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D A Vignali

Publications and source records attributed to D A Vignali.

At least 19 recordsLinked to original sources
PubMed2000

Multiplexed particle-based flow cytometric assays.

Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer. Several multiplexed bead systems are currently marketed by different vendors. We have used the Luminex FlowMetrix system which consists of 64 different bead sets manufactured with uniform, distinct proportions of red and orange fluorescent dyes (detected by FL2/FL3 on a FACScan). Each bead set forms the basis of an individual assay using a green fluorescent reporter dye (FL1). This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. They can also be developed into rapid, 'no wash' assays that can be completed in <2 h. This review traces the historical association between microspheres and flow cytometry, the development and use of particle-based flow cytometric assays, how they compare with current assays and potential future developments of this very exciting technology.

Cytokines↗
PubMed2000

On the dynamics of TCR:CD3 complex cell surface expression and downmodulation.

TCR downmodulation following ligation by MHC:peptide complexes is considered to be a pivotal event in T cell activation. Here, we analyzed the dynamics of TCR:CD3 cell surface expression on resting and antigen-activated T cells. We show that the TCR:CD3 complex is very stable and is rapidly internalized and recycled in resting T cells. Surprisingly, the internalization rate is not increased following TCR ligation by MHC:peptide complexes, despite significant TCR downmodulation, suggesting that constitutive internalization rather than ligation-induced downmodulation serves as the force that drives serial ligation. Furthermore, TCR downmodulation is mediated by the intracellular retention of ligated complexes and degradation by lysosomes and proteasomes. Thus, our data demonstrate that ligation induces TCR downmodulation by preventing recycling rather than inducing internalization.

Cell Line↗
PubMed1999

Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay.

Several methods have been developed to quantify cytokines and chemokines in biological fluids and tissue culture samples, including bioassays, enzyme-linked immunosorbent assay (ELISA), intracellular staining, ribonuclease protection assay (RPA) and polymerase chain reaction (PCR). However, each of these techniques possesses one or more significant limitations. Here, we describe a new multiplexed assay, using the FlowMetrix system, that can quantify multiple cytokines simultaneously in a small sample volume. This assay was found to be more accurate, sensitive and reproducible than the conventional microtitre ELISA procedure. Furthermore, the time and cost involved are comparable to, or less than, the ELISA. A key feature of the FlowMetrix assay is its ability to multiplex: here, we show that this assay can accurately quantitate 15 cytokines in a 100 microl sample volume while the same analysis by ELISA requires 1.5 ml (100 microl for each cytokine assay). By using this Flow Metrix assay, we could demonstrate that only T helper 1 (T(H)1)-deviated cells produce detectable levels of interleukin (IL)-2, while only T(H)2-deviated cells produce significant amounts of IL-4. Six other cytokines were produced by both T cell subsets, with the T(H)1 population producing more IL-3, granulocyte-monocyte colony stimulating factor (GM-CSF) and interferon (IFN)-gamma, and the T(H)2 population producing more IL-5, IL-10, and IL-13. Seven other cytokines were not produced in detectable amounts. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are available. It will also provide a more complete picture of the plethora of cytokines secreted during an immune response.

Animals↗
PubMed1999

Differential CD3 zeta phosphorylation is not required for the induction of T cell antagonism by altered peptide ligands.

T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 zeta-chain, pp21 and pp23. Replacement of residues in MHC-bound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3zeta phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3zeta mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3zeta is required for full agonist but not antagonist responses, and 3) differential CD3zeta phosphorylation is not a prerequisite for T cell antagonism.

Animals↗
PubMed1999

Profound enhancement of T cell activation mediated by the interaction between the TCR and the D3 domain of CD4.

CD4 plays an important role in the activation and development of CD4+ T cells. This is mediated via its bivalent interaction with MHC class II molecules and the TCR:CD3 complex through p56lck. Recent studies have implicated a third site of interaction between the membrane-proximal extracellular domains of CD4 and the TCR. Due to these multiple interactions, direct evidence for the functional importance of this extracellular association has remained elusive. Furthermore, the residues that mediate this interaction are unknown. In this study, we analyzed the function of 61 CD4 mutants. Alanine substitution of just 2 residues, either Q114/F182 or F182/F201, which are partially buried and located close to the D2/D3 interface, completely abrogated CD4 function. Direct evidence for the functional importance of TCR:CD4.D3 interaction was obtained using an anti-CD3fos:anti-CD4jun-bispecific Ab. Surprisingly, it induced strong T cell activation in hybridomas transfected with cytoplasmic-tailless CD4, despite the lack of association with either p56lck or MHC class II molecules. However, this effect was completely abrogated with the CD4 mutants Q114A/F182A or F182A/F201A. These data demonstrate that TCR:CD4.D3 interaction can have a profound effect on T cell activation and obviates the need for receptor oligomerization.

Amino Acid Sequence↗
PubMed1999

Immunoregulation of Th cells by naturally processed peptide antagonists.

Th cells recognize protein Ags as short peptides bound to MHC class II molecules. Altered peptide ligands can antagonize (inhibit) T cell responses to stimulatory peptides. Peptides generated by APC may contain peptide flanking residues (PFR), which lie outside the minimal binding epitope and can be recognized by the TCR. Our data show that PFR-dependent T cells were found to be potently antagonized by peptides that lack PFR and responded poorly to native protein or the immunogenic epitope delivered by a recombinant influenza virus. These data provide the first evidence that Ag processing generates both stimulatory and antagonist peptides from a single immunogenic epitope, an observation that may have important implications for T cell immunoregulation and autoimmunity.

Amino Acid Sequence↗
PubMed1997

T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage.

Naturally processed MHC class II-bound peptides possess ragged NH2 and COOH termini. It is not known whether these peptide flanking residues (PFRs), which lie outside the MHC anchor residues, are recognized by the TCR or influence immunogenicity. Here we analyzed T cell responses to the COOH-terminal PFR of the H-2A(k) immunodominant epitope of hen egg lysozyme (HEL) 52-61. Surprisingly, the majority of T cells were completely dependent on, and specific for, the COOH-terminal PFR of the immunogen. In addition, there were striking correlations between TCR V beta usage and PFR dependence. We hypothesize that the V alpha CDR1 region recognizes NH2-terminal PFRs, while the V beta CDR1 region recognizes COOH-terminal PFRs. Last, peptides containing PFRs were considerably more immunogenic and mediated a greater recall response to the HEL protein. These results demonstrate that PFRs, which are a unique characteristic of peptides bound to MHC class II molecules, can have a profound effect on TCR recognition and T cell function. These data may have important implications for peptide-based immunotherapy and vaccine development.

Amino Acid Sequence↗
PubMed1996

TCR-mediated adhesion of T cell hybridomas to planar bilayers containing purified MHC class II/peptide complexes and receptor shedding during detachment.

T cell recognition of foreign Ag/MHC class II complexes is sensitive down to approximately 100 complexes per cell or approximately 0.2 complexes/micron2. To better understand the physical basis of the recognition stage of Ag presentation, we examined adhesion of the lysozyme- specific T cell hybridoma, 3A9, to artificial bilayers containing covalent MHC class II/peptide complexes or adhesion molecules. Adhesion of 3A9 cells required a superphysiologic density of the MHC class II/peptide complex and was partly dependent on CD4; cells adhered but did not crawl. No adhesion was observed to bilayers containing MHC class II molecules without the lysozyme peptide. Activated 3A9 cells adhered and crawled on bilayers containing ICAM-1. The physical strength of contacts was tested with fluid shear. 3A9 cells adherent to bilayers containing MHC class II/peptide complexes shed their contact, which remained on the substrate and contained TCR. In contrast, 3A9 cells peeled from the ICAM-1 bilayer, and held firmly on LFA-1 bilayers; in a manner dependent on filamentous actin. When ICAM-1 and the MHC/peptide complexes were combined, the 3A9 cells adhered tightly and spread, but did not crawl, on the bilayers and TCR clustered at the center of the contact area. Physiologically, the TCR is unlikely to directly initiate adhesion. TCR clusters formed with the assistance of adhesion mechanisms may have to be shed to allow de-adhesion, and this may contribute to TCR down-regulation.

Animals↗
PubMed1996

Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells.

Signal transducers and activators of transcription (STATs) are activated by tyrosine phosphorylation in response to cytokines and mediate many of their functional responses. Stat4 was initially cloned as a result of its homology with Stat1 (refs 4, 5) and is widely expressed, although it is only tyrosine-phosphorylated after stimulation of T cells with interleukin (IL)-12 (refs 6,7). IL-12 is required for the T-cell-independent induction of the cytokine interferon (IFN)-gamma, a key step in the initial suppression of bacterial and parasitic infections. IL-12 is also important for the development of a Th1 response, which is critical for effective host defence against intracellular pathogens. To determine the function of Stat4 and its role in IL-12 signalling, we have produced mice that lack Stat4 by gene targeting. The mice were viable and fertile, with no detectable defects in haematopoiesis. However, all IL-12 functions tested were disrupted, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and Th1 differentiation.

Animals↗
PubMed1996

The two membrane proximal domains of CD4 interact with the T cell receptor.

During T cell activation, CD4 is intimately involved in colocalizing the T cell receptor (TCR) with its specific peptide ligand bound to class II molecules of the major histocompatibility complex (MHC). Previously, the COOH-terminal residues, Trp62/63, which flank the immunodominant epitope of hen egg lysozyme (HEL 52-61), were shown to have a profound effect on TCR recognition. CD4 maintains the fidelity of this interaction when short peptides are used. To determine which portion of CD4 was responsible for this effect, a series of CD4 mutants were made and transfected into CD4 loss variants of two HEL 52-61-specific T cell hybridomas. Surprisingly, some CD4 mutants that failed to interact with MHC class II molecules (D2 domain mutant) or with p56kk (cytoplasmic-tailless mutant) restored responsiveness. Nevertheless, a significant reduction in association between cytoplasmic-tailless CD4 and the TCR, as determined by fluorescence resonance energy transfer, was observed. Thus, neither colocalization of CD4 and the TCR nor signal transduction via CD4 was solely responsible for the functional restoration of these T cell hybridomas by wild-type CD4. However, substitution of the two membrane proximal domains of murine CD4 (D3 and D4) with domains from human CD4 or intercellular adhesion molecule 1 not only abrogated its ability to restore function, but also substantially reduced its ability to associate with the TCR. Furthermore, the mouse/human CD4 chimera had a potent dominant negative effect on T cell function in the presence of equimolar concentrations of wild-type CD4. These data suggest that the D3/D4 domains of CD4 may interact directly or indirectly with the TCR-CD3 complex and influence the signal transduction processes. Given the striking structural differences between CD4 and CD8 in this region, these data define a novel and unique function for CD4.

Amino Acid Sequence↗
PubMed1996

Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene.

Signal transducers and activators of transcription (Stats) are activated by tyrosine phosphorylation in response to cytokines, and are thought to mediate many of their functional responses. Stat6 is activated in response to interleukin (IL)-4 and may contribute to various functions including mitogenesis, T-helper cell differentiation and immunoglobulin isotype switching. To evaluate the role of Stat6, we generated Stat6-null mice (Stat6 -/-) by gene disruption in embryonic stem cells. The mice were viable, indicating the lack of a non-redundant function in normal development. Although naive lymphoid cell development was normal, Stat6 -/- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE. In contrast, IL-4-mediated proliferation was only partly affected.

Animals↗
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

The interaction between CD4 and MHC class II molecules and its effect on T cell function.

During T cell activation, CD4 and CD8 form a 'bridge' between the T cell receptor (TCR) and major histocompatibility complex (MHC) class II and class I molecules, respectively. Due to this intimate association, CD4 and CD8 are now termed co-receptors and considered an integral part of this multimolecular complex. In addition, interest in CD4 has been heightened by the discovery that it is, in part, the receptor for HIV. Although CD4 and CD8 appear to perform similar immune functions, they are structurally diverse suggesting that their mode of interaction with the TCR and MHC molecules may differ. This review will focus primarily on a series of studies which have attempted to map the residues which mediate CD4:MHC class II interaction. These data will be evaluated in light of our current understanding of CD8:MHC class I, and CD4:TCR interactions. In addition, a model to explain the structural and functional differences between CD4 and CD8 will be presented. Finally, the potential effect of these multiple interactions on T cell function will be discussed.

Amino Acid Sequence↗
PubMed1994

Negative and positive selection by HLA-DR3(DRw17) molecules in transgenic mice.

The establishment of HLA transgenic mice as models for autoimmune disorders requires that the HLA molecules can be efficiently recognized and mediate positive and negative selection of mouse T cells. This question was investigated in DR3(DRw17) transgenic mice back-crossed to the B10.Q(H-2q) strain which does not form mixed mouse-human class II heterodimers. Here we report that efficient negative selection on DR3(DRw17) molecules was observed for v beta 5, 11, and 13 subpopulations of CD4+T cells, but not for v beta 4, 7, 8, 9, and 10. v beta 5 and 11 cells are also negatively selected by mouse class II E molecules which is the structural homologue to DR molecules. Positive selection on DR3(DRw17) was only observed for v beta 6 cells but this was less efficient than positive selection of v beta 6 cells by E molecules. The data indicate that DR3(DRw17) molecules select similar subgroups of mouse T cells as E molecules although with slightly different efficiency.

Animals↗
PubMed1993

Interactions of CD4 with MHC class II molecules, T cell receptors and p56lck.

CD4 and CD8 are members of the immunoglobulin supergene family of proteins, and function as co-receptors with the T cell receptor (TCR) in binding MHC class II or class I molecules, respectively. Within this multimeric complex, CD4 interacts with three distinct ligands. CD4 interacts through its D1 and D2 domains with MHC class II proteins, through its D3 and D4 domains with T cell receptors, and through its cytoplasmic tail with p56lck, a src-related, protein tyrosine kinase. Each of these interactions is important in the function of CD4 and will be discussed in turn.

Amino Acid Sequence↗
PubMed1993

Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.

Alleles↗
PubMed1993

Minute quantities of a single immunodominant foreign epitope are presented as large nested sets by major histocompatibility complex class II molecules.

The processing and presentation of immunogenetic peptides is an obligate event in the generation of an immune response. However, the degree of complexity with which an immunogenic foreign epitope is presented is still unclear. This question was addressed by analyzing the naturally processed peptides generated from exogenously-derived hen egg white lysozyme (HEL) bound to the murine major histocompatibility complex (MHC) class II molecule, H-2Ak. Using reversed-phase chromatography (RPC), T cell hybridomas and mass spectrometry, 16 peptides were identified that contain the minimal MHC binding epitope 52-61. These peptides exhibited substantial N- and C-terminal extensions and ranged from 13-28 amino acids in length. In contrast, MHC class I molecules present peptides of 8-11 residues and each foreign epitope appears to be represented by only a single peptide. The data here also show that only approximately 0.8% of the total bound peptide was derived from this single HEL epitope. These findings provide direct evidence that relatively small amounts of processed peptide are required to stimulate an effective T cell response.

Amino Acid Sequence↗