Immunoseq

To determine T cell diversity and clonality, frozen EDTA-anticoagulated blood and FFPE tissue specimens were sent to Adaptive Biotechnologies (WA, USA) for ImmunoSEQ - T-cell Receptor Sequencing (TCR) sequencing. Extracted genomic DNA was amplified in a bias-controlled multiplex PCR, followed by high-throughput sequencing. Sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region for further analysis as previously described^{65 (link)–67 (link)}. Data was analyzed using the ImmunoSEQ Analyzer software platform. Morisita index is a modified correlation comparing both shared clones and relative abundance of clones between two samples. Values can range from 1 (identical) to 0 (disparate) when comparing repertoires^{68 (link)}. Peripheral T cells fraction is calculated as the total T cells divided by an estimate of the total number of nucleated cells based on mass. Higher T cell fraction indicates more recruitment and activation of T cells in peripheral blood. The pairwise comparison of shared clones between subjects assessed the overlap of the top 100 most abundant clones for all 10 possible pairs of blood samples within each treatment group (n = 5), with each pair resulting in a data point representing the number of shared top 100 clones. Dearth of “public clones” may indicate increase in number or diversity of tumor antigens within each subject. Simpson Clonality⁶⁹ quantifies the extent to which one or few clones dominate the repertoire. It ranges from 0 (even distribution) to 1 (monoclonal) and is robust to sequencing depth. Here Simpson Clonality is defined as the square root of Simpson’s index.

To determine T cell diversity and clonality, frozen EDTA-anticoagulated blood and FFPE tissue specimens were sent to Adaptive Biotechnologies (WA, USA) for ImmunoSEQ - T-cell Receptor Sequencing (TCR) sequencing. Extracted genomic DNA was amplified in a bias-controlled multiplex PCR, followed by high-throughput sequencing. Sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region for further analysis as previously described^{65 (link)–67 (link)}. Data was analyzed using the ImmunoSEQ Analyzer software platform. Morisita index is a modified correlation comparing both shared clones and relative abundance of clones between two samples. Values can range from 1 (identical) to 0 (disparate) when comparing repertoires^{68 (link)}. Peripheral T cells fraction is calculated as the total T cells divided by an estimate of the total number of nucleated cells based on mass. Higher T cell fraction indicates more recruitment and activation of T cells in peripheral blood. The pairwise comparison of shared clones between subjects assessed the overlap of the top 100 most abundant clones for all 10 possible pairs of blood samples within each treatment group (n = 5), with each pair resulting in a data point representing the number of shared top 100 clones. Dearth of “public clones” may indicate increase in number or diversity of tumor antigens within each subject. Simpson Clonality⁶⁹ quantifies the extent to which one or few clones dominate the repertoire. It ranges from 0 (even distribution) to 1 (monoclonal) and is robust to sequencing depth. Here Simpson Clonality is defined as the square root of Simpson’s index.