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J L Strominger

Publications and source records attributed to J L Strominger.

At least 199 records · Page 11Linked to original sources
PubMed1991

Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic defects defined by complementation analysis.

Expression of class II major histocompatibility complex antigens in defective B-lymphoblastoid cell lines from patients with class II antigen deficiency and from in vitro mutants generated with the same phenotype was studied. By heterogenetic fusion experiments, at least three, and probably four, complementation groups were defined. Furthermore, clone 13 (a DR-, DP-, but DQ+ cell line) appeared to belong to the RJ2.2.5 complementation group, for which all other members are DR-, DP-, and also DQ-. Thus, it is hypothesized that the cell lines of this group lack the activity of a gene that can differentially regulate the DR/DP and the DQ promoters.

B-Lymphocytes↗
PubMed1991

Transcription of a subset of human class II major histocompatibility complex genes is regulated by a nucleoprotein complex that contains c-fos or an antigenically related protein.

Transcriptional regulation of the human major histocompatibility complex class II genes requires at least two upstream elements, the X and Y boxes, located in the -50- to -150-base-pair region of all class II promoters. The DRA and DPB promoters contain phorbol ester-responsive elements overlapping the 3' side of their X boxes. Mutation of this sequence down-regulates the efficiency of the DRA promoter, suggesting that a positive regulator(s) binds to this site. In this report, anti-sense c-fos RNA and an anti-c-fos antibody were used to show that the product of the protooncogene c-fos or an antigenically related protein is a component of a complex that binds to the X box and is required for maximal transcription from the DRA and DPB promoters. As c-fos (or its related proteins) cannot bind alone to DNA, these results suggest that it may dimerize with other members of the JUN/AP-1 family, such as hXBP1, to participate in the activation of a subset of class II major histocompatibility complex genes.

Amino Acid Sequence↗
PubMed1991

Human X-box-binding protein 1 is required for the transcription of a subset of human class II major histocompatibility genes and forms a heterodimer with c-fos.

A complementary DNA encoding a member of the leucine-zipper class of proteins (human X-box-binding protein, hXBP-1) that binds to the 3' end of the conserved X box (X2) of the HLA-DRA major histocompatibility complex gene was recently described. Further gel-retardation analysis has demonstrated that hXBP-1 also binds to HLA-DPB X2 but not to other X2 sequences. Transient transfection of a mammalian expression vector with the hXBP-1 cDNA inserted in the antisense orientation represses the surface expression of HLA-DR and HLA-DP in Raji cells. Cotransfection of the antisense hXBP-1 vector with a HLA-DRA/chloramphenicol acetyltransferase (but not a HLA-DQB/chloramphenicol acetyltransferase) reporter plasmid decreases chloramphenicol acetyltransferase activity in Raji cells and in gamma-interferon-treated HeLa cells relative to cells cotransfected with a control antisense vector. Moreover, hXBP-1 is shown to form a stable heterodimer with the product of the c-fos protooncogene. These data suggest that the hXBP-1 c-fos heterodimer is critical for the transcription of a subset of the human class II major histocompatibility complex genes and that the regulatory mechanisms for the different class II genes are distinct.

Antisense Elements (Genetics)↗
PubMed1991

Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection.

During latent Epstein-Barr virus (EBV) infection of human B lymphocytes, six viral nuclear antigen (EBNAs) are expressed from long primary transcripts by means of alternative splicing and alternative polyadenylylation sites. These transcripts initiate from one of two promoters, Cp or Wp, that function in a mutually exclusive fashion. Wp is exclusively utilized during the initial stages of infection of primary B lymphocytes, followed by a switch to Cp usage. These studies have been extended to show that (i) a mutant EBV strain lacking the gene encoding EBNA 2 fails to switch from Wp to Cp usage in primary B lymphocytes, although the virus contains a functional Cp; (ii) a region from -429 to -245 base pairs upstream of Cp is essential for Cp activity in B lymphocytes, but only in the context of upstream and downstream sequences; (iii) this region contains an EBNA 2-dependent enhancer; and (iv) DNase I protection employing nuclear extracts from B and T lymphocytes revealed a B-cell-specific footprint in the region of the EBNA 2-dependent enhancer. These results support a model for viral promoter switching during the initial stages of infection in which Wp activity leads to the expression of EBNA 2, followed by activation of Cp through the EBNA 2-dependent enhancer.

Antigens, Viral↗
PubMed1991

Non-MHC-restricted target-cell lysis by a CD4-CD8- TCR alpha beta T-cell line, as well as by TCR gamma delta T-cell lines, results from lymphokine-activated killing.

A long-term CD4-CD8- TCR alpha beta human T-cell line, as well as similar CD4-CD8- TCR gamma delta T-cell lines for comparison, were generated from various tissues by negative selection using anti-CD4 and anti-CD8 monoclonal antibodies (MAbs) followed by positive selection with specific anti-TCR MAb and then repeated in vitro stimulation with interleukin-enriched media and lectin. These cell lines all demonstrated non-MHC-restricted cytolysis on a variety of human tumor cell lines. However, removal of lymphokines from the culture media for 24 hr abrogated most of the non-MHC-restricted target-cell lysis without affecting TCR alpha beta or TCR gamma delta cell viability or TCR function as determined by antibody-triggered redirected target-cell lysis. Subsequent re-exposure to lymphokines reconstituted non-MHC-restricted cytolysis by these cell lines. Thus, much of the non-specific, non-MHC-restricted cytolytic activity generated by CD4-CD8- TCR alpha beta or TCR gamma delta cells is secondary to lymphokine-activated killing (LAK) activity. These cells have potent LAK activity and may be prominent in LAK-cell populations. In addition, after lymphokine deprivation, both CD4-CD8- TCR alpha beta and TCR gamma delta cells showed residual activity against some tumor-cell targets, the nature of which remains to be defined.

Animals↗
PubMed1991

Signals for retention of transmembrane proteins in the endoplasmic reticulum studied with CD4 truncation mutants.

A mutant of CD4 (CD4.Q421stop), in which the cytoplasmic C-terminal 13 amino acids were truncated, was not expressed on the surface of HeLa cells after transfection but was retained in the endoplasmic reticulum (ER). Seven other truncation mutants of CD4 were expressed well on the cell surface, thus suggesting that the C-terminal amino acids of CD4.Q421stop (-Ser-Glu-Lys-Lys-Thr-Cys) may have the sequence information for ER retention. Further mutational study has revealed that two consecutive lysine residues at the third and fourth positions from the C-terminal end are sufficient for ER retention. Lysine at the fourth position, but not at the third position, from the C terminus can be replaced by arginine without disturbing ER retention. Furthermore, two lysine residues at the third and fifth positions from the C terminus also resulted in ER retention. Thus lysine at the third position and a positively charged amino acid either at the fourth or fifth position from the C terminus are sufficient for ER retention of this CD4 mutant, and possibly all transmembrane proteins. In addition to the requirement of specific amino acids at specific positions, the ER retention signal -Lys-Lys-Xaa-Xaa also requires a transmembrane region for function. By contrast -Lys-Asp-Glu-Leu, which targets soluble proteins to the lumen of the ER, does not function in the presence of a transmembrane region.

Amino Acid Sequence↗
PubMed1991

An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.

The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.

Antibodies, Monoclonal↗
PubMed1991

Association of four HLA class III region genomic markers with HLA haplotypes.

We have studied restriction fragment length polymorphism (RFLP) in the region 300 kb centromeric to the HLA-B locus. Four probes were used: one was genomic DNA derived from the tumor-necrosis factor (TNF)-beta gene, one was a cDNA for the BAT3 gene, and two single-copy genomic probes, R5A and M20A. The order of these markers from HLA-B towards the centromere is M20A, R5A, TNF and BAT3. The BAT3 and TNF-beta probes each detected two allelic bands with Taq I and Nco I digestion, respectively; the R5A and M20A probes each detected three polymorphic allelic bands with BstEII digestion. To determine if these restriction polymorphisms are preferentially associated with certain HLA-B and -DR haplotypes, a total of 153 HLA haplotypes was analyzed. The haplotypes A1, B8, DR3 and A3, B7, DR2 were each associated with a distinct combination of polymorphisms identified at these four sites, thereby demonstrating that the strong linkage disequilibrium characteristic of these haplotypes extends also to this segment of the class III region. In contrast, haplotypes that are not in positive linkage disequilibrium, such as A1,B8,DR4 and A2,B7,DR3, showed no preferential association with any of these polymorphisms. The antigens HLA-B27 and B35 were also found to be in positive linkage disequilibrium with RFLP patterns at three of these sites, and HLA-B14,B35,B44,Bw57 and Bw62 were found preferentially associated with polymorphisms at one or two of these sites, independent of the DR antigen present. These data further demonstrate that genetic linkage disequilibrium in the HLA class III region is complex and variable among different HLA haplotypes.

Chromosome Mapping↗
PubMed1990

Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire.

The germline repertoire of variable genes for the TCR-gamma/delta is limited. This, together with the availability of several V delta-specific and a C delta-specific mAbs, has made it possible to assess differences in the TCR-gamma/delta repertoire in man. TCR-gamma/delta cells expressing particular V gene segments have been previously shown to be localized in different anatomical sites. In this study, analysis of TCR-gamma/delta V gene segment usage performed on subjects from the time of birth through adulthood revealed striking age-related changes in the TCR-gamma/delta repertoire in peripheral blood. V delta 1+ gamma/delta T cells predominated in thymus as well as in peripheral blood at birth and then persisted as a relatively constant proportion of CD3+ PBL. However, V delta 2+ gamma/delta T cells that constitute a small proportion of the CD3+ cells in thymus and in peripheral blood at birth, then expand and account for the major population of gamma/delta T cells in PBL in adults. No parallel postnatal expansion of V delta 2+ cells in the thymus was observed, even when paired thymus-peripheral blood specimens were obtained on subjects between the ages of 3 d and 8 yr. The subset of V delta 2+ lymphocytes that was expanded in peripheral blood expressed high levels of CD45RO suggesting prior activation of these cells, consistent with the possibility that their expansion might have resulted from exposure to foreign antigens or superantigens. In contrast, V delta 1+ T cells in PBL showed no comparable increase in relative numbers and were either negative or expressed only low levels of CD45RO. Consistent with evidence for extrathymic peripheral expansion of selective TCR-gamma/delta subsets, no link between MHC haplotype and differences in the TCR-gamma/delta V gene usage between individuals was apparent, and identical twins displayed TCR-gamma/delta variable gene segment phenotypes that were strikingly different from one another. The elements that determine the TCR-gamma/delta repertoire in individuals are not known. It is possible that both thymic selection and extrathymic factors may influence the peripheral repertoire. Recently, TCR-gamma/delta+ lymphocytes have been shown to expand markedly in peripheral lymphoid tissues and infectious lesions in response to mycobacterial antigens, and a correlation between mycobacterial responses and TCR-gamma/delta V gene usage has been shown in mice. The data presented here demonstrated peripheral age-related changes in the gamma/delta repertoire and point to the importance of extrathymic expansion of specific gamma/delta subsets in generating the human TCR-gamma/delta repertoire.

Adult↗
PubMed1990

Structural nature of the interaction between T lymphocyte surface molecule CD4 and the intracellular protein tyrosine kinase lck.

The strong non-covalent interactions between T lymphocyte surface CD4 or CD8 molecules and the intracellular membrane-associated protein tyrosine kinase lck are likely to mediate the role of CD4 and CD8 molecules in the immune response. The delineation of the structural nature of the CD4/lck and CD8/lck complexes is important for the understanding of the biochemical and functional significance of the interactions. Complementary charged regions in the C-terminal intracytoplasmic portions of CD4 or CD8, and in the N-terminal region of protein tyrosine kinase lck were noted. Peptides spanning these regions, residues 417 to 429 of CD4 and 10 to 22 of lck, were found to specifically dissociate these two molecules in CD4/lck complexes. A structural model of the interaction that accounts for its high stability is proposed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Structural features of the cytoplasmic region of CD4 required for internalization.

CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.

Amino Acid Sequence↗
PubMed1990

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding.

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.

Alanine↗
PubMed1990

Regulation of the class II-associated invariant chain gene in normal and mutant B lymphocytes.

The invariant chain protein is intracellularly associated with class II major histocompatibility proteins. In many cases, the expression of these molecules appears to be regulated in a similar manner. Contained within the promoter of the invariant chain gene are sequences (X and I gamma 1) that are similar to the X and Y box elements of class II genes, suggesting that these sequences might be involved in its regulation. DNase I footprinting reveals additional cis-acting elements (I gamma 2 and I gamma 3) that contain sequence similarities to NF-kappa B and/or H2TF1/KBF1 recognition sequences. A series of fusion constructs with the chloramphenicol acetyltransferase reporter gene were used to analyze the role of these sequences (I gamma 1, I gamma 2, I gamma 3, and X and Y elements) in both normal and mutant B lymphocytes. These data suggest the likelihood of multiple X box proteins in B cells, which can act as both negative and positive regulatory factors.

Animals↗
PubMed1990

Maturation or differentiation of human thymocyte precursors in vitro?

The differentiation or maturation potential of human thymocyte precursors has been studied by using a population of CD3/TCR-, CD4-, CD8- ("triple negative") thymocytes isolated by negative selection (TCR, T-cell receptor). This cell population, however, also contained 30-50% previously undescribed cells expressing very low levels of CD3/TCR gamma delta (CD3/TCR gamma delta low; approximately 60% of which expressed the variable region gene V delta 1). Correspondingly, TCR gamma and TCR delta gene rearrangements (predominantly V delta 1/joining region J delta 1) and full-length TCR gamma and TCR delta transcripts (but only immature TCR beta and no TCR alpha mRNAs) were found. These cells mobilized Ca2+ in response to ligation of CD3 but not following ligation of TCR gamma delta. When cultured in the presence of interleukin 7 or interleukin 2, these thymocytes gave rise to 30-60% CD3/TCR gamma delta medium and high cells (60-70% expressing V delta 1) seen as discrete populations. Thus, the proportion and V delta phenotype of in vitro generated CD3/TCR gamma delta cells closely resembled those of CD3/TCR gamma delta low cells in freshly isolated "thymocyte precursor" preparations. Small numbers of TCR alpha beta + cells also appeared. It is thus uncertain whether maturation, differentiation, or both account for the appearance of mature CD3/TCR+ thymocytes, although the former appears most likely.

Antibodies, Monoclonal↗
PubMed1990

Interferon gamma response region in the promoter of the human DPA gene.

The interferon gamma (IFN-gamma) response region of the human class II major histocompatibility complex gene, DPA, has been localized to a 52-base-pair (bp) DNA fragment in the proximal promotor at -107 to -55 bp after transfection into HeLa cells of a series of 5', 3', and gap deletion mutants linked to a reporter gene, human growth hormone, as well as of synthetic oligonucleotides fused to the heterologous promoter thymidine kinase. The 52-mer sequence contains the X and Y box elements conserved in all class II genes; their presence is indispensable for IFN-gamma inducibility. Furthermore, an additional 5 bp immediately 5' of the X box of the DPA gene are necessary and sufficient for IFN-gamma induction. This region may contain an IFN-gamma response element. A closely related sequence has also been found in the vicinity of the critical deletion sites of three other well-studied class II gene promoters, all of which require a much longer sequence 5' of the X box. A fourth element, the W element, located about 15 bp 5' of the X box in all class II genes, is clearly of little importance in IFN-gamma inducibility of the DPA gene.

Base Sequence↗