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Multiple sclerosis (MS) is a presumed cell-mediated Th1-type autoimmune disease. Thus therapies which decrease T cells secreting IFN-gamma production or increase IL-4 production would be expected to have an ameliorating effect on MS. We have previously reported increased anti-CD3-induced IL-4 secretion by T cells in progressive MS patients treated with cyclophosphamide plus methylprednisolone (CY/MP) which was associated with eosinophilia. To investigate whether the increased IL-4 secretion was myelin antigen specific, we generated 3990 short-term T cell lines to myelin basic protein (MBP), proteolipid protein (PLP), or tetanus toxoid (TT) from 31 progressive MS patients: 11 MS patients treated with CY/MP, 10 MS patients treated with MP alone, and 10 untreated MS patients. We found increased frequencies of both MBP- and PLP-specific IL-4-secreting T cell lines in CY/MP-treated patients compared to untreated MS patients. However, no change in the frequency of TT-specific IL-4-secreting T cells was observed. MP treatment alone did not increase the frequency of antigen-specific IL-4-secreting T cell lines. These results demonstrate immune deviation favoring Th2-type responses specific to autoantigens following pulse cyclophosphamide therapy in MS patients.

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Oral administration of antigen is a long-recognized method of inducing systemic immune tolerance. In animals with experimental autoimmune disease, a major mechanism of oral tolerance involves the induction of regulatory T cells that mediate active suppression by secreting the cytokine TGF-beta 1. Multiple sclerosis (MS) is a presumed T cell-mediated Th1 type autoimmune disease. In this paper we investigated, in patients with MS, whether oral myelin treatment (myelin containing both MBP and PLP) induced antigen-specific MBP- or PLP-reactive T cells that were either Th2-like (secreted IL-4 or TGF-beta 1), or alternatively whether Th1 type sensitization occurred as measured by IFN-gamma secretion. Specifically, 4,860 short-term T cell lines were generated to either MBP, PLP or TT from 34 relapsing-remitting patients with MS; 17 were orally treated with bovine myelin daily for a minimum of two years as compared to 17 non-treated patients. We found a marked increase in the relative frequencies of both MBP- and PLP-specific TGF-beta 1 secreting T cell lines in the myelin-treated MS patients as compared to non-treated MS patients (MBP, p < 0.001; PLP, p < 0.003). In contrast, no changes in the frequency of MBP- or PLP-specific IFN-gamma or TT-specific TGF-beta 1 secreting T cells were observed. These results suggest that the oral administration of antigens generates antigen-specific TGF-beta 1 secreting T cells of presumed mucosal origin that may represent a distinct cytokine-secreting lineage of T cells (Th3). Since, in animal models, antigen-specific TGF-beta 1 secreting cells localize to the target organ and then suppress inflammation in the local microenvironment, oral tolerization with self-antigens may provide a therapeutic approach for the treatment of cell-mediated autoimmune disease which does not depend upon knowledge of the antigen specificity of the original T cell clone triggering the autoimmune cascade.

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Oral administration of antigen is a long recognized method of inducing systemic immune tolerance. In animals with experimental autoimmune disease, a major mechanism of oral tolerance triggered by oral administration of antigen involves the induction of regulatory T cells that mediate active suppression by secreting the cytokine TGF-beta 1. Multiple sclerosis (MS) is a presumed T cell-mediated Th1 type autoimmune disease. Here, we investigated whether in MS patients oral myelin treatment, containing both myelin basic protein (MBP) and proteolipid protein (PLP), induced antigen specific MBP or PLP reactive T cells that either secreted IL4, TGF-beta1, or alternatively did Th1 type sensitization occur as measured by IFN-gamma secretion. Specifically, 4,860 short-term T cell lines were generated to either MBP, PLP, or tetanus toxoid (TT) from 34 relapsing-remitting MS patients: 17 orally treated with bovine myelin daily for a minimum of 2 yr as compared to 17 nontreated patients. We found a marked increase in the relative frequencies of both MBP and PLP specific TGF-beta1-secreting T cell lines in the myelin treated MS patients as compared to non-treated MS patients (MBP P < 0.001, PLP P < 0.003). In contrast, no change in the frequency of MBP or PLP specific IFN-gamma or TT specific TGF-beta1 secreting T cells were observed. These results suggest that the oral administration of antigens generates antigen specific TGF-beta1 secreting Th3 cells of presumed mucosal origin that represent a distinct lineage of T cells. Since antigen-specific TGF-beta1 secreting cells localize to the target organ and then suppress inflammation in the local microenvironment, oral tolerization with self antigens may provide a therapeutic approach for the treatment of cell-mediated autoimmune disease which does not depend upon knowledge of the antigen specificity of the original T cell clone triggering the autoimmune cascade.

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Multiple sclerosis is a presumed autoimmune disease, associated with inflammation in the CNS white matter, mediated by autoreactive T cells. We previously reported that oral myelin tolerization of relapsing-remitting MS patients resulted in fewer attacks, as compared to a placebo-fed group. Here, we examined whether oral tolerization with bovine myelin resulted in altered autoreactive T-cell populations or altered T-cell fraction. We generated 4,620 T-cell lines from 34 relapsing-remitting MS patients (17 were fed bovine myelin daily), and each line was examined for proliferation to MBP, PLP, and TT and for secretion of IL-4, IFN-gamma, and TGF-beta1. The frequency of TGF-beta1-secreting T-cell lines after MBP and PLP stimulation in fed patients was greater than that of nonfed patients. These experiments demonstrate that oral tolerization with autoantigen results in altered cytokine secretion in a human autoimmune disease with the generation of TGF-beta1-secreting T cells that may regulate the inflammatory response at the site of the demyelinating lesions in multiple sclerosis. These data provide the first evidence of antigen-specific modification of cytokine secretion in a human autoimmune disease.

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We investigated the immune response to proteolipid protein (PLP), the most abundant central nervous system myelin protein in humans. A total of 8207 short-term T cell lines were generated from 49 individuals, 39 patients with multiple sclerosis and 10 control subjects. As we have reported previously, the frequency of PLP-reactive T cells did not differ between the two groups. To determine immunodominant PLP epitopes, proliferative responses of 971 PLP-specific lines were tested with 27 overlapping 20-amino acid peptides encompassing the human PLP sequence and the binding affinities of the PLP peptides to DRB5*0101 and DRB1*1501, DR2 MHC class II isotypes associated with multiple sclerosis, were determined. The T cell response after primary PLP stimulation was focused on two immunodominant epitopes comprising residues p30-49 and p180-199. These two fragments were recognized after processing of native protein by APCs and were situated in hydrophilic regions of PLP exhibiting only moderate affinity to DR2 molecules. In contrast, when T cells from DR2+ subjects were stimulated initially by individual synthetic peptides with either high or low affinity to DRB5*0101 and DRB1*1501 isotypes, additional cryptic epitopes were recognized. MHC restriction of lines specific for the cryptic PLP epitopes were related to binding affinity to DR2 isotypes. Our results indicate that protein Ags are recognized in vivo as immunodominant epitopes after Ag processing by APCs and as cryptic epitopes after processing, presumably by extracellular proteolytic enzymes.

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We demonstrate that cognate peptide ligands altered at T cell receptor (TCR) contact residues and bound to class II major histocompatability complex can change the cytokine pattern of mature T cell clones. Myelin basic protein peptide 85-99-reactive Th0 T cell clones were stimulated with altered peptide ligands, which acted both as TCR antagonist and induced new mRNA synthesis and protein secretion of TGF-beta 1, while no longer inducing mRNA synthesis or protein secretion of IL-2, IL-4, IL-10, and IFN gamma. The modified peptides failed to induce a detectable calcium flux, p56lck activation, or thymidine incorporation, yet triggered nearly equal amounts of IL-4 secretion in the presence of ionomycin. Antigen-induced modulation of T cell cytokine secretion may be important in regulating the immune response.

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An autopsied case of amyotrophic lateral sclerosis complicated by cervical syringomyelia was reported. The case was a 59-year-old man, who first noticed weakness of both lower extremities at 54-year-old. The weakness spread to both upper extremities within 2 years. Cervical myelography revealed multi-level cervical spondylosis and anterior fusion of C5-C7 was done. But the weakness and atrophy of proximal muscle, diminished deep tendon reflex on upper extremities, hyperreflexia and pathological reflexes on both legs, tongue fasciculation and respiratory muscle weakness developed successively, and the patient died of respiratory distress at 59-year-old. Autopsy revealed multiple independent four syrinxes located at the level between C2-C7. One of these syrinxes had ependymal cell lining and thought to be idiopathic syringomyelia. The other three syrinxes were considered to be the cavitation in association with cervical spondylotic myelopathy. Degeneration and decreasing of spinal anterior horn cells, atrophy of medullary pyramis and Bunina bodies were observed as features of typical amyotrophic lateral sclerosis. Cervical spondylosis as causative lesion of multiple syrinxes was discussed, and relationship between ALS and the syrinxes was not indicated clearly.

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