Use of the HLA-DR antigens incorporated into liposomes to generate HLA-DR specific cytotoxic T lymphocytes.
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Animals↗
Biomedical subjects
J L Strominger
Publications and source records attributed to J L Strominger.
Identification and mapping of polypeptides encoded by the P3HR-1 strain of Epstein-Barr virus.
The Epstein-Barr virus (EBV)-specified polypeptides induced upon viral replication in the P3HR-1 cell line have been examined by immunoprecipitation with a high-titer human anti-EBV serum. Twenty-five predominant polypeptides were identified in cell extracts, whereas 18 polypeptides were precipitated from cell-free translation reactions directed by total mRNA. Hybrid selection of mRNA to the BamHI DNA clones of the EBV genome and immunoprecipitation of the corresponding cell-free translation products revealed 98 EBV-specified polypeptides and their coding location along the viral genome. In addition, the viral polypeptides that bind reversibly to DNA-cellulose have been characterized and the deduced map locations of this functional group of EBV-specified polypeptides is presented.
Cell Line↗
Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2.
Identity of the Epstein-Barr virus (EBV) receptor with the complement receptor type 2 (CR2) was established in three sets of experiments using the monoclonal antibodies, HB-5 and anti-B2, which recognize a Mr 145,000 B-lymphocyte membrane protein that is CR2. First, the rank order for binding of fluoresceinated EBV to four lymphoblastoid cell lines (SB, JY, Raji, and Molt-4) was identical to the rank order for binding of HB-5 and anti-B2 by analytical flow cytometry. Second, pretreatment of cells with HB-5 followed by treatment with goat F(ab')2 fragments to mouse IgG blocked binding of fluoresceinated EBV on SB, a B-lymphoblastoid cell line. Virus attachment was not inhibited by HB-5 alone, second antibody alone, rabbit anti-C3b receptor, or UPC10 (an irrelevant monoclonal antibody). Third, transfer of CR2 from SB to protein A-bearing Staphylococcus aureus particles, to which HB-5 had been absorbed, conferred on them the specific ability to bind 125I-labeled EBV. We conclude that CR2 is the EBV receptor of human B lymphocytes.
Antibodies, Monoclonal↗
Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.
Immune interferon (IFN-gamma) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-gamma induces parallel expression of two other class II major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-gamma at similar rates but from initial levels that are 50% of maximal. After removal of IFN-gamma, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells.
Endothelium↗
Cloning and sequence analysis of the human major histocompatibility complex gene DC-3 beta.
The DC antigen is one of the class II major histocompatibility antigens involved in the regulation of the immune response. This molecule is a heterodimer composed of an alpha and a beta chain. Southern blot analysis of several homozygous cell lines shows that there are two DC beta genes. The DC-3 beta gene, corresponding to a polymorphic restriction fragment, was cloned and sequenced and found to exist in five exons spanning 8 kilobase pairs of DNA. These exons correspond to the functional domains of the DC beta protein. Comparison of the beta 1 domains of known DC beta chains shows that the polymorphism is clustered in four regions. A similar comparison of the mouse A beta sequences shows only two prominent diversity regions. The DC beta chain sequences are eight amino acids shorter than the A beta chain sequences due to the elimination of a small exon by an aberrant splice acceptor.
Base Sequence↗
Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.
The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells.
Animals↗
Antigenic stimulation regulates the level of expression of interleukin 2 receptor on human T cells.
Antigen-specific, interleukin 2 (IL-2)-dependent human T-cell lines and clones were utilized to study the relationship between IL-2 receptor expression and antigenic stimulation. T cells that had not been exposed to antigen for 2 wk or more expressed a stable low level of the IL-2 receptor. After reexposure to antigen, a 10- to 30-fold increase in the level of the IL-2 receptor was rapidly induced, with the peak level of IL-2 receptor expression occurring at 15-30 hr. This peak preceded the peak in cell proliferation [( 3H]thymidine incorporation), which was at 48-72 hr. Within 2-14 days after peak IL-2 receptor expression, it returned to a low base-line level. The transient elevation in IL-2 receptor level was antigen specific because it occurred in response to specific allogeneic stimulator cells but not after exposure to cells expressing irrelevant HLA allotypes. The levels of other cell-surface proteins, including those related to T-cell activation (HLA-DR, T10, 4F2, A-1A5) as well as T3, which has been proposed to be a component of the T-cell receptor complex for antigen, did not change in response to antigen exposure or deprivation. Because IL-2 was maintained at a consistently high level throughout these experiments, the antigen-induced changes in the IL-2 receptor appear to be independent of changes induced by IL-2 itself. Both cloned T cells and mixed populations containing T4 and T8 subsets showed similar IL-2 receptor responsiveness, indicating that this finding is generalizable to most, if not to all, antigen-responsive T cells.
Antibodies, Monoclonal↗
Orientation of loci within the human major histocompatibility complex by chromosomal in situ hybridization.
We have determined the localization and orientation of two genetic probes within the human major histocompatibility complex by chromosomal in situ hybridization. Our data indicate that a cloned genomic probe cross-hybridizing to HLA-A, -B, and -C heavy chain loci is homologous to sequences located on chromosome 6 at band p21.3 while a subclone of the genomic HLA-DR alpha-chain gene corresponding to the nonpolymorphic p34 protein is homologous to sequences in band 6p21.1. Our data suggest that this technique may permit the estimation of map distances between linked gene loci, assuming a uniform frequency of map units in the human genome. The relative positions of these genes was confirmed in a mother and son carrying a chromosome rearrangement involving 6p and 14p in which the sequences hybridizing to a DR alpha-chain genomic clone were found at the distal end of the 6p--chromosome [der(6)] while the sequences hybridizing to the HLA-A, -B, -C alpha-chain probe were found in the 14p+ chromosome [der(14)].
Chromosome Mapping↗
Simultaneous release of penicilloic acid and phenylacetyl glycine by penicillin-binding proteins 5 and 6 of Escherichia coli.
Penicillin-binding proteins (PBPs) 5 and 6 of Escherichia coli released the bound penicilloyl moiety at an intermediate rate relative to, e.g., Staphylococcus aureus PBPs 4 (rapid) and 1 or 2 (slow). Each of these E. coli PBPs released the bound penicilloyl moiety as both penicilloic acid (hydrolysis) and phenylacetyl glycine (scission of the C-5--C-6 bond followed by hydrolysis).
Bacterial Proteins↗
Trapping of the substrate-derived acyl enzyme intermediate of purified penicillin-binding protein 1a of Escherichia coli.
Purified penicillin-binding protein 1a of Escherichia coli formed an acyl enzyme intermediate with the highly reactive synthetic substrate diacetyl-L-lysyl-D-alanyl-D-lactate at acid pH, although in extremely low yields.
Acylation↗
Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction.
This study provides direct correlation via dual parameter flow cytometry (simultaneous assessment of immunofluorescence and DNA content) between mixed lymphocyte reaction (MLR) responder cell entry into the S/G2/M phases of the cell cycle with the kinetics of expression of two activation-associated cell surface proteins, Tac (IL 2 receptor) and 4F2 (unknown metabolic function). A small population of activated cells was identifiable by expression of both Tac and 4F2 antigens before peak DNA synthesis in the MLR. This population of activation antigen-positive cells expanded linearly in size from days 3 to 7 of culture. Treatment of immature MLR cultures with anti-4F2 Mab and complement (C) before DNA synthesis (treatment on day 3, peak DNA synthesis on days 5 to 6) resulted in blunted proliferation and activation antigen expression when the same culture was analyzed after maturation on day 6, indicating that the activated population had been previously detected and removed by anti-4F2 Mab + C. The 4F2 antigen was expressed on a greater percentage of cells in the MLR at all times (days 3 to 9) than was Tac, was present on virtually all S/G2/M phase responder cells, and a large fraction of cells remained intensely 4F2+ subsequent to peak DNA synthesis. In contrast, after initially preceding responder cell entry into the S phase of the cell cycle, the kinetics of Tac antigen expression closely paralleled the kinetics of responder cell proliferation. A subpopulation of cycling responder cells was noted in all MLR cultures studied that expressed Tac antigen weakly or not at all. Cells within both T4 and T8 cell subsets proliferate with similar kinetics in response to alloantigen. The possibility that activation antigens can be utilized to study effector cell generation in the MLR and that this flow cytometric technique may be utilized to analyze the response to various alloantigens is discussed.
Antigens, Surface↗
Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.
A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.
Animals↗
Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes.
It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.
Amino Acid Sequence↗
Characterization of a B lymphoblastoid cell line mutant that secretes HLA-A2.
HLA-A2 antigen mutants were obtained previously from the B lymphoblastoid cell line T5-1 by mutagenesis followed by immunoselection. Here we present biochemical studies of one particular mutant, clone 8.14.1. These cells synthesize two forms of HLA-A2: a minor form, which remains cell-associated at all times, and an abundant form, which is secreted. The former appears by SDS-PAGE to be slightly larger than T5-1 HLA-A2, whereas the latter appears to be 4000 to 5000 daltons smaller. In vitro translation and in vivo pulse-chase studies suggest that these species are not related to each other by post-translational processing. Proteolytic digestion studies localize the resulting structural alteration in the mobility difference between wild-type and secreted HLA-A2 to a region near the carboxy terminus of the HLA-A2 heavy chain; however, their extreme carboxy termini appear similar, if not identical. We suggest that the secreted form may result from a pattern of RNA splicing in which the exon encoding the hydrophobic, membrane-spanning region is frequently deleted.
Antibodies, Monoclonal↗
HLA-DR antigens of autologous melanoma and B lymphoblastoid cell lines: differences in glycosylation but not protein structure.
The HLA-DR antigen expressed on the surface of the human melanoma cell line SK-MEL-37 was characterized and compared with the HLA-DR antigen from the MU B lymphoblastoid cell line originating from the same individual. The HLA-DR heavy chain from SK-MEL-37 cells had an apparent mobility on SDS-PAGE slightly slower than that isolated from MU cells. In contrast, the HLA-DR light chains and the HLA-A,-B heavy chains from the two cell lines had identical mobilities. Double-labeled tryptic peptide mapping and limited N-terminal sequencing showed that the SK-MEL-37 HLA-DR antigen, like all previously examined B lymphoblastoid cell HLA-DR antigens, was homologous to the murine I-E/C subregion antigens and that the mobility difference of the SK-MEL-37 HLA-DR heavy chain was not attributable to differences in the primary structure of the polypeptide. Treatment of the cells with tunicamycin abolished the m.w. difference, suggesting that it was due to a change in glycosylation in SK-MEL-37. This was confirmed by analysis of the glycopeptides from pronase-digested HLA-DR light and heavy chains and HLA-A,B heavy chains purified from the two cell types. The results suggest 1) there is a difference in asparagine-linked oligosaccharide processing in the two cell types, with more of the larger complex glycans synthesized in the melanoma cells than in the B lymphoblastoid cells, 2) the effect is more pronounced with HLA-DR heavy chains than with HLA-DR light chains or HLA-A,B heavy chains, and 3) the oligosaccharide size difference is not solely due to sialic acid content.
Amino Acid Sequence↗
Organization of the transcriptional unit of a human class II histocompatibility antigen: HLA-DR heavy chain.
A total of 5724 base pairs of a recombinant phage DNA containing a human HLA-DR heavy chain gene including flanking regions has been analyzed. The regions corresponding to all the exons have been identified. The sites of initiation of transcription and polyadenylation have been determined. A large intron of 2399 base pairs separates the first exon containing the 5' untranslated region and the signal peptide from the second exon containing the N-terminal peptide domain.
Amino Acid Sequence↗
Primary structure of papain-solubilized human histocompatibility antigen HLA-B40 (-Bw60). An outline of alloantigenic determinants.
The primary structure of papain-solubilized human histocompatibility antigen HLA-B40 (-Bw60) has been determined. Its comparison with that of the cross-reactive HLA-B7 antigen allows for the first time a direct sequence comparison between two HLA-B locus products and an outline of the location of their alloantigenic sites. Overall sequence homology between HLA-B40 and HLA-B7 is 93%. Of 19 detected differences, 18 are located in the two amino-terminal domains (residues 1-182). Half of them are clustered in two short segments spanning residues 63-74 and 147-156, respectively, which suggests that they may encompass major sites of their alloantigenic determinants. The first of these segments is highly polymorphic in HLA and H-2 molecules. It is proposed that it may belong to a hypervariable region of class I histocompatibility antigens. The remaining substitutions are scattered through the N-terminal portions of the two external domains. Thus, in addition to the above-mentioned segments, other positions may contribute significantly to the antigenic polymorphism of these molecules.
Amino Acid Sequence↗
Localization of the sites of iodination of human beta 2-microglobulin: quaternary structure implications for histocompatibility antigens.
Human urinary beta 2-microglobulin (beta 2m) and pa-pain-solubilized human histocompatibility antigen HLA-B7 were iodinated with iodogen and the sites of iodination determined. In the case of free urinary beta 2m, four of the six tyrosines were modified to some degree. Two of these were heavily iodinated (tyrosine-63 and -67) while two were lightly iodinated (tyrosine-10 and -26). In the case of beta 2m iodinated in the intact HLA-B7 complex, only one of these tyrosines was modified substantially (tyrosine-67). beta 2m iodinated at either of the two major sites exchanged into the HLA-B7 complex, whereas beta 2m iodinated at either of the two minor sites did not exchange at all. The relationship of these findings to the quaternary structure of HLA is discussed.
Amino Acid Sequence↗