Journal: Frontiers in Immunology

Article Title: Comprehensive self-antigen screening to assess cross-reactivity in promiscuous T-cell receptors

doi: 10.3389/fimmu.2025.1719827

Figure Lengend Snippet: Schematic summary of Tope-seq platform. The parallel approaches for self-antigen minigene library development are illustrated in (a) . In one arm, the whole proteome coding (WPC) format library is created from primary amino acid sequences of all proteins identified in the human reference proteome accessed and fragmented in silico using a defined tiling scheme. The extracted amino acid sequences are then converted to DNA sequences by random codon back-translation and individually synthesized as single-stranded oligonucleotides on an array-based platform. After synthesis, DNA fragments are liberated from the array, pooled, and used to produce a lentiviral vector library. In another arm of library construction, a whole exome shotgun (WES) library was generated using primary genomic DNA from live normal human cells isolated and physically fragmented by sonication. The resultant double-stranded DNA fragments are then subjected to exome capture by RNA hybridization probes, end-polished, and adapterized for PCR amplification prior being used to produce lentiviral vector library. Library virus from either stream is used to transduce sAPC in preparation for Tope-seq based screening. The principle of the Tope-seq assay format is represented in (b) . Activated effector T cells transduced with the exogenous TCR-of-interest are co-cultured with sAPC harboring an integrated self-antigen minigene library. When TCR-T cells encounter a target cell containing a minigene coding for a cognate epitope, GZMB molecules are delivered to the offending target where they cleave target-encoded ECFP-EYFP fusion proteins separated by a GZMB substrate peptide linker. The resulting loss of FRET signal (or ‘FRET-shift’) upon cleavage is monitored in flow cytometry and the library cell population is isolated by FACS to capture the Shifted cells and the counterpart Unshifted fraction. Recovered cells are characterized by targeted NGS to identify the virally encoded minigenes present in the cells from both matched gates. To quantify minigene enrichment in the Tope-seq experiment, a bioinformatic pipeline outlined in (c) is used. For WPC library-based screening, matched Shifted and Unshifted gate minigene sequencing reads are paired-end merged, normalized to sample total read depth, and aligned to designed library reference sequences. Once processed, enrichment scores for each individual minigene in the library are calculated by determining the relative frequency of normalized counts in the Shifted gate (rf s ) as a proportion of the total across both gates. For WES libraries, processing is performed by mapping paired-end reads to the reference human genome and using a sliding reference peptide window to scan all k-mer size ranges (from 8 to 11 residues in length) across the coding genome. In-frame minigenes traversing each window are counted and normalized to read depth for each matched gate dataset prior to calculating rf s score for individual peptides, rather than producing minigene-specific scores. Where applicable, rf s scores from replicate experiments are calculated as the geometric mean of all measurements. We defined two separate read processing pipelines for the two formats of minigene libraries since WES libraries contain epitopes embedded within an undefined number of possible distinct minigene fragments, WES fragments are generally longer in length than would allow for an accurate paired-end merging step, randomly sheared WES fragments could be cloned into viral vector in one of six possible expression frames (whereas frame is precisely controlled in WPC format), and WPC minigenes are constructed with non-native codon usage.

Article Snippet: Lentiviral transfer plasmid was derived from the pCCL-c-MNDU3-X backbone (Addgene #81071).

Techniques: In Silico, Synthesized, Plasmid Preparation, Generated, Isolation, Sonication, Hybridization, Amplification, Virus, Transduction, Cell Culture, Flow Cytometry, Library Screening, Sequencing, Clone Assay, Expressing, Construct

Journal: Frontiers in Immunology

Article Title: Comprehensive self-antigen screening to assess cross-reactivity in promiscuous T-cell receptors

doi: 10.3389/fimmu.2025.1719827

Figure Lengend Snippet: Schematic summary of Tope-seq platform. The parallel approaches for self-antigen minigene library development are illustrated in (a) . In one arm, the whole proteome coding (WPC) format library is created from primary amino acid sequences of all proteins identified in the human reference proteome accessed and fragmented in silico using a defined tiling scheme. The extracted amino acid sequences are then converted to DNA sequences by random codon back-translation and individually synthesized as single-stranded oligonucleotides on an array-based platform. After synthesis, DNA fragments are liberated from the array, pooled, and used to produce a lentiviral vector library. In another arm of library construction, a whole exome shotgun (WES) library was generated using primary genomic DNA from live normal human cells isolated and physically fragmented by sonication. The resultant double-stranded DNA fragments are then subjected to exome capture by RNA hybridization probes, end-polished, and adapterized for PCR amplification prior being used to produce lentiviral vector library. Library virus from either stream is used to transduce sAPC in preparation for Tope-seq based screening. The principle of the Tope-seq assay format is represented in (b) . Activated effector T cells transduced with the exogenous TCR-of-interest are co-cultured with sAPC harboring an integrated self-antigen minigene library. When TCR-T cells encounter a target cell containing a minigene coding for a cognate epitope, GZMB molecules are delivered to the offending target where they cleave target-encoded ECFP-EYFP fusion proteins separated by a GZMB substrate peptide linker. The resulting loss of FRET signal (or ‘FRET-shift’) upon cleavage is monitored in flow cytometry and the library cell population is isolated by FACS to capture the Shifted cells and the counterpart Unshifted fraction. Recovered cells are characterized by targeted NGS to identify the virally encoded minigenes present in the cells from both matched gates. To quantify minigene enrichment in the Tope-seq experiment, a bioinformatic pipeline outlined in (c) is used. For WPC library-based screening, matched Shifted and Unshifted gate minigene sequencing reads are paired-end merged, normalized to sample total read depth, and aligned to designed library reference sequences. Once processed, enrichment scores for each individual minigene in the library are calculated by determining the relative frequency of normalized counts in the Shifted gate (rf s ) as a proportion of the total across both gates. For WES libraries, processing is performed by mapping paired-end reads to the reference human genome and using a sliding reference peptide window to scan all k-mer size ranges (from 8 to 11 residues in length) across the coding genome. In-frame minigenes traversing each window are counted and normalized to read depth for each matched gate dataset prior to calculating rf s score for individual peptides, rather than producing minigene-specific scores. Where applicable, rf s scores from replicate experiments are calculated as the geometric mean of all measurements. We defined two separate read processing pipelines for the two formats of minigene libraries since WES libraries contain epitopes embedded within an undefined number of possible distinct minigene fragments, WES fragments are generally longer in length than would allow for an accurate paired-end merging step, randomly sheared WES fragments could be cloned into viral vector in one of six possible expression frames (whereas frame is precisely controlled in WPC format), and WPC minigenes are constructed with non-native codon usage.

Article Snippet: Lentiviral transfer plasmids were derived from pCCL-c-MNDU3-X backbone (Addgene #81071).

Techniques: In Silico, Synthesized, Plasmid Preparation, Generated, Isolation, Sonication, Hybridization, Amplification, Virus, Transduction, Cell Culture, Flow Cytometry, Library Screening, Sequencing, Clone Assay, Expressing, Construct