A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines.

The minimal region of the human tumor necrosis factor alpha (TNF-alpha) gene promoter necessary for its transcriptional induction by phorbol esters (PMA) in human T and B lymphocyte cell lines has been localized between -52 and +89 nucleotides (nt) relative to the gene's transcriptional start site. Comparison of these sequences to those required to mediate virus or lipopolysaccharide (LPS) induction of the gene reveal significant differences, and thus, the sequence requirements for PMA induction are distinct from those that mediate induction by virus or LPS. Although three sites in the TNF-alpha promoter (kappa 1, kappa 2, and kappa 3) specifically bind the transcription factor NF-kappa B in lymphoid nuclear extracts, TNF-alpha mRNA induction by PMA does not correlate with NF-kappa B binding activities displayed by different T and B cell lines. Moreover, kappa 1-kappa 3 can each be deleted from the TNF-alpha promoter with little effect on the gene's inducibility by PMA. Therefore, TNF-alpha mRNA induction by PMA, like its induction by virus and LPS, is not primarily mediated by NF-kappa B, but rather is mediated through other sequences and protein factors. Surprisingly, multimers of kappa 1-kappa 3 can confer PMA inducibility on a heterologous promoter in a B (Raji), but not a T (HUT78) cell line. However they are not functional on a truncated TNF-alpha promoter, indicating that promoter context and cell type specificity influence the PMA inducible function of these NF-kappa B binding sites.

Novel surface antigen expressed on dividing cells but absent from nondividing cells.

Radioimmunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the distribution on human lymphoid cells of a previously undescribed surface antigen recognized by several heteroantisera. A glycoprotein with a 90,000 mol wt (under reducing conditions) was detected on all cell lines tested including T, B, null, and myeloid cell lines, although the amount of antigen present varied considerably. The antigen was absent from normal peripheral blood lymphocytes (PBL), B and T cells, monocytes, granulocytes, thymocytes, and erythrocytes. After stimulation with lectins or allogeneic B cells, the antigen was induced on PBL T cells. A limited number of leukemic T cells tested all expressed the antigen, as did melanoma cell line and human embryonic lung fibroblasts. Hence, the antigen was present only on dividing lymphoid cells and absent from nondividing cells, but was also present on the two examples of dividing non-lymphoid cells tested. Under nonreducing conditions, the 90,000-mol wt band normally present disappeared to be replaced by another at approximately 200,000 mol wt. The glycoprotein bound to lectins from lentil and ricin, but not to wheat germ agglutinin. It could be readily labeled metabolically by [35S]methionine or by surface iodination, and appeared to be a major membrane protein on some cell lines.

Demonstration of structural polymorphism among HLA-DR light chains by two-dimensional gel electrophoresis.

Human HLA-DR antigens were immunoprecipitated from Nonidet P-40 extracts of [35S]methionine-labeled B lymphoblastoid cell lines and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Two-dimensional (2-D) gel analyses, combining SDS-PAGE in the first dimension and IEF in the second dimension, revealed that the heavy (alpha) and light (beta) chains of each DRw specificity displays microheterogeneity of charge. However, the pattern of the heavy chain did not vary among different DRw specificities. In contrast, the light chains of different DRw types varied both in apparent size and charge distribution. Removal of sialic acids with neuraminidase or inhibition of glycosylation with tunicamycin reduced the microheterogeneity of both DR subunits. However, the heavy and light chains each still focused as two major bands, suggesting that other post-translational modifications contribute to the microheterogeneity or that there are two nonallelic DR-like molecules. After treatment with either neuraminidase or tunicamycin, the DR light chains, but not the heavy chains, were still structurally polymorphic. The DR light chains of serologically cross-reactive specificities displayed similar 2-D gel patterns suggesting that the structural polymorphism of the DR light chains is the basis for the serologically detected polymorphism of the HLA-DR antigens. Two additional polypeptides were observed in immunoprecipitates of DR antigens. These proteins, designated M1 and M2, both had a basic isoelectric point and were invariant among different cell lines. The protein M1 may be intracellular because it can not be immunoprecipitated from the cell surface.

Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor.

Hen egg lysozyme 52-61-specific CD4+ T cells responded by interleukin 2 (IL-2) secretion to any peptide containing this epitope regardless of length of NH2- and COOH-terminal composition. However, CD4- variants could only respond to peptides containing the two COOH-terminal tryptophans at positions 62 and 63. Substitutions at these positions defined patterns of reactivity that were specific for individual T cells inferring a T cell receptor (TCR)-based phenomenon. Thus, the fine specificity of major histocompatibility complex (MHC)-peptide recognition by the TCR was dramatically affected by CD4 and the COOH-terminal peptide composition. Peptides that failed to induce IL-2 secretion in the CD4- variants nevertheless induced strong tyrosine phosphorylation of CD3 zeta. Thus, whereas the TCR still recognized and bound to the MHC class II-peptide complex resulting in protein phosphorylation, this interaction failed to induce effective signal transduction manifested by IL-2 secretion. This provides a clear example of differential signaling mediated by peptides known to be naturally processed. In addition, the external domains of CD4, rather than its cytoplasmic tail, were critical in aiding TCR recognition of all peptides derived from a single epitope. These data suggest that the nested flanking residues, which are present on MHC class II but not class I bound peptides, are functionally relevant.

Selective release of some invariant chain-derived peptides from HLA-DR1 molecules at endosomal pH.

The predominant peptides bound to major histocompatibility complex class II molecules expressed on human B cells are derived from a relatively limited number of self proteins. To determine whether any of the prebound self peptides might be released in endosomes during recycling, water-soluble HLA-DR1 molecules were incubated with a high affinity synthetic peptide at pH 4.0 and 7.0 at 37 degrees C. The resulting bound peptide repertoire was then acid extracted, and separated by reversed-phase high performance liquid chromatography. Using a combination of mass spectrometry and ultraviolet spectroscopy, prebound self peptides and newly bound synthetic peptide were characterized. Most self peptides bound to HLA-DR1 were not appreciably released during extended exposure to pH 4.0. However, some invariant chain-derived peptides were uniquely released at this pH.

An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.

The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.

Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter.

Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells.

HLA-DR antigens have polymorphic light chains and invariant heavy chains as assessed by lysine-containing tryptic peptide analysis.

HLA-DR antigens were isolated from B lymphoblastoid cells labeled with either [3H]- or [14C]lysine. Double-label comparisons of the tryptic peptides by microbore ion-exchange chromatography led to the following conclusions: The heavy and light chains share few peptides, one of which is free lysine that is liberated from the polypeptide chains by trypsin. The heavy chains of different HLA-DR allospecificities are virtually identical in all cell lines examined. In contrast, the light chains of different HLA-DR allospecificities are nonidentical and, thus, must bear the alloantigenic determinant.

Evidence for a shared HLA-A intralocus determinant defined by monoclonal antibody 131.

We describe here a monoclonal antibody, 131, which appears to recognize a determinant shared by HLA-A locus-encoded gene products. Isoelectric focusing analysis demonstrates that 131 reacts with the products of at least seven different HLA-A alleles but none of the five HLA-B allelic products tested. Together with evidence provided by other studies, this finding indicates the existence of A-unique and B-unique determinants, which may have different biological functions. Monoclonal antibody probes, such as the one described here, specific for shared intralocus determinants, may be valuable for assessing these possible functional differences.

Identification of shared antigenic determinants of the putative human T lymphocyte antigen receptor.

A mouse antiserum, anti-gp40,49 was obtained by immunizing BALB/c mice with the putative T cell antigen receptor isolated from HPB-MLT cells. This antiserum reacted with peripheral blood lymphocyte (PBL) and a panel of immunocompetent T cell lines and clones in each case precipitating from lysates of cells labeled by surface iodination, a disulfide-linked dimer consisting of an alpha subunit Mr (46,000-49,000) and a beta subunit Mr (40,000-45,000). Variability in Mr of the two subunits, particularly of the beta (light) subunit, was observed when the receptors of immunocompetent T cell lines with different antigen specificities were compared. Anti-gp40,49 serum reacted selectively with the alpha subunit after reduction and alkylation of the protein complex. These results confirm the relationship between the gp40,49 protein complex of HPB-MLT cells and the putative T cell antigen receptor on normal immunocompetent T cells and indicate that the alpha subunit of the human receptor expressed shared determinant(s) that are immunogenic in the mouse. Some features of the T cell antigen receptor appear to be unusual in that even with a xenoantiserum against the purified molecule, only antibodies against clonotypic determinants could be detected at the cell surface by quantitative immunofluorescence analysis.

HLA-A2 mutants immunoselected in vitro. Definition of residues contributing to an HLA-A2-specific serological determinant.

The HLA-A2-specific mouse monoclonal antibody BB7.2 plus complement has been used to immunoselect variant clones of the lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, and -B27). Members of one class of variant clones appear to express cell surface HLA-A2 molecules that display reduced reactivity with the selecting antibody, but normal or near normal reactivities with some other HLA-A2-specific monoclonal antibodies and human alloantisera. The HLA-A2 heavy chains derived from two of these variant clones were characterized by comparative double-label tryptic peptide mapping in conjunction with microsequence analysis. These heavy chains were found to carry distinct mutations in the same peptide in the molecule. We conclude that residues within this short segment of the polypeptide contribute to an HLA-A2-specific serological determinant.

Suppression of in vitro Epstein-Barr virus infection. A new role for adult human T lymphocytes.

Studies have been performed on in vitro infection by Epstein-Barr virus (EBV) of subpopulations of human lymphocytes. B cells of adult peripheral or fetal cord blood transform with equal efficiency, whether assayed by DNA synthesis induction or by outgrowth of transformed lymphocytes. In contrast, unfractionated adult lymphocytes transform much less efficiently than those from fetal cord. Reconstitution experiments of different cell preparations indicated that this difference was due to a suppression of B-cell proliferation by adult Ig-negative lymphocytes which fetal Ig-negative lymphocytes were unable to perform. Separation of Ig-negative lymphocytes into various subpopulations revealed that the suppression was performed by T cells. Macrophages and null cells play little or no role in suppression. The relevance of this phenomenon to infection and recovery from EBV infection during and after infectious mononucleosis is discussed.

Cooperativity between the J and S elements of class II major histocompatibility complex genes as enhancers in normal and class II-negative patient and mutant B cell lines.

The class II major histocompatibility complex genes all contain in their proximal promoters three cis-elements called S, X, and Y that are conserved in both sequence and position, and a fourth element, J, conserved in sequence but not in position. J, X, and Y and, to some extent, S, have been shown to be functionally important in regulation of expression of these genes. In the present study, a protein factor that binds cooperatively to the S plus J elements of the promoter of the class II major histocompatibility complex gene DPA has been detected. Moreover, functional cooperativity between S and J in activation of the enhancerless -40 interferon-beta (-40 IFN-beta) promoter has been demonstrated. Finally, the latter assay appears to subdivide complementation group A of class II negative human B cell lines that includes both mutants generated in vitro and cells from patients with the bare lymphocyte syndrome (type II). In three of these cell lines, the enhancerless -40 IFN-beta promoter containing the S plus J elements was functionally active, while in the others it was inactive.

Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression.

The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.

Modulation of HLA class II antigen expression by transfection of sense and antisense DR alpha cDNA.

In the human there are three isotypic forms of MHC class II gene products (HLA-DR, -DQ, and -DP). The isotype-matched alpha-beta dimers are predominant but isotype-mismatched dimers can also be expressed (DR alpha-DQ beta). Here it is shown that the expression of the DR alpha-DQ beta dimer can be correlated to a high ratio of DR alpha/DR beta mRNA. The DR alpha chain expression was modulated by transfection of a sense and antisense DR alpha cDNA. Overexpression of DR alpha promoted the appearance of the DR alpha-DQ beta dimer. On the other hand, pre-existing DR alpha-DQ beta dimer expression was suppressed after antisense DR alpha cDNA transfection. Therefore, imbalanced expression of the alpha and beta chain from a given isotype could lead to the modification of HLA class II phenotype.

Induction and suppression of an autoimmune disease by oligomerized T cell epitopes: enhanced in vivo potency of encephalitogenic peptides.

T cell epitope peptides derived from proteolipid protein (PLP139-151) or myelin basic protein (MBP86-100) induce experimental autoimmune encephalomyelitis (EAE) in "susceptible" strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund's adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the peptide epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10.S mice, "resistant" to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50 microg of the PLP139-151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139-151 peptide accelerated rather than retarded the progression of disease.

Cell-free synthesis and processing of the heavy and light chains of HLA-DR antigens.

Antisera have been prepared against the separated glycoprotein chains (p29 and p34) of HLA-DR antigens (p29,34) isolated from membranes of the B lymphoblastoid cell line JY (DRw4,6). These antisera (anti-p29 and anti-p34) were characterized by immunoprecipitation of in vivo labeled, detergent-solubilized extracts of JY cells grown in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. Anti-p29 and anti-p34 specifically immunoprecipitated the precursors to p29 and p34, respectively, from the cell-free translation products of a rabbit reticulocyte lysate system supplemented with polyadenylic acid-containing messenger RNA (mRNA) isolated from JY cells. These precursors, pre-p34 and two pre-p29s, appeared to be 1,500-3,000 daltons larger than their counterparts from tunicamycin-treated cells and presumably were synthesized with N-terminal extensions (signal sequences). Processing of pre-p29 and pre-p34 occurred during cell-free translation in the presence of dog pancreatic microsomes, which resulted in cleavage of the polypeptide, addition of glycan moieties, and segregation of the two chains in the cisternal portion of the microsome. the precursors of p29 and p34 were not appreciably different in isoelectric points on two-dimensional gels from p26 and p28, the light and heavy chains from antigens arise from separate mRNA, and not as a single polypeptide, which is endoproteolytically cleaved.

Specificity of mouse cytotoxic T lymphocytes stimulated with either HLA-A and -B or HLA-DR antigens reconstituted into phospholipid vesicles.

Distinct populations of murine cytolytic T lymphocytes (CTL) were elicited from primed spleen cells by preparations of HLA-A and -B (HLA-A,B) or HLA-DR antigens reconstituted into phospholipid vesicles. These populations could be distinguished by both antiserum blocking and by patterns of cytolysis of a panel of target cells. Cytolysis by CTL stimulated with liposomes that contained HLA-A2 and HLA-B7 antigens could only be blocked by antiserum against HLA-A,B antigens but not by antiserum against HLA-DR antigens. The inverse pattern was seen with HLA-DR-stimulated CTL. When compared with a panel of target cells expressing various HLA-A,B or HLA-DR allospecificities, the strongest CTL reactivity was seen toward those cells that bore the same allospecificities as those presented on the liposomes. Target cells that expressed cross-reactive specificities and unrelated specificities were recognized much less well. The implications of the results for the mechanism of CTL stimulation by liposomes, as well as the relationship between allogeneic and xenogeneic CTL recognition, are discussed.

A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor.

The class II major histocompatibility complex (MHC) molecules function in the presentation of processed peptides to helper T cells. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen-presenting cell is its ability to express class II MHC molecules. Expression of these molecules is usually restricted to cells of the immune system and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. Human complementary DNA clones encoding a newly identified, cysteine-rich transcription factor, NF-X1, which binds to the conserved X-box motif of class II MHC genes, were obtained, and the primary amino acid sequence deduced. The major open reading frame encodes a polypeptide of 1,104 amino acids with a symmetrical organization. A central cysteine-rich portion encodes the DNA-binding domain, and is subdivided into seven repeated motifs. This motif is similar to but distinct from the LIM domain and the RING finger family, and is reminiscent of known metal-binding regions. The unique arrangement of cysteines indicates that the consensus sequence CX3CXL-XCGX1-5HXCX3CHXGXC represents a novel cysteine-rich motif. Two lines of evidence indicate that the polypeptide encodes a potent and biologically relevant repressor of HLA-DRA transcription: (a) overexpression of NF-X1 from a retroviral construct strongly decreases transcription from the HLA-DRA promoter; and (b) the NF-X1 transcript is markedly induced late after induction with interferon gamma (IFN-gamma), coinciding with postinduction attenuation of HLA-DRA transcription. The NF-X1 protein may therefore play an important role in regulating the duration of an inflammatory response by limiting the period in which class II MHC molecules are induced by IFN-gamma.