MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCII ⌬͞⌬ mice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of A␣ and A␤ knockout animals, notably a dearth of CD4 ؉ lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCII ⌬͞⌬ mice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering ''humanized'' mouse models of human MHC-II-associated autoimmune disorders.

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.

Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II–restricted presentation of central nervous system–derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2–peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2–peptide-specific antibodies from HLA-DR2–transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85–99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2–MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP–specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85–99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2–MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 2X%555 positioned within the catalytic domain shows very clear homology to E. coli aminopcptidase N and contains Zn* + ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta-and intramembraneous parts of the molecule have been added/preserve-d during development. It is speculated that this part carries the apical address.

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4-and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261-273 (CII 261-273). We have defined MHC and T cell receptor contacts in CII 261-273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-A b ␤ 0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.T and B cells represent two fundamentally different recognition modes of the specific immune system. Through alternating selection processes T cells are educated to recognize antigenic peptides presented in association with self-molecules of the major histocompatibility complex (MHC) on the surface of antigen-presenting cells. In contrast, B cells are not educated to be self-MHC-restricted and B-cell receptors (antibodies), whether soluble or in membrane-bound form, recognize threedimensional target structures. The distinctly different education of B and T cells explains why antibodies with the MHCrestricted specificity of T cells are rare and why it has been difficult to generate such specificities by conventional B-cell hybridoma techniques. We have taken advantage of the selection power of the phage display technology which makes it possible to test tens of millions of individual clones and have devised a method to generate recombinant antibodies recognizing predetermined peptide/MHC complexes. The speed and feasibility of this method makes it realistic to produce antibodies to a variety of specific peptide/MHC complexes which may be useful in studying MHC-restricted T-cell recognition and may lead to novel approaches in diagnostics and immunotherapy.MATERIALS AND METHODS MHC Purification. The AKR mouse-derived lymphoma RDM-4 was used for Kk production as described (1). In brief, Kk molecules were immunoaffinity purified from detergent cell lysates by using the monoclonal anti-Kk antibody 11.4-1 (American Tissue Type Culture Collection). The affinity columns were washed extensively and bound MHC class I molecules were eluted with 0.05 M diethylamine, pH 11/0.15 M sodium chloride/0.1% sodium azide/0.1% sodium deoxycholate, neutralized, and concentrated by vacuum dialysis. Human 82-microglobulin was obtained from the urine of uremic patients and purified to homogeneity by gel filtration and chromatofocusing (1).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.The influenza virus-derived nucleoprotein peptide NP50-57 (single-letter code, SDYEGRLI) and hemagglutinin peptide Ha255-262 (FESTGNLI) were synthesized manually on a RaMPS synthesizer (DuPont) using standard fluorenylmethoxycarbonyl protection strategy.Generation of Peptide/MHC Cl...

Previous work has demonstrated the presence of a self‐splicing intron in the large subunit ribosomal RNA coding region in some strains of the ciliate protozoan Tetrahymena. Sequence comparisons of the intron regions from six Tetrahymena species showed these to fall into three homology groups. In an attempt to evaluate the evolutionary origins of the intervening sequences, we have now determined complete small subunit ribosomal RNA gene sequences from 13 species of Tetrahymena and the absolute number of nucleotide differences between the sequences was used to construct a phylogenetic tree. This phylogeny was consistent with the groupings suggested by comparisons of other biochemical characters including cytoskeletal proteins, isozyme analyses, and restriction maps of complete rRNA transcription units. The homology groupings that were based upon the intron sequence data do not agree with the relationships inferred from the small subunit rRNA sequence data. These observations are taken to indicate that the intron character has been acquired independently in different species at a stage later than the branching out of the species.

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