COmbinatorial PEptide POoling Design for TCR specificity

T cell receptor (TCR) repertoire diversity enables the antigen-specific immune responses against the vast space of possible pathogens. Identifying TCR-antigen binding pairs from the large TCR repertoire and antigen space is crucial for biomedical research. Here, we introduce copepodTCR, an open-access tool to design and interpret high-throughput experimental TCR specificity assays.

copepodTCR implements a combinatorial peptide pooling scheme for efficient experimental testing of T cell responses against large overlapping peptide libraries, that can be used to identify the specificity of (or “deorphanize”) TCRs. The scheme detects experimental errors and, coupled with a hierarchical Bayesian model for unbiased interpretation, identifies the response-eliciting peptide sequence for a TCR of interest out of hundreds of peptides tested using a simple experimental set-up.

How to use

The experimental setup starts with defining the protein/proteome of interest and obtaining synthetic peptides tiling its space. Peptide sequences can be generated in silico from a protein of interest and then checked using functions from Peptides generation and assessment section.

This set of peptides, containing an overlap of a constant length, is entered into copepodTCR. Parameters for CPP scheme can be selected using functions from Peptide occurrence search. It creates a peptide pooling scheme (functions from Pooling) and, optionally, provides the pipetting scheme as 3D mask models which could be further 3D printed and overlay the physical plate or pipette tip box (3D models section).

Following this scheme, the peptides are mixed, and the resulting peptide pools tested in a T cell activation assay. The activation of T cells is measured for each peptide pool with the assay of choice, such as flow cytometry- or microscopy-based activation assays detecting transcription and translation of a reporter gene.

The experimental measurements for each pool are entered back into copepodTCR which employs a Bayesian mixture model to identify activated pools. Based on the activation patterns, it returns the set of overlapping peptides leading to T cell activation (Results interpretation with a Bayesian mixture model). Also they can be displayed using functions from Plotting results section.

For more details, refer to «copepodTCR: Identification of Antigen-Specific T Cell Receptors with combinatorial peptide pooling» (bioRxiv version).

Algorithm for CPP generation

Algorithms for CPP generation are described in “Unbiased and Error-Detecting Combinatorial Pooling Experiments with Balanced Constant-Weight Gray Codes for Consecutive Positives Detection” (arXive version). CodePUB python package accompanies the paper and provides all functions required to use the algorithm.