Identification of patient-specific CD4⁺ and CD8⁺ T cell neoantigens through HLA-unbiased genetic screens
T cells transduced with the CDK4-specific TCRs DMF4, DMF5 or 1D3 in T cell medium with 25 U ml. Cultures were resuspended on day 1 and 2 of the experiment. For screens using patient-derived MHC class II-restricted neoantigen-specific TCRs, the model library was transduced into patient-matched immortalized B cells , and library-expressing B cells were cocultured with donor CD4-specific TCR as described above.
The 7-nt stretch of N nucleotides indicates a unique barcode sequence used to enable the multiplexed preparation of sequencing libraries. Following PCR, samples were pooled equimolarly and run on a 1% agarose gel to separate minigene amplicons from potential primer dimers. Minigene amplicons were extracted from gel using the Monarch DNA Gel Extraction Kit and deep sequenced on an Illumina HiSeq 2500 Sequencing system .
For patient neoantigen screens, mutanome libraries were transduced into autologous immortalized B cells, followed by selection with puromycin. The cytotoxic potential of expanded patient TIL was confirmed before neoantigen screens by measuring their capacity to degranulate. To this end, CD4TIL were polyclonally stimulated using CD3/CD28 Dynabeads in T cell medium in the presence of Golgistop and an antibody against CD107 for 12 h.
To validate patient neoantigens identified in screens, minigenes encoding the screen hits, as well as their WT counterparts, were synthesized as individual gBlocks , cloned into the CD74 signal sequence-modified pMSCV-puroR-mCherry vector and transduced into immortalized patient B cells. Following selection with puromycin, minigene-transduced B cells were cocultured with expanded patient CD4
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