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67 protocols using ImmunoSEQ

Whole lymph node, heart, and thymic tissue were harvested from young (between 11 and 21 days old) Ctla4−/−, Ctla4+/−, and Ctla4+/+ littermate mice and snap frozen in liquid nitrogen. Tissue were homogenized using a TissueLyser II (Qiagen) and genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen). Genomic DNA was sequenced at survey level using ImmunoSeq (Adaptive Biotechnologies). Analysis of TCR sequencing data was performed using the Adaptive Biotechnologies Analyzer. A potential limitation of these analyses is the low infiltration of heart tissue in littermate controls. Clonality was defined as the inverse of normalized Shannon entropy. Shannon entropy values range from 0-1, with 1 representing a monoclonal population. Frequencies of individual TCRvβ segments were identified and exported on a per mouse basis for further statistical analysis. Mann-Whitney Test comparisons of TCR clonotypes were performed in MATLAB. ImmunoSeq data will be made publicly available through Adaptive Biotechnologies Analyzer.
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Tumor sequencing of the CDR3 regions of human TCR-β chains was performed by immunoSEQ as recommended (Adaptive Biotechnologies, Seattle, WA). Clonality, a measure of T cell reactivity, and richness, a measure of T cell diversity, were calculated using the Analyzer platform. T cell density in fresh-frozen tissue samples was calculated by normalizing TCR-β template counts to the total amount of DNA used for TCR sequencing assuming 6.6pg of DNA per cell [28 , 29 (link)].
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3

TCR Sequencing and Clonality Quantification in Neuroinflammation

TCR sequencing and clonality quantification was assessed in neuropathologist-selected (author BM) FFPE samples of highly inflamed and non-inflamed brain parenchyma using survey level immunoSEQ™ and the Immunoverse™ assay, as previously described (Adaptive Biotechnologies, ArcherDX)39 ,40 . Sequencing results were evaluated using the immunoSEQ analyzer version 3.0. Shannon entropy, a measure of sample diversity, was calculated on the clonal abundance of all productive TCR sequences in the data set. Shannon entropy was normalized by dividing Shannon entropy by the logarithm of the number of unique productive TCR sequences. This normalized entropy value was then inverted (1 − normalized entropy) to produce the clonality metric.
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DNA was extracted from peripheral blood mononuclear cells, then TCRβ CDR3 regions were amplified and sequenced using ImmunoSEQ (Adaptive Biotechnologies, Seattle, WA) from 400 ng of DNA template59 (link). Bias-controlled V and J gene primers were used to amplify rearranged V(D)J segments for high throughput sequencing at ~20x coverage. After correcting sequencing errors via a clustering algorithm, CDR3 segments were annotated according to the International ImMunoGeneTics collaboration, identifying which V, D, and J genes contributed to each rearrangement 60 (link).
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5

Profiling T-cell Receptor Diversity in Pediatric Epilepsy

Bulk genomic DNA was isolated from frozen blocks of fresh involved brain tissue from 13 of the 53 pediatric epilepsy surgery cases and whole blood from nine of these cases (Monarch® genomic DNA purification kit, New England Biolabs, Ipswich, MA). Vβ chain TCR sequences were obtained using the ImmunoSEQ® assay (Adaptive Biotechnologies, Seattle, WA). CDR3 sequences were manually curated to remove those that did not start with the third framework cysteine residue at position 104 (14 (link)). Clonality was calculated as described (15 (link)); comparison of proportions Chi-squared tests (16 (link)) were used to determine whether the frequency of the same Vβ CDR3 sequence was significantly different between brain and blood samples from the same patient. Whether Vβ CDR3 sequences corresponded to public T cell clones was determined by searching the immuneACCESS® database (Adaptive Biotechnologies) and similarity to know anti-viral T cell clonotypes was determined using the VDJdb browser (17 (link)). Venn diagrams and heat maps were made with online tools (bioinformatics.psb.ugent.be/webtools/Venn, software.broadinstitute.org/morpheus/). Venn diagrams and heat maps were exported to CorelDraw2017 as scalable vector graphics and portable document format files respectively (Corel Corporation, Ottawa, Canada).
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Genomic DNA from pre-infusion CART-PSMA-TGFβRDN cells and sorted post-infusion CAR T-cells was isolated using the DNeasy Blood and Tissue Kit (Qiagen). Initial flow cytometric assessment the T-cell repertoire by flow cytometry was conducted using the Beta Mark TCRVβ repertoire kit and Kaluza analysis software (Beckman Coulter Life Sciences). Subsequent TCRVβ deep sequencing was carried out by immunoSEQ (Adaptive Biotechnologies). Only productive TCR rearrangements were used in the evaluation of clonotype frequencies.
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DNA was extracted from FACS-sorted bone marrow B220+IgM cells and thymic CD4+CD8+CD3εlo cells using the Qiagen DNeasy Blood & Tissue Kit (Qiagen; 69506). High-throughput DNA sequencing of rearranged Igh and Tcrb genes was performed using ImmunoSEQ and was performed by Adaptive Biotechnologies. Gene segment usage was analyzed by ImmunoSEQ Analyzer software (Adaptive Biotechnologies) and R.
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DNA was extracted from human skin grafts using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) as per manufacturer’s instructions with overnight tissue digestion. TCRβ CDR3 regions were amplified and sequenced by ImmunoSEQ (Adaptive Biotechnologies, Seattle, WA) from 300 ng DNA template as previously described (27 ).
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TCR-Vβ deep sequencing was performed by immune repertoire sequencing (iRepertoire) on mRNA or immunoSEQ (Adaptive Biotechnologies) on genomic DNA isolated from FFPE tumor tissue, peripheral blood mononuclear cells and T cells. We reasoned that T cell receptors that are neoantigen-specific would be present at an appreciable frequency in the DKESEEEVS-stimulated IFNγ+ population and present in at least one of the patient tumor or blood populations. Of the 1525 T cell receptor beta chain CDR3s identified via our IFNγ capture assay, 121 overlapped with at least one of the patient sample populations. We tested whether the CDR3s identified in patient blood and tissue samples during immunotherapy were enriched for those in the IFNγ capture assay population, by comparing the overlap of patient sample CDR3s and IFNγ+ capture T cell CDR3s, compared to unselected (bulk population) CDR3s. Of the 763 clones present above median frequency in the IFNγ+ population, 71 were present in at least one of the blood sample populations. We conducted a permutation test sampling 763 random CDR3s from the bulk population and overlapping with the blood populations over 10,000 iterations, and found that only 16/10,000 (p = 0.016) iterations produced overlap greater than or equal to the observed overlap. Analyses were conducted in R version 3.5.0.
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Lungs were harvested at 14 weeks post Ad-cre instillation and dissociated. DNA was extracted from lung tissue using the Quick-DNA/RNA Microprep Plus Kit (Zymo Research) and 3 µg was sent for TCRB sequencing using immunoSEQ (Adaptive Biotechnologies).
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