Abstract
Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αβ T-cell receptors (TCRαβ) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαβs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαβ genes as Sleeping Beauty transposons and the determination of the introduced TCRαβs’ antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.
Highlights
Two lines of evidence suggest that the immune cell population in the tumor microenvironment is correlated with clinical outcome [1,2,3]
The library consists of 48 TCRα sequences as Sleeping Beauty (SB) plasmids, each assembled from an individual TCR alpha variable (TRAV) gene paired with a consensus TCR beta constant 2 (TRBC2) gene sequence
A library of 59 TCRβ sequences encoding consensus TCR alpha constant (TRAC) genes linked with 59 unique TCR beta variable (TRBV) genes by the furin-SGSG-P2A ribosome-skipping sequence was constructed (Fig 1A and 1B and S1 Fig)
Summary
Two lines of evidence suggest that the immune cell population in the tumor microenvironment is correlated with clinical outcome [1,2,3]. The therapeutic immune checkpoint blockade of CTLA-4 or PDL1/PD1 reinvigorates exhausted tumorinfiltrating lymphocytes (TILs) and has anti-tumor effects in a subset of patients [5]. Recognition of neoantigens by TILs is supported by clinical findings demonstrating that successful immune checkpoint blockade therapy is correlated with high mutation loads in tumor cells [7,8,9,10]. That CD8+PD1+ T cells are enriched in the tumor microenvironment supports a role for neoantigen-specific TILs as mediators of immune checkpoint blockade [11, 12]
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