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J L Strominger

Publications and source records attributed to J L Strominger.

At least 253 records · Page 14Linked to original sources
PubMed1988

Effect of mutations and variations of HLA-A2 on recognition of a virus peptide epitope by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) specific for influenza A virus were prepared from 15 donors. Those with HLA-A2 recognized autologous or HLA-A2-matched B-lymphoblastoid cells in the presence of synthetic peptide representing residues 55-73 or 56-68 of the virus matrix protein sequence. Influenza A virus-specific CTL from donors without HLA-A2 or with an HLA-A2 variant type failed to respond to this peptide. CTL lines specific for HLA-A2 plus peptide did not lyse peptide-treated target cells from HLA-A2 variant donors. They also failed to lyse peptide-treated cells with point mutations that had been inserted into HLA-A2 at positions 62-63, 66, 152, and 156 and, in some instances, mutations at positions 9 and 70. CTL lysed peptide-treated target cells with mutations in HLA-A2 at positions 43, 74, and 107. The results imply that this defined peptide epitope therefore interacts with HLA-A2 in the binding groove so that the long alpha-helices of HLA-A2 make important contact with the peptide at positions 66, 152, and 156. Different amino acids at position 9, which is in the floor of the peptide binding groove of HLA-A2 and the closely related position 70, modulate the peptide interaction so that some T-cell clones react and some do not.

Antigen-Antibody Complex↗
PubMed1988

Active suppression of major histocompatibility complex class II gene expression during differentiation from B cells to plasma cells.

Constitutive expression of major histocompatibility complex class II genes is acquired very early in B-cell ontogeny and is maintained up to the B-cell blast stage. Terminal differentiation in plasma cells is, however, accompanied by a loss of class II gene expression. In B cells this gene system is under the control of several loci encoding transacting factors with activator function, one of which, the aIr-1 gene product, operates across species barriers. In this report human class II gene expression is shown to be extinguished in somatic cell hybrids between the human class II-positive B-cell line Raji and the mouse class II-negative plasmacytoma cell line P3-U1. Since all murine chromosomes are retained in these hybrids and no preferential segregation of a specific human chromosome is observed, the results are compatible with the presence of suppressor factors of mouse origin, operating across species barriers and inhibiting class II gene expression. Suppression seems to act at the level of transcription or accumulation of class II-specific mRNA, since no human, and very few murine, class II transcripts are detectable in the hybrids.

Animals↗
PubMed1988

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line.

Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography. This is the first time all three subsets have been prepared as nonradioactive materials on a milligram scale. The yields of DR, DQ, and DP from 10 g of cells were approximately 12, 2, and 0.2 mg, respectively. Cross-contamination of the subsets was found to be less than 2% when assayed by measuring the binding of antigen-specific monoclonal antibodies to antigen immobilized on fixed erythrocytes. The three purified subsets were extensively characterized. They contained no detectable invariant chain. The three proteins were distinguished by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The denatured antigens were susceptible to partial removal of carbohydrate by endoglycosidase H and apparently complete removal of carbohydrate by endoglycosidase F. The isolated, denatured chains differed in their affinities for radiolabeled lectins, suggesting differences in carbohydrate structures. A water-soluble form of each antigen was prepared by a controlled papain digestion of the native antigen. Both native and denatured antigens were analyzed for their reactivities with a panel of class II antigen-specific monoclonal antibodies, allowing a precise definition of the specificities of the antibodies.

Alkylation↗
PubMed1987

Site-directed mutagenesis of class I HLA genes. Role of glycosylation in surface expression and functional recognition.

We have investigated the role of the carbohydrate moiety on the HLA-B7 molecule in mAb and CTL recognition using oligonucleotide-directed mutagenesis and gene transfer techniques. A conservative substitution of asparagine to glutamine at amino acid 86 in HLA-B7 was created to abolish the unique glycosylation site present on all HLA molecules. A second mutant B7 molecule was made by substituting asparagine-aspartic acid-threonine for the resident lysine-aspartic acid/lysine tripeptide at amino acids 176-178, thus creating an N-linked glycan at amino acid 176, which is additionally present on all known murine H-2 class I antigens. Upon gene transfer into mouse and human cell recipients, the HLA-B7M176+ mutant and normal HLA-B7 expressed identical levels of surface protein. However, the binding of two mAbs (MB40.2 and MB40.3) thought to recognize different epitopes of the HLA-B7 molecule was completely eliminated. In contrast, the HLA-B7M86- mutant displayed no surface expression (mouse L cells) or minimal surface expression (human RD cells or mouse L cells coexpressing human beta 2 microglobulin [beta 2m]) after indirect immunofluorescence (IIF) and flow cytometric analysis with a panel of 12 HLA-B7 mAb reactive with monomorphic and polymorphic determinants. Immunoprecipitation analysis demonstrated that intracellular denatured mutant protein was present. Tunicamycin treatment did not rescue the expression of HLA-B7M86- antigens to the cell surface; while interferon did induce higher levels of surface expression. Tunicamycin treatment also did not allow binding of the mAbs MB40.2 or MB40.3 to HLA-B7M176+ mutant antigens, suggesting that the carbohydrate moiety itself was not directly involved in the recognition or conformation of these mAb epitopes. Further mutation of the B7M86- molecule to create a glycan moiety at amino acid position 176 (B7M86-/176+) did not rescue normal levels of surface expression. Finally, neither mutation was seen to affect recognition by a panel of 12 allospecific CTL clones. The low expression of HLA-B7M86- on the surface of human cell transfectants was sufficient to achieve lysis, albeit at a reduced efficiency, and lysis could be increased by interferon induction of higher levels of expression. Thus, the carbohydrate moiety on HLA antigens plays a minimal or nonexistent role in recognition by available mAb and allospecific CTL clones.

Amino Acid Sequence↗
PubMed1987

The HLA-DQ beta gene upstream region contains an immunoglobulin-like octamer motif that binds cell-type specific nuclear factors.

A method is described for scanning relatively large fragments of DNA for sequences which bind nuclear factors. This method was used to identify an octamer (ATTTGTAT) in the DQ beta gene upstream region which differs from the immunoglobulin gene octamer (ATTTGCAT) by only 1 bp. The DQ beta gene octamer binds two proteins, one (B2) appears to be B cell specific while the other (B1) is not. These factors are either similar or identical to factors which bind to the octamer motif in immunoglobulin genes. All other class II MHC genes for which sequence information is available contain an octamer motif in their upstream region. Thus, the possibility that these sequences regulate B cell specific expression of class II MHC genes requires careful evaluation.

Base Sequence↗
PubMed1987

Characterization of the cell surface heterodimer VLA-4 and related peptides.

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.

Amino Acid Sequence↗
PubMed1987

Characterization and expression of the human alpha beta T cell receptor by using a framework monoclonal antibody.

A monoclonal antibody (mAb) with framework reactivity against the T cell receptor (TCR) alpha beta complex is characterized. The mAb, beta Framework 1 (beta F1) is capable of immunoprecipitating the TCR alpha beta complex from 125I-labeled human T cell tumors, immunocompetent T cell clones, and peripheral blood lymphocytes (PBL). beta F1 recognizes the separated TCR beta subunit in Western blotting. Because it does not bind to the surface of viable T cells but does react with the plasma membrane form of the TCR after treatment with membrane solubilizing agents, the beta F1 mAb reacts with a "hidden" determinant on the TCR beta subunit. After solubilization with 70% ethanol, the TCR alpha beta complex is shown to exist on greater than 92% of T3+ human PBL, whereas 2 to 8% of T3+ PBL do not react with the mAb. The beta F1 mAb demonstrates the existence of differently glycosylated surface 125I-labeled TCR alpha-chains (alpha, alpha', alpha") in association with a common TCR beta-chain on the HPB-MLT T cell leukemia. Reactivity of the beta F1 mAb on thymus tissue sections is similar to that of anti-Leu-4 (anti-T3). The beta F1 mAb should prove useful as a research tool for both the immunochemical characterization and isolation of virtually any alpha beta T cell receptor, whether from individual T cell clones or polyclonal populations of T lymphocytes. Recognition of T cell receptors in histologic tissue sections suggests that the beta F1 mAb may be useful in the clinical diagnosis of T cell lineage neoplasms. In failing to recognize all T3+ lymphocytes, it allows the identification of novel populations of T3+ lymphocytes that may express non-alpha, non-beta T cell receptors.

Antibodies, Monoclonal↗
PubMed1987

Detection of HLA-DP serological allodeterminants by the use of radioiodinated DP molecules.

HLA class II molecules were partially purified from cells of an HLA deletion mutant cell line, LCL721.82, that lost DR and DQ expression but retained DPw2 specificity and labeled with radioactive 125I. The radioiodinated preparation bound to DP-specific monoclonal antibody B7/21 as well as rabbit anti-HLA class II antiserum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the component involved in these bindings gave, unlike known HLA class II molecules, a sharp and dense band of approximately 60 kDa under nonreducing conditions and a single but diffuse band of approximately 30 kDa under reducing conditions. By screening 401 anti-HLA class II alloantisera, including those distributed in the 9th International Histocompatibility Workshop and also those locally available, eight were found to possess significant binding activity. Specificity analysis of these eight binding-positive antisera on a panel of DP-pretyped HLA homozygous typing cells revealed the presence of two clusters, one corresponding to an allodeterminant associated with DPw1, 2 and 3 and the other to that associated with DPw2 and 4. These two determinants were shown by the sequential binding test to reside on the B7/21-defined HLA class II molecules. Thus, two major conclusions were drawn: two distinct allodeterminants are carried by a single DP molecule; and these serologically detected DP allodeterminants are supertypic to cellularly defined DP allospecificities.

Antibodies, Monoclonal↗
PubMed1987

Cell-surface expression of H-2Db requires N-linked glycans.

The question of whether beta-2 microglobulin (B2m)-independent expression of the mouse major histocompatibility complex (MHC) antigen H-2Db results from the atypical glycosylation pattern associated with this MHC antigen (i.e., three glycans instead of two) has been addressed. Cell-surface expression of transfected H-2Db in the B2m deficient cell line R1E was completely abolished by the drug tunicamycin (Tm). Introduction of a functional B2m gene by transfection did not re-establish cell-surface expression of Db in the presence of Tm. Tm had no effect, however, on the expression of a truncated Db molecule lacking the alpha 1 and alpha 2 domains which is glycosylated at amino acid position 256, suggesting that the Db molecule, unlike other class I antigens, possesses an unstable conformation in the alpha 1 and/or alpha 2 domains which requires the attachment of glycans before it is transported to the cell surface. Once attached, however, glycans may confer a stable alpha 1/alpha 2 conformation apparently peculiar to Db which allows cell-surface expression in the absence of B2m.

Antigens, Surface↗
PubMed1987

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype.

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from "normal" class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i.e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.

Animals↗
PubMed1987

Structure and expression of HLA-DQ alpha and -DX alpha genes: interallelic alternate splicing of the HLA-DQ alpha gene and functional splicing of the HLA-DQ alpha gene using a retroviral vector.

The nucleotide sequences of the two closely related HLA-DQ alpha and HLA-DX alpha genes have been determined. Exons coding for the signal peptide, alpha 2 and transmembrane domains are 94-99% homologous, whereas the alpha 1 exon and the promoter region have diverged as much as or more than introns and the 3' untranslated region. The promoter regions of both genes contain two short sequences thought to be important for regulation of transcription by gamma-interferon. Transfection studies established that the DQ alpha and DQ beta genes encode the HLA-DQ antigen. Transcripts of varying length are produced from different alleles as the result of the use of alternate splice and polyadenylation signals at the 3' end of the DQ alpha gene. Thus typing at the DQ alpha locus can be achieved by Northern blot analysis. No transcript of DX alpha was detected in B lymphocytes. The DX alpha gene was accurately spliced when introduced into a retroviral vector, suggesting that the lack of expression of DX alpha is not due to aberrant splice signals.

Alleles↗
PubMed1987

Analysis of the DR beta chains from two DRw6 cell lines (WT46 and WT52): recombination in vivo may have generated new haplotypes.

The HLA-DRw6 haplotype of the class II major histocompatibility complex (MHC) antigens exhibits unusual complexity and cannot be uniquely typed serologically. The DR beta chains expressed by consanguineous homozygous DRw6 typing cells WT46 and WT52 were biochemically analyzed using three monoclonal antibodies (mAb) that recognize denatured DR beta chains. The results of isoelectric focusing and N-terminal sequencing demonstrate that each DRw6 B-cell line expresses two DR beta chains. Evidence of an exchange of mAb epitopes involving the two DR beta chains of one of these cell lines was obtained and may be explained by a recombinational mechanism involving reciprocal exchange of genetic segments of the DR beta chains, one of which may encode the putative DRw6 chain and the other the chain carrying the MT2 allotypic determinant. Since a recombinational hot spot has been shown to occur uniquely in the mouse MHC within the E beta gene, the occurrence of a recombination within the human homolog, DR(MT2) beta, could reflect some specific feature of this MHC region. Comparison of the DR beta chains of the WT46 and WT52 cell lines with those of a third DRw6 cell line, LB, suggests that two alleles of MT2 occur.

Alleles↗
PubMed1987

A highly diverged beta 1 exon in the DR region of the human MHC: sequence and evolutionary implications.

The DR subregion of the human major histocompatibility complex from a DR4 haplotype includes the well-characterized DR alpha, DR4 beta, DR(MT3)beta psi genes. In addition, the region between the DR alpha and the proximal DR(MT3)beta contains several copies of conserved DR beta-related sequences. These repeated elements, numbered II, III, and IV, include the DR beta signal sequence and a region located further upstream. Further examination of these conserved sequences showed that DR beta first intron sequences are present at the 3' ends of these repeats. Progressively longer portions of the DR beta first intron are conserved from repeat II to repeat IV, producing a gradient of conservation. The most complete repeat element of repeats II, III, and IV is associated with a lone beta 1 exon (DR beta 1). Upon sequencing, DR beta 1 was found to contain several deleterious mutations, indicating that it is nonfunctional. DR beta 1 has accumulated a large number of replacement substitutions and mutations at positions which are invariant in beta 1 domains from expressed DR beta genes: 77.8% of the nucleotide substitutions were replacement substitutions, and 41.5% of the amino acids at invariant positions have been altered. Calculations based on these figures suggest that DR beta 1 may have become inactive approximately 25 million years ago. There are, however, two histidine residues within a variable region which are unique to DR beta 1 and the DR4 beta gene, suggesting that they represent a gene pair which probably evolved by duplication of a single DR beta chain gene.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of Ly-1, a membrane glycoprotein of mouse T lymphocytes and a subset of B cells: molecular homology to its human counterpart Leu-1/T1 (CD5).

We report the isolation of cDNA clones of the mouse lymphocyte differentiation antigen Ly-1. One of these cDNA clones was confirmed to be full-length by DNA sequencing and by expression of Ly-1 by L cells transfected with this clone. Analysis of the predicted amino acid sequence indicated that the Ly-1 polypeptide is synthesized with a 23 amino acid leader and that the mature protein consists of an amino-terminal region of 347 amino acids, a transmembrane sequence of 30 residues, and a carboxyl-terminal region of 94 amino acids. The amino-terminal region appears to be divided into two subregions by a threonine- and proline-rich sequence of 23 amino acids that is highly conserved between Ly-1 and its human homologue Leu-1 (CD5) in position and amino acid composition. The first amino-terminal subregion of 111 amino acids is predicted to be arranged in a beta-pleated sheet structure of six strands. The entire amino-terminal region is rich in cysteine, with all of its 22 cysteine residues conserved between Ly-1 and Leu-1. The carboxyl-terminal region has no cysteines. Ly-1 and Leu-1 are 63% identical, with a gradient of identical residues from 43% for the first amino-terminal subregion to 58% for the second amino-terminal subregion and 90% for the carboxyl-terminal region. The predicted secondary structure of the first amino-terminal subregion and identities of certain conserved residues among most members of the immunoglobulin gene superfamily suggest that Ly-1 and Leu-1 are distant members of this family.

Amino Acid Sequence↗