Correlation between active rosette formation and delayed cutaneous hypersensitivity in experimental allergic encephalomyelitis

The effect of encephalitogenic myelin basic protein, BP, on active rosette-forming T cells (ARFC) was compared to that of nonencephalitogenic peptide S42, a synthetic analogue of the tryptophan region of BP. Depression of ARFC by these antigens was reversible within 24 h after a second dose of the antigen into the skin, or after in vitro incubation of lymphocytes with the sensitizing antigen (Ag-ARFC). The ratio of Ag-ARFC to ARFC rose with time following the sensitization but fell shortly before the clinical onset of experimental allergic encephalomyelitis in animals sensitized with BP. In contrast, the Ag-ARFC/ARFC ratios for animals sensitized with peptide S42 reached plateau levels from which they did not drop. The kinetics of the Ag-ARFC/ARFC responses paralleled those for delayed-type skin hypersensitivity (DTH) in the respective animals. The DTH responses rose following sensitization and fell shortly after the appearance of clinical signs of EAE. The results of this study provide in vitro and in vivo evidence for sensitization to myelin basic protein, and focus attention on the ARFC as a measure for an immunologically active cell population which may be quantitated by antigenic stimulation.Abbreviations used in this report EAE experimental allergic encephalomyelitis - DTH delayed-type skin hypersensitivity - ARFC active rosette-forming T cells - Ag-ARFC antigen-stimulated active rosette-forming T cells - TRFC total rosette-forming T cells     

Experimental allergic encephalomyelitis: activation of suppressor T lymphocytes by a modified sequence of the T effector determinant

Deletion of certain amino acid residues from the amino acid sequence of the encephalitogenic determinant for guinea pigs, H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, destroyed its ability to induce experimental allergic encephalomyelitis (EAE), a cell-mediated autoimmune disease of myelin. The administration of the modified determinant in the form of 4 repeating pentameric sequences, H-(Phe-Ser-Trp-Gln-Lys)4-Gly-OH, activated an antigen-specific T suppressor lymphocyte subset that rendered both presensitized donors and recipients of donor T lymphocytes unresponsive to an encephalitogenic challenge. Treatment of donors or recipients with cyclophosphamide before or after lymphocyte transfer, respectively, obliterated the ability of peptide S42-sensitized T lymphocytes to induce a state of unresponsiveness to an EAE-challenge. The results establish the existence of antigenic determinants for both immunoinduction and immunoregulation of EAE. The immunoregulatory determinant that activates antigen-specific and cyclophosphamide-sensitive suppressor T lymphocyte subset is sequestered within the disease-inducing or T effector determinant.     

Experimental allergic encephalomyelitis: basic protein regions responsible for delayed hypersensitivity

Three separate peptide regions were isolated from the chymotrypsin digest of the encephalitogenic basic protein from bovine myelin of the central nervous system. The peptides induced delayed type hypersensitivity (DTH) and elicited delayed skin reactivity in experimental animals. However, none of the isolated peptides was capable of inducing experimental allergic encephalomyelitis (EAE). The amino acid sequence of peptide CTP-3 (Gly-Ala-Glu-Gly-Gln-Lys-Pro-Gly-Phe-OH) and peptide CTP-la were found to overlap the C-terminal sequence of encephalitogenic peptides E (residue 112–125) and T8 (residue 65–74) of the basic protein, respectively. The third DTH inducing peptide, CB1-T1, (N-Acetyl-Ala-Ser-Ala-Gln-Lys-OH) was found to overlap the N-terminal sequence of the basic protein molecule. Common to the three DTH inducing peptides, to the basic protein and to the encephalitogenic peptides E-S and T8S is the X-X-X-Gln-Lys sequence. Isolation of the regions of the basic protein that are responsible for DTH provides antigens for the study of the mechanism of cellular immunity in EAE.     

Prevention of experimental allergic encephalomyelitis by nonencephalitogenic basic peptides

The biological properties of the chymotrypsin-treated encephalitogenic basic protein are described. The basic protein, isolated from bovine spinal cord, was digested with chymotrypsin and filtered through Sephadex gel resulting in three distinct well-separated peaks starting at the void volume of the column. Tubes common to each peak were combined into Fractions I, II, and III, respectively. More than 90% of the original protein was recovered in the three fractions. Fraction II, representing 76% of the original protein, was nonencephalitogenic when tested in guinea pigs at 0.010-, 0.025-, and 0.500-mg doses emulsified in complete Freund's adjuvant. Guinea pigs immunized with Fraction II were protected from EAE when challenged with encephalitogenic emulsions. A critical dose of 1.0 mg completely protected the animals from disease, while partial protection was obtained with lower doses. In addition to producing circulating antibodies, animals sensitized with Fraction II, the encephalitogenic tryptophan peptide or the basic protein displayed a delayed-type hypersensitivity response when skin tested with either of the three antigens. The positive skin reactivity in animals sensitized with Fraction II was not followed by EAE during 5 months of observation. In contrast, animals sensitized with extracts from bovine tissues other than the central nervous system were not protected from disease when challenged with encephalitogenic emulsions.The main finding here reported is the prevention of EAE with nonencephalitogenic peptides derived from the parent EAE-producing protein. The peptides retain the ability to induce delayed-type hypersensitivity and provide antigens to study the role of delayed hypersensitivity in experimental allergic encephalomyelitis.     

Experimental allergic encephalomyelitis in Lewis rats: immunoregulation of disease by a single amino acid substitution in the disease-inducing determinant

A single amino acid substitution in the sequence of the encephalitogenic determinant for Lewis rats destroyed its ability to induce experimental allergic encephalomyelitis (EAE) but generated a potent immunoregulatory sequence capable of suppressing the development of both clinical and histologic signs of EAE. The EAE-inducing determinant (synthetic peptide S6) H-Ala-Gln-Gly-His-Arg-Pro-Gln-Asp-Glu-Asn-OH (residues 75 to 84) of the bovine MBP induced clinical and histologic signs of EAE when it was administered at doses of 0.5 micrograms or higher. Gly substituted for the C-terminal Asn during the synthesis of peptide S6 generated the homologous sequence designated by peptide S79. Peptide S79 failed to induce either clinical or histologic signs of EAE even when it was administered at dosages up to 1000 times higher than those of S6. Similarly, rats pretreated with a single dose of S79 were not only unresponsive to an encephalitogenic challenge but also were capable of transferring unresponsiveness to syngeneic recipients with viable donor lymphocytes. The induction of unresponsiveness that was abrogated by pretreatment with cyclophosphamide suggests the development of an S79-sensitive lymphocyte subset that regulates MBP-induced EAE in Lewis rats.     

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.     

Encephalitogenic T cell clones with variant receptor specificity

We explored antigenic differences between guinea pig (GP)-basic protein (BP), rat (Rt)-BP, and respective peptides from the encephalitogenic region for Lewis rats by comparing the fine specificity of T lymphocyte lines and clones selected from animals primed with these Ag. Encephalitogenic T cell lines specific for GP-BP or Rt-BP predictably recognized the corresponding 72-89 and to a lesser degree the 72-84 (S55S) amino acid sequence. T cell lines selected from rats primed with GP-S55S responded preferentially to GP-S55S compared to other peptides. A T cell line raised to Rt-S55S, however, initially recognized the S55S and S72-89 peptides but were nearly unresponsive to the intact GP-BP or Rt-BP. T cell clones selected from the Rt-S55S line at that point had two distinct patterns of response: clones that recognized both of the BP and the S55S peptides adoptively transferred delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. These clones also recognized residues 69-81 (S67) but not peptide S75-89. In contrast, T cell clones that responded only to synthetic peptides GP-S55S and Rt-S55S but not to the parent BP adoptively transferred delayed-type hypersensitivity but not disease in Lewis rats. The same clones failed to respond to either the S67 or the S75-89 sequences. These results demonstrate that the encephalitogenic Rt-S55S sequence houses a minimum of two T cell epitopes with differing specificities and functions. One epitope is immuno-dominant and resembles the encephalitogenic region of the intact BP molecule. The second non-encephalitogenic epitope is restricted to the S55S sequences and is not shared by the parent BP, the S67, or the S75-89 sequences. Both types of Rt-S55S-specific clones differ in fine specificity from encephalitogenic clones selected from GP-BP immunized rats, thus indicating that uniformity of T cell recognition of the encephalitogenic epitope is not an absolute condition for T cells to be encephalitogenic.     

Measurement of cell-mediated inflammation in experimental murine autoimmune encephalomyelitis by radioisotopic labeling.

A radioisotopic index test was used to detect that time of onset and intensity of cell-mediated immune inflammation of experimental autoimmune encephalomyelitis (EAE) in mice. Mice were tested at various time intervals after an encephalitogenic immunization with mouse spinal cord to homogenate for delayed-type hypersensitivity (DTH) to myelin basic protein (MBP) by intradermal challenge with antigen in the ear pinna. After 25 hr, the intensity of DTH was measured by 125I-radiometry which depends upon the migration of 125I-UdR radiolabeled mononuclear cells into the antigen depot. Cells reactive to MBP were detected by the ear assay as early as 7 days after the initial encephalitogenic sensitization. The degree of cell-mediated immune inflammation in the brain and spinal cord during the evolution of EAE was also measured by a radioisotopic technique; increased 125I-UdR uptake could be detected in the brain 3 to 4 days before the onset of signs of EAE at days 11 to 12, whereas 125I-UdR in the spinal cord was detected only 1 day before, or concomitant with, the onset of signs of EAE. Both, or concomitant with, the onset of signs of EAE. Both the "ear" and "organ" radiometric index tests are useful in measuring the degree of cell-mediated inflammation in EAE, and supplement routine histopathological and observational assessments.     

Mast cell proteases liberate stable encephalitogenic fragments from intact myelin.

Protease-containing supernatants from activated rat mast cells were found to degrade purified rat myelin with a subsequent release of a stable encephalitogenic peptide. The two most abundant peptides were identified as residues 69-87 (GSLPQKSQRTQDENPVV) and residues 69-88 (GSLPQKSQRTQDENPVVH). While additional exposure to the mast cell supernatants removes the COOH terminal histamine from peptide 69-88 to yield peptide 69-87, additional proteolytic degradation of the 69-87 peptide was not detected. Immunization with this peptide emulsified in CFA caused the development of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In addition this 69-87 sequence was found to activate resting encephalitogenic myelin basic protein-reactive T cell lines to adoptively transfer clinical EAE. The release of stable encephalitogenic peptides from the myelin sheath by mast cell proteases may play a role in activation of encephalitogen-specific T cells during the progression of EAE.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.     

The role of the MHC class II transactivator in class II expression and antigen presentation by astrocytes and in susceptibility to central nervous system autoimmune disease

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4(+) T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4(+) Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-gamma-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-gamma activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.     

Encephalitogenic and proliferative responses of Lewis rat lymphocytes distinguished by position 75- and 80-substituted peptides of myelin basic protein

The encephalitogenic and proliferative responses of Lewis rat lymphocytes were defined by use of synthetic peptide GP68-84, representing the 68-84 sequence of guinea pig myelin basic protein (GPMBP), and otherwise identical peptides containing substitutions of either A75 or P80 residues. The comparative activities of these peptides were tested in the following bioassays: 1) active induction of experimental autoimmune encephalomyelitis (EAE), 2) potentiation of EAE transfer activity by MBP- or peptide-sensitized lymph node cells (LNC), 3) in vitro proliferation of MBP- or peptide-sensitized LNC, and 4) in vitro proliferation of an encephalitogenic T cell line. The GP68-84 peptide exhibited potent activity in all four bioassays. In contrast, [A75]GP68-84 and [P80]GP68-84 exhibited a selective loss of certain activities while retaining activity in other bioassays. For example, LNC were activated by culture with [A75]GP68-84 to express potentiated EAE transfer activity. Furthermore, [A75]GP68-84 and GP68-84 were equipotent in stimulating the proliferation of the encephalitogenic T cell line. However, [A75]GP68-84 was virtually inactive in assays measuring the induction of EAE or the proliferation of either GPMBP- or [A75]GP68-84-sensitized LNC. Conversely, the [P80]GP68-84 peptide actively induced EAE and potentiated EAE cellular transfer activity but was incapable of stimulating proliferation of either GPMBP-sensitized LNC or an encephalitogenic T cell line. When [P80]GP68-84 was used for sensitization, in vitro proliferation of LNC was stimulated, but only by MBP sequences containing a P80 substitution. Overall, these results indicate that at least two structurally distinct T cell determinants of GP68-84 regulate functionally diverse encephalitogenic and proliferative activities of EAE-associated T cells.     

Immunomorphologic features of experimental allergic encephalomyelitis induced by the administration of tryptophan peptide

Three patterns of EAE with different morbidity and mortality rates were induced in the guinea pigs inoculated with various doses of tryptophane peptide (TP) and complete Freund's adjuvant. TP-sensitized animals manifested the delayed type hypersensitivity (DTH) reactions to TP and circulating anti-TP and -BPF antibodies were not found, polypeptide fraction of myelin basic protein (BPF while a A correlation was revealed between the DTH-reactions and EAE development. Intracutaneous TP and BPF injections at the early period before the EAE onset resulted in reduction of morbidity rate from 90 to 50 per cent. For the first time with the help of modified Marchi method demyelination has been shown to be highly marked in CNS tissue process in CNS can be caused by cell-mediated immune of animals given TP. It is supposed that the demyelinating reaction to encephalitogenic fragment of BP molecule. The data indicate a possibility of EAE inhibition by means of TP and BPF injections in saline solution.     

A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.     

Experimental allergic encephalomyelitis: sequestered encephalitogenic determinant in the bovine myelin basic protein.

The presence of a sequestered encephalitogenic determinant for Lewis rats in the bovine myelin BP was demonstrated with synthetic peptide sequences prepared in our laboratory by the Merrifield solidphase method. The sequence of the encephalitogenic determinant (residues 75-84) from bovine BP (peptide S6), H-Ala-Gln-Gly-His-Arg-Pro-Gln-Asp-Glu-Asn-OH, is similar but not identical to the sequence reported for the guinea pig BP (peptide S53), H-Ser-Gln-(–)-(–)-Arg-Ser-Gln-Asp-Glu-Asn-OH. The presence or the absence of Gly-His from the sequence of either the bovine or the guinea-pig determinants did not alter their encephalitogenic potencies; however, the presence of Gly-His at positions 77 and 78 together with H-Gly-Ser-Leu-Pro-Gln-Lys- (residues 69-74) at the N-terminal end of the bovine determinant destroyed its encephalitogenic potency. In contrast to the absence of Gly-His from the potent encephalitogenic guinea-pig BP, guinea-pig fragment 44-89, and synthetic peptide S49, its presence in the bovine sequence prevents recognition of this determinant and renders the parent bovine BP, bovine fragment 44-89, and synthetic peptide S8 (residues 69-84) relatively non-encephalitogenic. The results of this study suggest that intramolecular interactions occur between adjacent amino acids, conferring secondary or tertiary structures upon this region of the bovine BP which renders the encephalitogenic determinant inaccessible for recognition by the host animal. The presence of sequestered disease-inducing determinants needs to be considered in choosing a particular BP for therapeutic use.     

Minimum structural requirements for encephalitogen and for adjuvant in the induction of experimental allergic encephalomyelitis.

Mycobacteria in the adjuvant used for induction of experimental autoimmune encephalomyelitis (EAE) in guinea pigs can be replaced by synthetic N-acetylmuramyl-l-alanyl-d-isoglutamine. A combination of synthetic encephalitogenic peptides and muramyl dipeptides induces EAE effectively at a dose on the microgram level. In this system, the synthetic heptapeptide, H-Trp-Gly-Ala-Glu-Gly-Gln-Arg-OH, with a sequence identical to those of residues 116 to 122 of the basic protein of human myelin, was the shortest peptide causing EAE. These compounds form a simple system which should be useful in studies on the mechanism of the cell-mediated autoimmune reaction.     

Characterization of the antigen specificity and TCR repertoire,and TCR-based DNA vaccine therapy in myelin basic protein-induced autoimmune encephalomyelitis in DA rats

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.     

Allergic encephalomyelitis: characterization of the determinants for delayed type hypersensitivity

Three non-encephalitogenic peptides derived from the encephalitogenic myelin basic protein of the central nervous system, produce delayed type hypersensitivity responses and elicit delayed skin reaction in guinea pigs sensitized with either peptide, the encephalitogenic tryptophan region (peptide E) or the basic protein. The amino acid sequence of the peptides is N-Acetyl-Ala-Ser-Ala-Gln-Lys-OH, forming the N-terminal region of the basic protein molecule, H-Gly-Ser-Leu-Pro-Gln-Lys-OH and H-Gly-Ala-Glu-Gly-Gln-Lys-OH representing residues number 69–74 and 117–122 of the basic protein respectively.     

De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG(35-55)-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4(+) Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4(+) T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP(178-191). Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG(35-55), comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG(35-55)-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.