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J L Strominger

Publications and source records attributed to J L Strominger.

At least 55 records · Page 3Linked to original sources
PubMed1999

Conformational variants of class II MHC/peptide complexes induced by N- and C-terminal extensions of minimal peptide epitopes.

Class II MHC molecules are known to exist in conformational variants. "Floppy" and "compact" forms of murine MHC molecules, for example, are discriminated by their migration behavior on SDS/PAGE and represent empty and ligand-loaded forms. Here we show that formation of distinctly faster-migrating ligand complexes (F-forms) rather than the normal compact (C-) forms of HLA-DR1 or -DR4 results from extensions of minimal peptide epitopes (such as HA306-318 or IC106-120) by approximately 10 amino acids at either the N or the C terminus. Two similar but distinct F-forms (FI and FII) were detected, depending on the site of the extension. Both F-forms were characterized by increased surface hydrophobicity and reduced SDS-stability. Native gel separations and size exclusion chromatography indicated that the F-forms had increased hydrodynamic radii compared with the C-form and an apparent size similar to that of empty MHC molecules. The regions on the ligand overhangs responsible for the effect began at a distance of approximately 5 amino acids on either side of the epitopes, comprised 4-8 amino acids (i.e., a total overhang of 9-14), and did not have a particular sequence preference. The possible functional significance of these forms is discussed.

Antigen Presentation↗
PubMed1999

Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity.

In addition to their role in peptide antigen presentation, class I MHC proteins also play a critical role in inhibiting natural killer (NK) cytotoxicity through interaction with NK inhibitory receptors. Thus, NK cells are cytotoxic to virus-infected and tumor cells that have lost class I MHC protein expression. However, the nature of the receptors involved in the triggering of lysis of target cells is poorly understood. CD16 (Fcgamma receptor III) has been described as a receptor expressed on NK cells that facilitates antibody-dependent cellular cytotoxicity (ADCC) by binding to the Fc portion of various antibodies. However, we show here that CD16 has a broader function and is directly involved in the lysis of some virus-infected cells and tumor cells, independent of antibody binding. The presence of a putative CD16 ligand on appropriate target cells has also been demonstrated by the use of a CD16-Ig fusion protein.

Antibodies, Monoclonal↗
PubMed1999

The transmembrane sequence of human histocompatibility leukocyte antigen (HLA)-C as a determinant in inhibition of a subset of natural killer cells.

Molecular interactions with the extracellular domains of class I major histocompatibility complex proteins are major determinants of immune recognition that have been extensively studied both physically and biochemically. However, no immunological function has yet been placed on the transmembrane or cytoplasmic amino acid sequences of these proteins despite strict conservation of unique features within each class I major histocompatibility complex locus. Here we report that lysis by a subset of natural killer (NK) cells inhibited by target cell expression of human histocompatibility leukocyte antigen (HLA)-Cw6 or -Cw7 was not inhibited by expression of chimeric proteins consisting of the extracellular domains of HLA-C and the COOH-terminal portion of HLA-G. Assays using transfectants expressing a variety of HLA-Cw6 mutants identified the transmembrane sequence and, in particular, cysteine at position 309 as necessary for inhibition of 68% (25/37) of NK cell lines and 23% (33/145) of NK clones tested. Moreover, these NK clones inhibited by target cell expression of HLA-Cw6 and dependent upon the transmembrane sequence were found not to express or to only dimly express NK inhibitory receptors (NKIR1) that are EB6/HP3E4-positive. Furthermore, assays using monoclonal antibody blocking suggest that an NK receptor other than NKIR1 or CD94 is responsible for recognition dependent upon the transmembrane sequence of HLA-Cw6.

Amino Acid Sequence↗
PubMed1999

Binding motifs of copolymer 1 to multiple sclerosis- and rheumatoid arthritis-associated HLA-DR molecules.

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1999

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin.

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.

Animals↗
PubMed1999

NOD background genes influence T cell responses to GAD 65 in HLA-DQ8 transgenic mice.

The major histocompatibility complex (MHC) genes play a significant role in the predisposition to insulin-dependent diabetes mellitus or type 1 diabetes. HLA-DQ8 (DQB1*0302, DQA 1*0301) genes have been shown to have the highest relative risk for human type 1 diabetes. To develop a "humanized" mouse model of diabetes, HLA-DQ8 was transgenically expressed in mice lacking endogenous class II genes. Since non-MHC background genes of the NOD influence the disease process, AP"/DQ8 mice were mated with the NOD strain and backcrossed to generate Abeta degree/DQ8/NOD mice. These mice have DQ8 as the sole MHC class II restriction element with NOD background genes at the N 2 generation. The DQ8 transgenic mice were used to identify T cell epitopes on glutamic acid decarboxylase (GAD 65), an important putative autoantigen in type 1 diabetes. The NOD background genes strongly influenced antigen processing, that is, different T cell epitopes were generated from the processing of GAD 65 in vivo in the Abeta degree/DQ8 and in the Abeta degree/DQ8/NOD mice.

Animals↗
PubMed1999

Noncanonical Valpha24JalphaQ T cells with conservative alpha chain CDR3 region amino acid substitutions are restricted by CD1d.

Human NKRP-1A (CD161)+ T cells include members of a family of CD4+ or CD4-/CD8- lymphocytes that utilize an invariant alpha chain in the T-cell receptor (TCR). The alpha chain consists of the Valpha24 segment joined to Jalpha18 (JalphaQ) (TCRAV24/AJ18). These families of T cells rapidly produce both interleukin-4 and interferon-gamma upon TCR cross-linking, and are restricted by CD1d. To determine the spectrum of allowable V/J rearrangements in the Valpha24+ CD4-/CD8- family, TCR Valpha24 chain transcripts derived from the total CD4-/CD8- population in peripheral blood mononuclear cells were sequenced. A second invariant rearrangement, Valpha24Jalpha45, was found in two donors. In addition, a subset of 15 clones with single amino acid substitutions in the CDR3 were identified and used to define CD1d restriction. All 15 variant clones were indistinguishable from invariant clones on the basis of surface phenotype and response to CD3 cross-linking. However, CD1d was the restriction element only for those clones with the conservative substitution of threonine or asparagine for serine at the V/J junction. Thus, the family of human CD161+ T cells can be extended to include a subset of Valpha24-JalphaQ rearrangements with a single amino acid substitution that defines a Valpha24 CDR3 residue critical for CD1d restriction.

Amino Acid Sequence↗
PubMed1999

The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells.

To avoid detection by CTL, HIV encodes mechanisms for removal of class I MHC proteins from the surface of infected cells. However, class I downregulation potentially exposes the virus-infected cell to attack by NK cells. Human lymphoid cells are protected from NK cell cytotoxicity primarily by HLA-C and HLA-E. We present evidence that HIV-1 selectively downregulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E. We then identify the residues in HLA-C and HLA-E that protect them from HIV down-regulation. This selective downregulation allows HIV-infected cells to avoid NK cell-mediated lysis and may represent for HIV a balance between escape from CTL and maintenance of protection from NK cells. These results suggest that subpopulations of CTL and NK cells may be uniquely suited for combating HIV.

Cells, Cultured↗
PubMed1999

Biotinylation of class I MHC molecules abrogates recognition by W6/32 antibody.

W6/32 is one of the most common monoclonal antibodies (mAb) used to characterize human class I major histocompatibility complex (MHC) molecules. It recognizes a conformational epitope on the intact MHC molecule containing both beta2-microglobulin (beta2-m) and the heavy chain. Labelling proteins by biotinylation is a very useful technique of for their detection, purification and analysis. A common method for biotinylating proteins is through the use of N-hydroxysuccinimide (NHS) biotin or Sulfo-NHS-biotin where the free amino groups on the protein are used for coupling the biotin moiety. However, W6/32 was unable to effectively immunoprecipitate biotinylated human class I MHC molecules including the human non-classical HLA-G molecule. FACScan analysis confirmed that biotinylating human class I MHC and HLA-G molecules prevents the recognition of these molecule by W6/32. In contrast, the recognition by another conformation-dependent monoclonal antibody, ME1, specific to HLA-B27 molecules, remained totally unaffected.

Antibodies, Monoclonal↗
PubMed1999

A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

Animals↗
PubMed1999

Binding of random copolymers of three amino acids to class II MHC molecules.

Copolymer 1 [Cop 1, poly(Y,E,A,K)] is a random synthetic amino acid copolymer of L-tyrosine, L-glutamic acid, L-alanine and L-lysine, effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Cop 1 binds promiscuously and very efficiently to purified human HLA-DR molecules within the peptide-binding groove. In the present study the binding of copolymers composed of three of the four amino acids found in poly(Y,E,A,K) to purified class II MHC molecules was examined. Poly(Y,A,K) and poly(Y,E,A,K) bound to purified human HLA-DR1 or -DR4 molecules with affinity higher than poly(Y,E,A), poly(E,A,K) or poly(Y,E,K), whereas poly(Y,E,A,K) and poly(E,A,K) were the better binders of HLA-DR2 molecules. On the other hand, poly(Y,E,A) and poly(Y,A,K) inhibited the binding of biotinylated poly(Y,E,A,K) to these molecules 10-fold more efficiently than poly(Y,E,K). Finally, poly(Y,E,A), poly(Y,A,K) and poly(E,A,K) were cross-reactive with poly(Y,E,A,K) using YEAK-specific T cell lines and clones of mouse or human origin.

Animals↗
PubMed1998

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones.

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of L-tyrosine, L-glutamic acid, L-alanine, and L-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261-273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261-273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Amino Acids↗
PubMed1998

Expression and crystallization of the complex of HLA-DR2 (DRA, DRB1*1501) and an immunodominant peptide of human myelin basic protein.

HLA-DR2 is associated with susceptibility to multiple sclerosis (MS). A peptide from human myelin basic protein (MBP, residues 85-99) was previously found to bind to purified HLA-DR2 (DRA, DRB1*1501) and to be recognized by human MBP-specific T cell clones. Soluble HLA-DR2 was expressed in the baculovirus system by replacing the hydrophobic transmembrane regions and cytoplasmic segments of DRalpha and DRbeta with leucine zipper dimerization domains from the transcription factors Fos and Jun. In the expression construct, the MBP(85-99) sequence was covalently linked to the N terminus of the mature DRbeta chain. The recombinant protein was secreted by Sf9 cells infected with the recombinant baculovirus and purified by affinity chromatography. The leucine zipper dimerization domains were then cleaved from the assembled HLA-DR2/MBP peptide complex with V8 protease, and the protein was further purified by anion-exchange HPLC. Analysis by HPLC gel filtration indicated that the HLA-DR2/MBP peptide complex did not have a tendency to aggregate. The purified HLA-DR2/MBP peptide complex readily crystallized by the hanging drop method in 15-18% polyethylene glycol 6000/100 mM glycine, pH 3.5. At a synchrotron radiation source, a crystal with a tetragonal space group diffracted to a resolution of 2.6 A. The expression of such homogenous HLA-DR/peptide complexes may facilitate cocrystallization with T cell receptors as well as other molecules involved in T cell receptor recognition and signaling.

Amino Acid Sequence↗
PubMed1998

Promiscuous binding of synthetic copolymer 1 to purified HLA-DR molecules.

Copolymer 1 (Cop 1) is a random synthetic amino acid copolymer of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine, effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Cop 1 binds promiscuously and very efficiently to living APCs of various HLA haplotypes. In the present study, a substantial part of the whole mixture of random polypeptides that compose Cop 1 was shown to bind to purified human HLA-DR1, DR2, and DR4 with high affinity in a temperature- and time (and, in the case of DR4, pH)-dependent manner, and was competitively inhibited by DR-restricted peptides, but not by peptide derivatives that bind with low affinity. Bacterial superantigens inhibited Cop 1 binding only at very high concentrations. The formation of the Cop 1-DR1 complex was also shown by SDS-PAGE. These findings represent the first direct evidence for interactions of Cop 1 with purified DR molecules, and suggest that its effectiveness in experimental allergic encephalomyelitis and multiple sclerosis may be directly related to its binding in the groove of HLA-DR proteins.

B-Lymphocytes↗