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To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2Kb basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2Kb class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2Kb gene is one of the elements that is required for Ad12-E1A-mediated negative repression.

Ag fragments derived from the cytosol are transported into the endoplasmic reticulum (ER) lumen, where they bind to nascent MHC class I molecules. However, it is not known whether only high affinity peptides enter the ER, or whether ER proteases must trim longer precursor peptides down to optimal size. To evaluate the feasibility of proteolytic fine trimming in vitro, soluble Kd and Kb were preincubated with peptides that bind to Kd or Kb and the mixture was exposed to three different proteases. Class I protected allele-specific peptides against proteolysis, whereas the other peptides were degraded to the amino acid level. When a Kd/S11E (SYIPSAEYILE) complex was immunoprecipitated after incubation with carboxypeptidase, both S11E and the optimal sized S9I (SYIPSAEYI) were found to be specifically bound to Kd. However, only S91 was recovered if S11E, Kd and carboxypeptidase were mixed at the same time, and there was no fine-trimming of bound S11E if high protease concentrations and short proteolysis times were used, which suggests that trimming occurs only in the unbound state. The amount of peptide that immunoprecipitated with Kd increased after carboxypeptidase treatment of Kd/S11E, implying that the peptide affinity had increased. Kd also protected S9I against proteolysis by a lysed microsome preparation, demonstrating that class I could also protect high affinity peptides in vivo. These results suggest that class I participates in the selection of high affinity peptides in the ER, by sampling transported unbound peptides are degraded by ER proteases or efflux back to the cytosol.

Both positive and negative regulatory elements responsive to the product of the adenovirus type 12 E1A gene are located in the promoter region of the gene for mouse H-2Kbm1 major histocompatibility complex (MHC) class I antigen (1). We have analyzed the negative regulatory element that is affected by E1A and identified a target CAA repeated motif, CAA(A)CAAA, within -1725 to -1705 and -1591 to -1568 in a 316-bp sequence located in the far upstream region of H-2Kb promoter (-1837 to -1522; +1 refers to the cap site). The extent of cell surface expression of the MHC class 1 antigen was significantly decreased in the case of transfectants obtained by introducing an expression plasmid that included MHC class 1 cDNA with the CAA repeated element, as compared with that of a plasmid with mutated CAA repeats. We have also characterized the nuclear proteins that bind to this motif. The analysis of the effects of mutations during competition assays of in vivo and gel retardation competition assays demonstrated that the CAA repeated element is essential not only for E1A-dependent repression of transcription but also for the cell surface expression of the product of the mouse H-2Kb class I gene, presumably through nuclear proteins that specifically bind to it.

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2Kd cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse beta 2-microglobulina (beta 2-ma). When infected with the heavy chain-encoding virus alone, H-2Kd was produced in a beta 2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, approximately 10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2Kd-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro, in cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 microM) of an H-2Kd-restricted octapeptide epitope from Plasmodium berghei. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse beta 2-m but not to cells expressing free heavy chain only.

The 2.0 kb-long nucleotide sequences of the promoter regions of two closely related class I genes of the mouse major histocompatibility complex (H-2Kb and H-2Kbm1) have been determined and compared. The promoter sequence of the H-2Kbm1 gene differs from that of the H-2Kb gene by a single deletion of a 'C' at position -456 in the upstream region of H-2Kbm1 gene. The actual existence of this deletion of a single base in genomic DNA has been verified by genomic DNA hybridization, using oligonucleotide probes specific for H-2Kbm1 or H-2Kb respectively. The effect on the enhancer activity of H-2Kbm1 promoter region of the difference at position -456 has been analyzed by the chloramphenicol acetyltransferase (CAT) assay, using appropriate DdeI fragments (-533 to -408 for H-2Kbm1; -534 to -408 for H-2Kb) cloned downstream of pH-2(367)CAT gene construct. The CAT activity determined by the H-2Kbm1 fragment was about 3-fold higher than that of H-2Kb, a result which probably accounts for the higher level of the H-2Kbm1 transcript and antigen in lymph node cells.

The mechanism of transcriptional regulation of the H-2Kbm1 major histocompatibility complex (MHC) class I gene by adenovirus type 12 E1A (Ad12-E1A) was studied in transfected rat embryonal fibroblasts. Results of long-term expression of the chloramphenicol acetyl transferase (CAT) gene placed under the control of the 5'-flanking region of the mouse MHC class I gene. H-2Kbm1, and the results of nuclear run-on transcription assays, yield evidence for both positive and negative regulation of H-2Kbm1 by E1A gene product. Deletion studies in the H-2Kbm1 promoter region revealed that a proximal 58 bp upstream sequence (-194 to -136, relative to the cap site) and a distal 316 bp sequence (-1837 to -1521) respectively contribute to positive and negative regulation mediated by the E1A gene product. Both regulatory elements of MHC class I gene promoter region are responsible for the differential expression of the H-2Kbm1 gene in Ad12 transformed cells. A nuclear factor binding to the negative element has been detected only in extracts derived from cells expressing Ad12-E1A.

Binding studies and competition experiments have shown that a monoclonal antibody (mAb) named 28-8-6 recognizes only 5 to 10% of the cell surface Dd molecules. The molecules detected by 28-8-6 mAb appear to be genuine H-2Dd antigens on the basis of their MW and isolectric points. In addition, the detectability of the subset of cell surface Dd molecules by 28-8-6 does not depend on their degree of glycosylation nor on the presence of mouse beta-2-microglobulin. Several interpretations are discussed. mAb 28-8-6 might detect a particular conformation or a particular chemical derivatization of otherwise normal H-2Dd molecules. Also, because the epitope recognized by 28-8-6 lies close to the peptide binding site, it is possible that mAb 28-8-6 recognizes a subset of Dd molecules bearing a certain category of self peptides.

The t-haplotypes of the house mouse are defined by several genetic events which involve about one-third of chromosome 17 and include the major histocompatibility complex (MHC). One of the primary features of complete t-haplotypes is the nearly complete suppression of recombination between a t-chromosome and a wild-type chromosome 17. If we assume that all genetic exchanges are abolished because of the inhibition of recombination, the class I genes of the MHC can be used as vicinity markers of t-haplotypes. We have studied the H-2K, D and L genes of 10 independant t-haplotypes and have found that their H-2K genes are rather polymorphic, although always very similar or identical to H-2K genes of laboratory mice. By contrast, the DL-Tla region is nearly constant. It is suggested that the t-haplotypes reveal the existence of several mechanisms involved in the evolution of the MHC, one of which is possibly the greater "mobility" of H-2K genes in the genome.

We have isolated a cDNA clone carrying a 1560 bp long insert which contains the entire coding and 3' untranslated regions of an H-2K(d) mouse histocompatibility antigen. Its sequence and overal features are described. They point to the existence of unique properties of DNA sequences associated with the H-2K(d) antigen.

A cDNA library constructed from liver mRNA of DBA/2 (H-2d) mice has been screened with H-2-specific probes. The nucleotide sequence of one clone (pH-2d-24) indicates that it derives from an H-2 gene with an unexpected exon-intron organization. Nucleotide sequence comparisons suggest that two distinct mRNAs are produced from a single H-2Kd gene by a mechanism involving the use of alternative splicing sites in its 5' region. pH-2d-24 carries an open reading frame encoding a thus-far-undescribed polypeptide product identical to an H-2Kd-molecule, except for the NH2-terminal half of the first domain.

The MRL/lpr (H-2k) inbred strain, a model for the autoimmune disease systemic lupus erythematosus, differs from the healthy inbred strain MRL +/+ (H-2k) by only 0.1% of its genome. Southern blot analysis using class I and class II probes confirmed the H-2k genotype of both strains. Among the Iak-positive peritoneal cells, cells with an unexpected expression of Iad specificities were detected in a radioimmunoassay using several monoclonal antibodies and one conventional antiserum. This was only found in aged (6- to 9-month-old) mice both in the MRL/lpr strain (32% Iad-positive mice) and in the MRL +/+ strain (42% Iad-positive mice). Furthermore, 24% of aged MRL/lpr mice exhibited strong spontaneous cytotoxic T lymphocyte (CTL) activities against P815 (H-2d) target cells, and 57% had a weaker but still detectable level of cytotoxicity. In contrast, such a CTL activity has never been found in the MRL +/+ strain. These results suggest that the anti-H-2d CTL plays a role in the onset of the autoimmune process in MRL/lpr mice.

mRNA coding for mouse major transplantation antigens of the d haplotype was partially purified, copied into double-stranded cDNA, and cloned in Escherichia coli. Clones were selected by their ability to hybridize specifically with mRNA coding for H-2K, D, or L antigens. One of these clones, pH-2d-1, carries a 1200-base-pair insert, comprising the noncoding region, including poly(A) at the 3' end and part of the coding region. A partial sequence of the latter region showed extensive homology with the known amino acid sequences of H-2Kb,Kk, and HLA-B7 antigens. From this comparison, it appears that the coding region extends from amino acid 133 in the second domain, through the third domain, to the cytoplasmic COOH-terminal region. A stretch of 24 hydrophobic or uncharged residues, located 31 amino acids from the COOH-terminal end, could represent the segment that spans the membrane. This is followed on the cytoplasmic side of the membrane by a cluster of basic amino acids and a possible phosphorylation site on a threonine residue.

The recent isolation, by recombinant DNA techniques, of cloned probes for mouse and human class I major transplantation antigens has initiated the molecular analysis of the corresponding genes. Mouse genes belong to a relatively large multigene family, whose members share extensive structural homologies. Sequence analyses suggest that some genes could have a mosaic structure. This feature might help us to understand one of the distinctive traits of these antigens: their large antigenic polymorphism.

The complete nucleotide sequences of three cDNAs coding for the C-terminal part of mouse histocompatibility (H-2) antigens, and for the 3' non coding regions of these clones have been determined. Comparison of the sequence indicates a large homology throughout the coding and non-coding regions and suggests the existence of a genetic mechanism which homogenizes nucleotide sequences among genes of the H-2 multigene family.

We have studied the pattern of expression of the Q10 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-I molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-I genes, namely Kd and 37, the latter gene located in the thymus leukemia region. We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for Q10 mRNA than for the others. Furthermore, we found that the Q10 gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the Q10 protein is involved in immune tolerance. However, the time course of expression of Q10 mRNA and its tissue distribution during embryogenesis suggest that the Q10 protein could play a role in the differentiation of hematopoietic stem cells.

Two recombinant mice have been recovered from the progeny of Ttf/tw32 + animals. They have lost the tw32 lethality factor(s) and gained tufted, presumably from the T chromosome. Southern blot analysis of class I genes of these two new partial tPA027 and tPA286 haplotypes indicates that they have retained at least part of the major histocompatibility complex of the tw32 chromosome (H-2 haplotype H-2w28). We have prepared a phage library of Eco RI-digested DNA from homozygous tPA027 animals. Upon screening the library with a cDNA probe specific for H-2K genes, we isolated a class I gene displaying all of the distinctive features of a genuine H-2K gene, and which could thus be defined as an H-2Kw28 gene. The H-2Kw28 gene is 92-95% homologous to H-2Kb and H-2Kd genes and differs significantly from the other class I genes sequenced so far. Homology with the H-2Kb sequence reaches nearly 100% in the 3' part of the H-2Kw28 gene. Moreover, the homology with an H-2Kq cDNA sequence reaches 99.8%. Several hypotheses can account for the near identity of H-2Kb, H-2Kq, and H-2Kw28 gene sequences: either recombination between H-2w28 and H-2b and H-2q sequences occurred before or at the time the strain was established, or the class I genes of the tw32 chromosome and the H-2b and H-2q genes found in inbred strains of mice have separated from each other rather recently.

A rabbit antiserum raised against papain-solubilized H-2 antigens has been used to investigate the eventual expression of H-2 antigens and related molecules on embryonal carcinoma cells and on other types of mouse cells. No material reacting with this serum could be detected on cells carrying the F9 antigen. It is concluded that no H-2 antigen or cross-reacting material is expressed on these cell types.