footpad-inoculated donors at the onset (day 11), or recovery (day 16), of the first attack. Popliteal LNC, especially from day 16 donors, provided significant and dose-dependent, but incomplete, protection of recipients from encephalitogenic challenge; maximal mean delay in EAE onset was 10 days later than controls, with subsequent paralysis reduced more than 6-fold. In contrast, particularly from day 11 donors, superficial inguinal LNC recipients developed actively induced disease of normal severity up to 4 days earlier than CR-EAE controls. Furthermore day 11 EAE splenocytes, but not day 16 ones, adoptively transferred disease into 50-88% of naive recipients. In separate studies, we demonstrated unresponsiveness to active induction of disease in all rats re-challenged during stable late remission, as well as in a minority of animals pretreated with antigen in incomplete Freund's adjuvant. These results suggest an organ-dependent and time-dependent balance between effector and suppressor populations in the model.     

The immune response to a subdominant epitope in myelin basic protein exon-2 results in immunity to intra-and intermolecular dominant epitopes

Experimental autoimmune encephalomyelitis was induced in SJL/J mice by adoptive transfer of a MBP exon-2 peptide-specific T cell line. The T cell line, when tested for antigen specificity, reacted strongly with exon-2 peptide, but not with MBP peptides pAc1-11, p43–88, p89–101 or PLP p139–151. The specificity of splenic or lymph node T cells isolated from mice with acute or first relapse EAE induced by adoptive transfer of the exon-2-specific T cell line was identical to the transferred line. Splenocytes or lymphocytes isolated from mice at the second relapse were reactive with MBP p43–88, p89–101 and PLP p139–151 in addition to exon-2 peptide and MBP peptide Ac1-11. T cell lines selected by culture with MBP exon-2 peptide or PLP p139–151 from splenic cells from mice with relapsing EAE were weakly encephalitogenic; however, T cell lines selected from the same mice with MBP pAc1-11 were not encephalitogenic. T cells from the exon-2 and p139–151 T cell lines primed recipients for rapid onset severe EAE, whereas the pAc1-11 T cell line did not. T cells from the exon-2-specific line did not express V_β17a⁺ TCR; however, peptide-specific T cell lines derived from the spleens of relapsing animals did express this TCR gene segment providing direct evidence of recruitment and sensitization of recipient T cells.