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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.

Understanding the immune response to myelin antigens in regard to the peptide/MHC/TCR complex is important in defining pathogenesis of demyelinating autoimmune diseases and in developing antigen-specific therapies. We previously reported that individual multiple sclerosis patients may use certain dominant TCR V beta chains to recognize immunodominant MBP peptides. In examining the TCR beta chain usage, we observed repeated TCR VDJ sequences among different T-cell lines isolated from the same patient. This suggested that a few expanded T-cell clones may dominate the immune response to immunodominant MBP peptides. Here, we report experiments where TCR rearrangements were used as a probe for the clonal origin of MBP specific T-cells cultured from blood lymphocytes of MS patients and normal subjects. In two patients with the DR2 haplotype that were analyzed in detail, the T-cell response to MBP was focused on the MBP (84-102) peptide and in vivo expanded population(s) dominated the response to the MBP (84-102) peptide. Two MBP (84-102) specific T-cell clones from a normal subject with the DR2 haplotype were also found to have identical TCR sequences. Clonality was proven by demonstrating that independent clones had identical TCR alpha and beta chain sequences as well as identical sequences of a TCR gamma chain or of a second TCR alpha chain rearrangement. These data suggest that the response to human MBP is dominated in at least some subjects by expanded clones that may persist in vivo for relatively long periods of time.

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.