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J L Strominger

Publications and source records attributed to J L Strominger.

At least 127 records · Page 7Linked to original sources
PubMed1995

HLA-A*0201 complexes with two 10-Mer peptides differing at the P2 anchor residue have distinct refolding kinetics.

The immune response to viruses partially depends on the biochemical interaction between viral peptides and histocompatibility molecules. In this study, the refolding of recombinant HLA-A*0201 heavy chain and beta 2-microglobulin (beta 2-m) in the presence of peptides from influenza B nucleoprotein (BNP), influenza A matrix protein, and HIV gp120 and their analogues was examined. The plateau value for the amount of refolded complex with three peptides, a 10-mer BNP 85-94 (A86) with alanine substituted for leucine at the P2 anchor residue and two BNP 8-mers, was significantly lower than the native peptide epitope BNP 85-94 or with other peptides tested. To attempt to understand the basis for the lower yield of complex, equilibrium dissociation constants (KdS) for the two 10-mers, BNP 85-94 (A86) and BNP 85-94, were determined from association and dissociation rates and from Scatchard plots, all measured at 10 degrees C. In addition, dissociation rates were measured at 0 degrees, 26 degrees, and 37 degrees C. Although the kinetics were similar at 0 degrees and 10 degrees, at 37 degrees these two complexes had distinct rates of dissociation, resulting in relatively stable or unstable complexes. The behavior of the unstable complexes paralleled the behavior of empty complexes described in vivo; they are unstable at physiologic temperature, produced in low yield, and stabilized by low temperature. Comparison of all of the kinetic data suggests that the equilibrium amounts of the two HLA/peptide complexes (plateau values) result from distinct reaction pathways, i.e., that the molecules that form stable complexes may undergo an additional reaction to those that form unstable complexes.

Amino Acid Sequence↗
PubMed1995

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs.

cDNAs coding for the HLA class II DR and DQ alpha and beta chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded alpha and beta chains of the DR3 or DR4 haplotypes, as well as the trans-encoded alpha and beta chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR alpha/DQ beta or DQ alpha/DR beta) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most "pan-DQ" mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain alpha beta heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.

Animals↗
PubMed1995

Selective steady-state and time-resolved fluorescence spectroscopy of an HLA-A2-peptide complex.

The human class I major histocompatibility complex (MHC) encoded molecule HLA-A2 loaded with the high-affinity peptide GILGRVFTL (p790), was studied by means of steady-state and picosecond fluorescence intensity and fluorescence anisotropy methods. The large number of tryptophan residues (W) (10 W/heavy chain, 2 W/beta 2m) as well as their fluorescence sensitivity to the microenvironment, determine the emission of the studied complex. The HLA-A2/peptide complex exhibits a considerable static inhomogeneous broadening (IB) of the W electronic spectra, which results in a dependence of the steady-state fluorescence spectrum on the excitation wavelength. The high concentration of W's chromophores and the spectral IB cause a directed non-radiative migration of electronic excitation energy by Foerster's mechanism from 'blue' W residues to 'red' ones. This phenomenon manifests itself in a nanosecond fluorescence spectral shift and an accelerated fluorescence depolarization at the red edge of the emission spectrum. Selective excitation at the red edge of the W absorption band (310 nm) provided a space selective reduction in the number of excited chromophores and enabled resolution of the emission of the 'red' subset of the protein's tryptophans. This avoided the non-radiative homo-energy transfer and enabled to study the fluorescence anisotropy decay kinetics of these residues without a distortion by the energy transfer (ET) process. Under these experimental conditions the fluorescence anisotropy decays practically from the limiting anisotropy value (0.3) for W in a bi-exponential process. The longer decay constant has a value larger than that expected for a global rotation of the HLA-A2/peptide complex suggesting that the protein molecules exist in an oligomeric form.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Direct binding of the Mtv7 superantigen (Mls-1) to soluble MHC class II molecules.

The superantigen encoded by the mouse mammary tumor virus (MMTV) is a potent stimulator of T cells when bound to MHC class II molecules. Recent data from this laboratory have shown that the Mtv7 superantigen, Mls-1, elicits a strong T cell response when presented by HLA-DR. To expand these observations further, we have produced the 28 kDa extracellular domain and the 18 kDa carboxy-terminal subfragment of the Mls-1 protein in E. coli and studied their interaction with human MHC class II molecules in vitro. In this report, we demonstrate direct binding of these recombinant forms of the Mls-1 protein to soluble HLA-DR1 and HLA-DR4, but not to HLA-A2. Our data imply a unique class II interaction site of retroviral superantigens that is not shared with bacterial superantigens.

Animals↗
PubMed1995

Macaques immunized with HLA-DR are protected from challenge with simian immunodeficiency virus.

Macaques immunized with uninfected human cells have been shown to be protected from challenge with simian immunodeficiency virus (SIV) propagated in human cells. To identify the potential antigens involved in this protection, macaques were immunized with uninfected human cells, sucrose density gradient-purified culture fluid from uninfected human cells (mock virus), beta-2 microglobulin (beta 2M), immunoaffinity-purified HLA class I and class II proteins from these human cells, and adjuvant. Although all macaques immunized with beta 2M and HLA class I developed high antibody titers to beta 2M, these animals were not protected from a subsequent challenge with infectious SIV grown in human cells. In contrast, the macaques immunized with class II protein (HLA-DR) and mock virus developed antibodies to class II protein and were protected from the intravenous infectious virus challenge. The class II protein- and mock virus-immunized animals which were protected from challenge were given boosters of the appropriate antigen and challenged with the same SIV propagated in macaque cells. All animals became infected, indicating that the protection seen with human class II protein did not extend to protection from infection with SIV containing macaque class II proteins. Since the virus released from SIV-infected macaque cells would contain macaque class II proteins, our results suggest that the initial SIV infected was completely prevented. In addition, the lack of protection from challenge with SIV propagated in macaque cells provided strong evidence that the protection was due to an immune response to the cellular proteins and not to epitopes cross-reactive between class II proteins and the viral proteins, since the identical virus proteins were present in both challenge stocks. These results are the first demonstration that immunization with a purified cellular protein can protect from virus infection.

Animals↗
PubMed1995

The Epstein-Barr virus BamHI F promoter is an early lytic promoter: lack of correlation with EBNA 1 gene transcription in group 1 Burkitt's lymphoma cell lines.

The Epstein-Barr virus BamHI F promoter (Fp) was previously identified as the putative EBNA 1 gene promoter in group 1 Burkitt's lymphoma (BL) cell lines. Fp has also been shown to be activated in Epstein-Barr virus-positive B-cell lines following induction of the viral productive cycle (A. L. Lear, M. Rowe, M. G. Kurilla, S. Lee, S. Henderson, E. Kieff, and A. B. Rickinson, J. Virol. 66:7461-7468, 1992). Here we demonstrate that Fp is exclusively a lytic promoter which was incorrectly identified as the EBNA 1 gene promoter in group 1 BL cell lines. It is shown that while Fp activity was observed in two group 1 BL cell lines, it could not be detected in a third group 1 BL cell line. Furthermore, the level of Fp activity detected in both group 1 and group 3 cell lines appeared to correlate only with the level of spontaneous lytic activity. Induction of the lytic cycle in group 1 or group 3 BL cell lines resulted in a dramatic increase in Fp-initiated transcripts but no detectable increase in EBNA 1 transcripts. Anti-immunoglobulin induction of the lytic cycle in the Akata group 1 BL cell line revealed that induction of Fp activity was detectable by 2 to 4 h after induction of the lytic cycle and was dependent on de novo protein synthesis. In addition, Fp reporter constructs transiently transfected into group 1 BL cell lines exhibited activity which was independent of the Fp initiation site, TATAA box, or other upstream sequences. The sequences required for efficient reporter gene activity mapped to a region ca. 210 bp downstream of the Fp cap site. Furthermore, Northern (RNA) blot analyses indicated that there are two Fp-initiated lytic transcripts between 9 and 15 kb in size, neither of which correspond to the known EBNA 1 transcripts present in group 1 BL cell lines.

Animals↗
PubMed1994

Functional three-domain single-chain T-cell receptors.

T-cell receptors (TCRs) are membrane anchored heterodimers structurally related to antibody molecules. Single-chain antibodies can be engineered by linking the two variable domains, which fold properly by themselves. However, proper assembly of the variable domains of a human TCR (V alpha and V beta) that recognize the HLA-DR2b/myelin basic protein-(85-99) peptide complex was critically dependent on the addition of a third domain, the constant region of the TCR beta chain (C beta), to the single-chain construct. Single-chain molecules with the three-domain design, but not those with the two-domain design, expressed in a eukaryotic cell as chimeric molecules linked either to glycosyl phosphatidylinositol or to the transmembrane/cytoplasmic domains of the CD3 zeta chain were recognized by a conformation-sensitive monoclonal antibody. The chimeric three-domain single-chain TCR linked to CD3 zeta chain signaled in response to both the specific HLA-DR/peptide and the HLA-DR/superantigen staphylococcal enterotoxin B complexes. Thus, by using this three-domain design, functional single-chain TCR molecules were expressed with high efficiency. The lipid-linked single-chain TCR was solubilized by enzymatic cleavage and purified by affinity chromatography. The apparent requirement of the constant domain for cooperative folding of the two TCR variable domains may reflect significant structural differences between TCR and antibody molecules.

Amino Acid Sequence↗
PubMed1994

Toxic shock syndrome toxin-1 complexed with a class II major histocompatibility molecule HLA-DR1.

The three-dimensional structure of a Staphylococcus aureus superantigen, toxic shock syndrome toxin-1 (TSST-1), complexed with a human class II major histocompatibility molecule (DR1), was determined by x-ray crystallography. The TSST-1 binding site on DR1 overlaps that of the superantigen S. aureus enterotoxin B (SEB), but the two binding modes differ. Whereas SEB binds primarily off one edge of the peptide binding site of DR1, TSST-1 extends over almost one-half of the binding site and contacts both the flanking alpha helices of the histocompatibility antigen and the bound peptide. This difference suggests that the T cell receptor (TCR) would bind to TSST-1:DR1 very differently than to DR1:peptide or SEB:DR1. It also suggests that TSST-1 binding may be dependent on the peptide, though less so than TCR binding, providing a possible explanation for the inability of TSST-1 to competitively block SEB binding to all DR1 molecules on cells (even though the binding sites of TSST-1 and SEB on DR1 overlap almost completely) and suggesting the possibility that T cell activation by superantigen could be directed by peptide antigen.

Bacterial Toxins↗
PubMed1994

A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor.

The class II major histocompatibility complex (MHC) molecules function in the presentation of processed peptides to helper T cells. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen-presenting cell is its ability to express class II MHC molecules. Expression of these molecules is usually restricted to cells of the immune system and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. Human complementary DNA clones encoding a newly identified, cysteine-rich transcription factor, NF-X1, which binds to the conserved X-box motif of class II MHC genes, were obtained, and the primary amino acid sequence deduced. The major open reading frame encodes a polypeptide of 1,104 amino acids with a symmetrical organization. A central cysteine-rich portion encodes the DNA-binding domain, and is subdivided into seven repeated motifs. This motif is similar to but distinct from the LIM domain and the RING finger family, and is reminiscent of known metal-binding regions. The unique arrangement of cysteines indicates that the consensus sequence CX3CXL-XCGX1-5HXCX3CHXGXC represents a novel cysteine-rich motif. Two lines of evidence indicate that the polypeptide encodes a potent and biologically relevant repressor of HLA-DRA transcription: (a) overexpression of NF-X1 from a retroviral construct strongly decreases transcription from the HLA-DRA promoter; and (b) the NF-X1 transcript is markedly induced late after induction with interferon gamma (IFN-gamma), coinciding with postinduction attenuation of HLA-DRA transcription. The NF-X1 protein may therefore play an important role in regulating the duration of an inflammatory response by limiting the period in which class II MHC molecules are induced by IFN-gamma.

Amino Acid Sequence↗
PubMed1994

Assembly and peptide binding of major histocompatibility complex class II heterodimers in an in vitro translation system.

In vitro transcription/translation of HLA-DR1 cDNAs in the presence of microsomal membranes was used to study the association of major histocompatibility complex class II molecules with peptide and invariant chain (Ii) in the endoplasmic reticulum (ER). HLA-DR alpha and HLA-DR beta subunits assembled into SDS-unstable heterodimers in the absence of exogenous peptide. The inclusion of synthetic peptides during the alpha/beta assembly process promoted their conversion to SDS-resistant heterodimers. Addition of Ii RNA during the translation of HLA-DR alpha and HLA-DR beta RNAs resulted in the formation of alpha/beta/Ii complexes. Peptide binding by class II molecules was detected even when excess Ii was present during alpha/beta assembly. These findings indicate that peptides can bind alpha/beta heterodimers in the ER microenvironment and suggest that peptides derived from cytosolic proteins that are presented by class II molecules at the cell surface may have bound to HLA-DR in the ER.

Amino Acid Sequence↗
PubMed1994

NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80.

Natural killer (NK) cell clones have been previously described which are inhibited by HLA-C alleles with Asn77-Lys80 (NK1-specific cells) or by HLA-C alleles with Ser77-Asn80 (NK2-specific cells). In the present work, the generation of NK cells with HLA-B-related specificities was attempted by stimulation of a Bw4 homozygous responder by a Bw6 homozygous donor. Two NK clones were found, which were inhibited by HLA-Bw4 (but not by HLA-Bw6) allotypes and by some HLA-A allotypes that share the Bw4 public epitope. Inhibition of NK cell-mediated lysis strongly correlated with the presence of an Ile residue at position 80 of the protective allele. These NK cell clones define a new specificity termed NK3.

Alleles↗
PubMed1994

Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli.

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

Amino Acid Sequence↗
PubMed1994

Selective release of some invariant chain-derived peptides from HLA-DR1 molecules at endosomal pH.

The predominant peptides bound to major histocompatibility complex class II molecules expressed on human B cells are derived from a relatively limited number of self proteins. To determine whether any of the prebound self peptides might be released in endosomes during recycling, water-soluble HLA-DR1 molecules were incubated with a high affinity synthetic peptide at pH 4.0 and 7.0 at 37 degrees C. The resulting bound peptide repertoire was then acid extracted, and separated by reversed-phase high performance liquid chromatography. Using a combination of mass spectrometry and ultraviolet spectroscopy, prebound self peptides and newly bound synthetic peptide were characterized. Most self peptides bound to HLA-DR1 were not appreciably released during extended exposure to pH 4.0. However, some invariant chain-derived peptides were uniquely released at this pH.

Amino Acid Sequence↗
PubMed1994

Calcineurin mediates human tumor necrosis factor alpha gene induction in stimulated T and B cells.

The tumor necrosis factor alpha (TNF-alpha) gene is rapidly transcribed in activated T cells via a calcium-dependent pathway that does not require de novo protein synthesis, but is completely blocked by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Here we show that calcineurin phosphatase activity is both necessary and sufficient for TNF-alpha gene transcription in T cells, and identify the factor that binds to the kappa 3 element of the TNF-alpha gene promoter as the target for calcineurin action. The ability of analogues of CsA and FK506 to block calcineurin phosphatase activity correlates completely with their ability to inhibit induction of TNF-alpha mRNA, induction of a TNF-alpha promoter reporter plasmid in transiently transfected T cells, and induction of the kappa 3 binding factor in an electrophoretic mobility shift assay. Moreover, a cDNA encoding the constitutively active form of calcineurin is sufficient to activate the TNF-alpha promoter and the kappa 3 element. TNF-alpha gene transcription is also highly inducible, CsA-sensitive, and protein synthesis-independent in B cells stimulated through their surface immunoglobulin receptors. Using the panel of CsA and FK506 analogues, we show that calcineurin participates in the induction of TNF-alpha transcription in activated B cells. These results extend our previous demonstration that the kappa 3 binding factor is related to NFATp, the preexisting subunit of nuclear factor of activated T cells, and suggest that calcineurin-mediated modification of the kappa 3 binding factor in T cells is of key importance in the induction of TNF-alpha transcription.

B-Lymphocytes↗
PubMed1994

Tumor necrosis factor alpha is an autocrine growth factor for normal human B cells.

Transcription of the human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest events that occurs after stimulation of B or T cells via their antigen receptors. Antibody directed at surface immunoglobulin (anti-Ig) on B cells has previously been shown to induce a rapid burst of TNF-alpha gene transcription, which can be blocked by the immunosuppressants cyclosporin A (CsA) and FK506. Here, TNF-alpha gene transcription is shown also to be highly and rapidly induced in human B cells after stimulation via the CD40 and interleukin 4 pathways, which similarly is inhibited by CsA and a panel of CsA or FK506 analogues that block calcineurin phosphatase activity. Endogenous TNF-alpha produced after stimulation was involved in B-cell proliferation since anti-TNF-alpha monoclonal antibody inhibited both anti-Ig- and anti-CD40-induced B-cell proliferative responses. Moreover, addition of TNF-alpha during stimulation resulted in augmentation of B-cell proliferation, which was also inhibited by anti-TNF-alpha monoclonal antibody. Although lymphotoxin alpha (LT-alpha) mRNA is induced by both pathways, it is not blocked by CsA, whereas LT-beta mRNA is constitutively expressed in B cells. Thus, TNF-alpha is a necessary autocrine growth factor for human B cells stimulated via two independent CsA-sensitive pathways and plays a role similar to that of interleukin 2 in T-cell proliferation. The autocrine nature of TNF-alpha in activated B cells implies a potential role for this cytokine in infection-related polyclonal B-cell expansion and in B-cell malignancies.

Antigens, CD↗
PubMed1994

Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor.

Hen egg lysozyme 52-61-specific CD4+ T cells responded by interleukin 2 (IL-2) secretion to any peptide containing this epitope regardless of length of NH2- and COOH-terminal composition. However, CD4- variants could only respond to peptides containing the two COOH-terminal tryptophans at positions 62 and 63. Substitutions at these positions defined patterns of reactivity that were specific for individual T cells inferring a T cell receptor (TCR)-based phenomenon. Thus, the fine specificity of major histocompatibility complex (MHC)-peptide recognition by the TCR was dramatically affected by CD4 and the COOH-terminal peptide composition. Peptides that failed to induce IL-2 secretion in the CD4- variants nevertheless induced strong tyrosine phosphorylation of CD3 zeta. Thus, whereas the TCR still recognized and bound to the MHC class II-peptide complex resulting in protein phosphorylation, this interaction failed to induce effective signal transduction manifested by IL-2 secretion. This provides a clear example of differential signaling mediated by peptides known to be naturally processed. In addition, the external domains of CD4, rather than its cytoplasmic tail, were critical in aiding TCR recognition of all peptides derived from a single epitope. These data suggest that the nested flanking residues, which are present on MHC class II but not class I bound peptides, are functionally relevant.

Amino Acid Sequence↗
PubMed1994

Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen.

The structure of a bacterial superantigen, Staphylococcus aureus enterotoxin B, bound to a human class II histocompatibility complex molecule (HLA-DR1) has been determined by X-ray crystallography. The superantigen binds as an intact protein outside the conventional peptide antigen-binding site of the class II major histocompatibility complex (MHC) molecule. No large conformational changes occur upon complex formation in either the DR1 or the enterotoxin B molecules. The structure of the complex helps explain how different class II molecules and superantigens associate and suggests a model for ternary complex formation with the T-cell antigen receptor (TCR), in which unconventional TCR-MHC contacts are possible.

Amino Acid Sequence↗