Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion

Abstract

The thymus contains a diversity of dendritic cells (DCs) that exist in defined locations and have different antigen-processing and -presenting features. This suggests that they play nonredundant roles in mediating thymocyte selection. In an effort to eliminate SIRPα+ classic DC2 subsets, we discovered that a substantial proportion expresses the surface lectin, CD301b, in the thymus. These cells resemble the CD301b+ type 2 immune response promoting DCs that are present in the skin-draining lymph nodes. Transcriptional and phenotypic comparison to other DC subsets in the thymus revealed that thymic CD301b+ cDCs represent an activated state that exhibits enhanced antigen processing and presentation. Furthermore, a CD301b+ cDC2 subset demonstrated a type 2 cytokine signature and required steady-state interleukin-4 receptor signaling. Selective ablation of CD301b+ cDC2 subsets impaired clonal deletion without affecting regulatory T cells (Treg cells). The T cell receptor α repertoire sequencing confirmed that a cDC2 subset promotes deletion of conventional T cells with minimal effect on Treg cell selection. Together, these findings suggest that cytokine-induced activation of DCs in the thymus substantially enforces central tolerance.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: CD301b+ cDCs are enriched among thymic SIRPα+ cDC2 subsets.
Fig. 2: Thymic CD301b+ cDC2 subsets express a distinct gene signature.
Fig. 3: Thymic CD301b+ cDC2 subsets have characteristics of enhanced antigen presentation.
Fig. 4: Thymic CD301b+ cDC2 subsets require type 2 cytokines.
Fig. 5: CD301b+ cDC2 subsets mediate clonal deletion.

Data availability

RNA-seq and scRNA-seq data are available in the NCBI’s GEO (https://www.ncbi.nlm.nih.gov/geo/) under accession nos. GSE198789 (RNA-seq data, Figs. 2a–c, 3a,b and 4a–d) and GSE198247 (scRNA-seq data, Fig. 2d,e and Extended Data Figs. 4b,c and 7b–e). The main data supporting the findings of the present study are available in the article's Extended Data Figures and Supplementary Data 1. Data are available from the corresponding authors upon appropriate and reasonable request.

Code availability

Computational codes used in the RNA-seq and scRNA-seq analyses supporting the findings of the present study are available in Methods. Additional information is available from the corresponding author on reasonable and appropriate request.

References

  1. Guilliams, M. et al. Unsupervised high-dimensional analysis aligns dendritic cells across tissues and species. Immunity 45, 669–684 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Li, J., Park, J., Foss, D. & Goldschneider, I. Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus. J. Exp. Med. 206, 607–622 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Klein, L., Kyewski, B., Allen, P. M. & Hogquist, K. A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nat. Rev. Immunol. 14, 377–391 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Steinman, R. M., Hawiger, D. & Nussenzweig, M. C. Tolerogenic dendritic cells*. Annu. Rev. Immunol. 21, 685–711 (2003).

    CAS  PubMed  Google Scholar 

  5. Lei, Y. et al. Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development. J. Exp. Med. 208, 383–394 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Bonasio, R. et al. Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus. Nat. Immunol. 7, 1092–1100 (2006).

    CAS  PubMed  Google Scholar 

  7. Vollmann, E. H. et al. Specialized transendothelial dendritic cells mediate thymic T-cell selection against blood-borne macromolecules. Nat. Commun. 12, 6230 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Breed, E. R., Watanabe, M. & Hogquist, K. A. Measuring thymic clonal deletion at the population level. J. Immunol. 202, 3226–3233 (2019).

    CAS  PubMed  Google Scholar 

  9. Marzo, A. L. et al. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6, 793–799 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Hataye, J., Moon, J. J., Khoruts, A., Reilly, C. & Jenkins, M. K. Naive and memory CD4+ T cell survival controlled by clonal abundance. Science 312, 114–116 (2006).

    CAS  PubMed  Google Scholar 

  11. Bautista, J. L. et al. Intraclonal competition limits the fate determination of regulatory T cells in the thymus. Nat. Immunol. 10, 610–617 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Takahama, Y., Shores, E. W. & Singer, A. Negative selection of precursor thymocytes before their differentiation into CD4+CD8+ cells. Science 258, 653–656 (1992).

    CAS  PubMed  Google Scholar 

  13. Lacorazza, H. D., Tucek-Szabo, C., Vasović, L. V., Remus, K. & Nikolich-Zugich, J. Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development. J. Immunol. 166, 3184–3193 (2001).

    CAS  PubMed  Google Scholar 

  14. Erman, B., Feigenbaum, L., Coligan, J. E. & Singer, A. Early TCRalpha expression generates TCRalphagamma complexes that signal the DN-to-DP transition and impair development. Nat. Immunol. 3, 564–569 (2002).

    CAS  PubMed  Google Scholar 

  15. Perry, J. S. A. et al. Distinct contributions of Aire and antigen-presenting-cell subsets to the generation of self-tolerance in the thymus. Immunity 41, 414–426 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hinterberger, M. et al. Autonomous role of medullary thymic epithelial cells in central CD4+ T cell tolerance. Nat. Immunol. 11, 512–519 (2010).

    CAS  PubMed  Google Scholar 

  17. van Meerwijk, J. P. M. et al. Quantitative impact of thymic clonal deletion on the T cell repertoire. J. Exp. Med. 185, 377–384 (1997).

    PubMed  PubMed Central  Google Scholar 

  18. Leventhal, D. S. et al. Dendritic cells coordinate the development and homeostasis of organ-specific regulatory T cells. Immunity 44, 847–859 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. MacNabb, B. W. et al. Negligible role for deletion mediated by cDC1 in CD8 + T cell tolerance. J. Immunol. 202, 2628–2635 (2019).

    CAS  PubMed  Google Scholar 

  20. Loschko, J. et al. Inducible targeting of cDCs and their subsets in vivo. J. Immunol. Methods 434, 32–38 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Kumamoto, Y. et al. CD301b+ dermal dendritic cells drive T helper 2 cell-mediated immunity. Immunity 39, 733–743 (2013).

    CAS  PubMed  Google Scholar 

  22. Kumamoto, Y., Denda-Nagai, K., Aida, S., Higashi, N. & Irimura, T. MGL2 dermal dendritic cells are sufficient to initiate contact hypersensitivity in vivo. PLoS ONE 4, e5619 (2009).

    PubMed  PubMed Central  Google Scholar 

  23. Kroger, C. J., Wang, B. & Tisch, R. Temporal increase in thymocyte negative selection parallels enhanced thymic SIRPalpha+ DC function. Eur. J. Immunol. 46, 2352–2362 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Vobořil, M. et al. Toll-like receptor signaling in thymic epithelium controls monocyte-derived dendritic cell recruitment and Treg generation. Nat. Commun. 11, 2361 (2020).

    PubMed  PubMed Central  Google Scholar 

  25. Baba, T., Nakamoto, Y. & Mukaida, N. Crucial contribution of thymic Sirpα+ conventional dendritic cells to central tolerance against blood-borne antigens in a CCR2-dependent manner. J. Immunol. 183, 3053–3063 (2009).

    CAS  PubMed  Google Scholar 

  26. Ardouin, L. et al. Broad and largely concordant molecular changes characterize tolerogenic and immunogenic dendritic cell maturation in thymus and periphery. Immunity 45, 305–318 (2016).

    CAS  PubMed  Google Scholar 

  27. Pos, W., Sethi, D. K. & Wucherpfennig, K. W. Mechanisms of peptide repertoire selection by HLA-DM. Trends Immunol. 34, 495–501 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Yang, S., Fujikado, N., Kolodin, D., Benoist, C. & Mathis, D. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science 348, 589–594 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Gao, Y. et al. Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells. Immunity 39, 722–732 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Murakami, R. et al. A unique dermal dendritic cell subset that skews the immune response toward Th2. PLoS ONE 8, e73270 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Connor, L. M., Tang, S.-C., Camberis, M., Le Gros, G. & Ronchese, F. Helminth-conditioned dendritic cells prime CD4+ T cells to IL-4 production in vivo. J. Immunol. 193, 2709–2717 (2014).

    CAS  PubMed  Google Scholar 

  32. Lee, Y. J., Holzapfel, K. L., Zhu, J., Jameson, S. C. & Hogquist, K. A. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat. Immunol. 14, 1146–1154 (2013).

    CAS  PubMed  Google Scholar 

  33. Wang, H. et al. Myeloid cells activate iNKT cells to produce IL-4 in the thymic medulla. Proc. Natl Acad. Sci. USA 116, 22262–22268 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Breed, E. R., Lee, S. T. & Hogquist, K. A. Directing T cell fate: how thymic antigen presenting cells coordinate thymocyte selection. Semin. Cell Dev. Biol. 84, 2–10 (2018).

    CAS  PubMed  Google Scholar 

  35. Malhotra, D. et al. Tolerance is established in polyclonal CD4+ T cells by distinct mechanisms, according to self-peptide expression patterns. Nat. Immunol. 17, 187–195 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. McCaughtry, T. M., Wilken, M. S. & Hogquist, K. A. Thymic emigration revisited. J. Exp. Med. 204, 2513–2520 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Williams, J. A. et al. Thymic medullary epithelium and thymocyte self-tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways. J. Immunol. 192, 630–640 (2014).

    CAS  PubMed  Google Scholar 

  38. Lio, C.-W. J., Dodson, L. F., Deppong, C. M., Hsieh, C.-S. & Green, J. M. CD28 facilitates the generation of Foxp3 cytokine responsive regulatory T cell precursors. J. Immunol. 184, 6007–6013 (2010).

    CAS  PubMed  Google Scholar 

  39. Vang, K. B. et al. Cutting edge: CD28 and c-Rel-dependent pathways initiate regulatory T cell development. J. Immunol. 184, 4074–4077 (2010).

    CAS  PubMed  Google Scholar 

  40. Tang, Q. et al. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J. Immunol. 171, 3348–3352 (2003).

    CAS  PubMed  Google Scholar 

  41. Guermonprez, P., Valladeau, J., Zitvogel, L., Théry, C. & Amigorena, S. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20, 621–667 (2002).

    CAS  PubMed  Google Scholar 

  42. Tatsumi, N., Codrington, A. L., El-Fenej, J., Phondge, V. & Kumamoto, Y. Effective CD4 T cell priming requires repertoire scanning by CD301b+ migratory cDC2 cells upon lymph node entry. Sci. Immunol. 6, eabg0336 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Mayer, J. U. et al. Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization. Nat. Immunol. 22, 1538–1550 (2021).

    CAS  PubMed  Google Scholar 

  44. Maier, B. et al. A conserved dendritic-cell regulatory program limits antitumour immunity. Nature 580, 257–262 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Cowan, J. E. et al. Differential requirement for CCR4 and CCR7 during the development of innate and adaptive αβT cells in the adult thymus. J. Immunol. 193, 1204–1212 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Hu, Z., Lancaster, J. N., Sasiponganan, C. & Ehrlich, L. I. R. CCR4 promotes medullary entry and thymocyte–dendritic cell interactions required for central tolerance. J. Exp. Med. 212, 1947–1965 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Freeman, G. J., Casasnovas, J. M., Umetsu, D. T. & Dekruyff, R. H. TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunol. Rev. 235, 172–189 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Kurd, N. S. et al. A role for phagocytosis in inducing cell death during thymocyte negative selection. eLife 8, e48097 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang, H. & Hogquist, K. A. CCR7 defines a precursor for murine iNKT cells in thymus and periphery. eLife 7, e34793 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. White, A. J. et al. A type 2 cytokine axis for thymus emigration. J. Exp. Med. 214, 2205–2216 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Kernfeld, E. M. et al. A single-cell transcriptomic atlas of thymus organogenesis resolves cell types and developmental maturation. Immunity 48, 1258–1270.e6 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Miller, C. N. et al. Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development. Nature 559, 627–631 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Gause, W. C., Wynn, T. A. & Allen, J. E. Type 2 immunity and wound healing: evolutionary refinement of adaptive immunity by helminths. Nat. Rev. Immunol. 13, 607–614 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Neill, D. R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464, 1367–1370 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Wong, P., Goldrath, A. W. & Rudensky, A. Y. Competition for specific intrathymic ligands limits positive selection in a TCR transgenic model of CD4+ T cell development. J. Immunol. 164, 6252–6259 (2000).

    CAS  PubMed  Google Scholar 

  56. Skon, C. N. et al. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells. Nat. Immunol. 14, 1285–1293 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Moon, J. J. et al. Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity 27, 203–213 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Xing, Y. & Hogquist, K. A. Isolation, identification, and purification of murine thymic epithelial cells. J. Visualized Exp. https://doi.org/10.3791/51780 (2014).

  59. Baller, J., Kono, T., Herman, A. & Zhang, Y. CHURP: a lightweight CLI framework to enable novice users to analyze sequencing datasets in parallel. in ACM International Conference Proceeding Series 1–5, https://doi.org/10.1145/3332186.3333156 (Association for Computing Machinery, 2019).

  60. Miller, C. H. et al. Eomes identifies thymic precursors of self-specific memory-phenotype CD8+ T cells. Nat. Immunol. 21, 567–577 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Lee, Y. J. et al. Tissue-specific distribution of iNKT cells impacts their cytokine response. Immunity 43, 566–578 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank J. Ding for technical assistance, J. Motl from the University Flow Cytometry Resource for cell sorting, T. Dileepan for providing tetramer reagents, the University of Minnesota Genomics Center for assistance with RNA-seq and the University of Minnesota Research Animal Resources for animal husbandry. This project was supported by the National Institutes of Health (grant nos. R37 AI39560 and P01 AI35296 to K.A.H., T32 AI007313 to K.M.A. and E.R.B., and F30 AI131483 and T32 GM008244 to E.R.B.).

Author information

Authors and Affiliations

Authors

Contributions

K.A.H. and E.R.B. conceived of, and obtained funding for, the project. E.R.B., M.V. and K.A.H. conceptualized and designed experiments and wrote the manuscript. E.R.B. and M.V. performed the experiments, with help from R.J.M. for DC antigen-presentation experiments, K.M.A. and L.Q. for TCR-seq experiment and analysis, H.W. for parabiosis experiments and O.C.S. for tetramer enrichment experiments. E.R.B. analyzed the experiments with help from R.J.M. for RNA-seq data analysis. C.H.O. and M.V. performed and analyzed scRNA-seq experiments. All authors provided feedback and approved the manuscript.

Corresponding author

Correspondence to Kristin A. Hogquist.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Immunology thanks Lauren Ehrlich 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.

Additional information

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

Extended data

Extended Data Fig. 1 Thymic APC Gating Strategy.

Flow cytometric gating strategy for identifying B cells and dendritic cell subsets. B cells were identified by the expression of CD19 and B220 (blue; top, middle). Plasmacytoid DC (pDC) were identified by the expression of B220, CD11c, and PDCA-1 (CD317) (salmon; bottom, middle). Conventional dendritic cells were identified by the expression of MHC II and CD11c (top, middle right). cDC1 were identified by the expression of XCR1 (magenta) and cDC2 were identified by the expression of SIRPα (teal; top, right). CD301b expression is shown on cDC1 (green; right) and cDC2 green; (green; bottom).

Extended Data Fig. 2 Ontogeny of bone marrow-derived APC subsets in the thymus.

(a-e) Frequency of thymic APC subsets (as indicated) among total thymocytes in 2d (n = 4), 1w (n = 7), 2w (n = 7), 3w (n = 3), and 8w-old (n = 6) C57BL/6 mice (gated as in Extended Data Fig. 1a). Each symbol represents an individual mouse. Male and female mice were used. Small horizontal lines indicate the mean, and error bars represent s.d. Data are pooled from three independent experiments.

Extended Data Fig. 3 Thymic eGFP expression in Mgl2DTReGFP mice.

(a) Representative flow cytometry of Mgl2-eGFP expression by CD45 EpCAM+ thymic epithelial cells (TECs). Cell were MACS enriched as CD3 CD4 CD19TER119. Mgl2WT mouse was used as control. (b) Representative flow cytometry of Mgl2-eGFP expression by CD11c+ cells in the thymus. The Mgl2-eGFP+ cells represent B220 XCR1 SIRPα+ cells that can be divided by the expression of MHC II and CCR7 to two main populations (as in Fig. 1h). (c) Representative flow cytometry of Mgl2-eGFP expression and CD301b protein staining of thymic SIRPα+ DCs divided according to the MHC II and CCR7 expression to DC2 (bottom) and mDC2 (top). Mgl2WT and CD0301b-PE FMO were used as control. Numbers adjacent to outlined areas represent percent cells in each. Six to ten-week-old male and female mice were used. Data are representative of at least three independent experiments.

Extended Data Fig. 4 Identification of clusters in scRNA sequence data of thymic myeloid (CD11c+ CD11b+) cells.

(a) Flow cytometric gating strategy for sorting the thymic CD11c+ and CD11b+ populations for single-cell RNA (scRNA) sequencing. Cells were MACS enriched for CD90.2 cells. Dead cells were gated out using Viability Dye Aqua 510 and Cytox. CD90.2 CD11c+ and CD11b+ cells were sorted, captured with a 3’ Single Cell V5 chemistry platform, and sequenced. (b) UMAP plot showing the analysis of 10,234 transcriptome events identifying 22 color-coded clusters that were divided into 10 main populations marked by dashed lines. (c) Feature plots showing normalized expression of signature genes associated with clusters defined in b. Seven-week-old male mice were used.

Extended Data Fig. 5 No depletion of thymic epithelial cells in Mgl2DTR-eGFP mice.

(a) Quantification of total numbers of CD301b+ cDC2 from BALB/c/BYJ (n = 3), IL-4 KO (n = 4) and IL-4Ra KO (n = 4) mice. (b) Experimental strategy for selective depletion of CD301b expressing cells in Mgl2DTR-eGFP mice. (c) Representative flow cytometric gating strategy of thymic epithelial cell (TEC) populations. Cells were MACS enriched as CD3 CD4 CD19TER119. TECs were gated as CD45EpCAM+, then divided to cortical TECs (cTECs) and medullary TECs (mTECs) according to the expression of Ly51 and UEA, respectively. mTECs were then divided based on the Ly6d and MHC II expression to mTECsLow (black), mTECsHigh (blue), Pre-post-Aire (red), and Post-Aire (green). (d) Quantification of numbers of TEC populations (gated as in c) from diphtheria toxin (DTx) treated Mgl2WT (gray dots) (n = 5) or Mgl2DTR-eGF (green dots) (n = 5). Six to ten-week-old male and female mice were used. Small horizontal lines indicate mean, and error bars represent s.d. Data are representative of at least 3 independent experiments (c) or are pooled from at least 2 independent experiments (a, d). ns=not significant, *P < 0.05, ***P < 0.001. One-way ANOVA test with Tukey’s multiple comparisons test was used.

Extended Data Fig. 6 Selectivity of thymic myeloid cell depletion in Mgl2DTR-eGFP mice.

(a) Experimental strategy for selective depletion of cells in Mgl2DTR-eGFP mice. (b) Representative flow cytometric gating strategy of thymic cell populations. Cells were enriched for CD90.2 negative cells to eliminate thymocytes. Macrophages were identified by the expression of CD64. Neutrophils were identified by the expression of CD11b and Ly6g. Eosinophils were gated as CD11b+ SiglecF+. B cells were identified as CD11cB220+ and plasmacytoid DCs (pDC) as CD11c+ B220+. Monocytes were gated as Ly6c+ CD11c CD11b+ and cDCs as MHCII+ CD11c+. (b) Quantification of cell numbers of thymic populations (gated as in a) from diphtheria toxin (DTx) treated Mgl2WT (gray dots) or Mgl2DTR-eGFP (green dots) (n = 9). (c) Quantification of thymic cDC numbers (gated as in Supplementary Figure 1) and thymic cDC subpopulations (gate as in Fig. 1h) from diphtheria toxin (DTx) treated Mgl2WT (gray dots) (n = 9) or Mgl2DTR-eGFP (green dots) (n = 9). Six to ten-week-old male and female mice were used. Small horizontal lines indicate mean, and error bars represent s.d. Data are representative of at least 3 independent experiments (b) or are pooled from at least 3 independent experiments (c, d). ns=not significant, *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA test with Tukey’s multiple comparisons test was used.

Extended Data Fig. 7 scRNA sequencing analysis of thymic myeloid cell depletion in Mgl2DTR-eGFP mice.

(a) Experimental strategy for selective depletion of cells in Mgl2DTR-eGFP mice. (b) The CD11c+ and CD11b+ cells (gated as in Supplementary figure 4) from diphtheria toxin (DTx) treated Mgl2WT (left plot) or Mgl2DTR-eGFP (right plot) were FACS-sorted, captured with a 3’ Single Cell V5 chemistry platform, and sequenced. Cell hashing was used to distinguish the genotypes of origin. The non-myeloid and granulocyte populations was bioinformatically depleted from the analysis. UMAP plots showing the analysis of 13,129 transcriptome events (Mgl2WT = 8,175, Mgl2DTR-eGFP = 4,959) and identified 8 major clusters marked by dashed lines. The clusters showing the most diffence in abundance of events between the genotypes (cDC2 and mDC2) are marked by bold navy and yellow lines. (c) Enumeration of clusters frequencies from CD11c/CD11b+ cells identified in b. (d) Feature plots showing normalized expression of Mgl2, Mki67 and Ccr7 genes in clusters identified in b. (e) Feature plots comparing the Mgl2 expression between Mgl2WT and Mgl2DTR-eGFPmice. Seven-week-old male mice were used.

Extended Data Fig. 8 Clonal deletion gating strategy.

Flow cytometry gating strategy for identifying thymocytes undergoing clonal deletion or death by neglect. Signaled and non-signaled cells were identified based on expression of CD5 and TCRb (middle, left; green gate (signaled), gray gate (non-signaled). Thymocytes undergoing death by neglect were identified from non-signaled cells based on expression of cleaved caspase-3 (bottom, left; gray gate). Cortical and medullary thymocytes were identified based on expression of CCR7 (middle, middle). Medullary CD4 thymocytes undergoing clonal deletion were identified from signaled CCR7+ CD4+ cells based on expression of cleaved caspase-3+ (bottom, right; red gate). Numbers adjacent to outlined areas represent percent cells in each.

Extended Data Fig. 9 Frequencies of thymic T cell populations.

(a–c) Total CD4 T cells in mice with selective deficiencies. The administration of DTx into Mgl2DTR-eGFP mice was done as in Fig. 5d. (d and e) Frequency of CD5+ TCRb+ cleaved caspase 3+ thymocytes among CCR7+ CD4 T cells in in mice with selective deficiencies (gated as in Extended Data Fig. 8). (f) Frequency of CD5+ TCR+ cleaved caspase 3+ thymocytes among DP T cells in Mgl2WT (n = 9) or Mgl2DTR-eGFP (n = 8). The administration of DTx into Mgl2DTR-eGFP mice was done as in Fig. 5d. (a-e) AireWT (n = 5) or AireKO (n = 6), Batf3WT (n = 9) or Batf3KO (n = 11) and Mgl2WT (n = 8) or Mgl2DTR-eGFP (n = 6) mice were used. (g) Frequency of CD25+FOXP3 and CD25FOXP3+ Treg cell progenitors (TRP) and (h) nascent CD25+ FOXP3+ CD73and recirculating CD25+ FOXP3+ CD73+ Treg cells in Mgl2WT (n = 9) or Mgl2DTR-eGFP (n = 8) mice following 9 days of diphtheria toxin treatment. The administration of DTx into Mgl2DTR-eGFP mice was done as in Fig. 5d. Each symbol represents an individual mouse. Six to twelve-week-old male and female mice were used. Small horizontal lines indicate the mean and error bars represent s.d. ns=not significant, *P < 0.05, **P < 0.01. Data are pooled from at least three independent experiments. Unpaired Mann-Whitney test was used.

Extended Data Fig. 10 RNA sequencing of TCRs from Mgl2DTR-eGFP mice.

(a) Heatmap analysis of CPM (counts per million reads mapped) of CDR3 peptides that were deferentially expressed between CD4+ Tconv thymocytes from Mgl2WTTcra+/−TclibTgFoxp3eGFP and Mgl2DTR-eGFPTcra+/−TclibTgFoxp3eGFP mice (n = 4 mice per genotype). The plot also displays the expression of those CDR3 peptides in CD4+ Treg thymocytes from the same mice. The Log10 FDR (False discovery rate) of for each CDR3 peptide CPM is shown.

Supplementary information

Reporting Summary

Supplementary Data 1

Comparison of gene expression profiles from bulk RNA-seq among thymic DC subsets.

Supplementary Table 1

List of antibodies used.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Breed, E.R., Vobořil, M., Ashby, K.M. et al. Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion. Nat Immunol 23, 1042–1051 (2022). https://doi.org/10.1038/s41590-022-01218-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-022-01218-x

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing