George A. Hashim

Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis

T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.

T CELL RECEPTOR PEPTIDES IN TREATMENT OF AUTOIMMUNE DISEASE : RATIONALE AND POTENTIAL

The natural tendency in T cell‐mediated autoimmune conditions to develop focused antigen‐specific responses that over‐utilize certain T cell receptor (TCR) V region segments prompts the induction of anti‐TCR‐specific T cells and antibodies that can inhibit the pathogenic T cells and promote recovery from disease. This natural regulatory network can be manipulated by injecting synthetic peptide vaccines that correspond to segments of the over‐expressed V genes. In experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), the pathogenic T cells are directed at myelin components, including basic protein (MBP). In some strains such as the Lewis rat and the PL/J mouse, the encephalitogenic BP‐specific T cells overexpress a particular V region gene (Vβ8.2) in their TCR. In vivo administration of Vβ8.2 peptides in rats or mice can prevent and treat EAE by boosting regulatory anti‐Vβ8.2‐specific T cells that inhibit but do not delete the encephalitogenic specificities. This regulation is mediated by soluble factors, suggesting that the presence of regulatory TCR‐specific T cells within the target organ (the central nervous system) may inhibit not only the stimulating Vβ8.2+ T cells, but also bystander T cells bearing different V genes. Parallel studies in MS patients have revealed striking V gene biases among BP‐specific T cell clones from some patients that provided a rationale for TCR peptide therapy. Injection of Vβ5.2 and Vβ6.1 peptides boosted the frequency of TCR peptide‐specific T cells and reduced responses to BP, in some cases with clinical benefit, indicating the presence of an anti‐TCR regulatory network in humans that may also be manipulated with TCR peptide therapy.

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.

TCR peptide therapy decreases the frequency of encephalitogenic T cells in the periphery and the central nervous system

The V beta 8 CDR2 consensus peptide, residues 44-54, is highly effective in the treatment of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To monitor immunological changes during EAE resulting from TCR peptide therapy, the frequencies of encephalitogenic and regulatory T cells were quantitated in lymph nodes, blood, and spinal cord. The frequency of T cells specific for basic protein and its major encephalitogenic epitope, residues 72-89, increased during EAE to about 1 cell per 100,000 lymph node or blood cells at the peak of clinical disease, and then declined. In contrast, the frequency of these T cells in spinal cord was highest, 50 per 100,000, prior to onset of clinical signs, and then decreased rapidly prior to spontaneous recovery. Injection of 100 micrograms of TCR V beta 8-44-54 peptide caused a decrease within 1-5 days in the frequencies of guinea pig basic-protein (GP-BP) and 72-89-reactive T cells in blood and spinal cord, and in the total number of infiltrating cells in spinal cord. In lymph nodes, 72-89-reactive T cells decreased as T cells specific for a protective epitope, residues 55-69 of GP-BP increased, suggesting epitope switching at the site of GP-BP immunization. Conversely, the frequency of T cells specific for the V beta 8-44-54 peptide increased, especially in blood and spinal cord, whereas T cell frequencies to control antigens were unchanged. These data document the critical presence of encephalitogenic T cells within the spinal cord during clinical EAE, and demonstrate that rapid and profound changes in T cell frequencies in the periphery and spinal cord are triggered by TCR peptide therapy.

Myelin lipophilin-induced demyelinating disease of the central nervous system.

Purified lipophilin, a hydrophobic lipoprotein of myelin, induces a cell-mediated demyelinating disease of the central nervous system similar to experimental allergic encephalomyelitis (EAE) induced by the myelin basic protein (MBP). Guinea pigs challenged with lipophilin (emulsified with CFA) developed clinical and histological signs of disease indistinguishable from those developed by animals similarly challenged with MBP. Both lipophilin and MBP induced and elicited delayed-type hypersensitivity in animals challenged with respective antigens. Tryptophan, an essential component of the MBP-determinant for disease in guinea pigs, is required for the encephalitogenicity of lipophilin.

Preferential peptide specificity and HLA restriction of myelin basic protein-specific T cell clones derived from MS patients

A panel of 17 myelin basic protein (MBP)-specific T lymphocyte clones were generated from four multiple sclerosis (MS) patients. All T cell clones expressed CD4 phenotype and 14 clones exhibited substantial cytotoxic activity on MBP-coated target cells. T cell recognition sites of the clones on human MBP were identified by using MBP fragments and synthetic peptides. Despite the fact that at least three epitopes were defined, these T cell clones displayed a striking bias to the C-terminal peptide 149-171 independent of differences in HLA-DR and DQ expression. In addition, the T cell responses of the clones appeared to be restricted by HLA-DR molecules irrespective of peptide specificities. The present study suggests an immunodominant property of the C-terminal peptide for HLA-DR-restricted T cell responses to MBP. However, its association with encephalitogenicity in humans and its potential pathologic importance in MS await further clarification.

Hydrolysis of myelin basic protein with brain acid proteinase

Summary The encephalitogenic basic protein from bovine spinal cord was treated with purified bovine brain proteinase (cathepsin D, EC 3.4.4.23) yielding 3–4 peptide fragments. An encephalitogenic fragment (peptide III-a, 8645 daltons) was purified by repetitive filtration on Sephadex gel. The purity of the peptide was established by polyacrylamide gel electrophoresis at acid and alkaline pH and by amino acid and end group analysis. The N- and C-terminal amino acid sequence of peptide III-a shows that the brain acid proteinase hydrolyzes the Phe-Phe linkage (residues 89–90) releasing the C-terminal 81 residues of the basic protein molecule.

Myelin basic protein-stimulated rosette-forming T cells in multiple sclerosis

A subpopulation of T lymphocytes sensitized to human myelin basic protein in peripheral blood of patients with multiple sclerosis, central nervous system (CNS) tumors, and cerebrovascular accidents was demonstrated by the antigen-stimulated, rosette-forming T cell assay. A significant increase in the percent of active rosette-forming T cells was detected after in vitro exposure of peripheral blood lymphocytes to human myelin basic protein but not to histones. In contrast, peripheral blood lymphocytes from healthy controls and from patients with benign and malignant breast diseases were unresponsive to stimulation by either antigen. These results demonstrate a functionally active T-lymphocyte subpopulation sensitized to myelin basic protein in patients with multiple sclerosis and in patients with certain other CNS diseases.

Antigen-stimulated rosette formation by T lymphocytes in experimental allergic encephalomyelitis

Peripheral blood lymphocytes form rosettes in the presence of heterologous etythrocytes. Spontaneous or active rosette formation has been reported to be a measure of circulating and immunologically functional thymus-dependent lymphocytes. The present study utilizes the rosette assay to measure changes in the circulating T cells of guinea pigs sensitized with encephalitogenic myelin basic protein (BP) or with nonencephalitogenic peptide S42 known to induce cellular transformation in experimental allergic encephalomyelitis, a cell-mediated disorder of the central nervous system. The results show a significant depression in the number of active but not in the total number of rosette-forming T lymphocytes from the peripheral blood of antigen-sensitized animals. This reduction, which was not related to the encephalitogenic property of the BP, was readiiy reversible by incubating lymphocytes with the sensitizing antigen but not with histone. Under these conditions, lymphocytes from unsensitized control animals were unresponsive to stimulation by any of the antigens used. The antigenstimulated rosette assay described in this report provides a specific assay for sensitization to basic protein in BP-related demyelinating diseases.

Substrate specificity of rat brain calcium-activated and phospholipid-dependent protein kinase

A synthetic peptide ArgThrProProProSerGly with sequence similar to the threonine sites of phosphorylation in both myelin basic protein and simian virus 40 T antigen could be phosphorylated in vitro by a purified rat brain Ca2+-activated and phospholipid-dependent protein kinase, protein kinase C. The apparent Km and Vm values of this heptapeptide for the enzyme were determined to be 240 microM and 60 nmol/min/mg, respectively. Up to 0.8 mol 32P could be incorporated into the peptide, mainly at the threonine residue. Substitution of the L-threonine residue in the heptapeptide by its D-enantiomer abolished the phosphorylatability of the peptide by protein kinase C. However, this (D)Thr-containing peptide could act as a competitive inhibitor for the kinase with an apparent Ki value of approximately 320 microM. These findings suggest that a triprolyl sequence may act as a recognition site for protein kinase C.