Biomedical subjects

C Beeson

Publications and source records attributed to C Beeson.

At least 19 recordsLinked to original sources

PubMed2001

Utility of azapeptides as major histocompatibility complex class II protein ligands for T-cell activation.

Major histocompatibility complex class II (MHC II) protein binding and antigen specific activation of CD4+ "helper" T cells are demonstrated with peptides composed of the antigenic hen egg ovalbumin 325-339 peptide (OVA) substituted with azaamino acids. AzaAla and azaGly substitutions were made at 10 sequential peptide positions (326Ala-335Asn) that lie in the binding groove. The peptide positions substituted with azaamino acids encompass almost the entire binding groove, including positions where the identity of the amino acid side chain is known to have the most significant effect on MHC binding and the least effect on T-cell recognition. In addition, the T-cell contact 333Glu was substituted with azaGlu to generate a partial agonist ligand for the 3DO-54.8 T-cell hybridoma. Binding to MHC II protein was assayed by measuring the kinetic stability of complexes formed between detergent-solubilized MHC II I-A(d) protein and fluorescein-labeled OVA peptides using a fluorescence-HPLC assay. T-cell activation was also evaluated for aza-substituted peptides with azaamino acid substitutions at the peptide positions known to interact with the MHC II protein. All aza-substituted peptides showed detectable MHC binding, and some were found to show T-cell activation potency equal to the native peptide. Several of these were also found to be weak or partial agonists. Our results demonstrate that azaamino acids substituted into an antigenic peptide cause a subtle, global effect on peptide conformation that can be used to design altered peptide ligands (APL) as T-cell partial agonists. These may have potential as T-cell epitopes for synthetic vaccines and therapeutic agents for autoimmune diseases.

Amino Acid Sequence↗

PubMed2001

Energetic asymmetry among hydrogen bonds in MHC class II*peptide complexes.

Comparison of crystallized MHC class II*peptide complexes has revealed that, in addition to pocket interactions involving the peptide side chains, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds between genetically conserved amino acid residues in the class II molecule and the main chain of the peptide. Many class II*peptide structures have two sets of symmetrical hydrogen bonds at the opposite ends of the class II antigen-binding groove (beta-His-81, beta-Asn-82 vs. alpha-His-68, alpha-Asn-69). In this study, we alter these peripheral hydrogen bonds and measure the apparent contribution of each to the kinetic stability of peptide* II complexes. Single conservative amino substitutions were made in the I-A(d) protein to eliminate participation as a hydrogen bonding residue, and the kinetic stability of a diverse set of peptides bound to the substituted I-A(d) proteins was measured. Although each hydrogen bond does contribute to peptide binding, our results point to the striking conclusion that those hydrogen bonds localized to the amino terminus of the peptide contribute profoundly and disproportionately to the stability of peptide interactions with I-A(d). We suggest that the peripheral hydrogen bonds at the amino terminus of the bound peptide that are conserved in all class II*peptide crystal structures solved thus far form a cooperative network that critically regulates peptide dissociation from the class II molecule.

Amino Acid Sequence↗

PubMed2000

Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy.

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

Animals↗

PubMed1999

Ovalbumin(323-339) peptide binds to the major histocompatibility complex class II I-A(d) protein using two functionally distinct registers.

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

Animals↗

PubMed1999

Detection of oxygen consumption of cultured adherent cells by bead injection spectroscopy.

This paper describes a method for detecting oxygen consumption of adherent cell cultures. The sensing is based on oxygen-dependent quenching of the phosphorescence of a Pt-porphyrin complex immobilized on microcarrier beads, which are used as the cell culture substrate. Bead injection, a recent variant of the flow injection technique, is used to pack an aliquot of the beads into a small sensing layer that can be easily and rapidly renewed. The technique is tested on a model system of Chinese Hamster Ovary M1 cells grown on Cytodex-3 microcarrier beads. Cellular respiration is monitored through O2 consumption measured across a period of 3 min. The method is validated by detecting the impairment of aerobic metabolism caused by 1.5 mM amobarbital. Further, it is shown to have enough precision to distinguish even more subtle changes, such as the increase in oxygen consumption caused by stimulation of the muscarinic m1 receptor with 100 microM carbachol.

Animals↗

PubMed1999

Cutting edge: a single, essential hydrogen bond controls the stability of peptide-MHC class II complexes.

The binding of peptides to MHC class II molecules is mediated in part by a conserved array of intermolecular hydrogen bonds. We have evaluated the consequences of disrupting the hydrogen bond between beta-His-81 of the class II molecule and bound peptide. These studies revealed that peptide dissociation rates were accelerated by factors ranging to 200-fold. The sensitivity of a peptide to loss of the hydrogen bond is inversely correlated with the inherent kinetic stability of the peptide-MHC complex. The same relationship has been observed between inherent kinetic stability and the susceptibility to DM. Given that the rate enhancement observed for MHC class II I-Ad protein mutated at position 81 in the beta-chain is comparable with DM-catalyzed rates for other class II molecules, we suggest that DM could function by stabilizing a peptide-MHC intermediate in which one or more hydrogen bonds between the peptide and MHC, such as that contributed by the beta-His-81 hydrogen bond, are disrupted.

Amino Acid Sequence↗

PubMed1999

A partially agonistic peptide acts as a selective inducer of apoptosis in CD8+ CTLs.

We have analyzed the effect of partially agonistic peptides on the activation and survival of CTL clones specific for a highly immunogenic HLA A11-restricted peptide epitope derived from the EBV nuclear Ag-4. Several analogues with substitutions of TCR contact residues were able to trigger cytotoxic activity without induction of IL-2 mRNA and protein or T cell proliferation. Triggering with these partial agonists in the absence of exogenous IL-2 resulted in down-regulation of the cytotoxic potential of the specific CTLs. One analogue selectively triggered apoptosis as efficiently as the original epitope, subdividing the partial agonists into apoptosis-inducing and noninducing ligands. Analysis of early T cell activation events, induction of Ca2+ influx, and acid release did not reveal significant differences between the two types of analogue peptides. These results demonstrate that some partial agonists can dissociate the induction of CTL death from CTL activation. Peptides with such properties may serve as useful tools to study signal transduction pathways in CD8+ lymphocytes and as therapeutic agents modulating natural immune responses.

Apoptosis↗

PubMed1999

Highly cross-reactive T cell responses to myelin basic protein epitopes reveal a nonpredictable form of TCR degeneracy.

We identified two nonoverlapping epitopes in myelin basic protein presented by I-Au that are responsible for mediating tolerance induction to this self-Ag. A large number of T cells expressing diverse TCRs are strongly cross-reactive to both epitopes. Surprisingly, the TCR contact residues in each peptide are highly dissimilar. Furthermore, functional TCR contacts cannot be interchanged between the two epitopes, indicating that the TCR contacts in each peptide can only be recognized within the context of the other amino acids present in that peptide's sequence. This observation indicates that both buried and exposed residues of each peptide contribute to the sculpting of completely distinct antigenic surfaces. We propose that the cross-reactive TCRs adopt mutually exclusive conformations to recognize these dissimilar epitopes, adding a new dimension to TCR degeneracy. This unpredictable TCR plasticity indicates that using just the TCR contacts on a single epitope to define other cross-reactive peptides will identify only a subset of the complete repertoire of cross-reactive epitopes.

Amino Acid Sequence↗

PubMed1999

Individual hydrogen bonds play a critical role in MHC class II: peptide interactions: implications for the dynamic aspects of class II trafficking and DM-mediated peptide exchange.

Determination of the crystal structure of class II: peptide complexes has shown that in addition to pocket interactions involving the side chains of the peptide, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds which are contributed by genetically conserved amino acid residues in the class II molecule to the main chain of the peptide. Our experiments have revealed an unexpectedly large contribution of hydrogen bonds at the periphery of the MHC peptide binding pocket to MHC class II function. Kinetic studies have shown that peptide dissociation rates are profoundly accelerated by loss of a single hydrogen bonding residue. The magnitude of the effects seen with the loss in potential for a single hydrogen bond support a co-operative model in which individual bonds between class II and peptide are dependent on the integrity of neighboring interactions. Collectively our studies have revealed that MHC class II structure, peptide binding and intracellular trafficking events are critically dependent on the integrity of the hydrogen bonding network between class II molecules and its bound peptide.

Animals↗

PubMed1998

Evidence that the autoimmune antigen myelin basic protein (MBP) Ac1-9 binds towards one end of the major histocompatibility complex (MHC) cleft.

The NH2-terminal peptide of myelin basic protein (MBP) bound to the class II major histocompatibility complex (MHC) protein I-Au is an immunodominant epitope in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. However, the MBP-I-Au complex is very unstable. To investigate this, we performed site-directed mutagenesis of the I-Au MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demonstrate that the MBP peptide's key residue for MHC binding, lysine 4, is buried in the P6 pocket of I-Au, which is predominantly hydrophobic. This implies that the MBP-I-Au complex differs from more stable complexes in two respects: (a) the peptide leaves the NH2-terminal region of the MHC peptide-binding cleft unoccupied; (b) the peptide is not anchored by typical favorable interactions between peptide side chains and MHC pockets. To test these hypotheses, a modified MBP peptide was designed based on molecular modeling, with the aim of producing strong I-Au binding. Extension of the NH2 terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain in the P6 pocket with an aromatic anchor, results in >1,000-fold increased binding stability. These results provide an explanation for the unusual peptide-MHC-binding kinetics of MBP, and should facilitate an understanding of why mice are not tolerant to this self-peptide- MHC complex.

Amino Acid Sequence↗

PubMed1998

Differential tolerance is induced in T cells recognizing distinct epitopes of myelin basic protein.

Experimental allergic encephalomyelitis (EAE) is induced by T cell-mediated immunity to central nervous system antigens. In H-2u mice, EAE is mediated primarily by T cells specific for residues 1-11 of myelin basic protein (MBP). We demonstrate that differential tolerance to MBP1-11 versus epitopes in MBP121-150 is induced by expression of endogenous MBP, reflecting extreme differences in stability of peptide/MHC complexes. The diverse MBP121-150-specific TCR repertoire can be divided into three fine specificity groups. Two groups were identified in wild-type mice despite extensive tolerance, but the third group was not detected. Activated MBP121-150-specific T cells induce EAE in wild-type mice. Thus, encephalitogenic T cells that escape tolerance either recognize short-lived peptide/MHC complexes or express TCRs with unique specificities for stable complexes.

Animals↗

PubMed1998

Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected].

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

Amino Acid Sequence↗

PubMed1997

Specific T cell recognition of kinetic isomers in the binding of peptide to class II major histocompatibility complex.

Helper T cells are triggered by molecular complexes of antigenic peptides and class II proteins of the major histocompatibility complex. The formation of stable complexes between class II major histocompatibility complex proteins and antigenic peptides is often accompanied by the formation of a short-lived complex. In this report, we describe T cell recognition of two distinct complexes, one short-lived and the other long-lived, formed during the binding of an altered myelin basic protein peptide to I-Ak. One myelin basic protein-specific T cell clone is triggered by only the short-lived complex, and another is triggered by only the stable complex. Thus, a single peptide bound to a particular class II molecule can activate different T cells depending on the conditions of the binding reaction.

Animals↗

PubMed1997

Kinetics and extent of T cell activation as measured with the calcium signal.

We have characterized the calcium response of a peptide-major histocompatibility complex (MHC)-specific CD4(+) T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell-antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

Amino Acid Sequence↗

PubMed1997

Screening for novel drug effects with a microphysiometer: a potent effect of clofilium unrelated to potassium channel blockade.

Changes in cellular metabolism in response to pharmacological compounds can be detected using a biosensor known as a microphysiometer, which measures the rate at which cells release acidic metabolites. We have applied this technique to screen for effects of cation channel blockers on the metabolism of a variety of human and murine cell lines. At concentrations sufficient for cation channel blockade, most of these drugs have little or no effect on cellular metabolism as measured by acid release. In contrast, the potassium channel blocker clofilium triggers sustained increases in acid release at low (> or = 3 microM) concentration. Acid release persists in media containing high (150 mM) extracellular potassium. This release is not triggered by chemically similar potassium channel blockers. Thus these metabolic effects reflect a potent and specific function of clofilium which is unrelated to potassium channel blockade. Attempts to identify physiological correlates to this response revealed that low concentrations of clofilium but not other potassium channel blockers cause lymphoma apoptosis. These findings demonstrate that effects of clofilium found in other studies may not be due to changes in plasma membrane potassium conductance.

Adrenergic beta-Antagonists↗

PubMed1996

Early biochemical signals arise from low affinity TCR-ligand reactions at the cell-cell interface.

The kinetics of acid release by a mixture of T cells and antigen presenting cells were measured with a microphysiometer during a brief exposure to antigenic peptides. We find that some of the early biochemical events that lead to cellular proliferation cause a specific increase in the rate of acid release. The duration of this increase in acid release reflects the life-time of the peptide-MHC complexes. Peptides that form long-lived complexes produce a response that is stable for more than an hour. Serial TCR engagement is suggested by the observation that the amplitude of this stable response can be rapidly shifted up or down with additional agonist peptide or with antibodies that block T cell receptor binding. Cells briefly exposed to a peptide that forms short-lived peptide-MHC complexes produce a response that decays rapidly as peptide is washed away. A quantitative analysis of the kinetics of this decay in acidification demonstrates that intercellular TCR-ligand reactions are rapid, reversible, and of low apparent affinity with < 20% of peptide-MHC ligand bound to a TCR at any one time. These results demonstrate that the fraction of peptide-MHC ligands bound to TCRs at the cell-cell interface is no higher than anticipated from the affinities observed in solution for isolated TCRs and ligands.

Amino Acid Sequence↗

PubMed1996

Kinetic discrimination in T-cell activation.

We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

Animals↗

PubMed1996

Altered T cell receptor ligands trigger a subset of early T cell signals.

TCR ligands are complexes of peptides and MHC proteins on the surfaces of APCs. Some of these ligands cause T cell proliferation (agonists), while others block it (antagonists). We compared the acid release, calcium flux, and proliferation response of helper T cells to a variety of ligands. We found that all agonist ligands but not most antagonist ligands trigger acid release, a general indicator of early cellular activation. Only a subset of ligands triggering acid release cause sustained calcium flux, and only a subset of these ligands cause T cell proliferation. Antagonist ligands and anti-CD4 antibodies both effectively block T cell proliferation. However, significantly greater antagonist ligand or antibody concentrations are required to block acid release and initial calcium influx. These data demonstrate a hierarchy of early T cell signaling steps and show that altered TCR ligands can initiate some steps while blocking the completion of others.

Amino Acid Sequence↗