RESUMO
The ability to display functional T-cell receptors (TCR) on the surface of bacteriophage could have numerous applications. For instance, TCR phage-display could be used to develop new strategies for isolating TCRs with unique specificity or it could be used to carry out mutagenesis studies on TCR molecules for analyzing their structure-function. We initially selected a TCR from the murine T-cell hybridoma, DO11.10, as our model system, and genetically engineered a three domain single-chain TCR (scTCR) linked to the gene p8 protein of the Escherichia coli bacteriophage fd. Immunoblotting studies revealed that (1) E. coli produced a soluble scTCR/p8 fusion protein and (2) the fusion protein was packaged by the phage. Cellular competition assays were performed to evaluate the functionality of the TCR and showed the DO11.10 TCR-bearing phage could significantly inhibit stimulation of DO11.10 T hybridoma cells by competing for binding to immobilized MHC/peptide IA(d)/OVA(323-339). Flow cytometric analysis was carried out to evaluate direct binding of DO11.10 TCR-bearing phage onto the surface of cells displaying either IAd containing irrelevant peptide or OVA peptide. The results revealed binding of DO11.10 TCR-bearing phage only on cells expressing IA(d) loaded with OVA peptide showing TCR fine specificity for peptide. To illustrate the generality of TCR phage-display, we also cloned and displayed on phage a second TCR which recognizes a peptide fragment from human tumor suppressor protein p53 restricted by HLA-A2. These findings demonstrate functional TCR can be displayed on bacteriophage potentially leading to the development of novel applications involving TCR phage-display.
Assuntos
Inovirus/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Escherichia coli/virologia , Humanos , Hibridomas , Inovirus/genética , Camundongos , Ratos , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Especificidade por Substrato , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/fisiologiaRESUMO
A patient with a renal arteriovenous fistula is described. She was though to have valvular aortic stenosis because of a history of rheumatic fever, symptoms of congestive heart failure and syncope, and the presence of a harsh systolic murmur with a thrill in the aortic area. Cardiac catheterization revealed a left-to-right shunt of 8.7 L/min. Ligation of the fistula resulted in complete relief of the symptoms and attenuation of the murmur.
