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E Joly

Publications and source records attributed to E Joly.

At least 19 recordsLinked to original sources
PubMed2001

Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach.

BACKGROUND: Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E. RESULTS: An in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies. CONCLUSIONS: Compared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes.

Amino Acid Motifs↗
PubMed2001

Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner.

Upon encounter of a CTL with a target cell carrying foreign Ags, the TCR internalizes with its ligand, the peptide-MHC class I complex. However, it is unclear how this can happen mechanistically because MHC molecules are anchored to the target cell's surface via a transmembrane domain. By using antigenic peptides and lipids that were fluorescently labeled, we found that CTLs promptly capture target cell membranes together with the antigenic peptide as well as various other surface proteins. This efficient and specific capture process requires sustained TCR signaling. Our observations indicate that this process allows efficient acquisition of the Ag by CTL, which may in turn regulate lymphocyte activation or elimination.

3T3 Cells↗
PubMed2001

Analysis of neural stem cells by flow cytometry: cellular differentiation modifies patterns of MHC expression.

Neural stem cells are currently considered very hopeful candidates for cell replacement therapy in neurodegenerative pathologies such as Parkinson's disease. Here we show that different cell types derived from neurospheres amplified in vitro can be identified by FACS analysis relying solely on physical parameters (FSC/SSC) or autofluorescence. Additionally, after treatment with a panel of inflammatory cytokines, neurospheres and their differentiated progeny were shown to express MHC antigens which could potentially cause transplant rejection. Astrocytes expressed the highest levels of MHC. Hence removing such cells prior to transplantation could potentially optimise transplant survival.

Animals↗
PubMed2001

Two different, highly exposed, bulged structures for an unusually long peptide bound to rat MHC class I RT1-Aa.

The rat MHC class Ia molecule RT1-Aa has the unusual capacity to bind long peptides ending in arginine, such as MTF-E, a thirteen-residue, maternally transmitted minor histocompatibility antigen. The antigenic structure of MTF-E was unpredictable due to its extraordinary length and two arginines that could serve as potential anchor residues. The crystal structure of RT1-Aa-MTF-E at 2.55 A shows that both peptide termini are anchored, as in other class I molecules, but the central residues in two independent pMHC complexes adopt completely different bulged conformations based on local environment. The MTF-E epitope is fully exposed within the putative T cell receptor (TCR) footprint. The flexibility demonstrated by the MTF-E structures illustrates how different TCRs may be raised against chemically identical, but structurally dissimilar, pMHC complexes.

Adenosine Triphosphatases↗
PubMed2001

Genetic control of peripheral TCRAV usage by representation in the preselection repertoire and MHC allele-specific overselection.

TCRAV segments contribute significantly to MHC restriction as illustrated by their general preference for either the CD4 or CD8 T cell subset and additional, MHC allele-specific overselection during T cell differentiation. The 10-fold over-representation of the TCRAV8S2 (VA8S2) segment in CD8 over CD4 T cells by the RT1(f) haplotype of LEW.1F rats provides the most striking example of MHC allele-specific overselection of a VA segment reported so far. Also in alloreactivity, VA8S2(+) CD8 cells from RT1(f-) rats are preferentially expanded by RT1(f+) stimulators. We have identified the class I molecule, A(f), mediating VA8S2 overselection and report that it differs only in four amino acids at the MHC-TCR interface from the class I molecule A(a), which is neutral with regard to selection of VA8S2. We also provide an extensive survey of the TCRAV8 family and show that among 14 functional VA8 segments in LEW rats, the dramatic A(f)-dependent overselection is unique for VA8S2. Surprisingly, VA8S2 expression in CD8 T cells of RT1(f+) rats derived from a Sprague-Dawley stock was only 3% as compared to the 12% observed in LEW.1F. The VA8S2 segment of Sprague-Dawley (VA8S2(SD)) differs from VA8S2 of the LEW background (VA8S2(l)) in only two amino acids, one of which is located in CDR2 and could thus participate in allele-specific recognition of A(f). However, analysis of the pre- and postselection thymic repertoires of Sprague-Dawley and LEW.1F rats and of the repertoire of CD8 cells from both strains expanded in the alloreactive response to RT1(f) revealed that the difference in VA8S2 representation between the two backgrounds is explained by differential availability in the preselection repertoires and not by a difference in overselection. Sequence comparisons of A(f) and A(a) and of both VA8S2 segments suggest a predominant role of CDR1 in hyper-reactivity to A(f). Thus, the VA composition of the mature TCR repertoire is influenced by TCRA: locus polymorphisms at two levels: the regulation of VA usage in the preselection repertoire and the composition of structural elements which contribute to specific VA-MHC interactions during thymic selection.

Alleles↗
PubMed2000

Peptide specificity of RT1-A1(c), an inhibitory rat major histocompatibility complex class I natural killer cell ligand.

The rat major histocompatibility complex class Ia allelomorph RT1-A1(c) is a potent ligand for the recently identified inhibitory rLy-49 receptor, STOK-2. With the ultimate objective of studying the interactions of these molecules using structural and functional methods, we undertook a detailed study of its peptide specificity. The study revealed that designing an "ideal peptide" by choosing the most abundant residues in the "binding motif" obtained by pool sequencing does not necessarily yield an optimal binding peptide. For RT1-A1(c), as many as four positions, P2, P4, P5, and P9, were detected as putative anchors. Since this molecule displays a preference for highly hydrophobic peptides, we tested binding of peptides derived from the known leader peptide sequences of other rat histocompatibility complex class I molecules. One such peptide, found to bind well, requiring 1.6 microm peptide to achieve 50% stabilization, was searched for in vivo. Natural RT1-A1(c) binding peptides were purified from rat splenocytes and characterized by mass spectrometry using a combined matrix-assisted laser desorption ionization/time-of-flight and quadrupole time-of-flight approach. Results showed that the signal sequence-derived peptide was not detectable in the purified peptide pool, which was composed of a complex spectrum of peptides. Seven of these self-peptides were successfully sequenced.

Animals↗
PubMed2000

Normal polyclonal immunoglobulins ('IVIg') inhibit microglial phagocytosis in vitro.

Phagocytosis removes pathogens and tissue debris during inflammatory reactions, but also plays an important role in autoimmune reactions. The main phagocytes in the central nervous system (CNS) are microglial cells that are activated during CNS inflammation. In the treatment of inflammatory demyelinating diseases like multiple sclerosis (MS), administration of intravenous immunoglobulins (IVIg) has become a promising immunomodulatory therapy. Although a large number of potential mechanisms for the effects of IVIg has been suggested, the precise mode of action in CNS inflammation is unknown. We assessed the influence of IVIg on phagocytosis and endocytosis in microglia in vitro. IVIg had little effect on non-specific phagocytosis of latex particles in untreated microglia, while there was a dose-dependent inhibition in microglia activated with LPS and IFNgamma. Endocytosis of soluble myelin basic protein (MBP) was downregulated by IVIg in both untreated and activated microglia. The effect was mediated by an F(ab')(2) preparation of immunoglobulins, suggesting that Fc receptor-mediated phagocytosis is not involved. Intact IVIg, but not F(ab')(2) fragments also suppressed Fc receptor-mediated phagocytosis of opsonised erythrocytes in both untreated and activated microglia. These results show that IVIg can inhibit the phagocytic activity of microglia via different mechanisms. Such an effect could contribute to the immunomodulatory capacity of IVIg in inflammatory CNS diseases.

Animals↗
PubMed2000

Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.

Animals↗
PubMed2000

NK cells modulate MHC class I expression on tumor cells and their susceptibility to lysis.

Cytotoxicity and production of cytokines are two important functions of NK cells. These two different NK functions were studied in a syngeneic rat model in relation to MHC class I expression. We focussed on the mechanism by which NK cells modulate MHC class I expression on target cells and how this interferes with NK cell-mediated lysis. Using transfection experiments an inhibitory role on NK cell cytotoxicity for expression of target cells of RT1.A, rat MHC class I, was found. Co-culturing syngeneic tumor cells and NK cells resulted in enhanced MHC class I expression on the surviving tumor cell fraction, which was less susceptible to NK lysis. Increased tumor cell MHC class I was due to production of a soluble factor by NK cells, most likely interferon gamma. The regulatory function of NK cells shows here, that the enhancing of MHC class I expression on tumor cells in vitro and in vivo, results in downregulation of their target cell killing, but at the same time may facilitate the cytotoxic T cell function.

Animals↗
PubMed2000

Analysis of peptide length preference of the rat MHC class Ia molecule RT1-A(u), by a modified random peptide library approach.

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

Animals↗
PubMed2000

Effects of Ca(2+) channel activity on renal hemodynamics during acute attenuation of NO synthesis in the rat.

In cultured vascular muscle cells, nitric oxide (NO) has been shown to inhibit voltage-dependent Ca(2+) channels, which are involved in renal blood flow (RBF) autoregulation. Therefore, our purpose was to specify in vivo the effects of this interaction on RBF autoregulation. To do so, hemodynamics were investigated in anesthetized rats during Ca(2+) channel blockade before or after acute NO synthesis inhibition. Rats were treated intravenously with vehicle (n = 10), 0.3 mg/kg body wt N(G)-nitro-L-arginine-methyl ester (L-NAME; n = 7), 4.5 microg. kg body wt(-1). min(-1) nifedipine (n = 8) alone, or with nifedipine infused before (n = 8), after (n = 8), or coadministered with L-NAME (n = 10). Baseline renal vascular resistance (RVR) averaged 14.0 +/- 1.2 resistance units and did not change after vehicle. RVR increased or decreased significantly by 27 and 29% after L-NAME or nifedipine, respectively. Nifedipine reversed, but did not prevent, RVR increase after or coadministered with L-NAME. RBF autoregulation was maintained after L-NAME, but the autoregulatory pressure limit (P(A)) was significantly lowered by 15 mmHg. Nifedipine pretreatment or coadministration with L-NAME limited P(A) resetting or suppressed autoregulation at higher doses. Results were similar with verapamil. Intrarenal blockade of Ca(2+)-activated K(+) channels also prevented autoregulatory resetting by L-NAME (n = 8). These findings suggest NO inhibits voltage-dependent Ca(2+) channels and thereby modulates RBF autoregulatory efficiency.

Animals↗
PubMed1999

RT1-U: identification of a novel, active, class Ib alloantigen of the rat MHC.

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.

Amino Acid Sequence↗
PubMed1998

An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes.

The QuikChangeTM protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by using a second 'standard' oligonucleotide. By a further modification, we have also used the QuikChangeTM approach to exchange DNA sequences between closely related genes.

Amino Acid Substitution↗
PubMed1998

Efficient generation of major histocompatibility complex class I-peptide complexes using synthetic peptide libraries.

The use of synthetic random peptide libraries is a powerful technology for the study of many aspects of antigen presentation and peptide selection by major histocompatibility complex (MHC) molecules. Here we have used them in conjunction with a recombinant system to determine the peptide binding motifs of three classical class I MHC molecules of the laboratory rat: RT1-Aa, RT1-Au, and RT1-A1c. Described is a method for producing large amounts of soluble class I heavy and light chains in bacteria. Refolding RT1-Aa heavy chain (HC) with rat beta2-microglobulin (beta2m) in the presence of a specific peptide and the subsequent purification of the complex yielded conformationally correct material. This was assessed by gel chromatography, SDS-polyacrylamide gel electrophoresis, isoelectric focussing gel electrophoresis, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter analysis employing a previously unreported method utilizing a His-Tag affinity silica. By refolding RT1-Aa HC and rat beta2m around a random nonapeptide library and subjecting the resulting complex to acid elution of the bound peptides and pool sequencing, the peptide binding motif for this MHC class I molecule was determined. Results corresponded well with those previously determined from naturally bound peptides and in addition gave a clear and unambiguous signal for the C-terminal anchor residue. This method was then applied to determine the previously undescribed binding motifs for RT1-Au and RT1-A1c. For both molecules, the whole motif was confirmed from naturally bound peptides. We propose this method as an alternative way to obtain the whole class I MHC peptide motif, particularly when a specific antibody is unavailable and/or natural expression of the class I molecule of interest is low.

Animals↗
PubMed1998

Co-evolution of rat TAP transporters and MHC class I RT1-A molecules.

The genes for rat major histocompatibility complex (MHC) class I molecules are associated either with those for the A allele of the transporter associated with antigen processing (TAP-A), which can transport peptides with basic carboxy-terminal residues, or with those for TAP-B, which cannot [1-5]. To explore whether these associations have a functional basis, we compared the sequences of 13 rat MHC class la RT1-A cDNAs from nine MHC haplotypes. Of seven TAP-A- linked RT1-A molecules, six possess strongly acidic F pockets, and these bind a high proportion of peptides with basic carboxy-terminal residues. The F pockets of TAP-B-linked molecules, by contrast, were more basic. Furthermore, we identified six positions at the 'righthand end' of the peptide-binding groove, at which a majority of TAP-B-linked molecules diverge from the consensus sequence for class la molecules whereas, at these positions, all the TAP-A-linked molecules reflect the consensus sequence. Our results suggest that the linked rat class la and TAP genes have co-evolved to maximize the supply of appropriate peptides to the presenting molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries.

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.

Animals↗