Dataset Information

Quantitative Characterization of the T Cell Receptor Repertoire of Naive and Memory Subsets Using an Integrated Experimental and Computational Pipeline Which Is Robust, Economical, and Versatile.

ABSTRACT: The T cell receptor (TCR) repertoire can provide a personalized biomarker for infectious and non-infectious diseases. We describe a protocol for amplifying, sequencing, and analyzing TCRs which is robust, sensitive, and versatile. The key experimental step is ligation of a single-stranded oligonucleotide to the 3' end of the TCR cDNA. This allows amplification of all possible rearrangements using a single set of primers per locus. It also introduces a unique molecular identifier to label each starting cDNA molecule. This molecular identifier is used to correct for sequence errors and for effects of differential PCR amplification efficiency, thus producing more accurate measures of the true TCR frequency within the sample. This integrated experimental and computational pipeline is applied to the analysis of human memory and naive subpopulations, and results in consistent measures of diversity and inequality. After error correction, the distribution of TCR sequence abundance in all subpopulations followed a power law over a wide range of values. The power law exponent differed between naïve and memory populations, but was consistent between individuals. The integrated experimental and analysis pipeline we describe is appropriate to studies of T cell responses in a broad range of physiological and pathological contexts.

SUBMITTER: Oakes T

PROVIDER: S-EPMC5643411 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Quantitative Characterization of the T Cell Receptor Repertoire of Naïve and Memory Subsets Using an Integrated Experimental and Computational Pipeline Which Is Robust, Economical, and Versatile.

Oakes Theres T Heather James M JM Best Katharine K Byng-Maddick Rachel R Husovsky Connor C Ismail Mazlina M Joshi Kroopa K Maxwell Gavin G Noursadeghi Mahdad M Riddell Natalie N Ruehl Tabea T Turner Carolin T CT Uddin Imran I Chain Benny B

Frontiers in immunology 20171012

The T cell receptor (TCR) repertoire can provide a personalized biomarker for infectious and non-infectious diseases. We describe a protocol for amplifying, sequencing, and analyzing TCRs which is robust, sensitive, and versatile. The key experimental step is ligation of a single-stranded oligonucleotide to the 3' end of the TCR cDNA. This allows amplification of all possible rearrangements using a single set of primers per locus. It also introduces a unique molecular identifier to label each st ...[more]

PMID: 29075258

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Project description:Nanobodies (Nbs) have recently emerged as a promising class of antibody fragments for biomedical and therapeutic applications. Despite having marked physicochemical properties, Nbs are derived from camelids and may require "humanization" to improve translational potentials for clinical trials. Here we have systematically analyzed the sequence and structural properties of Nbs based on NGS (next-generation sequencing) databases and high-resolution structures. Our analysis reveals substantial framework diversities and underscores the key differences between Nbs and human Immunoglobulin G (IgG) antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen-binding, providing insights into Nb humanization. Based on big data analysis, we developed " Llamanade '', a user-friendly, open-source to facilitate rational humanization of Nbs. Using Nb sequence as input, Llamanade provides information on the sequence features, model structures, and optimizes solutions to humanize Nbs. The full analysis for a given Nb takes less than a minute on a local computer. To demonstrate the robustness of this tool, we applied it to successfully humanize a cohort of structurally diverse and highly potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a web server to support the development of a rapidly expanding repertoire of therapeutic Nbs into safe and effective trials.Author summaryCamelid Nbs are characterized by small size, excellent pharmacological properties and high flexibility in bioengineering for therapeutic development. However, Nbs are "xeno" antibodies, which require "humanization" to improve their translational potential. Currently, there is a lack of systematic investigation of Nbs to rationally guide humanization. No dedicated software has been developed for this purpose. Here, we report the development of Llamanade , an open-source computational pipeline and the first dedicated software to facilitate rational humanization of Nbs. To subjectively evaluate Llamanade , we used it to humanize a cohort of structurally diverse and ultrapotent antiviral Nbs against SARS-CoV-2. Robust humanization by Llamanade significantly improved the humanness level of Nbs to closely resemble fully human IgGs. Importantly, these highly humanized antiviral Nbs remained excellent solubility and comparably high bioactivities to the non-humanized Nb precursors. We envision that Llamanade will help advance Nb research into therapeutic development.

Dataset Information

Quantitative Characterization of the T Cell Receptor Repertoire of Naive and Memory Subsets Using an Integrated Experimental and Computational Pipeline Which Is Robust, Economical, and Versatile.

Publications

Quantitative Characterization of the T Cell Receptor Repertoire of Naïve and Memory Subsets Using an Integrated Experimental and Computational Pipeline Which Is Robust, Economical, and Versatile.

Similar Datasets

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