Biomedical subjects

J L Strominger

Publications and source records attributed to J L Strominger.

At least 37 records · Page 2Linked to original sources

PubMed2000

Assembly and dissociation of human leukocyte antigen (HLA)-A2 studied by real-time fluorescence resonance energy transfer.

Class I major histocompatibility complex (MHC) heterodimer, composed of human leukocyte antigen (HLA)-A2 heavy chain and human beta(2)-microglobulin (beta(2)m), was produced by denaturation and gel filtration of the recombinant water-soluble HLA-A2/beta(2)m/peptide ternary complex in 8 M urea Tris-HCl buffer, followed by refolding of the separated chains without peptide. Peptide affinity and kinetics of the ternary complex formation and dissociation were investigated in real time by monitoring the fluorescence resonance energy transfer (FRET) from intrinsic HLA-A2 heavy-chain tryptophans to a dansyl fluorophore conjugated to the bound peptide. Peptide binding to the heterodimer was a second order process with rate constants linearly dependent upon temperature in Arrhenius coordinates over 0-20 degrees C. The binding rate constant of pRT6C-dansyl [ILKEPC(dansyl)HGV] at 37 degrees C evaluated by extrapolation of the Arrhenius plot was (2.0 +/- 0.5) x 10(6) M(-1) s(-1). Association of the heavy chain with beta(2)m was a first order process, apparently controlled by a conformational transition in the heavy chain. One of these conformations bound to beta(2)m to form the heavy chain/beta(2)m heterodimer whereas the second conformer oligomerized. Peptide dissociation from the ternary complex was a first-order reaction over the temperature range 20-37 degrees C, suggesting that the ternary complex also exists in two conformations. Taken together, the present data suggest that association of beta(2)m changes the HLA-A2 heavy-chain conformation thereby promoting peptide binding. Peptide dissociation from the ternary complex induces dissociation of the heavy-chain/beta(2)m heterodimer thereby causing oligomerization of the heavy chain. The lability of the HLA-A2/beta(2)m heterodimer and the strong tendency of the "free" heavy chain to oligomerize may provide an efficient mechanism for control of antigen presentation under physiological conditions by reducing the direct loading of HLA with exogenous peptide at the cell surface.

Dimerization↗

PubMed2000

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes.

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

Diabetes Mellitus, Type 1↗

PubMed2000

Induction and suppression of an autoimmune disease by oligomerized T cell epitopes: enhanced in vivo potency of encephalitogenic peptides.

T cell epitope peptides derived from proteolipid protein (PLP139-151) or myelin basic protein (MBP86-100) induce experimental autoimmune encephalomyelitis (EAE) in "susceptible" strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund's adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the peptide epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10.S mice, "resistant" to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50 microg of the PLP139-151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139-151 peptide accelerated rather than retarded the progression of disease.

Animals↗

PubMed2000

Perfusion chromatography for very rapid purification of class I and II MHC proteins.

Major histocompatibility complex (MHC) proteins are surface glycoproteins that are strongly associated with either self or foreign peptides. Their interaction with the T-cell receptor on the T-cells initiates an immune response and help in discriminating between self and non-self, respectively. We describe here a novel means of rapidly purifying human MHC molecules on either small scale or large scale from the cell lysate of lymphoblastoid B cell line and from insect cell culture supernatants by using affinity perfusion chromatography. As representative cases HLA-B2705, a class I MHC molecule, and HLA-DR1, a class II MHC molecule were purified from EBV-transformed human lymphoblastoid B cells, LG2. Soluble HLA-DR1 was also purified from the cell culture supernatant of insect cells. The peptides eluted from the purified HLA-B2705 were pool sequenced and found to have the same motif as has previously been published. This new method provides a very rapid means of purifying MHC protein molecules, applicable to both large scale and small scale purification, which in turn greatly enhances the accuracy of further analysis of the associated peptides through mass spectrometry.

Animals↗

PubMed2000

Definition of polymorphic residues on killer Ig-like receptor proteins which contribute to the HLA-C binding site.

Killer cell immunoglobulin-like receptors (KIR) bind HLA class I proteins in an allele- and locus-specific manner. This report describes the use of transfectants expressing recombinant chimeric proteins, comprising the extracellular portions of KIR molecules and the transmembrane and cytoplasmic tails of CD3-zeta, to create an in vitro system in which signaling is readily measured and that preserves the specificity of the KIR / HLA-C interaction. The identity of the amino acid residues on the KIR molecule important for binding to the HLA protein is not well understood; although some KIR2D residues involved in HLA-C recognition have been identified, their relative importance and whether other amino acids contribute to binding was unclear. This novel system was used to study, by site-directed mutagenesis, the role of various amino acids in KIR binding to HLA-C ligand. The data presented here show that while multiple polymorphic residues contribute to the HLA-C binding site on KIR proteins, two clusters of polymorphic residues define the group allotype specificity of HLA-C binding to a KIR2D molecule.

Animals↗

PubMed2000

Antigen-specific elimination of T cells induced by oligomerized hemagglutinin (HA) 306-318.

In a previous study we reported that oligomerized T cell epitopes "superactivated" CD4+ T cells. These oligomers, consisting of 12-16 copies of a peptide epitope derived from the hemagglutinin protein of influenza virus (HA306-318), induced a specific T cell response in amounts as little as 5 pg/ml. We now show that the improved antigenicity of these multimerized epitopes can also be utilized to induce "high zone tolerance". Tolerization, similar to activation, occurred at about 3 logs lower concentration of oligomer than of peptide. HA306-318-specific T cell cultures became nonresponsive to stimulation with peptide after incubation with 0.5-5 microg/ml HA306-318 12-mer. The nonresponsiveness was accompanied by a drastic down-regulation of the TCR and by T cell elimination by apoptotic cell death. In contrast, stimulation with peptide even at 50 microg/ml led to temporary induction of anergy. Consequently, induction of tolerance with the oligomer was permanent and no recovery of the cultures was seen in recall experiments 12-14 days after high zone exposure to the 12-mer.

Antigens, Viral↗

PubMed2000

Synthetic peptides that inhibit binding of the collagen type II 261-273 epitope to rheumatoid arthritis-associated HLA-DR1 and -DR4 molecules and collagen-specific T-cell responses.

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), a disease that is linked to HLA-DR2 (DRB1*1501). Another copolymer [poly (Y, A, K)] was also identified that binds to rheumatoid arthritis (RA)-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules and inhibits the response of HLA-DR1- and -DR4-restricted T cell clones to an immunodominant epitope of collagen type II (CII) 261-273 (a candidate autoantigen in RA). In the present study various peptides have been synthesized based on binding "motifs" of Cop 1 for HLA-DR1 and -DR4 molecules. Those peptides with K at P-1 or K at P8 were particularly effective as inhibitors of binding of CII 261-273, of Cop 1 and of the influenza virus hemagglutinin peptide 306-318 to these class II proteins. Moreover, several of them were also potent inhibitors of the CII 261-273-reactive T cell clones. These findings suggest that small peptides or their more stable derivatives may be able to substitute for copolymers in the treatment of RA, and by implication of MS.

Animals↗

PubMed2000

Expression of the CD80 and CD86 molecules enhances cytotoxicity by human natural killer cells.

In contrast to the inhibitory pathway of NK cell regulation, much less is known about stimulatory or activation signals in NK cells. Both CD80 and CD86 function as costimulatory molecules in T-cell cytotoxicity. Several previous reports, most of them in the murine system, have indirectly or directly indicated the possible role of B7 molecules (CD80 and CD86) triggering NK cell-mediated cytotoxicity in vitro. Nevertheless, only little is known about the role of these molecules on human target cells. Therefore, anti-CD80 and anti-CD86 mAbs were used in blocking experiments and both were shown to inhibit lysis by human NK cells. The degree of inhibition observed was variable. 64% of these NK clones were strongly inhibited by both anti-CD80 and anti-CD86 (Type 1). A small number (19%) were only moderately inhibited by both of these antibodies (Type 2), and 17% of these NK clones were inhibited strongly by anti-CD86 but weakly or not at all by anti-CD80 (Type 3). To further examine the importance of these proteins, B7.1 (CD80) and B7.2 (CD86) genes were transfected into the mouse mastocytoma P815 cell line that could not be killed by the human NK cells. These transfectant cell lines were then tested in cytotoxicity assays using a number of human NK lines. Expression of the CD80 and CD86 molecules resulted in enhanced lysis of P815 by most of the NK lines tested. Thus, both CD80 and CD86 molecules are involved in triggering of human NK cells.

Animals↗

PubMed2000

N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors.

The possible role of carbohydrate in the interaction of HLA-C with a human inhibitory natural Killer cell Immunoglobulin-like Receptor with two Ig domains, KIR2DL1, was investigated. Transfectants of 721.221 (a class I MHC-negative human B cell line) expressing only HLA-Cw4 or -Cw6 or their respective non-glycosylated mutants (N86Q, S88A) were made. The binding of a KIR2DL1-Ig fusion protein to the non-glycosylated mutant HLA-Cw4- or -Cw6-expressing cells was markedly decreased compared to the wild type-expressing cells. The ability to induce an inhibitory signal in the NK tumor line YTS transfected with KIR2DL1 was also impaired in the nonglycosylated mutant expressing cells. Furthermore, in a second functional assay, mutant HLA-Cw4 and -Cw6 molecules had impaired ability to induce signal transduction in BW cells expressing a KIR2DL1-CD3 zeta chain chimeric protein. Thus, the deletion of the N-linked glycosylation signal in HLA-Cw4 and -Cw6 greatly reduced recognition by KIR2DL1. Alternative interpretations of the data are discussed.

Amino Acid Substitution↗

PubMed2000

Using DNA chips to unravel the genetics of type 1 diabetes.

Thousands of genes are currently being discovered by sequencing the human genome. Of these, hundreds if not thousands fall into regions of the genome identified by genetic studies as linked to the development of type 1 diabetes. Inheritance patterns for these regions suggest that diabetes results from the combinatorial interaction of susceptibility loci. The study of such complex events will require technologies that can simultaneously evaluate expression profiles and allelic differences for all these genes in order to dissect the mechanisms responsible for the development of disease. We will argue that DNA microarrays are the natural vehicle for the exploration of diabetes-related gene clusters, and the application of these arrays to understanding diabetes is discussed.

Diabetes Mellitus, Type 1↗

PubMed2000

Co-expression of HLA DR3 and DQ8 results in the development of spontaneous insulitis and loss of tolerance to GAD65 in transgenic mice.

Specific HLA DQ and DR alleles have been associated with susceptibility to type 1 diabetes. HLA DQ8 and DQ2 have been shown to strongly predispose to disease and to be in linkage disequilibrium with at-risk DR4 and DR3 alleles, respectively. Inheritance of a mixed DR3/DR4 haplotype confers the greatest risk. A double transgenic mouse expressing both DR3 and DQ8 was generated to investigate potential major histocompatibility complex class II interactions. The DR3/DQ8 transgenic mice developed a spontaneous loss of tolerance to GAD65, in which the T-cell response to GAD65 was restricted by HLA DR. Although the mice also showed spontaneous insulitis, they did not progress to overt diabetes. Mice expressing either transgene (DQ8 or DR3) alone showed mild infiltration of their islets, which disappeared when DQ8 or DR3 was co-expressed with a resistant DR2 allele or the neutral DQ6 allele. Therefore, in a fashion analogous to human diabetes, the murine model demonstrated a requirement for a combination of at-risk DR and DQ allotypes for the initiation of spontaneous autoimmunity.

Animals↗

PubMed1999

Extracellular antigen processing and presentation by immature dendritic cells.

In antigen presentation to CD4(+) T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded onto class II MHC proteins for subsequent transport to the surface and presentation to T cells. Here, we provide evidence for an additional antigen-processing pathway in immature dendritic cells (DC). Immature DC express at the cell surface empty or peptide-receptive class II MHC molecules, as well as H-2M or HLA-DM. Secreted DC proteases act extracellularly to process intact proteins into antigenic peptides. Peptides produced by such activity are efficiently loaded onto cell surface class II MHC molecules. Together these elements comprise an unusual extracellular presentation pathway in which antigen processing and peptide loading can occur entirely outside of the cell.

Animals↗

PubMed1999

The human natural killer cell immune synapse.

Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.

Actins↗

PubMed1999

Trafficking of spontaneously endocytosed MHC proteins.

Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. beta(2)-microglobulin (beta(2)m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell surface and endocytosed class I MHC protein. Intracellular beta(2)m colocalized with fluorophore-conjugated transferrin, implying that class I MHC protein endocytosed into early endosomes. These endosomes containing fluorescent beta(2)m were found close to or within the Golgi apparatus, marked by fluorescent ceramide. Even after 24 hr of incubation, very little fluorescent beta(2)m was found in intracellular organelles stained by DiOC(6), marking the endoplasmic reticulum, or fluorophore-conjugated low density lipoprotein, marking late endosomes and lysosomes. Fluorophore-conjugated superantigens (staphylococcal enterotoxin A and B), presumed to enter cells bound to class II MHC protein, also were found to endocytose into beta(2)m-containing early endosomes. Staining with mAb and use of transfectants expressing MHC protein attached to green fluorescent protein confirmed the presence of intracellular compartments rich in both class I and II MHC protein and demonstrated that class I and II MHC protein also colocalize in discrete microdomains at the cell surface. These cell surface microdomains also contained transferrin receptor and often were juxtaposed to cholesterol-rich lipid rafts. Thus, class I and II MHC protein meet in microdomains of the plasma membrane and endocytose into early endosomes, where both may acquire and present exogenous antigen.

B-Lymphocytes↗

PubMed1999

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.

Allosteric Regulation↗

PubMed1999

Differential responses of invariant V alpha 24J alpha Q T cells and MHC class II-restricted CD4+ T cells to dexamethasone.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.

Adjuvants, Immunologic↗

PubMed1999

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.

Amino Acid Sequence↗

PubMed1999

Heterophile antibodies segregate in families and are associated with protection from type 1 diabetes.

Markedly elevated levels of serum IL-4 were reported previously in 50% of a small group of type 1 diabetes nonprogessors. To determine the patterns of expression for this phenotype, a larger cohort of 58 families containing type 1 diabetic patients was examined. Analysis of the two-site ELISA assay used to measure serum IL-4 revealed evidence for heterophile antibodies, i.e., nonanalyte substances in serum capable of binding antibodies mutivalently and providing erroneous analyte (e.g., IL-4) quantification. Interestingly, relatives without type 1 diabetes were significantly more likely to have this phenotype than were patients with the disease (P = 0.003). In addition, the trait appears to have clustered within certain families and was associated with the protective MHC allele DQB1*0602 (P = 0.008). These results suggest that heterophile antibodies represent an in vivo trait associated with self-tolerance and nonprogression to diabetes.

Alleles↗