John R. Richert - US grants

No Abstract Provided

myelin; nervous system disorder chemotherapy; viral myelinopathy; monoclonal antibody; immunotherapy; multiple sclerosis; clone cells; major histocompatibility complex; leukocyte activation /transformation; T lymphocyte; hybridomas; antibody specificity; surface antigens; histocompatibility typing; human tissue; immunochemistry; enzyme linked immunosorbent assay; nervous system disorder diagnosis;

Myelin basic protein (BP) is a potential autoimmune target in multiple sclerosis (MS) and in post-viral demyelinating states. Encephalitogenic sites on the BP molecule vary from species to species and even from strain to strain. Therefore, it is impossible to determine from animal studies which sites on the molecule may be encephalitogenic in humans. We have generated 40 BP-specific T cell clones from the peripheral blood of a patient with MS and are using them to identify T cell recognition sites on the molecule. Current data indicate that there are at least ten epitopes on the BP molecule that are recognized by human T cells. We will further define the epitopes recognized by our current clones and will generate new clones from additional patients with MS, from patients with post-viral demyelinating syndromes, and from normal subjects. Initial studies with each new clone will examine the proliferative responses against large fragments of the BP molecule. These fragments will be generated by limited thrombin digestion at position 97-98 of the human molecule and limited pepsin digestion at position 88-89 of the guinea pig molecule. Subsequent studies will evaluate the pattern of responses against a panel of xenogeneic BPs of known amino acid sequence. Once these studies have narrowed down the potential sites of T cell recognition, small peptic fragments and synthetic peptides will be used to fully define the epitopes in question. These clones will subsequently be used to evaluate cross- reactivity between BP and viral antigens, to examine genetic restriction patterns involved in BP recognition by T cells, to compare the patterns of reactivity among individual subjects and between patient groups, to evaluate for BP-specific cytotoxic activity, and to generate anticlonotypic monoclonal antibodies which may be used to identify idiotype-bearing T cells in MS pathologic material and which may be useful in suppressing anti- BP responses in vitro and in vivo. The clones may also be used to "vaccinate' patients with MS, again in order to suppress a potential anti-BP immune response, with the possibility of successfully treating the disease.

Antigen-specific suppression of the human T cell response to myelin basic protein (MBP) represents a potential therapeutic modality for multiple sclerosis (MS). This has been accomplished in experimental allergic encephalomyelitis by vaccination with synthetic peptides corresponding to sequences on particular V-beta T cell receptor (TCR) gene products expressed by rat encephalitogenic T cells. However, the human T cell response to MBP appears to be considerably more diverse than in the animal model, with multiple epitopes, major histocompatibility complex (MHC) restriction elements, and TCR molecules being involved. For a similar therapeutic strategy to be successful in MS, a small number of conserved sequences must be utilized by the expressed TCRs. Theoretically, the chance for success would be optimized if the conserved sequences included sites of TCR binding to the peptide-MHC complex. The proposed studies will determine the residues on the TCR that bind to the MBP-MHC complex. Full length cDNAs corresponding to the TCR alpha and beta genes expressed by human MBP-specific T cell clones will be transfected into mutant T cell lines that lack one or both TCR genes. MBP- reactivity of the transfectants will be assayed by: l) production of IL2- specific message, 2) IL2 secretion measured by bioassay, and 3) CD69 expression. Site-directed mutagenesis studies will determine which residues on the TCR alpha and beta chains are required for recognition of the MBP-MHC complex and if conservative vs. non-conservative substitutions generate different patterns of reactivity. These studies will provide valuable information on the fine specificities involved in T cell recognition of peptide antigens, will allow subsequent evaluation of the 3-dimensional configurations of the TCR molecules at the sites of binding to the MBP-MHC complex, and will determine if therapeutic maneuvers aimed at the antigen (Ag)-MHC binding site on the TCR are feasible in attempts to suppress the immune response to MBP.

Anchored polymerase chain reaction (PCR) will be used to isolate and amplify the T cell receptor (TCR)-alpha and beta genes expressed by a panel of myelin basic protein (MBP)-specific human T cell clones derived from patients with multiple sclerosis (MS) and controls. T cell clones that have previously been generated, and new clones to be isolated from peripheral blood, will be assayed for epitope specificity on the MBP molecule. Genetic (HLA) restriction studies will be performed to determine which major histocompatibility complex determinants govern antigen recognition by the clones. The MS and control populations will be selected to include subjects with varied HLA haplotypes and ethnic backgrounds. The amplified genes will be sequenced in order to identify the utilized V- alpha (Valpha), Jalpha, V-beta (VB), DB, and JB gene segments and junctional residues. TCR gene segment usage will be correlated with epitope specificity and HLA restriction patterns exhibited by the T cell clones. Potential disease-specific correlations will also be evaluated. When previously undescribed TCR gene segments, or predominant use of known gene segments, are detected, a rapid initial determination will be made with regard to the disease-specificity of the finding. This will be accomplished by performing anchored PCR on cDNA derived from peripheral blood lymphocyte preparations obtained from large populations of MS patients and controls. The amplified material will be screened with oligonucleotides specific for the gene segment in question or with cDNA from the T cell clones that expressed the gene segment. The results will be used to determine the feasibility of proceeding with clinical trials in which MS patients are treated with regimens designed to inhibit immune responses mediated by specific TCR-V, D, or J gene segments in an effort to specifically suppress the immune response to MBP. Such a therapeutic approach would be encouraged by a finding of restricted heterogeneity of TCR gene segment usage. Alternatively, if a great degree of TCR heterogeneity is found, T cell vaccination is likely to provide a more reasonable approach to the suppression of MBP-specific T cell responses in vivo.

DESCRIPTION: Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that is thought to be a consequence of a microbial infection occurring in a genetically susceptible host in the mid-teenage years, leading to an autoimmune process that is manifested by clinical disease some years later. MS patients tend to be immune hyper-responders to a variety of microbial and self antigens, suggesting an element of immune dysregulation in the disease. Little is known about the mechanisms that lead to this pathological inflammatory response. Differential display was used to screen peripheral blood mononuclear cells (PBMC) from identical twins who are discordant for MS. One of the mRNAs detected from the normal twin but not from the MS twin codes for the bifunctional transcription factor Sp3. Although Sp3 was initially described as a dominant suppressor of gene transcription, it now appears to be a bifunctional molecule whose various isoforms may serve as either suppressor or activators of transcription. Genes whose transcription has been shown to be repressed by Sp3 include c-myc, HIV-1 LTR, dihydrofolate reductase, and elastin, while Sp3 activates TGF-beta, cyclin-dependent kinase inhibitor p21, and pyruvate kinase M genes. Using RT-PCR, Sp3 cDNA was amplified from 83% of control subjects, including those with rheumatoid arthritis and lupus, but was detected in only 21% of MS patients (p<0.001). The lack of Sp3 mRNA could not be explained on the basis of abnormally rapid degradation. We propose that transcription of the Sp3 gene is blocked in immune cells from most MS patients and that this leads to altered expression of one or more gene products involved in the development of CNS inflammation in the disease. The principal investigator hypothesizes that MS immune cells harbor one or more factors, possibly of microbial origin, that bind to the regulatory elements of the Sp3 gene and block its transcription. The 5' flanking regions of the Sp3 gene will be sequenced from our recently isolated Sp3 genomic clone. Nuclear and cytoplasmic extracts from PBMC will be screened by gel shift assays and DNaseI footprint analysis for factors present in MS but not control cells, that bind to one or more regulatory elements of the Sp3 gene. These factors will be purified and initial identification will be carried out by N-terminal amino acid analysis. In addition, purified human herpesvirus-6 and cytomegalovirus polypeptides will be studied for their ability to bind to Sp3 regulatory elements. Subsequent studies will determine if these factors/peptides exhibit an inhibitory influence on Sp3 gene transcription.