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Landscape of tumor-infiltrating T cell repertoire of human cancers

Abstract

We developed a computational method to infer the complementarity-determining region 3 (CDR3) sequences of tumor-infiltrating T cells in 9,142 RNA-seq samples across 29 cancer types. We identified over 600,000 CDR3 sequences, including 15% that were full length. CDR3 sequence length distribution and amino acid conservation, as well as variable gene usage, for infiltrating T cells in many tumors, except in brain and kidney cancers, resembled those for peripheral blood cells from healthy donors. We observed a strong association between T cell diversity and tumor mutation load, and we predicted SPAG5 and TSSK6 as putative immunogenic cancer/testis antigens in multiple cancers. Finally, we identified three potential immunogenic somatic mutations on the basis of their co-occurrence with CDR3 sequences. One of them, a PRAMEF4 mutation encoding p.Phe300Val, was predicted to result in peptide binding strongly to both MHC class I and class II molecules, with matched HLA types in its carriers. Our analyses have the potential to simultaneously identify immunogenic neoantigens and tumor-reactive T cell clonotypes.

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Figure 1: Distribution of αβ T cell variable gene usage and γδ T cell abundance across multiple cancer types.
Figure 2: Length and amino acid conservation of β- and δ-chain CDR3 sequences in tumor-infiltrating T cells.
Figure 3: Public and private β-CDR3 amino acid sequences have different lengths and proportions of hydrophobic residues.
Figure 4: The diversity of T cell clonotypes positively associates with cancer somatic mutation load.
Figure 5: Association of T cell diversity with expression of cancer/testis antigens identifies SPAG5 and TSSK6 as vaccine targets.
Figure 6: Nonsynonymous mutations co-occur with CDR3 motifs.

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Acknowledgements

We thank G. Freeman for helpful discussion during manuscript preparation. We also acknowledge the following funding sources for supporting our work: NCI grant 1U01 CA180980, National Natural Science Foundation of China grant 31329003 and a Chinese Scholarship Council Fellowship. This work was supported in part by NIH/NCI DF/HCC Kidney Cancer SPORE P50 CA101942 to S.S. and T.K.C.

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Authors and Affiliations

Authors

Contributions

B.L. conceived this project, developed the CDR3 calling method, processed the data sets and performed statistical analysis. T.L. performed statistical analysis, generated a subset of the figures and helped write the manuscript. B.W., J.W. and R.D. helped with analysis of CDR3 sequences. S.A.S. performed analyses using POLYSOLVER. Q.C. helped analyze the data. J.-C.P., S.S. and T.K.C. conducted experimental validation. F.S.H., C.W. and N.H. conceived some of the analyses and contributed to the manuscript. X.S.L. and J.S.L. supervised the whole study and wrote the manuscript with B.L.

Corresponding authors

Correspondence to Jun S Liu or X Shirley Liu.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Workflow of CDR3 sequence assembly from RNA-seq data.

Paired-end short-read RNA-seq data were mapped to human reference genome hg19, and unmapped reads in the TCR regions were extracted for pairwise comparison. CDR3 sequences were assembled from disjoint read sets and annotated using IMGT nomenclatures.

Supplementary Figure 2 Number of reads/contigs at each step of the CDR3 assembly method.

For a selected sample, we demonstrate the number of reads or contigs kept at each step of our method. The numbers are included at the bottom of each text box. The selected sample represents the median library size of TCGA tumors with the median number of assembled CDR3 sequences.

Supplementary Figure 3 Method evaluation using TCGA tumors profiled with both TCRβ sequencing and RNA-seq.

Left, relationship between CDR3 transcripts called from TCR-seq and RNA-seq data. Middle, distribution of clonal frequencies of RNA-seq assemblies and TCR-seq transcripts. Right, another visualization of the clonal frequency distribution: the x axis shows the quantiles of clonal frequencies from immunoSeq data, and the y axis shows the fraction of above-quantile TCR transcripts called from RNA-seq data.

Supplementary Figure 4 Schematics of two simulation approaches used to validate the method developed in this work.

Descriptions for each approach can be found in the Online Methods.

Supplementary Figure 5 Performance evaluation of the CDR3 assembly algorithm using an in silico mixture experiment.

Our method was applied to data sets produced by the second simulation approach (Online Methods), and the CDR3 calls were compared to the gold standard TCR sequencing reads. (a) At different levels of T cell infiltration, our method recovered 4–6% of the infiltrating T cell repertoire, with 94–98% accuracy. (b) The called CDR3 sequences (infiltration level of 60%) were enriched for T cells with high clonal frequency. (c) Quantile–quantile plot showing that the clonal frequency for called CDR3 sequences is skewed to the higher end in comparison to the background distribution.

Supplementary Figure 6 Evaluation of the CDR3 assembly algorithm at high coverage and comparison with iSSAKE.

Both methods were applied to analyze the data sets produced from the first simulation approach (Online Methods). Called CDR3 sequences were compared to the 100 simulated transcripts, and true or false positive rates were calculated. False positive calls were defined as contigs that did not contain the CDR3 region. Standard deviation was estimated using 100 simulations at each given level of coverage. The true positive rate was the number of unique correct calls divided by 100, and the false positive rate was the number of unique incorrect calls divided by the total number of CDR3 calls. (a,b) These results were visualized as box plots for our method (a) and iSSAKE (b). We did not include a precision recall curve at each coverage setting because there was not a continuous threshold that would affect the performance in our algorithm.

Supplementary Figure 7 Differential usage of TRAV and TRBV genes in lower-grade glioma and kidney clear cell cancer.

(ac) Bar plots of TRAV and TRBV gene usage in glioma (a,b) and kidney tumors (c) are presented. TRAV and TRBV genes are in the same order as in Figure 1a,b, and the fractions were calculated in the same way.

Supplementary Figure 8 Distribution of read counts for assembled CDR3 contigs.

Read counts for each CDR3 contig were obtained from the assembly algorithm. When shared across multiple contigs, the count for a read was evenly split between each contig.

Supplementary Figure 9 Association of CPK with genes involved in cytolysis.

The expression levels of previously defined cytolytic genes19,27 were associated with CPK. The heat map displays values from partial Spearman’s correlation corrected for tumor purity. Cancers with fewer than ten samples were excluded. Statistical significance was evaluated using partial Spearman’s correlation test.

Supplementary Figure 10 Scatterplot between CPK and mutation load.

Each point on the plot represents a cancer sample, with color referring to the corresponding disease type. The statistical significance of the association was evaluated using Spearman’s correlation. This represents a complementary analysis to Figure 4b.

Supplementary Figure 11 Fraction of public β-CDR3 sequences across cancer types.

For each cancer type, the fraction was calculated as the number β-CDR3 sequences in the final public sequence set divided by the number of total distinct β-CDR3 sequences. All fractions were then mean centered, with the mean being the number of total public β-CDR3 sequences divided by the number of total distinct β-CDR3 sequences. Significance was evaluated using the binomial test, with the mean being the expected frequency and counts for public and total β-CDR3 sequences for each cancer as observations.

Supplementary Figure 12 MHC I binding predictions for SPAG5 and TSSK6 protein sequences.

Complete amino acid sequences for SPAG5 and TSSK6 were obtained from the NCBI protein database. All tiling nine-amino-acid sequences were analyzed by NetMHC4.0 for MHC I binding predictions. The peptides with strong binding (rank <0.5%) to an MHC I allele are underlined, and the corresponding MHC I allele is labeled by color. Only common MHC I alleles (HLA-A01:01, HLA-A02:01, HLA-A03:01, HLA-A07:02 and HLA-B08:01) with high population frequencies are displayed in the plot for visualization purposes.

Supplementary Figure 13 MHC II binding predictions for peptides produced from PRAMEF4 F300V.

MHC II binding was predicted by NetMHC-II 2.2. Fifteen-amino-acid sequences are the standard input for the webserver, and one mutated peptide was predicted to bind to three MHC II alleles with high affinity.

Supplementary Figure 14 Box plot of PRAMEF4 expression levels in multiple cancer types and paired normal tissues.

Testicular cancer (TGCT) is highlighted by the blue box. Numbers of outliers are included in red along the top of the plot.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1–3. (PDF 2171 kb)

Supplementary Data

Distinct deidentified CDR3 sequence calls generated in this study in fasta format. (ZIP 4151 kb)

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Li, B., Li, T., Pignon, JC. et al. Landscape of tumor-infiltrating T cell repertoire of human cancers. Nat Genet 48, 725–732 (2016). https://doi.org/10.1038/ng.3581

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