ABSTRACT
The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.
Subject(s)
Anthrax/drug therapy , Antigens, Bacterial/toxicity , Bacillus anthracis/pathogenicity , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/toxicity , Hydroxamic Acids/pharmacology , Models, Molecular , Animals , Antigens, Bacterial/metabolism , Bacillus anthracis/metabolism , Bacterial Toxins/metabolism , Ciprofloxacin/therapeutic use , Crystallography , Cytotoxicity Tests, Immunologic , DNA Primers , Drug Therapy, Combination , Hydroxamic Acids/metabolism , Hydroxamic Acids/therapeutic use , Macrophages/metabolism , Mice , Mice, Inbred BALB C , RabbitsABSTRACT
L-826,141 [4-(2-(3,4-bis-difluromethoxyphenyl)-2-(4-(1,1,1, 3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl)-3-methylpyridine-1-oxide] is a selective and potent inhibitor of phosphodiesterase 4 (PDE4) with an IC(50) value of 0.26 to 2.4 nM for inhibition of the catalytic activity of PDE4A, B, C, and D. The cAMP elevation that can be maintained by PDE4 inhibitors attenuates the signaling cascades that lead to the production of certain cytokines. In cellular-based assays, L-826,141 transcriptionally down-regulates production of tumor necrosis factor (TNF)-alpha in peripheral blood mononuclear cell and whole blood assays with IC(50) values of 31 and 310 nM, respectively. Profiling the effect of this compound on various cytokines in the signaling cascade attenuated by cAMP elevation demonstrates that L-826,141 is also a potent inhibitor of interleukin (IL)-12, granulocyte macrophage-colony stimulating factor, and interferon (IFN)gamma (IC(50) values of 0.3-0.9 microM) as well as TNF-alpha formation. We have also shown that the PDE4 inhibitors rolipram and L-826,141 are potent inhibitors of CD3-plus CD28-stimulated IL-2 production in naive human T cells. To address the effect of PDE4 inhibitors on cytokine release from T helper (Th)1 and Th2 effector cells, we used a well characterized model in which T cells are derived from ovalbumin (323-339)-specific T cell receptor transgenic mice. L-826,141 inhibits Th0-mediated IL-2 production with an IC(35) value of 25 nM and Th1-mediated IFNgamma production with an IC(30) value of 46 nM. In contrast, L-826,141 had no significant inhibitory effect (IC(30) value > 2.5 microM) on Th2 cell-mediated IL-4 nor IL-13 production. Together, these data demonstrate that specific inhibition of PDE4 preferentially blocks the production of Th1 versus Th2 effector cytokines in vitro.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Cytokines/antagonists & inhibitors , Immunosuppressive Agents/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Pyridines/pharmacology , Th1 Cells/metabolism , Th2 Cells/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Animals , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cytokines/metabolism , Dose-Response Relationship, Drug , Humans , Mice , Mice, Inbred BALB C , Mice, Transgenic , Phosphodiesterase Inhibitors/chemistry , Pyridines/chemistry , Th1 Cells/drug effects , Th2 Cells/drug effectsABSTRACT
Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.
Subject(s)
Antigen-Presenting Cells/immunology , Epitopes, T-Lymphocyte/metabolism , Immunodominant Epitopes/metabolism , Amino Acid Sequence , Animals , Antigen Presentation , Antigen-Presenting Cells/metabolism , Cell Line , Epitopes, T-Lymphocyte/immunology , HLA Antigens/chemistry , HLA Antigens/immunology , HLA Antigens/metabolism , Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Humans , Immunization, Passive , Immunoglobulin G/administration & dosage , Immunoglobulin G/immunology , Immunoglobulin kappa-Chains/immunology , Immunoglobulin kappa-Chains/metabolism , Immunoglobulins/immunology , Immunoglobulins/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Peptides/immunology , Peptides/metabolism , Protein Binding/immunology , Protein Structure, Tertiary , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.
Subject(s)
Protein-Tyrosine Kinases/analysis , Amino Acid Sequence , Animals , Chemistry Techniques, Analytical/methods , Drosophila , Humans , Indicators and Reagents , Jurkat Cells , Miniaturization , Molecular Sequence Data , Substrate Specificity , TitrimetryABSTRACT
HLA-DM catalyzes the release of invariant chain fragments from newly synthesized major histocompatibility complex (MHC) class II molecules, stabilizes empty class II molecules, and edits class II-associated peptides by preferentially releasing those that are loosely bound. The ability of HLA-DM to carry out these functions in vitro is pH dependent, with an optimum at pH 4.5-5.5 and poor activity at pH 7. The structural basis for these properties of HLA-DM is unknown. Sequence homology suggests that HLA-DM resembles classical, peptide-binding MHC class II molecules. In this study, we examined whether HLA-DM has a secondary structure composition consistent with an MHC fold and whether HLA-DM changes conformation between pH 5 and pH 7. Far-UV circular dichroism (CD) spectra of recombinant soluble HLA-DM (sDM) indicate that HLA-DM belongs to the alpha/beta class of proteins and structurally resembles both MHC class I and class II molecules. The CD peak around 198 nm increases upon going from neutral to endosomal pH and drops sharply upon denaturation below pH 3.5, distinguishing at least three states of sDM: the denatured state and two highly similar folded states. Fluorescence emission spectra show a slight blue-shift and a approximately 20% drop in intensity at pH 5 compared with pH 7. Unfolding experiments using guanidinium chloride show that the stability of sDM is somewhat reduced but not lost at pH 5. These results indicate that sDM undergoes a pH-dependent conformational change between neutral and endosomal pH. The change seems to involve both hydrogen bonding patterns and the hydrophobic core of sDM and may contribute to the pH dependence of DM activity.
Subject(s)
HLA-D Antigens/chemistry , Histocompatibility Antigens Class II , Recombinant Proteins/chemistry , Circular Dichroism , Guanidine/pharmacology , HLA-D Antigens/drug effects , HLA-D Antigens/genetics , Humans , Hydrogen-Ion Concentration , Protein Denaturation , Protein Structure, Secondary , Recombinant Proteins/drug effects , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Tryptophan/chemistryABSTRACT
The three-dimensional structure of the soluble ecto-domain of HLA-DM has been determined to 2.5 A resolution by X-ray crystallography. HLA-DM has both peptide exchange activity and acts as a chaperone to peptide-free class II MHC molecules. As predicted, the structure is similar to that of classical class II MHC molecules except that the peptide-binding site is altered to an almost fully closed groove. An unusual cavity is found at the center of the region that binds peptides in class II MHC molecules, and a tryptophanrich lateral surface is identified that is a candidate both for binding to HLA-DR, to effect catalysis, and to HLA-DO, an inhibitor.
Subject(s)
HLA-D Antigens/chemistry , Antigen Presentation/physiology , Antigens/metabolism , Catalysis , Crystallography, X-Ray , HLA-DR Antigens/chemistry , Histocompatibility Antigens Class II/metabolism , Humans , Molecular Sequence Data , Peptides/immunology , Protein Conformation , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.
Subject(s)
Arthritis/etiology , Autoimmune Diseases/etiology , Collagen/immunology , HLA-DR4 Antigen/physiology , Amino Acid Sequence , Animals , CD4 Antigens/physiology , Cattle , HLA-DR4 Antigen/genetics , Humans , Immunization , Mice , Mice, Transgenic , Molecular Sequence Data , T-Lymphocytes/immunologyABSTRACT
Genetic predisposition to rheumatoid arthritis (RA) is linked to the MHC class II allele HLA-DR4. The charge of the amino acid at DRbeta71 in the peptide-binding site appears to be critical in discriminating DR molecules linked to increased disease susceptibility. We have determined the 2.5 A x-ray structure of the DR4 molecule with the strongest linkage to RA (DRB1*0401) complexed with a human collagen II peptide. Details of a predicted salt bridge between lysine DRbeta71 and aspartic acid at the P4 peptide position suggest how it may participate in both antigen binding and TCR activation. A model is proposed for the DR4 recognition of collagen II (261-273), an antigen immunodominant in human-transgenic mouse models of RA.
Subject(s)
Collagen/immunology , HLA-DR4 Antigen/ultrastructure , Alleles , Amino Acid Sequence , Animals , Arthritis, Rheumatoid/immunology , Binding Sites , Collagen/chemistry , Collagen/ultrastructure , Crystallography, X-Ray , Dimerization , Enterotoxins/chemistry , Epitope Mapping , HLA-DR4 Antigen/genetics , Humans , Mice , Mice, Transgenic , Models, Molecular , Peptides/chemistry , Peptides/immunology , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins , Superantigens/chemistryABSTRACT
Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.
Subject(s)
Arthritis, Experimental/immunology , B-Lymphocytes/immunology , Collagen/immunology , HLA-DR1 Antigen/biosynthesis , T-Lymphocytes/immunology , Adjuvants, Immunologic , Amino Acid Sequence , Animals , Antibody Formation , Arthritis, Experimental/genetics , Arthritis, Experimental/pathology , Base Sequence , DNA Primers , Disease Susceptibility , Exons , HLA-DR1 Antigen/chemistry , HLA-DR1 Antigen/genetics , Humans , Immunity, Cellular , Joints/immunology , Joints/pathology , Mice , Mice, Transgenic , Molecular Sequence Data , Peptide Fragments/analysis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesisABSTRACT
The identification of class II binding peptide epitopes from autoimmune disease-related antigens is an essential step in the development of antigen-specific immune modulation therapy. In the case of type 1 diabetes, T cell and B cell reactivity to the autoantigen glutamic acid decarboxylase 65 (GAD65) is associated with disease development in humans and in nonobese diabetic (NOD) mice. In this study, we identify two DRB1*0401-restricted T cell epitopes from human GAD65, 274-286, and 115-127. Both peptides are immunogenic in transgenic mice expressing functional DRB1*0401 MHC class II molecules but not in nontransgenic littermates. Processing of GAD65 by antigen presenting cells (APC) resulted in the formation of DRB1*0401 complexes loaded with either the 274-286 or 115-127 epitopes, suggesting that these naturally derived epitopes may be displayed on APC recruited into pancreatic islets. The presentation of these two T cell epitopes in the islets of DRB1*0401 individuals who are at risk for type 1 diabetes may allow for antigen-specific recruitment of regulatory cells to the islets following peptide immunization.
Subject(s)
Autoantibodies/analysis , Diabetes Mellitus, Type 1/immunology , Epitopes/analysis , Glutamate Decarboxylase/immunology , HLA-DR Antigens/genetics , T-Lymphocytes/immunology , Alleles , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , Cell Line , Diabetes Mellitus, Type 1/genetics , Epitopes/chemistry , Genes, MHC Class II , Glutamate Decarboxylase/biosynthesis , HLA-DR Antigens/biosynthesis , HLA-DRB1 Chains , Humans , Lymphocyte Activation , Mice , Mice, Inbred NOD , Mice, Transgenic , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunologySubject(s)
Encephalomyelitis, Autoimmune, Experimental/etiology , Animals , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Humans , Immune Tolerance , Mice , Mice, Transgenic , Myelin Basic Protein/immunology , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunologyABSTRACT
The evolution of Lyme arthritis in DR4-transgenic mice infected with Borrelia burgdorferi was studied because chronic Lyme arthritis in humans is associated with an increased frequency of the HLA-DR4 allele. B10 nontransgenic and DR4-transgenic mice expressing chimeric human-mouse major histocompatibility complex class II genes in which the human alpha 1 and beta 1 domains of DR4Dw4 replaced the corresponding domains of the mouse I-E(d) were inoculated with B. burgdorferi and examined at up to 180 days for infection and disease. All mice were infected throughout the 180 days, and arthritis evolved to equal severity in transgenic and control mice within 30 days and resolved by day 120. Both groups of mice developed high antibody titers to B. burgdorferi, but antibodies to outer surface proteins A and B were not readily detectable. The DR4Dw4 transgene did not predispose mice to the development of chronic Lyme arthritis.
Subject(s)
HLA-DR4 Antigen/biosynthesis , Lyme Disease/physiopathology , Alleles , Animals , Antibody Formation , Borrelia burgdorferi Group/immunology , Borrelia burgdorferi Group/pathogenicity , Enzyme-Linked Immunosorbent Assay , Genes, MHC Class II , HLA-DR4 Antigen/genetics , Humans , Lyme Disease/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, TransgenicABSTRACT
A method to predict quantitatively peptide binding to HLA DRB1*0401 has been developed using a data set of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide back-bone. The prediction assumed that the relative role of each of the peptide side chains could be treated independently and could be measured by assaying each of the 20 naturally occurring amino acids at the central 11 positions of a 13-residue peptide previously shown to contain the minimal requirements for high-affinity binding to HLA-DR proteins. The resultant database was shown to have predictive value when tested on a set of 13 unrelated peptides known to bind DRB1*0401 with a wide range of apparent affinity. The database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401-restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted and shown to bind the class II protein with high affinity.
Subject(s)
HLA-DR Antigens/chemistry , HLA-DR Antigens/metabolism , Peptides/immunology , Amino Acid Sequence , Animals , HLA-DR Antigens/isolation & purification , HLA-DRB1 Chains , Humans , Hybridomas/immunology , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , Molecular Sequence Data , Protein Binding/immunology , T-Lymphocytes/immunologyABSTRACT
Human leukocyte antigen (HLA)-DM is an unconventional major histocompatibility complex (MHC) class II heterodimer that is important for B-cell-mediated antigen processing and presentation to MHC class II-restricted T cells. HLA-DM is encoded by two genes, DMA and DMB, which map to the MHC class II region, and shares some homology with MHC class I and class II proteins. Here we define the biochemical role of HLA-DM. Recombinant soluble HLA-DM heterodimers have been purified from culture supernatants of insect cell transformants. At pH 5.0, they induce the dissociation of a subset of peptides bound to HLA-DR, including a nested set of class-II-associated invariant chain peptides (CLIP). This process liberates HLA-DR and leads to the enhanced binding of exogenous peptides.
Subject(s)
Antigens, Differentiation, B-Lymphocyte , B-Lymphocytes/immunology , HLA-D Antigens/physiology , HLA-DR Antigens/metabolism , Oligopeptides/metabolism , Amino Acid Sequence , Animals , Cell Line , Drosophila , HLA-DR Antigens/chemistry , Histocompatibility Antigens Class II/metabolism , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Conformation , Transformation, GeneticABSTRACT
A method to quantitatively predict peptide binding to HLA DRB1*0401, B1*0101, and B1*1501 has been developed using a dataset of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide backbone. The prediction assumed that the relative role of each of the peptide sidechains could be treated independently and could be measured by assaying each of the twenty naturally occurring amino acids at the central eleven positions of a 13 residue peptide previously shown to contain the minimal requirements for high affinity binding to HLA DR proteins. Three separate databases were generated. They were shown to have predictive value when tested on a set of 13 unrelated peptides known to bind the DR proteins with a wide range of apparent affinity. The DRB1*0401 database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401 restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted, and shown to bind the class II protein with high affinity.
Subject(s)
HLA-DR1 Antigen/metabolism , Peptides/metabolism , Amino Acid Sequence , Animals , Humans , Hybridomas/immunology , Mice , Models, Chemical , Myelin Basic Protein/chemistry , Myelin Basic Protein/immunology , Myelin Basic Protein/metabolism , Peptides/chemistry , Peptides/immunology , Protein Binding/immunology , Serum Albumin/chemistry , Serum Albumin/immunology , Serum Albumin/metabolism , T-Lymphocytes/immunologyABSTRACT
Hydrogen bonding between conserved amino acids in the HLA DR and the peptide backbone of the ligand both provide the majority of free energy of binding and force the peptide ligands to adopt a similar extended conformation. Consequently the corresponding side chains of all peptides interact with similar pockets in the binding site. For peptides of a common length the contribution of the peptide backbone can be treated as a constant and the differential affinity can be viewed as a simple sum of the side chain interactions. These can be quantified by measuring the effects of each of the naturally occurring amino acids in the context of a simplified polyalanine backbone containing an aromatic amino acid to orient the peptide unequivocally in the binding site. The dataset of the relative contributions can be used to predict quantitatively the affinity of any peptide sequence.
Subject(s)
HLA-DR Antigens/chemistry , Peptides/metabolism , Amino Acid Sequence , Humans , Molecular Sequence Data , Protein Binding , Protein ConformationABSTRACT
Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying human immunology. However, attempts to obtain human class II-restricted T cell responses in such transgenic mice have had only limited success, possibly due to an inability of mouse CD4 to interact efficiently with human MHC class II molecules. To circumvent this problem, we constructed recombinant MHC class II genes in which the peptide-binding domain was derived from human DR sequences whereas the CD4-binding domain was derived from mouse I-E sequences. Purified chimeric human/mouse MHC class II molecules were capable of specifically binding DR-restricted peptides. Human B cell transformants that expressed these chimeric MHC class II molecules could present peptide antigens to human T cell clones. Expression of these chimeric class II molecules in transgenic mice led to the intrathymic deletion of T cells expressing superantigen-reactive V beta gene segments, indicating that the chimeric class II molecules could influence the selection of the mouse T cell repertoire. These transgenic mice were fully capable of mounting human DR-restricted immune responses after challenge with peptide or whole protein antigens. Thus, the chimeric class II molecules can serve as functional antigen presentation molecules in vivo. In addition, transgenic mice expressing chimeric class II molecules could be used to generate antigen-specific mouse T cell hybridomas that were capable of interacting with human antigen-presenting cells.
Subject(s)
Histocompatibility Antigens Class II/physiology , Recombinant Fusion Proteins/physiology , T-Lymphocytes/immunology , Animals , Base Sequence , Humans , Hybridomas/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/geneticsABSTRACT
We constructed a transgenic mouse model that mimics the human autoimmune disease multiple sclerosis in its spontaneous induction and pathology. Transgenic mice were constructed expressing genes encoding a rearranged T cell receptor specific for myelin basic protein (MBP). T cell tolerance was not induced in the periphery, and functional, autoreactive T cells were found in the spleen and lymph nodes of these mice. Transgenic mice developed experimental allergic encephalomyelitis (EAE) following immunization with MBP and adjuvant plus pertussis toxin as well as with administration of pertussis toxin alone. Spontaneous EAE can develop in transgenic mice housed in a non-sterile facility but not in those maintained in a sterile, specific pathogen-free facility. This model system affords a unique opportunity to dissect the genetic and environmental variables that may contribute to the development of spontaneous autoimmune disease.
Subject(s)
Autoimmunity , Myelin Basic Protein/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Animals , Base Sequence , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Immune Tolerance , Lymphocyte Activation , Mice , Mice, Transgenic , Molecular Sequence Data , Multiple Sclerosis/genetics , Multiple Sclerosis/immunology , Oligodeoxyribonucleotides/chemistry , Polymerase Chain Reaction , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
Experimental allergic encephalomyelitis (EAE) is a model system for T cell-mediated autoimmune disease. Symptoms of EAE are similar to those of multiple sclerosis (MS) in humans. EAE is induced in susceptible animal strains by immunization with myelin basic protein (MBP) and potent adjuvant. The major T cell response to MBP in B10.PL mice is directed towards an NH2-terminal epitope and involves T cells expressing either V beta 8.2 or V beta 13 gene segments. Animals treated with a TCR V beta 8-specific mAb have a reduced incidence of EAE. We report here that the in vivo administration of a combination of anti-V beta 8.2 and anti-V beta 13 mAbs results in a long-term elimination of T cells involved in the response to MBP. When given before MBP immunization, anti-TCR antibody treatment leads to nearly complete protection against EAE. Antibody treatment also results in a dramatic reversal of paralysis in diseased animals. Thus, treatment with a combination of V beta-specific antibodies is a very effective therapy for the prevention and treatment of EAE. It is hoped that the future characterization of TCR V gene usage in human autoimmune diseases may lead to similar strategies of immune intervention.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Receptors, Antigen, T-Cell/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Autoimmune Diseases/immunology , Cell Separation , Encephalomyelitis, Autoimmune, Experimental/therapy , Flow Cytometry , Lymph Nodes/cytology , Lymphocyte Activation/immunology , Mice , Mice, Inbred Strains , Myelin Basic Protein/administration & dosage , Myelin Basic Protein/immunology , Paralysis/rehabilitation , Receptors, Antigen, T-Cell, alpha-betaABSTRACT
The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.