Immunoseq assay

Neoantigen-specific TCRs were identified using the modified MANAFEST assay as previously described.^{19 20} (link) The productive frequency of those TCR identified as known neoantigen-reactive T-cell clonotype sequences was compared between Control and CD40L conditions. TCR repertoire analysis from post-REP TIL was performed by immunosequencing of CDR3 regions of human TCR-beta chains using immunoSEQ assay (human TCR-beta deep resolution service—Adaptive Biotechnologies, Seattle, Washington, USA). Samples were obtained from consenting patients treated at Moffitt Cancer Center. Further details are provided in the online supplemental methods.

We extracted gDNA from bulk PBMCs, purified T cells and patient tissue samples using a DNA extraction kit (Qiagen). We used Dropsense 96 to quantify samples, dilute standard concentrations, and prepare libraries. We then generated sample data using an immunoSEQ Assay (Adaptive Biotechnologies), as previously described^{1 (link)}. In brief, we amplified the somatically rearranged TCRB CDR3 using a two-step, amplification bias-controlled multiplex PCR reaction, that contains forward and reverse amplification primers specific to every known V and J gene segment and amplifies the CDR3 receptor hypervariable locus, followed by a second PCR to add barcode and Illumina adapter sequences^{33 (link)}. Reference gene primers in the PCR further quantify total nucleated cells available for sequencing, and thus measure the fraction of T cells in each sequenced sample. The CDR3 and reference gene libraries were then sequenced, raw sequence reads demultiplexed per Adaptive’s barcode sequences, and demultiplexed reads processed to remove adapter and primer sequences and identify and remove primer dimer, germline and contaminant sequences. Relative frequency ratios between similar clones and a modified nearest-neighbour algorithm were used to cluster the filtered results, and merge closely related sequences to correct for sequencing induced technical errors. The output sequences were then annotated to the V, D, and J genes and N1 and N2 regions that constitute each unique CDR3, and the corresponding encoded CDR3 amino acid sequence. We defined annotated genes per the IMGT database (https://www.imgt.org). The output TCR V CDR3 sequences were then normalized and corrected for residual multiplex PCR amplification bias and quantified versus synthetic TCRB CDR3 sequence analogues^{34 (link)}.

DNA from FFPE tumor samples with different IGG signature levels was purified with the QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany) according to manufacturer’s instructions and BCR and TCR were sequenced from 23 and 33 DNA samples, respectively, using the immunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). The somatically rearranged Homo sapiens TCR locus complementarity-determining region 3 (CDR3) and BCR immunoglobulin heavy chain (IgH) locus CDR3 were amplified from DNA samples using a two-step, amplification bias-controlled multiplex PCR approach. CDR3 and reference gene libraries were sequenced on an Illumina (San Diego, CA) instrument according to the manufacturer’s instructions. Raw sequence reads were demultiplexed according to Adaptive’s proprietary barcode sequences. Demultiplexed reads were further processed to remove adapter and primer sequences, and identify and remove primer dimer, germline, and other contaminant sequences. The filtered data were clustered using both the relative frequency ratio between similar clones and a modified nearest-neighbor algorithm, to merge closely related sequences to correct for technical errors introduced through PCR and sequencing. The resulting sequences allowed annotation of the V, D, and J genes and the N1 and N2 regions constituting each unique CDR3 and the translation of the encoded CDR3 amino acid sequence. Gene definitions were based on annotation in accordance with the IMGT database (www.imgt.org). The set of observed biological TCR and BCR IgH CDR3 sequences was normalized to correct for residual multiplex PCR amplification bias and quantified against a set of synthetic CDR3 sequence analogues. Data were analyzed using the immunoSEQ Analyzer toolset.