Thymic regulatory T cells arise via two distinct developmental programs


The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Two thymic Treg progenitor cell populations exist.
Fig. 2: CD25+ TregP cells and Foxp3lo TregP cells are distinct thymic Treg cell lineages.
Fig. 3: CD25+ and Foxp3lo TregP cells are in discrete selection stages.
Fig. 4: Treg cells and Foxp3lo TregP cells show different localization in the thymus.
Fig. 5: Foxp3lo TregP cells are dependent on NFκB1 activation and the Foxp3 regulatory element Cns.
Fig. 6: Itk–/– mice show increased Treg cell production from both TregP cell pathways via distinct molecular mechanisms.
Fig. 7: CD25+ and Foxp3lo TregP cells have distinct cytokine responsiveness.
Fig. 8: CD25+ and Foxp3lo TregP cells are functionally distinct.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Single-cell RNA-seq data were deposited at Gene Expression Omnibus, with the following accession code: GSE123067.


  1. 1.

    Lio, C. W. & Hsieh, C. S. A two-step process for thymic regulatory T cell development. Immunity 28, 100–111 (2008).

    CAS  Article  Google Scholar 

  2. 2.

    Burchill, M. A. et al. Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity 28, 112–121 (2008).

    CAS  Article  Google Scholar 

  3. 3.

    Mahmud, S. A. et al. Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells. Nat. Immunol. 15, 473–481 (2014).

    CAS  Article  Google Scholar 

  4. 4.

    Burchill, M. A., Yang, J., Vogtenhuber, C., Blazar, B. R. & Farrar, M. A. IL-2 receptor β-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J. Immunol. 178, 280–290 (2007).

    CAS  Article  Google Scholar 

  5. 5.

    Yao, Z. et al. Nonredundant roles for Stat5a/b in directly regulating Foxp3. Blood 109, 4368–4375 (2007).

    CAS  Article  Google Scholar 

  6. 6.

    Tai, X. et al. Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals. Immunity 38, 1116–1128 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Hsieh, C. S. et al. Recognition of the peripheral self by naturally arising CD25+CD4+ T cell receptors. Immunity 21, 267–277 (2004).

    CAS  Article  Google Scholar 

  8. 8.

    Fenton, R. G., Marrack, P., Kappler, J. W., Kanagawa, O. & Seidman, J. G. Isotypic exclusion of γδ T cell receptors in transgenic mice bearing a rearranged β-chain gene. Science 241, 1089–1092 (1988).

    CAS  Article  Google Scholar 

  9. 9.

    Jorgensen, J. L., Esser, U., Fazekas de St Groth, B., Reay, P. A. & Davis, M. M. Mapping T-cell receptor-peptide contacts by variant peptide immunization of single-chain transgenics. Nature 355, 224–230 (1992).

    CAS  Article  Google Scholar 

  10. 10.

    Pacholczyk, R., Ignatowicz, H., Kraj, P. & Ignatowicz, L. Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells. Immunity 25, 249–259 (2006).

    CAS  Article  Google Scholar 

  11. 11.

    Wong, J. et al. Adaptation of TCR repertoires to self-peptides in regulatory and nonregulatory CD4+ T cells. J. Immunol. 178, 7032–7041 (2007).

    CAS  Article  Google Scholar 

  12. 12.

    Beura, L. K. et al. Normalizing the environment recapitulates adult human immune traits in laboratory mice. Nature 532, 512–516 (2016).

    CAS  Article  Google Scholar 

  13. 13.

    Moran, A. E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

    CAS  Article  Google Scholar 

  14. 14.

    Howie, D. et al. MS4A4B is a GITR-associated membrane adapter, expressed by regulatory T cells, which modulates T cell activation. J. Immunol. 183, 4197–4204 (2009).

    CAS  Article  Google Scholar 

  15. 15.

    Xing, Y., Wang, X., Jameson, S. C. & Hogquist, K. A. Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling. Nat. Immunol. 17, 565–573 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Boursalian, T. E., Golob, J., Soper, D. M., Cooper, C. J. & Fink, P. J. Continued maturation of thymic emigrants in the periphery. Nat. Immunol. 5, 418–425 (2004).

    CAS  Article  Google Scholar 

  17. 17.

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

    CAS  Article  Google Scholar 

  18. 18.

    Zhan, Y., Bourges, D., Dromey, J. A., Harrison, L. C. & Lew, A. M. The origin of thymic CD4+CD25+ regulatory T cells and their co-stimulatory requirements are determined after elimination of recirculating peripheral CD4+ cells. Int. Immunol. 19, 455–463 (2007).

    CAS  Article  Google Scholar 

  19. 19.

    Paessens, L. C., Singh, S. K., Fernandes, R. J. & van Kooyk, Y. Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes. Mol. Immunol. 45, 42–48 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Gerner, M. Y., Kastenmuller, W., Ifrim, I., Kabat, J. & Germain, R. N. Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity 37, 364–376 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Fontenot, J. D. et al. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329–341 (2005).

    CAS  Article  Google Scholar 

  22. 22.

    Bettini, M. L. et al. Loss of epigenetic modification driven by the Foxp3 transcription factor leads to regulatory T cell insufficiency. Immunity 36, 717–730 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Darce, J. et al. An N-terminal mutation of the Foxp3 transcription factor alleviates arthritis but exacerbates diabetes. Immunity 36, 731–741 (2012).

    CAS  Article  Google Scholar 

  24. 24.

    Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Feng, Y. et al. A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance. Nature 528, 132–136 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Huang, H. et al. Fine-mapping inflammatory bowel disease loci to single-variant resolution. Nature 547, 173–178 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Huang, J., Ellinghaus, D., Franke, A., Howie, B. & Li, Y. 1000 Genomes-based imputation identifies novel and refined associations for the Wellcome Trust Case Control Consortium phase 1 Data. Eur. J. Hum. Genet. 20, 801–805 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Onengut-Gumuscu, S. et al. Fine mapping of type 1 diabetes susceptibility loci and evidence for colocalization of causal variants with lymphoid gene enhancers. Nat. Genet. 47, 381–386 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Schaeffer, E. M. et al. Tec family kinases modulate thresholds for thymocyte development and selection. J. Exp. Med. 192, 987–1000 (2000).

    CAS  Article  Google Scholar 

  31. 31.

    Huang, W., Jeong, A. R., Kannan, A. K., Huang, L. & August, A. IL-2-inducible T cell kinase tunes T regulatory cell development and is required for suppressive function. J. Immunol. 193, 2267–2272 (2014).

    CAS  Article  Google Scholar 

  32. 32.

    Wu, J. N. et al. Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection. J. Immunol. 176, 6681–6689 (2006).

    CAS  Article  Google Scholar 

  33. 33.

    Weinreich, M. A., Odumade, O. A., Jameson, S. C. & Hogquist, K. A. T cells expressing the transcription factor PLZF regulate the development of memory-like CD8+ T cells. Nat. Immunol. 11, 709–716 (2010).

    CAS  Article  Google Scholar 

  34. 34.

    Huang, W., Huang, F., Kannan, A. K., Hu, J. & August, A. ITK tunes IL-4-induced development of innate memory CD8+ T cells in a γδ T and invariant NKT cell-independent manner. J. Leukoc. Biol. 96, 55–63 (2014).

    Article  Google Scholar 

  35. 35.

    Burchill, M. A., Yang, J., Vang, K. B. & Farrar, M. A. Interleukin-2 receptor signaling in regulatory T cell development and homeostasis. Immunol. Lett. 114, 1–8 (2007).

    CAS  Article  Google Scholar 

  36. 36.

    Vang, K. B. et al. IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development. J. Immunol. 181, 3285–3290 (2008).

    CAS  Article  Google Scholar 

  37. 37.

    Watanabe, N. et al. Hassall’s corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature 436, 1181–1185 (2005).

    CAS  Article  Google Scholar 

  38. 38.

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

    CAS  Article  Google Scholar 

  39. 39.

    Bornstein, C. et al. Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells. Nature 559, 622–626 (2018).

    CAS  Article  Google Scholar 

  40. 40.

    Gerbe, F. et al. Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites. Nature 529, 226–230 (2016).

    CAS  Article  Google Scholar 

  41. 41.

    McGeachy, M. J., Stephens, L. A. & Anderton, S. M. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J. Immunol. 175, 3025–3032 (2005).

    CAS  Article  Google Scholar 

  42. 42.

    Shahinian, A. et al. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261, 609–612 (1993).

    CAS  Article  Google Scholar 

  43. 43.

    Sha, W. C., Liou, H. C., Tuomanen, E. I. & Baltimore, D. Targeted disruption of the p50 subunit of NF-κB leads to multifocal defects in immune responses. Cell 80, 321–330 (1995).

    CAS  Article  Google Scholar 

  44. 44.

    Liao, X. C. & Littman, D. R. Altered T cell receptor signaling and disrupted T cell development in mice lacking itk. Immunity 3, 757–769 (1995).

    CAS  Article  Google Scholar 

  45. 45.

    Peterson, E. J. et al. Coupling of the TCR to integrin activation by Slap-130/Fyb. Science 293, 2263–2265 (2001).

    CAS  Article  Google Scholar 

  46. 46.

    Sonoda, K. H., Exley, M., Snapper, S., Balk, S. P. & Stein-Streilein, J. CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site. J. Exp. Med. 190, 1215–1226 (1999).

    CAS  Article  Google Scholar 

  47. 47.

    Shinkai, Y. et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867 (1992).

    CAS  Article  Google Scholar 

  48. 48.

    Nelson, R. W. et al. T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity. Immunity 42, 95–107 (2015).

    CAS  Article  Google Scholar 

  49. 49.

    Blair-Handon, R., Mueller, K. & Hoogstraten-Miller, S. An alternative method for intrathymic injections in mice. Lab. Anim. (NY) 39, 248–252 (2010).

    Article  Google Scholar 

  50. 50.

    Ruscher, R., Kummer, R. L., Lee, Y. J., Jameson, S. C. & Hogquist, K. A. CD8αα intraepithelial lymphocytes arise from two main thymic precursors. Nat. Immunol. 18, 771–779 (2017).

    CAS  Article  Google Scholar 

  51. 51.

    Haribhai, D. et al. A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity. Immunity 35, 109–122 (2011).

    CAS  Article  Google Scholar 

  52. 52.

    Perry, J. S. 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  Article  Google Scholar 

  53. 53.

    Lin, W. et al. Regulatory T cell development in the absence of functional Foxp3. Nat. Immunol. 8, 359–368 (2007).

    CAS  Article  Google Scholar 

  54. 54.

    McDavid, A. et al. Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments. Bioinformatics 29, 461–467 (2013).

    CAS  Article  Google Scholar 

  55. 55.

    Spanier, J. A., Nashold, F. E., Mayne, C. G., Nelson, C. D. & Hayes, C. E. Vitamin D and estrogen synergy in Vdr-expressing CD4+ T cells is essential to induce Helios+FoxP3+ T cells and prevent autoimmune demyelinating disease. J. Neuroimmunol. 286, 48–58 (2015).

    CAS  Article  Google Scholar 

  56. 56.

    Mottet, C., Uhlig, H. H. & Powrie, F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170, 3939–3943 (2003).

    CAS  Article  Google Scholar 

Download references


We thank G. Hubbard, A. Rost, A. Meskic, D. Duerre and H. Wiesolek for technical assistance; T. Martin, N. Shah, J. Motl and P. Champoux for cell sorting and maintenance of the Flow Cytometry Core Facility at the University of Minnesota (5P01AI035296); S. Hamilton, M. Pierson and funding from the University of Minnesota academic health center for maintaining the NME mouse facility; P. Fink for providing initial Rag2-GFP thymi; B. Burbach and Y. Shimizu for Adap–/– mice; M. Jenkins for the MOG:I-Ab tetramer; and C. Katerndahl and L. Heltemes-Harris for helpful commentary and reviewing the manuscript. D.L.O. and S.A.M. were supported by an immunology training grant (no. 2T32AI007313). S.A.M. was also supported by an individual predoctoral F30 fellowship from the National Institutes of Health (NIH; no. F30DK096844). J.A.S. was supported by University of Minnesota Medical Foundation grant no. UMF0020624 and NIH grants nos 5U24AI118635 and R01AI106791. Y.Z. was supported by NIH grant no. R01AI107027. U.B. and C.B.W. were supported by grants from the Children’s Hospital of Wisconsin, and C.B.W. was also supported by NIH grant no. R01AI085090-07A1. A.M. and M.S.A were supported by NIH grant no. DP3DK111914-01. A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is an investigator at the Chan Zuckerberg Biohub. M.A. was supported by NIH grant no. R01AI115716. M.S.A. was supported by NIH grant no. R37 AI097457. A.A. was supported by NIH grants nos AI108958, AI120701, AI126814 and AI129422 to A.A. and W.H. W.H. was supported by NIH grant no. AI29422 (to W.H. and A.A.), a Careers in Immunology Fellowship from the American Association of Immunologists, a Faculty Development Award and a competitive research grant from Louisiana State University, and a pilot award from the LSU-Tulane Center for Experimental Infectious Diseases Research funded by NIH grant no. GM110760. M.A.F. was supported by NIH grants nos AI124512, AI113138, AI061165, CA154998, CA151845 and CA185062.

Author information




D.L.O. designed and conducted experiments and wrote the manuscript. S.A.M, L.E.S, J.B.W., J.A.S., D.R.S., R.R., W.H., I.P., C.N.M., C.H., J.C.J, P.A., U.B., R.S.L., C.M.H. and Y.Z. performed some experiments or analyzed data and contributed intellectually to the work. M.A., M.S.A., A.A., A.M., Y.Z., and C.B.W. provided key reagents and/or animals and intellectual contributions. M.A.F. designed experiments, supervised research and assisted in the preparation of this manuscript. All authors read the manuscript and helped with final revisions.

Corresponding author

Correspondence to Michael A. Farrar.

Ethics declarations

Competing interests

A.M. is a co-founder of Spotlight Therapeutics. A.M. has served as an advisor to Juno Therapeutics and is a member of the scientific advisory board at PACT Pharma. The Marson laboratory has received sponsored research support from Juno Therapeutics, Epinomics and Sanofi, and a gift from Gilead. A.A. has received sponsored research support from 3 M. MAF has received sponsored research support from Merck.

Additional information

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

Integrated supplementary information

Supplementary Figure 1 Conversion of distinct TregP cell subsets into mature Treg cells in vitro.

Sorted Treg cell lineage subsets were stimulated for 3 days in the indicated concentrations of IL-2 and analyzed for the % of cells which converted into (left and middle panel) or remained (right panel) CD25+Foxp3+ mature Treg cells. Data represents 1 experiment, n = 2 (CD25+ TregP cells- untreated, 0.2 Uml–1 IL2; Foxp3lo TregP cells- untreated, 100 Uml–1 IL-2; Treg cells- 0.04 Uml–1 IL-2, 0.2 Uml–1 IL-2, 1 Uml–1 IL-2), n = 1 (Foxp3lo TregP cells- 0.04 Uml–1 IL-2; Treg cells- untreated), n = 3 (CD25+ TregP cells- 0.04 Uml–1 IL-2, 1 Uml–1 IL-2, 100 Uml–1 IL-2; Foxp3lo TregP cells- 0.2 Uml–1 IL-2, 1 Uml–1 IL-2; Treg cells- 100 Uml–1 IL-2) technical replicates. Bars represent mean ± SD.

Supplementary Figure 2 Germ-free and NME mice show no defect in either thymic TregP cell pathway.

a) Representative flow plots of SPF, germ-free reconstituted and germ-free mice thymi and quantification of the percent of each Treg cell lineage subset within CD4+CD73- thymocytes. b) Representative flow plots and quantification of SPF, germ-free reconstituted and germ free mice spleens showing the percent of CD4+ lymphocytes which are Foxp3+. a,b) Data is representative of 1 experiment, n = 6 SPF mice, 5 germ-free reconstituted mice, and 6 germ-free mice. Data was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. c) Representative flow plots of CD4+CD73- thymocytes from SPF mice or mice with normalized microbial experience (NME) and quantification of the percent within each Treg cell lineage population. Data is representative of 2 experiments, n = 3 SPF mice and 5 NME mice. Data was analyzed by two-sided unpaired t test. All bars represent mean ± SD. *P<0.05, **P<0.005, ns- not significant.

Supplementary Figure 3 Single-cell RNA-seq of thymic Treg cell lineage.

a) Violin plots (left) or feature plots (right) displaying single-cell expression for either Foxp3-GFP reporter (top) or Il2ra (bottom) for each cluster from the single-cell RNA-seq data set presented in Fig. 2e,f. b) Data from an independent repeat of 10x Genomics single-cell RNA-seq. Heatmap displays the top 10 differentially regulated genes in each cluster from the data set presented in Fig. 2e,f. c) Violin plots displaying single-cell expression for Bcl2l11 (left) and Nr4a1 (right) for each cluster from the single-cell RNA-seq data set presenting in Fig. 2e,f. a-c) Data is representative from 3 independent experiments, n = 3 mice.

Supplementary Figure 4 Maturation analysis of thymic Treg cell populations.

a) Thymocytes of the indicated subsets were analyzed for expression of HSA and Qa-2. Gates are drawn to demonstrate the frequency of cells within each maturation state. Data is representative of 1 experiment, n = 2 mice. b) Thymocytes of the indicated subsets were analyzed for CD69 and MHC-I expression. Gates are drawn to demonstrate the frequency of cells within each maturation state. Data is representative of 1 experiment, n = 3 mice.

Supplementary Figure 5 Frequency of contaminating recirculating cells in thymic Treg cell lineage subsets.

Thymocytes of the indicated subsets were analyzed for RAG2-GFP expression. Gates were drawn to determine the frequency of RAG2-GFP- (recirculating) and RAG2-GFP+ (newly developing) fractions of cells within each subset. Displayed are concatenated data from 3 thymi. Results are representative of 7 experiments, n = 9 mice.

Supplementary Figure 6 Enhancer deletions do not cause reduced levels of Foxp3 or CD25.

a) Quantification of Foxp3-gMFI in mature, CD73+ thymic Treg cells in WT, Foxp3-GFPKIN or Foxp3 Cns3-/- mice. All data points are normalized to the Foxp3-GFPKIN average within each experiment. Data is representative of 4 experiments, n = 8 wild-type mice, 14 GFPKIN mice and 14 Cns3-/- mice. Data was analyzed by a one-way ANOVA with Tukey’s multiple comparisons test. b) Quantification of CD25-gMFI in CD73- thymic Treg cells in WT or EDEL (Il2ra CaRE4-/-) mice in the non-obese diabetic (NOD) background. Data is representative of 3 experiments, n = 10 wild-type NOD mice and 10 EDEL NOD mice. Data was analyzed by a two-sided Mann-Whitney test. a,b) Bars represent mean ± SD. *P<0.05, ns- not significant.

Supplementary Figure 7 Treg cells derived from IL2 and IL4 exhibit distinct phenotypes.

a,b) Flow plots of the indicated TregP cell subsets following 3 days of stimulation with the indicated cytokines. c,d) Quantification of the gMFI of CD25 or Foxp3 within mature Treg cells (CD25+Foxp3+) generated from the indicated cytokine conditions. Data was analyzed by two-sided Kruskal-Wallis test. Data represents 3 experiments, n = 9 (CD25+ TregP cells- 1 Uml–1 IL2, 1 ng/mL IL4), n = 8 (CD25+ TregP cells- 100 ng/mL IL4, 1 Uml–1 IL2 + 1 ng/mL IL4, 1 Uml–1 IL2 + 100 ng/mL IL4; Foxp3lo TregP cells- 1 Uml–1 IL2, 1 Uml–1 IL2 + 1 ng/mL IL4, 1 Uml–1 IL2 + 100 ng/mL IL4), n = 7 (Foxp3lo TregP cells- 1 ng/mL IL4, 100 ng/mL IL4) or 1 experiment, n = 3 (CD25+ TregP cells- 0.5 ng/mL IL4, 1 Uml–1 IL2 + 0.5 ng/mL IL4; Foxp3lo TregP cells- 1 Uml–1 IL2 + 0.5 ng/mL IL4), n = 2 (Foxp3lo TregP cells- 0.5 ng/mL IL4) replicates. Bars represent mean ± SD. *P<0.05; **P<0.005; ***P<0.0001.

Supplementary Figure 8 Treg cells derived from Foxp3lo TregP cells protect against transfer induced colitis.

Data depicts % starting weight from Foxp3lo TregP cell transfer over the time-course of colitis experiment. Graph represents 2 experiments, n = 4 mice per group. Data was analyzed by two-sided multiple t test with Holm-Sidak method. *adjusted p value<0.05. Bars represent mean ± SEM.

Supplementary information

Supplementary Figures 1-8

Supplementary Table 1 and Supplementary Note

Reporting Summary

Supplementary Table 2

Differentially expressed genes between CD25+ TregP and Foxp3lo TregP cells from single-cell RNA-seq data sets. This table displays differentially expressed genes between CD25+ and Foxp3lo TregP cells in 2 independent single-cell RNA-seq data sets. The list is annotated by protein type and if it is a known gene correlated with negative selection

Supplementary Video 1: Kinetics of Treg cell development through RAG2-GFP expression

The video displays expression of CD25 and Foxp3 for cells falling within different bins of RAG2-GFP expression, from high RAG2-GFP to RAG2-GFP low (RAG2-GFP negative cells were excluded here). Data is from 3 concatenated thymi and is representative of 6 additional independent experiments

Bioinformatics code

Code used to analyze single cell RNA-seq data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Owen, D.L., Mahmud, S.A., Sjaastad, L.E. et al. Thymic regulatory T cells arise via two distinct developmental programs. Nat Immunol 20, 195–205 (2019).

Download citation

Further reading


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing