scholarly journals Screening for Potential HLA-DRB1-Restricted T-Cell Epitopes in Factor VIII Using Peptide Microarrays

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1506-1506
Author(s):  
Debargh Dutta ◽  
Maochang Liu ◽  
Devi Gunasekera ◽  
Kenneth B. Lewis ◽  
Eddie A. James ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Factor Viii ◽  
Conflicts Of Interest ◽  
Positive Control ◽  
Amino Acid Residues ◽  
T Cell Epitopes ◽  
Computer Prediction ◽  
Microarray Scanner ◽  
Ic50 Values

Abstract Therapeutic factor VIII (FVIII) infusions provoke CD4 T-cell proliferation and cytokine secretion leading to neutralizing anti-FVIII antibodies (inhibitors) in a substantial fraction of hemophilia A patients, leading to bleeding that is difficult to control. T-cell stimulation occurs when FVIII peptides bind to HLA-DRB1 proteins and are presented on the surface of antigen presenting cells, followed by T-cell recognition of these peptide-HLA-DRB1 complexes. Our laboratory is interested in identifying immunodominant, HLA-restricted T-cell epitopes in FVIII, in order to identify and then modify these immunological “hotspots” that cause inhibitors to develop. We have previously mapped several immunodominant HLA-restricted T-cell epitopes in FVIII recognized by the immune systems of inhibitor subjects, using HLA-DRB1 tetramers loaded with 20-mer FVIII peptides. However, because FVIII is such a large protein (>2,000 amino acid residues), methods more efficient than systematic mapping using overlapping peptides spanning its sequence would be useful. This study focuses on the binding of FVIII peptides to recombinant extracellular domains of 10 HLA-DRB1 proteins, which together represent HLA-DRB1 alleles found in >50% of the U.S population. PepStarTM (JPT Peptide Technologies) microarrays containing triplicate sets of FVIII peptides immobilized on glass slides via a flexible linker were incubated with HLA-DRB1 proteins at several dilutions, followed by incubation with a fluorescent-labeled antibody that recognizes the peptide-bound conformations of HLA-DRB1 proteins, and the slides were washed and read on a microarray scanner. The intensities of fluorescent signals from the triplicate sets of spots were quantified and also displayed as heat maps. Fluorescent signals ranged from background levels (indicating no binding) up to the detector saturation range. Between 20-40% of the FVIII peptides produced fluorescent signals indicating peptide binding to each of the HLA-DRB1 proteins tested, including positive control peptides containing FVIII and non-FVIII epitopes with known HLA-DRB1 restriction. The percentage of apparently high-affinity binding interactions was much lower. T-cell epitopes are generally 10-14 amino acid residues long. Therefore, strong signals from pairs of 20-mer peptides with a 12-residue overlapping sequence localized the FVIII sequence fitting into the HLA-DRB1 binding groove to the overlapping region, while disparate signals from other pairs of overlapping sequences localized the HLA-DRB1 binding sequence to the non-overlapping regions. For subsets of the FVIII peptides, the signal strengths were compared to (a) affinities (IC50 values) derived from quantitative, competition peptide-HLA-DRB1 binding assays, and (b) results of computer prediction algorithms MHCPanII and Propred. Peptides that bound strongly to one or more HLA-DRB1 protein were prioritized for further testing. This microarray-based screening procedure is an efficient method to generate manageable lists of potential T-cell epitopes in large antigenic proteins such as FVIII, where systematic epitope mapping using HLA-DRB1 tetramers loaded with peptides spanning the entire sequence is not feasible, due to both the high cost of such experiments and the blood volumes that can reasonably be requested from patient volunteers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Thyrotropin-Receptor and Thyroid Peroxidase-Specific T Cell Clones and Their Cytokine Profile in Autoimmune Thyroid Disease1

1997 ◽  
Vol 82 (11) ◽  
pp. 3655-3663
Author(s):  
Maria Elena Fisfalen ◽  
Ellen M. Palmer ◽  
Gijs A. van Seventer ◽  
Keyoumars Soltani ◽  
Yoshikuni Sawai ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Thyroid Tissue ◽  
Cytokine Profile ◽  
Thyroid Peroxidase ◽  
Amino Acid Residues ◽  
T Cell Epitopes ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

We studied the cytokine profile and the immune responses to thyroid antigens of specific T cell clones (TCC) isolated from patients with Hashimoto’s thyroiditis (HT) and Graves’ disease (GD). Antigen-specific TCC were reactive to thyroid peroxidase (TPO), thyroglobulin (Tg) or human recombinant TSH-receptor extracellular domain (TSH-R), and/or their respective peptides. Of the 43 clones derived from HT patients, 65% were reactive to TPO, and 59% of the 32 clones derived from GD patients were reactive to TSH-R. TPO epitopes 100–119 and 625–644 were recognized by 75% of HT-derived clones, whereas TSH-R epitopes 158–176, 207–222, and 343–362/357–376 were recognized by 85% of GD-derived TCC. The TCC were classified according to their cytokine profile into T helper cell (Th)0 [secreting interleukin (IL)-4, IL-5, interferon (IFN)-γ], Th1 (secreting IFN-γ) and Th2 (secreting IL-4 and/or IL-5). Tumor necrosis factor-β and IL-10 were produced by all subsets. The specific TCC were predominantly Th1-like cells in HT, and were Th0- and Th1-like cells in GD. Fifty three percent of Th0 clones were derived from GD patients and were reactive to TSH-R, whereas 50% of Th1 clones were derived from HT patients and were reactive to TPO or Tg. Most Th2 clones (82%) were reactive to TPO and were established from peripheral blood. All these clones produced IL-5, and 64% produced IL-4 and IL-10. Interestingly, IFN-γ was highly produced by TPO- or Tg-specific clones established from HT thyroid tissue. These results confirm at the clonal level our previous studies regarding T cell epitopes on TPO and TSH-R molecules and support the concept that immunodominant T cell epitopes are located on amino acid residues 100–119 and 625–644 of TPO in HT and amino acid residues 158–176, 207–222 and 343–362/357–376 of TSH-R in GD. Our studies also demonstrate that thyroid-specific T cells can be classified into Th0, Th1, and Th2 subsets. TPO- or Tg-specific clones with Th1 phenotype appear to be involved in the pathogenesis of HT, mediating thyroid tissue destruction, whereas TSH-R clones with Th0 phenotype may induce thyroid-stimulating autoantibodies in GD.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

scholarly journals The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif

2017 ◽  
Vol 21 (9) ◽  
pp. 2117-2128 ◽  
Author(s):  
Shui-yi Hu ◽  
Qiu-hua Gu ◽  
Jia Wang ◽  
Miao Wang ◽  
Xiao-yu Jia ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
T Cell Epitopes ◽  
Amino Acid Motif ◽  
Critical Amino Acid ◽  
Goodpasture Antigen

Using random forest to classify T-cell epitopes based on amino acid properties and molecular features

2013 ◽  
Vol 804 ◽  
pp. 70-75 ◽  
Author(s):  
Jian-Hua Huang ◽  
Hua-Lin Xie ◽  
Jun Yan ◽  
Hong-Mei Lu ◽  
Qing-Song Xu ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Random Forest ◽  
T Cell Epitopes ◽  
Acid Properties ◽  
Molecular Features ◽  
Amino Acid Properties

scholarly journals Extensive Polymorphism and Evidence of Immune Selection in a Highly Dominant Antigen Recognized by Bovine CD8 T Cells Specific for Theileria annulata

2011 ◽  
Vol 79 (5) ◽  
pp. 2059-2069 ◽  
Author(s):  
Niall D. MacHugh ◽  
William Weir ◽  
Alison Burrells ◽  
Regina Lizundia ◽  
Simon P. Graham ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Theileria Annulata ◽  
T Cell Epitopes ◽  
Major Histocompatibility Complex Haplotype ◽  
Content Type ◽  
Cell Responses ◽  
Restricted Epitope

ABSTRACTAlthough parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasiteTheileria annulatainfects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses toT. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates ofT. annulatademonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.

Involvement of class II β-chain amino acid residues 85 and 86 in T-cell allorecognition

1990 ◽  
Vol 27 (3) ◽  
pp. 240-253 ◽  
Author(s):  
David D. Eckels ◽  
Mary J. Geiger ◽  
Thomas W. Sell ◽  
Jack A. Gorski
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Class Ii ◽  
Amino Acid Residues ◽  
Β Chain

scholarly journals A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1929-1936 ◽  
Author(s):  
JW Precup ◽  
BC Kline ◽  
DN Fass
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Protein Fragments ◽  
Thrombin Cleavage ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.

scholarly journals Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast.

1991 ◽  
Vol 174 (2) ◽  
pp. 425-434 ◽  
Author(s):  
K Falk ◽  
O Rötzschke ◽  
K Deres ◽  
J Metzger ◽  
G Jung ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Synthetic Peptides ◽  
Cell Epitope ◽  
Reversed Phase ◽  
T Cell Epitope ◽  
Amino Acid Residues ◽  
Allele Specific ◽  
Infected Cells ◽  
Natural Peptides

Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position.

scholarly journals The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

2004 ◽  
Vol 377 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Stéphanie MOUHAT ◽  
Amor MOSBAH ◽  
Violeta VISAN ◽  
Heike WULFF ◽  
Muriel DELEPIERRE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Scorpion Toxin ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Voltage Gated ◽  
Toxin Binding ◽  
Ic50 Values

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 µg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 µM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

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