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Eomes identifies thymic precursors of self-specific memory-phenotype CD8+ T cells

Nature Immunology volume 21, pages 567–577 (2020)Cite this article

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Abstract

Unprimed mice harbor a substantial population of ‘memory-phenotype’ CD8+ T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8+ memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8+ T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, which suggests potential roles in antitumor immunity and the response to immunotherapy.

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Fig. 1: The TCR repertoire of memory-phenotype CD8+ T cells is recurrent and distinct from that of naive-phenotype CD8+ T cells.
Fig. 2: CD8-MP differentiation is a TCR-directed process.
Fig. 3: CD8-MP T cell clones exhibit reactivity to MHC class I–restricted self-ligands presented by splenic dendritic cells.
Fig. 4: Thymocytes expressing CD8-MP TCRs upregulate Eomes during thymic maturation.
Fig. 5: Eomes identifies polyclonal thymic precursors of CD8-MP T cells.
Fig. 6: CD8-MP cells infiltrate TRAMP prostate tumors and express high densities of PD-1.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. The TCR sequence data are available at the Gene Expression Omnibus (GEO) repository under accession number GSE145365. The script used for TCR sequence analysis is available at https://github.com/soccin/MILLER_SAVAGE_CD8MP.

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Acknowledgements

We thank A. Bendelac for critical reading of the manuscript. We thank S. Kasal, A. Bendelac, V. Leone, E. Chang, Z. Earley, B. Jabri, E. Hegermiller and B. Kee for sharing reagents and resources. We thank M. Nussenzweig at Rockefeller University for Rag2-GFP mice. We thank T. Walzer at Inserm for Eomes-GFP mice. Flow cytometry and FACS were performed at the University of Chicago Cytometry and Antibody Technology Facility. This work was funded by R01-AI110507 (to P.A.S.). C.H.M. was supported by a National Institutes of Health/National Cancer Institute F30 predoctoral fellowship (F30-CA236061). V.L. was supported by a National Institutes of Health/National Cancer Institute F30 predoctoral fellowship (F30-CA217109). C.H.M and V.L. were supported by the Univeristy of Chicago Medical Scientist Training Program (T32-GM007281). D.K. was supported by T32-AI007090.

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Authors and Affiliations

  1. Department of Pathology, University of Chicago, Chicago, IL, USA

    Christine H. Miller, David E. J. Klawon, Sharon Zeng, Victoria Lee & Peter A. Savage

  2. Bioinformatics Core, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Nicholas D. Socci

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Contributions

C.H.M. designed the study, performed experiments, interpreted data and wrote the manuscript; D.E.J.K. performed experiments; S.Z. performed computational and statistical analysis of TCR sequence data; V.L. performed experiments and provided technical and conceptual advice; N.D.S. performed computational and statistical analysis of TCR sequence data; P.A.S. designed the study, interpreted data and wrote the manuscript. All authors contributed to discussion.

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Correspondence to Peter A. Savage.

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Extended data

Extended Data Fig. 1 The frequency of CD8-MP cells is not diminished in germ-free mice.

a, Representative flow-cytometric analysis of CD44 vs. CD122 expression by CD8β+ T cells from the spleens of 16-week-old B6 specific pathogen free (SPF) and germ-free (GF) mice. The percentage of cells falling in the indicated gates is denoted. Data are representative of two independent experiments. b, Summary plot of the frequency of CD44hiCD122+ expressing TCRβ+ CD8β+ T cells from the indicated lymphoid sites in 6 to 16-week-old SPF or GF mice. iabLN: inguinal, axial, brachial lymph nodes; cLN: cervical lymph nodes; mLN: mesenteric lymph nodes; pLN: periaortic lymph nodes. Each symbol represents an individual mouse. Mean ± SEM is indicated. n = 6, Spleen, iabLN, cLN, mLN; n = 3, pLN. At each lymphoid site, the frequency of CD8-MP cells was compared between the SPF and GF mice using one-way ANOVA with Bonferroni post-test analysis, comparing all pairs of columns (ANOVA p < 0.0001, F = 17.43, df = 53). Adjusted p-values from the Bonferroni post-test are depicted: Spleen, *p = 0.0133; iabLN, n.s. p = 0.3669; cLN, n.s. p > 0.9999; mLN, n.s. p > 0.9999; pLN, n.s. p > 0.9999. Data are pooled from two independent experiments. (n.s., not significant).

Extended Data Fig. 2 CD8-MP and CD8-Naive CDR3α chain hydrophobicity and length analysis.

CD8-MP (CD8β+ CD44hiCD122+) and CD8-Naive (CD8β+ CD44loCD122–) T cells were purified by FACS from the pooled spleen and lymph nodes of 9-week-old TCRβtg males and subjected to complete TCRα sequencing using the iRepertoire platform. N = 5 for CD8-MP and CD8-Naive samples. TCRα chains were assessed solely based on their predicted CDR3 segment, regardless of V-region usage. a, Histograms depicting grand average of hydropathy (GRAVY) values for CDR3 regions of the CD8-MP (red) and CD8-Naive (black) subsets for each mouse, n = 5 mice. Significance testing was performed with the paired, two-tailed Wilcoxon signed-rank test (W) and the paired, two-tailed Kolmolgorov-Smirnov test (K-S). Mouse 1 (CD8-MP CDR3 n = 11999, CD8-Naive CDR3 n = 29048), p = 0.2449 (W) and p = 1 (K-S); Mouse 2 (CD8-MP CDR3 n = 3327, CD8-Naive CDR3 n = 14736), p = 0.0511 (W) and p = 1 (K-S); Mouse 3 (CD8-MP CDR3 n = 2159, CD8-Naive CDR3 n = 26649), **p = 0.0017 (W) and p = 1 (K-S); Mouse 4 (CD8-MP CDR3 n = 2848, CD8-Naive CDR3 n = 24501), ***p < 0.0001 (W) and p = 1 (K-S); Mouse 5 (CD8-MP CDR3 n = 13524, CD8-Naive CDR3 n = 25367), **p = 0.0010 (W) and p = 1 (K-S). (n.s. not significant). b, Histograms depicting CDR3 lengths of the CD8-MP (red) and CD8-Naive (black) subsets for each mouse, n = 5 mice. Significance testing was performed with the paired, two-tailed Wilcoxon signed-rank test (W) and the paired, two-tailed Kolmolgorov-Smirnov test (K-S). Mouse 1 (CD8-MP CDR3 n = 11999, CD8-Naive CDR3 n = 29048), p = 0.2031 (W) and p = 1 (K-S); Mouse 2 (CD8-MP CDR3 n = 3327, CD8-Naive CDR3 n = 14736), p = 0.2031 (W) and p = 1 (K-S); Mouse 3 (CD8-MP CDR3 n = 2159, CD8-Naive CDR3 n = 26649), p = 0.4258 (W) and p = 1 (K-S); Mouse 4 (CD8-MP CDR3 n = 2848, CD8-Naive CDR3 n = 24501), p = 1 (W) and p = 1 (K-S); Mouse 5 (CD8-MP CDR3 n = 13524, CD8-Naive CDR3 n = 25367), p = 0.9102 (W) and p = 1 (K-S). (n.s. not significant).

Extended Data Fig. 3 Phenotype of TCRrg and filler CD8+ T cell populations.

(Top) Representative flow cytometric analysis of Thy1.1 vs. CD45.1 expression by TCRβ + CD8β+ cells from the spleens of indicated TCRrg mice. The percentage of cells falling in the indicated gates is denoted. (Bottom) Representative flow cytometric analysis of CD44 vs. CD122 expression by Thy1.1+ TCRrg or CD45.+ “filler” TCRβ+ CD8β+ cells from the spleens of indicated TCRrg mice. The percentage of cells falling in the indicated gates is denoted. Data are representative of six independent experiments.

Extended Data Fig. 4 A greater fraction of CD8-MP TCRrg cells adopt the CD44hiCD122+ phenotype at lower clonal frequencies.

(Top) Representative flow cytometric analysis of Thy1.1 vs. CD45.1 expression by TCRβ+ CD8+ cells and (Bottom) CD44 vs. CD122 expression by TCRβ+ CD8+ Thy1.1+ cells from “low frequency” TCRrg mice expressing the indicated TCRs, assessed 7 weeks after bone marrow reconstitution. It should be noted that the expression of the Thy1.1 reporter varies in different TCRrg mice, but the expression of TCRβ is uniform and comparable to that of endogenous cells (not shown). The percentage of cells falling in the indicated gates is denoted. Data are representative of four independent experiments.

Extended Data Fig. 5 CD8-MP TCRrg cells are broadly distributed across lymphoid sites.

Summary plots of the frequency of Thy1.1 expressing TCRrg cells of TCRβ+ CD8β+ T cells, normalized to the spleen from the indicated lymphoid sites 6–7 weeks after bone marrow reconstitution of the indicated “low frequency” TCRrg mice. Frequencies at different lymphoid sites were normalized to the spleen to control for varying engraftment of TCRrg bone marrow across different mice. iabLN: inguinal, axial, brachial lymph nodes; cLN: cervical lymph nodes; mLN: mesenteric lymph nodes; pLN: periaortic lymph nodes. Each symbol represents an individual TCRrg mouse. n = 3, GSSrg; n = 4, DTGrg; n = 4, SAVrg; n = 3, SATrg; n = 4, SMNrg; n = 3, RDTrg; n = 4, LNNrg; n = 4, DYQrg. Mean ± SEM is indicated. Data is pooled from four independent experiments.

Extended Data Fig. 6 Thymocytes expressing CD8-MP-skewed TCRs exhibit hallmarks of elevated TCR signaling.

a, Left: Representative flow cytometric analysis of CD73 expression by TCRβ+ CD8SP thymocytes from a 7-week-old Rag2-GFP mouse. Right: Expression of Rag2-GFP on the CD73– and CD73+ CD8SP thymocyte populations. The percentage of cells falling in the indicated gates is denoted. Data are representative of two independent experiments. b, Representative flow-cytometric analysis of CD44 and CD122 expression by indicated CD73– TCRβ+ Thy1.1+ CD8SP CD8-MP TCRrg thymocytes 6 weeks after bone marrow reconstitution. The percentage of cells falling in the indicated gates is denoted. Data are representative of five independent experiments. c, Representative flow cytometric analysis of Ki67 expression by TCRβ+ CD8β+ Thy1.1+ cells from the thymi of TCRrg mice expressing the indicated CD8-MP TCRs (top) and CD8-Naive TCRs (bottom), analyzed 6 weeks after bone marrow reconstitution. The percentage of cells in the indicated gates is denoted. Data are representative of five independent experiments. d, Left: Summary plot of pooled data from (c) showing the frequency of TCRβ+ CD8β+ Thy1.1+ cells that are positive for Ki67 staining for the listed T cell clone. Right: Data from the left panel were pooled from the CD8-MP TCRs and the CD8-Naive TCRs. Each symbol represents an individual TCRrg mouse. n = 11, CD8-MP TCRrg mice; n = 10, CD8-Naive TCRrg mice. Mean ± SEM is indicated. **p = 0.0017, two-tailed nonparametric Mann–Whitney test. Data are pooled from five independent experiments. e, Representative flow cytometric analysis of CD5 expression by TCRβ+ CD8β+ Thy1.1+ cells from the thymi of TCRrg mice expressing the indicated CD8-MP and CD8-Naive TCRs, analyzed 6 weeks after bone marrow reconstitution. The percentage of cells in the indicated gates is denoted. Data are representative of six independent experiments. f, Left: Summary plot of pooled data from (e) showing the normalized mean florescence intensity (MFI) of CD5 in TCRrg thymocytes compared to CD8SP thymocytes from a B6 thymus. Right: Data from the left panel were pooled from the CD8-MP TCRs and the CD8-Naive TCRs. Each symbol represents an individual TCRrg mouse. n = 13, CD8-MP TCRrg mice; n = 12, CD8-Naive TCRrg mice. Mean ± SEM is indicated. ***p < 0.0001, two-tailed nonparametric Mann–Whitney test. Data are pooled from six independent experiments. g, Representative flow cytometric analysis of CD4 vs. CD8, PD-1 vs. CD69, and cleaved Caspase3 expression by TCRβ+ CD8β+ Thy1.1+ cells from the thymi of TCRrg mice expressing the indicated CD8-MP TCRs and CD8-Naive TCRs, analyzed 6 weeks after bone marrow reconstitution. The percentage of cells in the indicated gates is denoted. Data are representative of three independent experiments.

Extended Data Fig. 7 Gating strategy to analyze TCRrg cells.

Lymphocytes were gated on forward and side scatter and doublets were removed by gating on FSC-H by FSC-A. TCRβ+ T cells were then gated for the expression of CD8β+ and CD4–. CD8β+ T cells were then gated for the expression of Thy1.1. This TCRrg cell population was used for subsequent phenotyping stains used throughout the paper. The percentage of cells in the indicated gates is denoted. Data are representative of six independent experiments.

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Supplementary Table 1

Complete TCRα sequencing frequency table.

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Miller, C.H., Klawon, D.E.J., Zeng, S. et al. Eomes identifies thymic precursors of self-specific memory-phenotype CD8+ T cells. Nat Immunol 21, 567–577 (2020). https://doi.org/10.1038/s41590-020-0653-1

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