Survey immunoseq assay, by Adaptive Biotechnologies - Product details

1

Profiling Tumor-Infiltrating T Cell Repertoire

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Intra-tumoral TCR clones were evaluated by next-generation sequencing of the baseline tumor as well as matched baseline-resistant tumors for cases 295, 459 and 926. TCR-β CDR3 regions were amplified using the survey ImmunoSeq assay in a multiplex PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies)^{64 (link)}. Productive TCR sequences were further analyzed, and clone counts were based on CDR3 amino acid sequences (Supplementary Table 9). Dominant TCR clones were assessed by estimating the proportion of TCR repertoire constituted by the top 5% of unique clones; for these analyses, TCR repertoires were filtered for clones representing at least 0.01% of the repertoire. For each sample, a clonality metric was estimated to quantitate the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. For differential abundance analysis between baseline and on-therapy tumors, we selected the most expanded and most regressed TCR clonotypes, corresponding to fold changes in productive frequency of TCR clones with a false discovery rate (FDR) < 0.01 (Fisher’s exact test) and requiring at least 0.01% relative repertoire abundance at baseline or resistance time points.

Forde P.M., Anagnostou V., Sun Z., Dahlberg S.E., Kindler H.L., Niknafs N., Purcell T., Santana-Davila R., Dudek A.Z., Borghaei H., Lanis M., Belcaid Z., Smith K.N., Balan A., White J.R., Cherry C., Ashok Sivakumar I.K., Shao X.M., Chan H.Y., Singh D., Thapa S., Illei P.B., Pardoll D.M., Karchin R., Velculescu V.E., Brahmer J.R, & Ramalingam S.S. (2021). Durvalumab with platinum-pemetrexed for unresectable pleural mesothelioma: survival, genomic and immunologic analyses from the phase 2 PrE0505 trial. Nature Medicine, 27(11), 1910-1920.

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TCR Sequencing and Clonality Quantification

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TCR sequencing and clonality quantification was performed as previously described^{16 (link),17 (link)} from tumour samples preserved using RNAlater (Qiagen) and stored at −80°C. DNA was isolated by mincing followed by extraction utilizing a DNeasy kit (Qiagen). Melanin was then removed from visibly pigmented melanoma samples using a PCR Inhibitor Removal kit (Zymo Research). TCRβ CDR3 regions were amplified and sequenced using the survey ImmunoSeq assay in a multiplexed PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies, Seattle, WA). Reads of length 87 bp were obtained using the Illumina HiSeq System. For each sample, Shannon entropy was calculated on the clonal abundance of all productive TCR sequences in the dataset. Shannon entropy was normalized to the range [0–1] by dividing Shannon entropy by the logarithm of the number of unique productive TCR sequences in the dataset. This normalized entropy value was then inverted (1 − normalized entropy) to produce our clonality metric.

Tumeh P.C., Harview C.L., Yearley J.H., Shintaku I.P., Taylor E.J., Robert L., Chmielowski B., Spasic M., Henry G., Ciobanu V., West A.N., Carmona M., Kivork C., Seja E., Cherry G., Gutierrez A., Grogan T.R., Mateus C., Tomasic G., Glaspy J.A., Emerson R.O., Robins H., Pierce R.H., Elashoff D.A., Robert C, & Ribas A. (2014). PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature, 515(7528), 568-571.

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Evaluation of TCR Clones in Tumor Tissue

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TCR clones were evaluated in tumor tissue by NGS. DNA from tumor samples was isolated by using the Qiagen DNA FFPE kit. TCR-β CDR3 regions were amplified using the survey ImmunoSeq assay in a multiplex PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies)^{62 (link)}. Productive TCR sequences were further analyzed. For each sample, a clonality metric was estimated to quantitate the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Clonality values range from 0 to 1, where values approaching 1 indicate a nearly monoclonal population (Supplementary Table 13).

Anagnostou V., Niknafs N., Marrone K., Bruhm D.C., White J.R., Naidoo J., Hummelink K., Monkhorst K., Lalezari F., Lanis M., Rosner S., Reuss J.E., Smith K.N., Adleff V., Rodgers K., Belcaid Z., Rhymee L., Levy B., Feliciano J., Hann C.L., Ettinger D.S., Georgiades C., Verde F., Illei P., Li Q.K., Baras A.S., Gabrielson E., Brock M.V., Karchin R., Pardoll D.M., Baylin S.B., Brahmer J.R., Scharpf R.B., Forde P.M, & Velculescu V.E. (2020). Multimodal genomic features predict outcome of immune checkpoint blockade in non-small-cell lung cancer. Nature cancer, 1(1), 99-111.

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TCR Sequencing and Clonality Quantification

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TCR sequencing and clonality quantification was performed as previously described^{16 (link),17 (link)} from tumour samples preserved using RNAlater (Qiagen) and stored at −80°C. DNA was isolated by mincing followed by extraction utilizing a DNeasy kit (Qiagen). Melanin was then removed from visibly pigmented melanoma samples using a PCR Inhibitor Removal kit (Zymo Research). TCRβ CDR3 regions were amplified and sequenced using the survey ImmunoSeq assay in a multiplexed PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies, Seattle, WA). Reads of length 87 bp were obtained using the Illumina HiSeq System. For each sample, Shannon entropy was calculated on the clonal abundance of all productive TCR sequences in the dataset. Shannon entropy was normalized to the range [0–1] by dividing Shannon entropy by the logarithm of the number of unique productive TCR sequences in the dataset. This normalized entropy value was then inverted (1 − normalized entropy) to produce our clonality metric.

Tumeh P.C., Harview C.L., Yearley J.H., Shintaku I.P., Taylor E.J., Robert L., Chmielowski B., Spasic M., Henry G., Ciobanu V., West A.N., Carmona M., Kivork C., Seja E., Cherry G., Gutierrez A., Grogan T.R., Mateus C., Tomasic G., Glaspy J.A., Emerson R.O., Robins H., Pierce R.H., Elashoff D.A., Robert C, & Ribas A. (2014). PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature, 515(7528), 568-571.

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Quantitative TCR Profiling of Tumor Samples

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TCR clones were evaluated in tumor tissue by next generation sequencing, with an input material of 500ng of genomic DNA for each sample analyzed. TCR-β CDR3 regions were amplified using the survey ImmunoSeq assay in a multiplex PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies)⁶² (link). Productive TCR sequences were further analyzed (Table S1H). For each sample, a clonality metric was estimated in order to quantitate the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Clonality values range from 0 to 1, where values approaching 1 indicate a nearly monoclonal population (Table S1H). For differential abundance analysis between baseline and on-therapy tumors, we selected the most expanded and most regressed TCR clonotypes, corresponding to fold changes in productive frequency of TCR clones with an FDR < 0.001. These TCR clones are referred to as expanded and regressed.

Anagnostou V., Bruhm D.C., Niknafs N., White J.R., Shao X.M., Sidhom J.W., Stein J., Tsai H.L., Wang H., Belcaid Z., Murray J., Balan A., Ferreira L., Ross-Macdonald P., Wind-Rotolo M., Baras A.S., Taube J., Karchin R., Scharpf R.B., Grasso C., Ribas A., Pardoll D.M., Topalian S.L, & Velculescu V.E. (2020). Integrative Tumor and Immune Cell Multi-omic Analyses Predict Response to Immune Checkpoint Blockade in Melanoma. Cell Reports Medicine, 1(8), 100139.

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Profiling Tumor T-Cell Repertoire

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DNA was extracted from 30 mg of thymic lymphoma tissue using the MagAttract HMW DNA Kit (Qiagen, 67563), and submitted to Adaptive Biotechologies to sequence the TCRβ using the ImmunoSeq survey assay, and the data were analyzed using the ImmunoSeq Analyzer (Adaptive Biotechnologies) to determine the clonogenicity of the tumor DNA as well as the nature of the V(D)J rearrangement and whether it leads to a productive template for functional TCRβ.

Feng X., Lu H., Yue J., Schneider N., Liu J., Denzin L.K., Chan C.S., De S, & Shen Z. (2019). Loss of Setd4 delays radiation-induced thymic lymphoma in mice. DNA repair, 86, 102754.

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TCRβ Repertoire Analysis of Tconv and Treg Cells

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TCR sequence analysis was performed on individual mice. Genomic DNA was extracted from sorted Foxp3^GFP+ and Foxp3^GFP− CD4⁺TCR⁺ T cells isolated from pancreatic islets of pre-diabetic NOD.Foxp3^GFP mice at 14-16 weeks of age. Purification of gDNA was performed using QIAamp DNA Micro Kit. TCRβ repertoires of Tconv and Treg populations were assessed by ImmunoSEQ Survey assay (Adaptive Biotechnologies). Data were analyzed by ImmunoSEQ Analyzer version 2.0.

Kong Y., Jing Y., Allard D., Scavuzzo M.A., Sprouse M.L., Borowiak M., Bettini M.L, & Bettini M. (2022). A dormant T cell population with autoimmune potential exhibits low self-reactivity and infiltrates islets in type 1 diabetes. European journal of immunology, 52(7), 1158-1170.

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Survey immunoseq assay

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7 protocols using survey immunoseq assay

Profiling Tumor-Infiltrating T Cell Repertoire

TCR Sequencing and Clonality Quantification

Evaluation of TCR Clones in Tumor Tissue

TCR Sequencing and Clonality Quantification

Quantitative TCR Profiling of Tumor Samples

Profiling Tumor T-Cell Repertoire

TCRβ Repertoire Analysis of Tconv and Treg Cells

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