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The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state

Nature Immunology volume 24, pages 487–500 (2023)Cite this article

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Abstract

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

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Fig. 1: Recurrent CD4+ Tconv clones are detected in non-lymphoid organs of Treg cell-depleted mice.
Fig. 2: Multiple recurrent prostate-infiltrating CD4+ Tconv clones exhibit hallmarks of steady-state activation and reactivity to MHC-II-restricted self-ligands.
Fig. 3: Group 3 clones express common hallmarks of TFH cells.
Fig. 4: T cells expressing Group 3 TCRs do not produce common helper T effector cytokines or promote spontaneous germinal center formation.
Fig. 5: Following Treg cell ablation, Group 3 clones are enriched in non-lymphoid organs and produce IFN-γ.
Fig. 6: In the thymus, Group 3 Tconv clones display hallmarks of elevated self-reactivity.
Fig. 7: The endogenous CD4+ Tconv repertoire harbors T cells displaying hallmarks of Group 3 clones.

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Data availability

RNA-seq data are available at the NIH Gene Expression Omnibus under accession code GSE190127. All other source data, including TCR sequencing data, are provided with this manuscript. Source data are provided with this paper.

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Acknowledgements

This work was funded by the following sources (to P.A.S.): R01-CA160371, R01-AI110507, U01-AI154560, a Cancer Research Institute Investigator Award, and the University of Chicago Comprehensive Cancer Center. V.L. was supported by a National Institutes of Health (NIH)/National Cancer Institute (NCI) F30 predoctoral fellowship (F30-CA217109). D.R. was supported by an NIH/NCI F30 predoctoral fellowship (F30-CA247264). C.H.M. was supported by an NIH/NCI F30 predoctoral fellowship (F30-CA236061). V.L, D.R., and C.H.M. were supported by the University of Chicago Medical Scientist Training Program (T32-GM007281). J.L.C, M.T.W., and D.E.J.K were supported by T32-AI007090. M.M-C was supported in part by the University of Illinois Center for Clinical and Translational Science (NCATS grant UL1TR002003). N.D.S. was supported by Memorial Sloan Kettering Cancer Center (support grant P30-CA008748). This work was also supported by R01-AI143778 and R01-AI150860 awarded to M.R.C. Flow cytometry and cell sorting were performed at the Cytometry and Antibody Technology Facility at University of Chicago (RRID SCR_017760), which receives financial support from the Cancer Center Support grant (P30-CA014599).

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Author notes

  1. Jaime L. Chao

    Present address: Department of Immunology, University of Washington, Seattle, WA, USA

  2. Domenick E. Kennedy

    Present address: Drug Discovery Science and Technology, AbbVie, North Chicago, IL, USA

  3. These authors contributed equally: Victoria Lee, Donald M. Rodriguez.

Authors and Affiliations

  1. Interdisciplinary Scientist Training Program, University of Chicago, Chicago, IL, USA

    Victoria Lee, Donald M. Rodriguez & Christine H. Miller

  2. Committee on Immunology, University of Chicago, Chicago, IL, USA

    Nicole K. Ganci, Jaime L. Chao, Matthew T. Walker & David E. J. Klawon

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

    Sharon Zeng, Mary H. Schoenbach & Peter A. Savage

  4. Section of Rheumatology, Department of Medicine and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL, USA

    Junting Ai, Domenick E. Kennedy & Marcus R. Clark

  5. Research Informatics Core, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA

    Mark Maienschein-Cline

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

    Nicholas D. Socci

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Contributions

V.L., D.R., and P.A.S. conceived the project, designed experiments, and performed data analysis. N.G. designed and performed experiments and performed data analysis. V.L., D.R., S.Z., J.L.C., M.T.W., C.H.M., D.E.J.K., M.H.S., and J.A. performed the experiments. D.E.K. and M.M.-C. performed computational and statistical analysis of RNA-seq data. N.D.S. performed computational and statistical analysis of TCR sequence data. M.R.C. contributed to experimental design and data interpretation. P.A.S., V.L., and D.R. wrote the manuscript.

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Correspondence to Peter A. Savage.

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Nature Immunology thanks Shohei Hori, Ellen Robey, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Recurrent CD4+ Tconv clones detected in the prostate of Treg cell-depleted mice are found at low frequencies amongst Tconv and Treg subsets of other reference datasets.

Heat map displaying the 20 TCRα sequences from Fig. 1C and their corresponding log10 frequency in six groups of samples, as indicated. From left to right, these groups are Tconv cells from the prostate of Treg-depleted Foxp3DTR-EGFP males (n = 5), Tconv cells from the salivary glands of Treg-depleted Foxp3DTR-EGFP males (n = 5), Tconv cells from the prostate of untreated Aire−/− males (n = 9), Tconv cells from the prostate of untreated TRAMP males (n = 5), Tconv cells from the pooled secondary lymphoid organs (SLOs) of untreated wild-type Foxp3GFP males (n = 4), and Treg cells from the SLOs of untreated wild-type Foxp3GFP males (n = 4). Data from the latter two groups and Aire−/− group are taken from ref. 21. Each column represents one biological sample. ND = not detected.

Extended Data Fig. 2 Representative flow cytometric analysis of TCRrg mice expressing Group 1 vs. Group 3 TCRs.

Two Group 1 TCRs (ANT and DAS) and two Group 3 TCRs (SAS and SKV) were expressed as TCRrg mice. >6 weeks after bone marrow reconstitution, TCRrg T cells were directly phenotyped using flow cytometry. a, Representative gating strategy schematic and flow cytometric analysis of Foxp3, CD44, and CD69 expression by splenic Group 1 (top) and Group 3 (bottom) TCRrg T cells (top row, TCRβ+CD4+Thy1.1+) or respective host T cells (TCRβ+CD4+CD45.1+). T cells were selected by gating on TCRβ+B220neg, followed by CD4+CD8βneg, then by Thy1.1+CD45.1neg (for TCRrg T cells) or Thy1.1negCD45.1+ (for host T cells). b, Summary plot of data from a showing the frequency of splenic Group 1 (black symbols) and Group 3 (gray symbols) TCRrg T cells (TCRβ+CD4+Thy1.1+) within TCRβ+CD4+ gate. Summary plot of data from a showing the frequency of B cells (B220+) within CD45.1+ gate. Summary plot of data from a showing the frequency of host CD8α+ cells (CD45.1+CD8α+) within CD45+ gate. Summary plot of data from a showing the frequency of host CD4+ cells (CD45.1+CD4+) within CD45+ gate. Summary plot of data from a showing the frequency of Treg cells (Foxp3+) within host CD4+ gate. Summary plot of data from a showing the percentage of host Tconv cells (CD45.1+CD4+Foxp3neg) expressing CD69. Summary plot of data from a showing the percentage of host Tconv cells (CD45.1+CD4+Foxp3neg) expressing CD44. Summary plot of data from a showing the percentage of host CD8α+ cells (CD45.1+CD8α+) expressing CD69. Summary plot of data from a showing the percentage of host CD8α+ cells (CD45.1+CD8α+) expressing CD44. n = 3–4 per TCRrg. Data are pooled from two independent experiments. Each symbol depicts cells from an individual TCRrg mouse. Bold horizontal lines represent means. Error bars represent means ± SEM. p values were calculated using Student’s t-test (two-tailed) for pooled Group 1 clones versus pooled Group 3 clones. ns, p ≥ 0.05, not significant. Source data contain exact p values and group sizes.

Source data

Extended Data Fig. 3 Multiple recurrent prostate-infiltrating CD4+ Tconv clones exhibit hallmarks of steady-state activation.

Three Group 1 and four Group 3 TCRs were expressed as TCRrg mice. >6 weeks after bone marrow reconstitution, TCRrg T cells were directly phenotyped using flow cytometry. a, Summary plot of data from Fig. 2a, b showing the percentage of CD69 expression by Group 1 (black symbols) and Group 3 (gray symbols) TCRrg T cells (TCRβ+CD4+Thy1.1+) isolated from the indicated regional lymph nodes of primary TCRrg mice (n = 1–7 per TCRrg). Data are pooled from eleven independent experiments. Each symbol depicts cells from an individual TCRrg mouse. iLN, inguinal lymph nodes; aLN, axillary lymph nodes; bLN, brachial lymph nodes; cLN, cervical lymph nodes; pLN, para-aortic lymph nodes; mLN, mesenteric lymph nodes. b, Representative flow cytometric analysis of Ki67 expression by Group 1 (top) and Group 3 (bottom) TCRrg T cells (TCRβ+CD4+Thy1.1+) isolated from the spleen of primary TCRrg mice. SP, spleen. c, Summary plot of data from b showing the percentage of Ki67 expression by Group 1 (black symbols) and Group 3 (gray symbols) TCRrg T cells (TCRβ+CD4+Thy1.1+) isolated from the spleen or pooled lymph nodes of primary TCRrg mice (n = 1–8 per TCRrg). Data are pooled from eight independent experiments. Each symbol depicts cells from an individual TCRrg mouse. SP, spleen; LN, pooled lymph nodes (which include inguinal, axillary, brachial, cervical, para-aortic, and mesenteric lymph nodes). Bold horizontal lines represent means. Error bars represent means ± s.e.m. Two-sided p values were calculated using Student’s t-tests for pooled Group 1 clones versus pooled Group 3 clones. ns, p ≥ 0.05. ns, not significant. Source data contain exact p values and group sizes.

Source data

Extended Data Fig. 4 Recurrent CD4+ Tconv clones detected in the prostate of Treg cell-depleted mice can be binned into three groups based on hallmarks of steady-state activation and reactivity to MHC-II-restricted self-ligands.

Three Group 1, six Group 2, and four Group 3 TCRs as defined in the text and Supplementary Table 2 were cloned and expressed as TCRrg mice. TCRrg T cells were purified and subjected to in vitro reactivity assays. a, Purified TCRβ+CD4+Thy1.1+ TCRrg T cells expressing the indicated Group 1 (top), Group 2 (middle), and Group 3 (bottom) clones were labeled with CellTrace Violet (CTV) and co-cultured with splenic CD11c+ dendritic cells (DCs) and mouse recombinant IL-2 (mrIL-2) for 5 days (see Methods). Representative flow cytometric analyses of CTV dilution are shown. b, Summary plot of data from a depicting the percentage of CTV-diluted Group 1 (black symbols), Group 2 (white symbols), and Group 3 (gray symbols) TCRrg T cells (n = 2–10 per TCRrg). Data are pooled from eight independent experiments. Each symbol represents an individual co-culture. c, Histograms displaying representative flow cytometric analysis of proliferation by Group 1 CTV-labeled TCRrg cells (TCRβ+CD4+Thy1.1+) co-cultured with splenic CD11c+ DCs and mrIL-2 ± protein extracts isolated from prostatic lysates of untreated Foxp3WT or Treg-depleted Foxp3DTR-EGFP males for 5 days (see Methods). Far left column, TCRrg cells co-cultured with mrIL-2 in the absence of CD11c+ DCs served as negative controls. Far right column, TCRrg cells co-cultured with anti-CD3ε/anti-CD28 MACSiBead particles (1:1) and mrIL-2 served as positive controls. Bottom row, purified TCRβ+CD4+ TCRtg T cells expressing the prostate-specific MJ23 transgenic TCR (MJ23tg) served as secondary positive controls, indicated by turquoise-colored histograms. d, Summary plot of data from c depicting the percentage of CTV-diluted Group 1 TCRrg (black symbols) and MJ23tg T cells (turquoise symbols) (n = 2–8 per TCRrg or TCRtg). Data are pooled from three independent experiments. Symbols represent individual co-cultures. In b and d, means ± s.e.m. are indicated. For b, two-sided p values were calculated using Kruskal–Wallis tests and Dunn’s multiple comparison tests, with clones pooled by group. For d, two-sided p values were calculated using Kruskal–Wallis tests and Dunn’s multiple comparison tests separately for each clone, comparing each experimental condition to the ‘No DC’ control. ns, p ≥ 0.05, not significant. Source data contain exact p values and group sizes.

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Extended Data Fig. 5 Group 3 clones express common hallmarks of T follicular helper cells.

As shown in Fig. 3, comparative RNA-Seq analysis of Group 3 SAS and Group 1 ANT TCRrg cells. The SAS and ANT TCRs were cloned and expressed as TCRrg mice (see Methods). TCRrg T cells were purified from the pooled secondary lymphoid organs (SLOs) of primary TCRrg mice and subjected to bulk RNA sequencing (see Methods). Only biological samples containing ≥ 1 × 105 TCRrg T cells were included for analysis (n = 4 mice per TCR). a, Gating strategy schematic for the fluorescence-activated cells sorting (FACS) of TCRrg T cells for RNA sequencing. TCRrg T cells were selected by gating on Dumpneg (B220negCD11bnegCD11cnegF4/80neg), followed by CD4+CD8βneg, then by Thy1.1+CD45.1neg. b, RNA-seq volcano plot depicting differential gene expression for Group 3 SAS and Group 1 ANT TCRrg cells. The –log10 q-value versus log2 fold-change is depicted for genes with average log2 CPM ≥ −1. Blue and red dots denote genes over-represented in Group 1 ANT and Group 3 SAS TCRrg T cells, respectively, with FDR ≤ 0.05. Labels denote top genes that are over- or underrepresented, q ≤ 1 × 10−50 (82 genes). Data are pooled from two independent experiments. q values were generated using edgeR (see Methods). c, Principal component analysis (PCA) of mRNA expression in Group 1 ANT and Group 3 SAS TCRrg T cells, presented as log-scaled normalized expression (log2 CPM). Each dot corresponds to an independent biological sample. Green and magenta dots denote samples isolated from Group 1 ANT and Group 3 SAS TCRrg mice, respectively. n = 4 per TCR. Data are pooled from two independent experiments.

Extended Data Fig. 6 The frequency and phenotype of Group 3 SAS T cells is not impacted following transfer to B cell-depleted recipients.

The Group 1 ANT TCR and Group 3 SAS TCRs were cloned and expressed as TCRrg mice. 1 × 105 FACS-purified TCRrg cells were transferred into treated recipient mice. 7 d prior to TCRrg cell transfer, recipient mice were treated with anti-CD20 or control antibody (see Methods). Donor cell frequency and phenotype was assessed 7 d post-transfer. a, Representative flow cytometric analysis showing the efficiency of B cell depletion in control and anti-CD20 treated recipient mice. SP, spleen; LN, pooled lymph nodes; PP, Peyer’s Patches. b, Summary plot of data from a showing the frequency of B cells (CD19+TCRbneg) recovered from the indicated sites of control- and anti-CD20-treated recipient mice (n = 10–12 per condition). Data pooled from two independent experiments. Each symbol depicts cells from an individual TCRrg mouse. c, Representative flow showing the frequency of CD45.2+CD45.1neg donor TCRrg cells recovered from the spleen of control- and anti-CD20-treated recipient mice. d, Summary plot of data from c showing the frequency of ANTrg and SASrg donor T cells in the spleen of recipient mice treated with control or anti-CD20 antibody (n = 5–7 per condition). Data pooled from two independent experiments. e, Representative flow analysis of donor TCRrg T cells expressing the indicated TCRs in either control or anti-CD20 treated recipients. Histograms of expression of the indicated markers are shown for the indicated TCR and treatment group. The percentage of cells falling within the indicated gates is denoted. f, Summary plots of data from e. For control treated mice and anti-CD20 treated mice, the percentage of cells expressing the indicated marker is plotted for the indicated TCR (n = 5–7 per condition). Data pooled from two independent experiments. Bold horizontal lines represent means. Error bars represent means ± s.e.m. Two-sided p values were calculated using unpaired Student’s t-tests and corrected for multiple comparisons using the Holm-Šídák method. ns, p ≥ 0.05, not significant. Source data contain exact p values and group sizes.

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Extended Data Fig. 7 The Group 3 TCR SKV does not facilitate thymic Treg cell development at low clonal frequencies.

The Group 3 SKV TCR was cloned and expressed as low-frequency TCRrg mixed bone marrow chimeras (see Methods). TCRrg T cells were directly phenotyped using flow cytometry. Thymocytes were gated on CD73neg cells to distinguish newly developed thymocytes from recirculating mature T cells in the thymus59. a, Representative flow cytometric analysis of thymic (top, CD73negTCRβ+Thy1.1+) and splenic (bottom, TCRβ+CD4+Thy1.1+) TCRrg T cell frequency recovered from low-frequency TCRrg mixed bone marrow chimeras. b, Representative flow cytometric analysis of Foxp3 vs. CD4 expression by thymic CD4SP (top, CD73neg TCRβhiCD4+CD8βnegThy1.1+) and splenic CD4+ (bottom, TCRβ+CD4+Thy1.1+) TCRrg T cells from low-frequency TCRrg mixed bone marrow chimeras. Far right column, thymic CD4SP (CD73negTCRβhiCD4+CD8βneg) and splenic CD4+ (TCRβ+CD4+) T cells from untreated B6.SJL mice served as positive controls. c, Representative flow cytometric analysis of Foxp3 vs. CD69 (top) and Ki67 (bottom) expression by splenic CD4+ (TCRβ+CD4+Thy1.1+) TCRrg T cells from low-frequency TCRrg mixed bone marrow chimeras. Far right column, splenic CD4+ (TCRβ+CD4+) T cells from untreated B6.SJL mice served as controls. Data is representative of at least two independent experiments. Representative flow plots are ordered from highest (left) to lowest (right) thymic TCRrg frequency.

Extended Data Fig. 8 Additional phenotyping of TCRrg thymocytes expressing Group 1 vs. Group 3 TCRs.

Two Group 1 TCRs (ANT and DAS) and two Group 3 TCRs (SAS and SKV) were cloned and expressed as TCRrg mice. Thymi from TCRrg mice were isolated and depleted of CD8α-expressing cells prior to phenotyping by flow cytometry. Thymocytes isolated from polyclonal wild-type (WT) mice served as comparative controls. a, Representative flow cytometric analysis of thymocytes expressing the indicated TCRs. Normalized histograms of number of cells vs. expression of the indicated markers are shown for CD4+CD8+ DP cells (top row, gated on CD73negTCRβhiThy1.1+CD4+CD8β+) or CD4SP cells (bottom row, gated on CD73negTCRβhiThy1.1+CD4+CD8βneg). The percentage of cells falling within each indicated gate is denoted. b, Summary plots of data from a. For CD4+CD8+ DP cells (top row) or CD4SP cells (bottom row), the percentage of polyclonal WT (open symbols), Group 1 (black symbols), or Group 3 (gray symbols) expressing the indicated markers is depicted (n = 3–4 per TCRrg, n = 2 per polyclonal WT group). Data are pooled from two independent experiments. Each symbol represents an individual mouse. Bold horizontal lines represent means. Error bars represent means ± s.e.m. Two-sided p values were calculated using Student’s t-tests for pooled Group 1 clones versus pooled Group 3 clones. ns, p ≥ 0.05, not significant. Source data contain exact p values and group sizes.

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Extended Data Fig. 9 Group 3 SAS T cells proliferate and adopt a TFH-like phenotype following intravenous transfer of CD4SP thymocytes.

The Group 1 ANT TCR and Group 3 SAS TCRs were cloned and expressed as TCRrg mice. Thymi from TCRrg mice were isolated and depleted of CD8α-expressing cells. 5 × 106 SASrg thymocytes or 5 × 105 ANTrg thymocytes were transferred intravenously into B6.SJL recipient mice and analyzed at day 4 or day 11 post-transfer via flow cytometry. a, Representative analysis of CD45.2+CD45.1neg donor TCRrg cells recovered from the spleens of recipient mice at the indicated time points. The percentage of cells falling within the indicated gates are denoted. b, Summary plots of data shown in a (n = 6 per condition). Data are pooled from two independent experiments. Each symbol represents an individual mouse. c, Representative flow cytometric analysis of donor T cells (TCRβ+CD4+CD45.2+Thy1.1+) expressing the indicated TCRs. Normalized histograms of number of cells vs. expression of the indicated markers are shown for the donor cell thymocyte suspension (‘input thymocytes’, top row), cells recovered from the spleen of recipients at day 4 (middle row), and cells recovered from the spleen of recipients at day 11 (bottom row). The percentage of cells falling within the indicated gates is denoted. Splenocytes isolated from polyclonal wild-type (WT) mice served as comparative controls. d, Summary plots of data from c. For donor ANTrg T cells (black symbols) and donor SASrg T cells (gray symbols), the percentage of cells expressing the indicated markers is plotted for the indicated time points (n = 2–6 per condition). Data are pooled from two independent experiments. Each symbol represents an individual mouse. Bold horizontal lines represent means. Error bars represent means ± s.e.m. Two-sided p values were calculated using Student’s t-tests and corrected for multiple comparisons using the Holm-Šídák method. ns, p ≥ 0.05, not significant. Source data contain exact p values and group sizes.

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Extended Data Table 1 Gene set enrichment analysis of Group 1 and Group 3 TCRrg Tconv cells
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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1 Complete TCRα sequencing frequency table. For samples subjected to complete TCRα sequencing (Methods), the frequencies of each TCR CDR3α segment, as a percentage of all TCRα sequence reads within a given sample, are listed. For this table, TCRs were catalogued solely based on predicted TCR CDR3α sequence, regardless of TRAV usage. The table lists the Sample IDs (PR 1–5, SG 1–5 and TRAMP 1–5) for the complete TCRα sequencing dataset, the organ from which T cells were isolated (PR and SGs), the genotype (Foxp3DTR-EGFP or TRAMP), the treatment (Treg-depleted or untreated), and the T cell subset (Tconv cells). For each biological sample, the total number of in-frame TCR sequence reads and the number of unique TCR sequence reads are also listed. Supplementary Table 2 Selection of recurrent prostate-infiltrating CD4+ Tconv clones for TCR retrogenic studies. Thirteen of the top 20 most abundant recurrent TCRα sequences expressed by Tconv cells in the prostate of Treg cell-depleted mice were selected for TCR retrogenic studies. TCRs were selected based on differential TRAV and TRAJ usage. Each TCRα chain is denoted using a three-letter code representing residues 3–5 of the predicted CDR3α chain. The full TCRα sequence information is listed, including TRAV and TRAJ usage. The 13 selected TCRα clonotypes can be binned into three groups based on hallmarks of steady-state activation and reactivity to MHC-II-restricted self-ligands (Extended Data Figs. 3 and 4). Supplementary Table 3 List of primers used for the generation of TCR retrogenic constructs.

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Lee, V., Rodriguez, D.M., Ganci, N.K. et al. The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state. Nat Immunol 24, 487–500 (2023). https://doi.org/10.1038/s41590-023-01425-0

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