Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

Abstract

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systemizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

T cells specific for α-myosin drive immunotherapy-related myocarditis

“…TCR sequences and cloning. TCR sequences were generated from CDR3 regions, V genes and J genes using Stitchr 17 . Alpha genes and beta genes were separated using a T2A sequence.…”

“…CDR3 amino acid sequences, V genes, and J genes are shown in Table 1. These TCRs were reconstructed using Stitchr, cloned and retrovirally transduced into Jurkat nuclear factor of activated T cells (NFAT)-GFP reporter cells [17][18][19] . Syngeneic bone marrow derived dendritic cells were used as antigen presenting cells (APCs).…”

Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination

“…Twelve TCR clonotypes were picked from donor 321-05 for cloning based on two criteria; the clone was detected at multiple time points after vaccination, and it had matching scPCR and bulk sequencing results from the sorted TFH cells. VDJ nucleotide sequences of the picked clones were reconstructed using stiTChR 42 with the TCRα and TCRβ chains linked with a P2A auto-cleavage site. To generate APCs expressing specific HLA, the nucleotide sequences of alpha and beta chains for donor-specific HLA class II haplotypes were curated using (Allele Search Tool < IMGT/HLA < IPD < EMBL-EBI) and linked via T2A site to form the full HLA constructs (HLA-DR, HLA-DP, and HLA-DQ).…”

Anaplastic lymphoma kinase-special immunity and immunotherapy

“…With the revolutionary breakthroughs in the field of TCR therapy in recent years, an increasing number of ALK epitopes/ peptides may become suitable targets for directed immunotherapy (109,110). An ongoing study is screening for autologous or allogeneic T cell receptor-transgenic T cells to test against ALK+/-patient-derived and cancer cell lines using in vitro and in vivo models to assess the potential utility of cytotoxic TCR-directed immunotherapies (111).…”