myelin-basic-protein and Cell-Transformation--Viral

Autoimmune diseases in humans represent an immune attack on self tissue. Current therapies for almost all autoimmune diseases utilize potent and nonspecific immunosuppressive regimens. These therapies are complicated by their side effects and also place the patient at increased risk for opportunistic infections and malignancies. Our current understanding of immune mechanisms underlying autoimmune diseases remains limited. Ongoing studies include identifying genes that predispose an individual to developing autoimmunity, identification of autoantigens that trigger or perpetuate autoimmunity, and studies of immune cell interactions that lead to immune response. Although it may be many years before a full understanding of autoimmunity is obtained, treatment in animal models of autoimmune disease and some human clinical trials have begun to study alternative treatment approaches to therapy of autoimmune disease. Future therapies for autoimmune diseases should target the inappropriate autoimmune response. This article will describe the use of gene therapy in the treatment of autoimmune disease. We believe that autoimmunity can be ameliorated by delivering trans-acting immunoregulatory molecules by retrovirally transduced autoantigen specific T cells that home to lesions of autoimmunity. Until recently, there has not been a practical alternative to systemic delivery of immunoregulatory molecules, however systemic delivery suffers from toxic side effects and dangerous global immunosuppression. In order to study immune regulation using retroviral transduction for local delivery of immunoregulatory products, we used myelin basic protein (MBP) reactive T cell hybridomas in the murine model of multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE). In this report, we show that MBP reactive T cell hybridomas transduced to express IL-4 or TNF, ameliorated or exacerbated disease, respectively. Additionally, the effects of these cells were dependent on T cell receptor (TCR) expression, indicating that the effects were due to homing of the T cells and the local delivery of cytokines. We believe that gene therapy, allowing local delivery of immunoregulatory proteins by autoantigen specific T cells, represents an interesting potential therapy for autoimmune disease.

Topics: Adoptive Transfer; Animals; Cell Transformation, Viral; Cytokines; Drug Delivery Systems; Encephalomyelitis, Autoimmune, Experimental; Epitopes, T-Lymphocyte; Hybridomas; Interleukin-4; Mice; Myelin Basic Protein; Receptors, Antigen, T-Cell; Retroviridae; Tumor Necrosis Factor-alpha

Recently it has been shown that purified complexes of major histocompatibility (MHC) class II and antigenic peptide can recognize T-cell receptors (TCRs) on virally transformed CD4+ T-cells in vitro. It is not clearly understood whether peptide bound to purified MHC II molecules (MHC-P), or to MHC II molecules on the surface of antigen-presenting cells (APC-peptide), initiate similar or different signals in transformed human T-cells. To address this question, the expression of protein tyrosine kinases (PTKs), their phosphorylation, and the effect of various kinase inhibitors were investigated using transformed T- and B-cells. HLA-DR2- and MBP(84-102)-restricted cloned T-cells (SS8T) immortalized with herpes saimiri virus (HSV) and DR2-expressing lymphoblastoid B cells transformed with Epstein-Barr virus (EBV) were utilized in this study. The expression and phosphorylation of three major PTKs (1ck-56, fyn-59, zap-70) involved in signaling through the TCR were analyzed by enhanced chemiluminescence blots. T-cells exposed to soluble MHC-P complex did not show altered expression of 1ck-56 protein. In contrast, a decrease in 1ck expression was observed in SS8T cells when TCRs were engaged with APC-peptide. Upon interaction with the TCR, both MHC-P complex and APC-peptide showed increased fyn-59 protein expression and phosphorylation. In our experiments using immortalized T- and B-cells, the expression of zap-70 protein remained unchanged. When T-cells were exposed to herbimycin and H-7, inhibitors of PTKs and protein kinase C (PKC) pathways, respectively, a dose-dependent decrease in gamma-IFN levels was observed with both systems. However, in the presence of genestein, another PTK inhibitor, such decrease in gamma-IFN was observed only in the case in which T-cells were exposed to MHC-P complexes. These results together suggest that the occupancy of TCRs in transformed T-cells by soluble MHC-P complex and APC-peptide differs with respect to the 1ck expression, although both can induce signals that lead to increased fyn activity and its phosphorylation. In addition, genestein showed differential inhibitory effect on gamma-IFN production by T-cells exposed to APC-peptide and MHC-P complexes, suggesting that the TCR occupancy by MHC-P complex and APC-peptide have subtle differences in PTK pathways.

Topics: Amino Acid Sequence; Antigen-Presenting Cells; B-Lymphocytes; Cell Transformation, Viral; Enzyme Inhibitors; Histocompatibility Antigens Class II; Humans; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Molecular Sequence Data; Myelin Basic Protein; Peptide Fragments; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; ZAP-70 Protein-Tyrosine Kinase

Staurosporine, a potent protein kinase inhibitor, has been shown to arrest the growth of a number of normal cell types in the G1 phase of the cell cycle, while having little effect on several transformed lines. We wished to determine whether increased resistance to staurosporine was a common feature of virus-immortalized human cells and whether this phenotype was an early event following the expression of SV40 tumor antigens. Human foreskin keratinocytes immortalized by the SV40 DNA tumor virus displayed an increased resistance to staurosporine-induced growth arrest when compared with normal parental cells, as has been seen in human diploid fibroblasts. Keratinocytes immortalized by human papillomaviruses, or by just the human papillomavirus E6 and E7 oncogenes were also staurosporine resistant, suggesting that this phenotype often accompanies the immortalization of human cells by DNA tumor viruses. Acquisition of staurosporine resistance was a late event during immortalization, because precrisis human diploid fibroblasts that expressed the SV40 large T and small t antigens were not resistant to staurosporine. The same parental cells that were fully immortalized by SV40 were resistant. Staurosporine resistance was not the result of increased activities and/or expression of cyclin A, cyclin-dependent kinase (cdk) 2, cdk4, or the mitogen-activated kinases ERK1 and ERK2. Although increased activities and/or expression of cyclin A and cdk2 and cdk4 proteins, but not ERK1 or ERK2, were associated with immortalization, similar increases were found in staurosporine-sensitive precrisis cells expressing SV40 tumor antigens.

Topics: Antigens, Polyomavirus Transforming; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Cyclin-Dependent Kinases; Cyclins; Diploidy; Drug Resistance; Fibroblasts; Histones; Humans; Keratinocytes; Myelin Basic Protein; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Phenotype; Polyomaviridae; Repressor Proteins; Simian virus 40; Skin; Staurosporine

A small fraction of affinity-purified MHC class II molecules are known to bind antigenic peptides in vitro. No simple method with acceptable recovery exists for separation of complexes of a known antigenic epitope and MHC class II from empty MHC class II and complexes of MHC class II and endogenously bound peptide. Here we describe an one step metal chelate affinity chromatography method to purify complexes of MHC class II and antigenic peptide of known composition. Complexes of human HLA-DR2 (DRB1*1501/DRB5*0101) and a peptide analog from human myelin basic protein MBP(84-102) containing a 6 histidine tag (6 x His) and a tyrosine residue at the N-terminus end [6 x His-MBP(83-102)Y83] were prepared and purified. The absence of residual free 6 x His-MBP peptide in the complex preparations were confirmed by gel filtration and TLC analyses. The purified complexes were applied onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose affinity support and 6 x His-tagged peptide class II complexes were selectively eluted with imidazole-containing buffer. The quantitation of bound peptide in the eluted complexes showed 100% occupancy of HLA-DR2 (DRB1*1501/DRB5*0101) with [6 x His-MBP(83-102)Y83] peptide with a recovery of 50-75%. The presence of a single peptide entity in the eluted complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid-extracted supernatant and by amino acid sequencing analyses. As expected, no endogenous polypeptide was detected in the Ni2+.NTA eluted complexes when analyzed by two-dimensional IEF gel electrophoresis. Finally, we demonstrate that both MBP(84-102) and [6 x His-MBP(83-102)Y83] peptides were equally capable of stimulating restricted T cell line in the presence of autologous antigen presenting cells (APCs). These results demonstrate that metal chelate affinity chromatography can be used to prepare MHC class II-peptide complexes containing single peptide. Such complexes of class II molecules containing known peptide have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of the trimolecular interaction between MHC class II, antigenic peptide and T cell receptor (TCR).

Topics: Amino Acid Sequence; Antigen-Presenting Cells; Antigens; Cell Line; Cell Transformation, Viral; Chelating Agents; Chromatography, Affinity; Chromatography, High Pressure Liquid; Electrophoresis, Gel, Two-Dimensional; HLA-DR Antigens; Humans; Lymphocyte Activation; Molecular Sequence Data; Myelin Basic Protein; Nickel; Nitrilotriacetic Acid; Peptides; T-Lymphocytes

Herpesvirus saimiri has recently been shown to immortalize human T cells. It was unknown, however, whether Herpesvirus saimiri transformation affects T-cell receptor (TCR) expression and signal transduction. In the present study, we have transformed CD4+ human T-cell clones specific for human myelin basic protein. The transformed T cells were grown in interleukin 2 and divided in the absence of antigen and antigen-presenting cells. They retained the membrane phenotype of activated T cells and secreted the cytokines interferon gamma and lymphotoxin, but interleukin 4 was not detected. Further, the transformed T cells continued to express the original TCR as demonstrated by TCR variable-region-V beta-specific monoclonal antibodies and TCR sequencing. Antigen-specific recognition and signal transduction by the TCR were demonstrated by myelin-basic-protein-induced HLA-DR-restricted secretion of interferon gamma and lymphotoxin and by myelin-basic-protein-specific proliferation. Antigen specificity and reactivity have been maintained for > 1 year after transformation. Transformation with Herpesvirus saimiri now allows the production of virtually unlimited numbers of (auto)antigen-specific T cells expressing functional TCR and a stable membrane phenotype. This technology will facilitate studies of the pathogenesis of putative autoimmune diseases, such as multiple sclerosis, and may be of help in TCR-targeted immunotherapy.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Base Sequence; CD2 Antigens; CD4-Positive T-Lymphocytes; CD58 Antigens; Cell Transformation, Viral; Cytokines; DNA Primers; Herpesvirus 2, Saimiriine; Humans; Lymphocyte Activation; Membrane Glycoproteins; Molecular Sequence Data; Myelin Basic Protein; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Immunologic

We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34 degrees C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39 degrees C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39 degrees C for > 9 days compared with cells maintained at 34 degrees C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers--galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 18.5-, and 21.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a "mature" oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of "maturation." This cell line has now been passaged > 40 times with fidelity of phenotype and genotype.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Animals, Newborn; Antigens, Polyomavirus Transforming; Base Sequence; Brain; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Crosses, Genetic; Mice; Mice, Inbred BALB C; Mice, Neurologic Mutants; Molecular Sequence Data; Myelin Basic Protein; Myelin Proteins; Myelin Sheath; Oligodendroglia; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Biosynthesis; Proteolipids; Recombinant Fusion Proteins; RNA, Messenger; Simian virus 40; Temperature

Immunization of experimental animals with myelin basic protein (MBP) or with specific MBP encephalitogenic determinants induces an autoimmune central nervous system (CNS) disease, experimental allergic encephalomyelitis, often studied as a model for human demyelinating disorders. This study examines the antigenic determinants of MBP recognized by human T cells using overlapping, synthetic peptides and T cell lines and clones isolated from four HLA-typed, neurologically normal subjects. T cell lines and clones isolated from individual subjects recognized at least one and as many as five distinct T cell determinants. In some instances the peptides recognized included determinants previously shown to induce experimental allergic encephalomyelitis (EAE) in experimental animals. In this group of four subjects, some determinants of MBP, including residues 5-25, 35-47, 65-75, and 81-100, were recognized by T cells derived from more than one individual suggesting that these regions may be particularly immunogenic for humans.

Topics: Antigen-Presenting Cells; B-Lymphocytes; Cell Transformation, Viral; Clone Cells; Epitopes; Herpesvirus 4, Human; Humans; Myelin Basic Protein; Peptides; T-Lymphocytes; T-Lymphocytes, Cytotoxic

Antibody-producing B lymphocytes were polyclonally activated and transformed, by Epstein-Barr virus (EBV), into multiple B lymphoblastoid cell lines in a microculture system. The frequencies of B precursor cells producing antibodies to myelin basic protein (MBP) and measles virus were analyzed in peripheral blood of patients with multiple sclerosis (MS) and control subjects. Measles virus-specific B cells were detected at a significantly higher frequency in MS patients (n = 10, P less than 0.005) than patients with other neurological diseases (n = 10) and normal subjects (n = 10). In contrast, the frequencies of B cells producing anti-MBP antibodies and natural antibodies did not differ statistically among the three groups tested (P greater than 0.05). In addition, the anti-MBP antibodies produced by a panel of stable B cell lines obtained were found to react selectively with an epitope(s) within the C-terminal half fragment 90-171 of the human MBP molecule. In our experiments, no antibody cross-reactivity between MBP and measles virus could be detected in a total of 2760 B cell cultures.

Topics: B-Lymphocytes; Cell Transformation, Viral; Cells, Cultured; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Herpesvirus 4, Human; Histones; Humans; Immunoglobulin M; In Vitro Techniques; Measles virus; Multiple Sclerosis; Mumps virus; Myelin Basic Protein; Tetanus Toxoid

In this study we describe for the first time the production of stable human B cell lines and clones that secrete IgM antibody specific for human myelin basic protein. The technique based on limiting dilutions of Epstein-Barr virus-transformed peripheral B cells from patients with multiple sclerosis, precluded the need for preselecting or stimulating antigen-specific B cells. Most of the cell lines were stable for at least 6 months in continuous culture and produced 5-12 micrograms/ml antibody after 2 weeks in culture. The myelin basic protein-specific B cells were surface IgM positive, and occurred with a frequency of approximately 1/2500 mononuclear cells in peripheral blood. The successful selection and quantitation of specific B cell clones described here suggests that this technique is well suited for evaluating B cell responses to known and suspected antigens and autoantigens.

Topics: Antibody Specificity; B-Lymphocytes; Cell Line; Cell Transformation, Viral; Clone Cells; Herpesvirus 4, Human; Humans; Immunoglobulin M; Kinetics; Myelin Basic Protein

Spleen-cell sensitivity to encephalitogenic factor (EF) was measured with the macrophage migration inhibition (MMI) test over a period of time in hamsters inoculated with SV40-transformed tumour cells and in rats treated with 4-dimethylamino-3'-methylazobenzene.Spleen cells from hamsters receiving 10 or 10(3) SV40 tumour cells gave inhibition of macrophage migration with EF at a significance level of P<0·05 21 days after implantation. Spleen cells from animals receiving 10(5) tumour cells gave inhibition at a significance level of P<0·001 after the same interval.Spleen-cell sensitivity to EF, and the abrogation of this sensitivity by serum, was investigated over a period of time in rats undergoing hepatocarcinogenesis. Sensitivity to EF was seen in 2/10 animals (20%) with minimal lesions of the liver, in 2/16 animals (12%) with proliferative changes and/or cholangiofibrosis, in 7/15 animals (46%) with dysplastic lesions of portal-tract epithelial cells and in all 5 animals with cholangiocarcinoma. None of a control group of 10 animals showed any response to EF. Autologous serum abrogated the spleen-cell response to EF in one sensitized animal with proliferative changes and cholangiofibrosis, in all 7 sensitized animals with dysplastic hepatic lesions and in 4/5 sensitized animals with cholangiocarcinoma. Autologous serum had no effect on macrophage migration in the 10 control animals.These findings indicate that a progressive increase in sensitization to EF occurs during carcinogenesis and is evident at the point of preneoplastic dysplasia. This has an obviously important bearing on the clinical use of such tests.

Topics: Animals; Cell Migration Inhibition; Cell Transformation, Viral; Hypersensitivity, Delayed; Immunity, Cellular; Liver Neoplasms; Macrophages; Myelin Basic Protein; Neoplasms, Experimental; Rats; Spleen; Time Factors