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 systematizing 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.

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.

The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ—) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ— lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δECγTMγIC (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ— T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ— patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.