scholarly journals Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

2009 ◽  
Vol 14 (2) ◽  
pp. 173-180 ◽  
Author(s):  
Mikkel Harndahl ◽  
Sune Justesen ◽  
Kasper Lamberth ◽  
Gustav Røder ◽  
Morten Nielsen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Binding Assay ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hla Class I Molecules ◽  
Hla Binding ◽  
Hla Molecules

The Human MHC Project aims at large-scale description of peptide-HLA binding to a wide range of HLA molecules covering all populations of the world and the accompanying generation of bioinformatics tools capable of predicting binding of any given peptide to any given HLA molecule. Here, the authors present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen™). Compared with an enzyme-linked immunosorbent assay—based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range, better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. ( Journal of Biomolecular Screening 2009:173-180)

scholarly journals Analysis of physical interactions between peptides and HLA molecules and application to the detection of human immunodeficiency virus 1 antigenic peptides.

1990 ◽  
Vol 172 (3) ◽  
pp. 889-899 ◽  
Author(s):  
J Choppin ◽  
F Martinon ◽  
E Gomard ◽  
E Bahraoui ◽  
F Connan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Binding Assay ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Antigenic Peptides ◽  
Peptide Binding Assay ◽  
Hiv 1 ◽  
Hla Molecules

The physical association of 40 antigenic peptides and purified HLA class I and class II molecules was monitored using a direct peptide binding assay (PBA) in solid phase and an inhibition peptide binding assay (IPBA) in which the competing peptide was present in a soluble phase. We also examined the ability of different peptides to inhibit the lytic activity of human antiviral cytolytic T cells towards cells incubated with the corresponding target peptide. Our results showed that: (a) Binding of a given human T cell-recognized peptide to several HLA class I and class II molecules occurred frequently. Nevertheless, preferential binding of peptides to their respective restriction molecules was also observed. (b) Binding of HLA molecules to peptides recognized by murine T cells occurred less frequently. (c) 11 of 24 (46%) randomly selected HIV-1 peptides contained agretopic residues allowing their binding to HLA molecules. (d) The kinetics of HLA/peptide association depended on the peptide tested and were faster than or similar to those reported for Ia molecules. Dissociation of these complexes was very low. (e) Peptide/HLA molecule binding was dependent on length, number of positive charges, and presence of hydrophobic residue in the peptide. (f) A correlation was demonstrated between a peptide inhibitory effect in the IPBA and its blocking effect in the cytolytic test. Our data indicated that the restriction phenomenon observed in T cell responses was not strictly related to either an elective HLA/peptide association, or a high binding capacity of a peptide to HLA molecules. These data also showed that the PBA and IPBA are appropriate for the detection of agretopic residues within HIV-1 proteins.

Competition‐Based Cellular Peptide Binding Assay for HLA Class I

2004 ◽  
Vol 61 (1) ◽  
Author(s):  
Jan H. Kessler ◽  
Willemien E. Benckhuijsen ◽  
Tuna Mutis ◽  
Cornelis J.M. Melief ◽  
Sjoerd H. Burg ◽  
...  
Keyword(s):  
Binding Assay ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Peptide Binding Assay

Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3168-3175
Author(s):  
Cécile Gouttefangeas ◽  
Marianne Diehl ◽  
Wieland Keilholz ◽  
Rainer Frank Hörnlein ◽  
Stefan Stevanović ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Hla Class I ◽  
T Cell Response ◽  
Third Party ◽  
Class I ◽  
Cytotoxic T Cell ◽  
Endogenous Peptides ◽  
Hla Class I Molecules ◽  
Hla Molecules

The origin and the function of HLA class I molecules present on the surface of human platelets are still unclear. In particular, it is controversial which fraction of these class I molecules represents integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of these platelet-derived peptides reveals that they originate mainly from ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives from the GPIX protein, which is specifically expressed by platelets and their precursors. Platelet HLA molecules are unstable in vitro at 37°C, but can be partially stabilized by addition of exogenous β2-microglobulin and HLA class I binding peptide, suggesting that platelets cannot load HLA molecules with endogenous peptides. In in vitro experiments platelets were used to stimulate peripheral blood mononuclear cells. No allospecific cytotoxicity was observed after primary stimulation, or secondary restimulation, with allogenic resting or activated platelets, even in the presence of additional third-party helper activity. These data indicate that HLA class I molecules from platelets cannot directly induce allogenic CD8+ cytotoxic T-cell response in vitro.

An HLA class I peptide-binding assay based on competition for binding to class I molecules on intact human B cells

1995 ◽  
Vol 44 (4) ◽  
pp. 189-198 ◽  
Author(s):  
S.H. van der Burg ◽  
E. Ras ◽  
J.W. Drijfhout ◽  
W.E. Benckhuijsen ◽  
A.J.A. Bremers ◽  
...  
Keyword(s):  
B Cells ◽  
Binding Assay ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Human B Cells ◽  
Peptide Binding Assay

scholarly journals Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry

2021 ◽  
Vol 22 (19) ◽  
pp. 10503
Author(s):  
Elena Lorente ◽  
Miguel Marcilla ◽  
Patricia G. de la Sota ◽  
Adriana Quijada-Freire ◽  
Carmen Mir ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Virus Infection ◽  
High Throughput ◽  
Antigen Processing ◽  
Hla Class I ◽  
T Cell Response ◽  
Class I ◽  
Hla Class I Molecules ◽  
Infected Cells ◽  
Hla Ligandome

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

scholarly journals The pockets guide to HLA class I molecules

2021 ◽  
Author(s):  
Andrea T. Nguyen ◽  
Christopher Szeto ◽  
Stephanie Gras
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Side Chains ◽  
Binding Prediction ◽  
Peptide Residue ◽  
Hla Class I Molecules ◽  
Binding Pockets

Human leukocyte antigens (HLA) are cell-surface proteins that present peptides to T cells. These peptides are bound within the peptide binding cleft of HLA, and together as a complex, are recognised by T cells using their specialised T cell receptors. Within the cleft, the peptide residue side chains bind into distinct pockets. These pockets ultimately determine the specificity of peptide binding. As HLAs are the most polymorphic molecules in humans, amino acid variants in each binding pocket influences the peptide repertoire that can be presented on the cell surface. Here, we review each of the 6 HLA binding pockets of HLA class I (HLA-I) molecules. The binding specificity of pockets B and F are strong determinants of peptide binding and have been used to classify HLA into supertypes, a useful tool to predict peptide binding to a given HLA. Over the years, peptide binding prediction has also become more reliable by using binding affinity and mass spectrometry data. Crystal structures of peptide-bound HLA molecules provide a means to interrogate the interactions between binding pockets and peptide residue side chains. We find that most of the bound peptides from these structures conform to binding motifs determined from prediction software and examine outliers to learn how these HLAs are stabilised from a structural perspective.

scholarly journals Evidence of widespread binding of HLA class I molecules to peptides.

1990 ◽  
Vol 172 (3) ◽  
pp. 827-834 ◽  
Author(s):  
J A Frelinger ◽  
F M Gotch ◽  
H Zweerink ◽  
E Wain ◽  
A J McMichael
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Assay ◽  
Solid Phase ◽  
Hla Class I ◽  
Class I ◽  
T Cell Epitopes ◽  
Single Class ◽  
Hla Class I Molecules ◽  
Solid Phase Binding ◽  
Hiv 1

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

scholarly journals HLA-derived peptides which inhibit T cell function bind to members of the heat-shock protein 70 family.

1996 ◽  
Vol 183 (2) ◽  
pp. 339-348 ◽  
Author(s):  
E Nossner ◽  
J E Goldberg ◽  
C Naftzger ◽  
S C Lyu ◽  
C Clayberger ◽  
...  
Keyword(s):  
T Cell ◽  
Heat Shock ◽  
T Lymphocytes ◽  
Heat Shock Protein ◽  
Cell Function ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Inhibitory Effects ◽  
Hla Class I Molecules

Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.

scholarly journals Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality

Blood ◽  
2013 ◽  
Vol 122 (22) ◽  
pp. 3651-3658 ◽  
Author(s):  
Joseph Pidala ◽  
Tao Wang ◽  
Michael Haagenson ◽  
Stephen R. Spellman ◽  
Medhat Askar ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Peptide Binding ◽  
Hla Class I ◽  
Class I ◽  
Graft Versus Host ◽  
Hla Class I Molecules ◽  
Key Points ◽  
Binding Pockets ◽  
Binding Residues

Key Points Amino acid substitution at peptide-binding residues of the HLA class I molecule is associated with graft-versus-host disease and mortality. Avoidance of donor-recipient combinations that result in amino acid substitution at peptide-binding residues may improve transplant outcomes.

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