TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures

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High-throughput 3′ single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3′-barcoded scRNA-seq samples. This approach is compatible with common 3′ scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.

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Fig. 1: Strategy for TCR recovery from 3′-barcoded single-cell sequencing libraries.
Fig. 2: Recovery of OT-I Tcra and Tcrb CDR3 sequences.
Fig. 3: scRNA-seq and TCR analysis of HPV-E7-immunized mice.
Fig. 4: ScRNA-seq and TCR analysis of peanut-dependent activated T cells from one of the individuals with an allergy to peanut (patient 77) combined with pseudotemporal analysis.

Data availability

FASTQ file format data related to murine samples are available through GEO and BioProject under accession numbers GSE136028 and PRJNA560970. FASTQ file format data related to human samples is available through dbGaP under accession number phs001897.v1.p1. Source data files and associated metadata tables for Figs. 24 are available on, or upon request. MSigDB results for Extended Data Fig. 1c, Extended Data Fig. 5a and Supplementary Fig. 3h are available as Supplementary Table 12. Full results from the differential gene expression comparison shown in Extended Data Fig. 2c are available as Supplementary Table 13. All recovered CDR3 sequences, and their frequencies, of TCR α and β chains from E7-immunized mice and patients with peanut allergy are available as Supplementary Tables 6 and 10. Gene expression matrices for E7-immunized mice and patients with peanut allergy are available as Supplementary Data 1 and 2.

Code availability

R scripts for generating all analysis, Matlab scripts for processing TCR sequencing data, as well as all updates, are available at, or upon reasonable request.


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We thank P. Blainey and M. Birnbaum for fruitful discussions. This work was supported in part by the Koch Institute Support (core) NIH Grant P30-CA14051 from the National Cancer Institute, as well as the Koch Institute - Dana-Farber/Harvard Cancer Center Bridge Project. This work was also supported by the Food Allergy Science Initiative at the Broad Institute and the NIH (5P01AI039671, 5U19AI089992, U19AI095261). A.K.S. was supported by the Searle Scholars Program, the Beckman Young Investigator Program, the Pew-Stewart Scholars Program for Cancer Research, a Sloan Fellowship in Chemistry, the NIH (1DP2GM119419, 2U19AI089992, 2R01HL095791, 1U54CA217377, 2P01AI039671, 5U24AI118672, 2RM1HG006193, 1R33CA202820, 1R01AI138546, 1R01HL134539, 1R01DA046277, 1U2CCA23319501) and Agilent Technologies.

Author information

A.A.T., T.M.G, A.K.S. and J.C.L. developed the concepts and designed the study. A.A.T. and T.M.G. performed the experiments. A.A.T. prepared the manuscript with input from all authors. A.A.T., T.M.G., B.M. and D.M.M. performed bioinformatics analyses. N.K.M. performed E7 immunization of mice. W.G.S. designed the clinical study and provided samples from peanut-allergic patients. B.R. performed stimulation and sorting of PBMCs from allergic patients.

Correspondence to Alex K. Shalek or J. Christopher Love.

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Competing interests

A.A.T., T.M.G., J.C.L. and the Massachusetts Institute of Technology have filed a patent application (patent no. PCT/US2018/013443) that relates to T cell receptor recovery, compositions of matter and the outlined experimental and computational methods and uses thereof. J.C.L. and A.K.S. are co-founders and shareholders of Honeycomb Biotechnologies, Inc. T.M.G. is currently an employee of Honeycomb Biotechnologies, Inc.

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Peer review information Laurie A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Stimulated and ex vivo cells are transcriptionally distinct.

a, tSNE visualization of all cells colored based on stimulation condition (n = 6,912 stimulated cells, dark grey; 7,512 ex vivo cells, light grey). b, Proportions of stimulated and ex vivo cells in each of the computationally determined clusters shown in Fig. 3a. Dash line indicates expected proportions assuming even distributions of cells from both conditions. c, Enriched MsigDb signatures of the four modules of genes identified in Fig. 3d. FDR q-values represent Benjamini and Hochberg-corrected, one-tailed hypergeometric P values. 50, 49, 35, and 48 genes are included in Module 1, 2, 3, and 4, respectively for enrichment calculation. See Supplementary Table 12 for more details. Data represent combined data from four independent experiments of four mice total (a-c).

Extended Data Fig. 2 Group 1 and 2 clonotypes differ in expansion and gene expression upon stimulation.

a, Clonal sizes of Group 1 and 2 clonotypes in the stimulated and ex vivo conditions shown in Fig. 3d. P value calculated by two-sample Mann-Whitney U test (Stimulated: n = 74 clonotype groups in Group 1; 37 Group 2 clonotypes. ex vivo: n = 82 Group 1 clonotypes; 40 Group 2 clonotypes). Box and whisker plots indicate the (box) 25th and 75th percentile along with (whisker) + /− 1.5*interquartile range. Violin plots represent estimated density of clonotypes. b, Gene expression fold changes between stimulated and ex vivo cells in (x-axis) Group 1 clonotypes and (y-axis) Group 2 clonotypes. Each point represents a shared gene across Group 1 and 2 clonotypes. Red line indicates fitted linear model. P value calculated by one-tailed F statistics (F(1,5908)) of the linear regression. n = 5908 genes. c, Volcano plots of differentially expressed genes between Group 1 and 2 clonotypes in the (left) stimulated and the (right) ex vivo conditions. P values were determined using a two-tailed likelihood ratio test, and adjusted by Bonferroni correction. Top 10 genes with positive or negative fold changes are labeled. Cells in Group 1 and 2 have been downsampled to 300 each (n = 300 cells for each of the groups). See Supplementary Table 13 for more details.

Extended Data Fig. 3 Analysis of shared clonotypes across four E7-HPV immunized mice.

a, Venn diagram of shared unique Tcrb clones across the four mice. b, Amino acid logo plot of TCRβ sequences that show high similarity among public clones shared by at least three of four animals. Individual sequences are shown in c. c, TCRα and β matching of highly similar clonotypes found in public clones shared by at least three of four animals (Supplementary Table 9). Bold outline indicates dual TCRα chains found in the same cells (Supplementary Table 3). Structural amino acids shown in grey. Number of cells shown in parenthesis. TCRα and β sequences that were detected in less than two cells were excluded for visualization.

Extended Data Fig. 4 Distinct patterns of gene expression correlate with pseudotime.

a, Expression of top 100 most significant genes visualized across pseudotime. Genes were clustered via Ward.D2 based on their patterns of expression. Data represent an individual experiment with 1847 single-cells from one patient (patient 77).

Extended Data Fig. 5 Psuedotime correlates with effector T cell signatures.

a, MsigDB analysis of genes enriched early (cluster 3 in Extended Data Fig 4a; n = 38 genes) or late (cluster 4 in Extended Data Fig 4a; n = 123 genes) on the pseudotemporal trajectory. Description indicates cell state enriched with the corresponding gene set in comparison to another cell state. FDR q-values represent Benjamini and Hochberg-corrected, one-tailed hypergeometric P values. See Supplementary Table 12 for more details. b, Pseudotime distribution of expanded clones shown in Fig. 4d. Number of cells for each clonotype group indicated in parenthesis. A total of 16 clonotype groups are shown. All box and whisker plots indicate the (box) 25th and 75th percentile along with (whisker) + /− 1.5*interquartile range (b). Data represent an individual experiment with 1847 single-cells from one patient (patient 77; a,b).

Supplementary information

Supplementary Information

Supplementary Figs. 1–5 and Tables 1,2,4,5,8 and 9.

Reporting Summary

Supplementary Tables

Supplementary Tables 3,6,7 and 10–13.

Supplementary Data 1

Digital gene expression matrices for data shown in Fig. 3.

Supplementary Data 2

Digital gene expression matrices for data shown in Fig. 4.

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Tu, A.A., Gierahn, T.M., Monian, B. et al. TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures. Nat Immunol 20, 1692–1699 (2019) doi:10.1038/s41590-019-0544-5

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