Skip to main content

Thank you for visiting 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.

Immune homeostasis enforced by co-localized effector and regulatory T cells


FOXP3+ regulatory T cells (Treg cells) prevent autoimmunity by limiting the effector activity of T cells that have escaped thymic negative selection or peripheral inactivation. Despite the information available about molecular factors mediating the suppressive function of Treg cells, the relevant cellular events in intact tissues remain largely unexplored, and whether Treg cells prevent activation of self-specific T cells or primarily limit damage from such cells has not been determined. Here we use multiplex, quantitative imaging in mice to show that, within secondary lymphoid tissues, highly suppressive Treg cells expressing phosphorylated STAT5 exist in discrete clusters with rare IL-2-positive T cells that are activated by self-antigens. This local IL-2 induction of STAT5 phosphorylation in Treg cells is part of a feedback circuit that limits further autoimmune responses. Inducible ablation of T cell receptor expression by Treg cells reduces their regulatory capacity and disrupts their localization in clusters, resulting in uncontrolled effector T cell responses. Our data thus reveal that autoreactive T cells are activated to cytokine production on a regular basis, with physically co-clustering T cell receptor-stimulated Treg cells responding in a negative feedback manner to suppress incipient autoimmunity and maintain immune homeostasis.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: pSTAT5+ Treg clusters in lymph nodes.
Figure 2: Preferential role of migratory dendritic cells in Treg clustering.
Figure 3: Highly suppressive phenotype of clustered Treg cells.
Figure 4: Role of Treg TCR expression in clustering and suppression.
Figure 5: Role of IL-2 feedback in Treg-mediated suppression.


  1. Benoist, C. & Mathis, D. Treg cells, life history, and diversity. Cold Spring Harb. Perspect. Biol. 4, a007021 (2012)

    Google Scholar 

  2. Josefowicz, S. Z., Lu, L. F. & Rudensky, A. Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012)

    CAS  Article  Google Scholar 

  3. Xing, Y. & Hogquist, K. A. T-cell tolerance: central and peripheral. Cold Spring Harb. Perspect. Biol. 4, (2012)

  4. Vignali, D. A., Collison, L. W. & Workman, C. J. How regulatory T cells work. Nature Rev. Immunol. 8, 523–532 (2008)

    CAS  Article  Google Scholar 

  5. Shevach, E. M. Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity 30, 636–645 (2009)

    CAS  Article  Google Scholar 

  6. Germain, R. N. Maintaining system homeostasis: the third law of Newtonian immunology. Nature Immunol. 13, 902–906 (2012)

    CAS  Article  Google Scholar 

  7. 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 

  8. Gerner, M. Y., Torabi-Parizi, P. & Germain, R. N. Strategically localized dendritic cells promote rapid T cell responses to lymph-borne particulate antigens. Immunity 42, 172–185 (2015)

    CAS  Article  Google Scholar 

  9. Radtke, A. J. et al. Lymph-node resident CD8α+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses. PLoS Pathog. 11, e1004637 (2015)

    Article  Google Scholar 

  10. Fontenot, J. D., Rasmussen, J. P., Gavin, M. A. & Rudensky, A. Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nature Immunol. 6, 1142–1151 (2005)

    CAS  Article  Google Scholar 

  11. D’Cruz, L. M. & Klein, L. Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nature Immunol. 6, 1152–1159 (2005)

    Article  Google Scholar 

  12. Cheng, G., Yu, A. & Malek, T. R. T-cell tolerance and the multi-functional role of IL-2R signaling in T-regulatory cells. Immunol. Rev. 241, 63–76 (2011)

    CAS  Article  Google Scholar 

  13. Smigiel, K. S. et al. CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets. J. Exp. Med. 211, 121–136 (2014)

    CAS  Article  Google Scholar 

  14. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006)

    ADS  CAS  Article  Google Scholar 

  15. Hofer, T., Krichevsky, O. & Altan-Bonnet, G. Competition for IL-2 between regulatory and effector T Cells to chisel immune responses. Front. Immunol. 3, 268 (2012)

    Article  Google Scholar 

  16. Naramura, M., Hu, R. J. & Gu, H. Mice with a fluorescent marker for interleukin 2 gene activation. Immunity 9, 209–216 (1998)

    CAS  Article  Google Scholar 

  17. Setoguchi, R., Hori, S., Takahashi, T. & Sakaguchi, S. Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 201, 723–735 (2005)

    CAS  Article  Google Scholar 

  18. Almeida, A. R., Zaragoza, B. & Freitas, A. A. Indexation as a novel mechanism of lymphocyte homeostasis: the number of CD4+CD25+ regulatory T cells is indexed to the number of IL-2-producing cells. J. Immunol. 177, 192–200 (2006)

    CAS  Article  Google Scholar 

  19. Amado, I. F. et al. IL-2 coordinates IL-2-producing and regulatory T cell interplay. J. Exp. Med. 210, 2707–2720 (2013)

    CAS  Article  Google Scholar 

  20. Powell, J. D., Ragheb, J. A., Kitagawa-Sakakida, S. & Schwartz, R. H. Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy. Immunol. Rev. 165, 287–300 (1998)

    CAS  Article  Google Scholar 

  21. Chinen, T., Volchkov, P. Y., Chervonsky, A. V. & Rudensky, A. Y. A critical role for regulatory T cell-mediated control of inflammation in the absence of commensal microbiota. J. Exp. Med. 207, 2323–2330 (2010)

    CAS  Article  Google Scholar 

  22. Mempel, T. R., Henrickson, S. E. & Von Andrian, U. H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–159 (2004)

    ADS  CAS  Article  Google Scholar 

  23. van Panhuys, N., Klauschen, F. & Germain, R. N. T-cell-receptor-dependent signal intensity dominantly controls CD4+ T cell polarization in vivo. Immunity 41, 63–74 (2014)

    CAS  Article  Google Scholar 

  24. Wang, H., Lim, D. & Rudd, C. E. Immunopathologies linked to integrin signalling. Semin. Immunopathol. 32, 173–182 (2010)

    Article  Google Scholar 

  25. Deaglio, S. et al. Adenosine generation catalysed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J. Exp. Med. 204, 1257–1265 (2007)

    CAS  Article  Google Scholar 

  26. Kobie, J. J. et al. T regulatory and primed uncommitted CD4 T cells express CD73, which suppresses effector CD4 T cells by converting 5′-adenosine monophosphate to adenosine. J. Immunol. 177, 6780–6786 (2006)

    CAS  Article  Google Scholar 

  27. Pandiyan, P., Zheng, L., Ishihara, S., Reed, J. & Lenardo, M. J. CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nature Immunol. 8, 1353–1362 (2007)

    CAS  Article  Google Scholar 

  28. Read, S. et al. Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo. J. Immunol. 177, 4376–4383 (2006)

    CAS  Article  Google Scholar 

  29. Wing, K. et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271–275 (2008)

    ADS  CAS  Article  Google Scholar 

  30. Levine, A. G., Arvey, A., Jin, W. & Rudensky, A. Y. Continuous requirement for the TCR in regulatory T cell function. Nature Immunol. 15, 1070–1078 (2014)

    CAS  Article  Google Scholar 

  31. Vahl, J. C. et al. Continuous T cell receptor signals maintain a functional regulatory T cell pool. Immunity 41, 722–736 (2014)

    CAS  Article  Google Scholar 

  32. Liao, W., Lin, J. X. & Leonard, W. J. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 38, 13–25 (2013)

    CAS  Article  Google Scholar 

  33. Lu, L. F. et al. Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity 30, 80–91 (2009)

    CAS  Article  Google Scholar 

  34. O’Gorman, W. E. et al. The initial phase of an immune response functions to activate regulatory T cells. J. Immunol. 183, 332–339 (2009)

    Article  Google Scholar 

  35. Tang, Q. et al. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nature Immunol. 7, 83–92 (2006)

    CAS  Article  Google Scholar 

  36. Tadokoro, C. E. et al. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J. Exp. Med. 203, 505–511 (2006)

    CAS  Article  Google Scholar 

  37. Ertürk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature Protocols 7, 1983–1995 (2012)

    Article  Google Scholar 

Download references


We thank Y. Belkaid for providing germ-free mice. We also would like to thank members of the Lymphocyte Biology Section, Laboratory of Systems Biology for their helpful comments during the course of these studies and critical input during preparation of this manuscript. This work was supported by the Intramural Research Program of NIAID, NIH and by the US National Institutes of Health (R37AI034206 to A.Y.R.; T32GM007739 to A.G.L.), the Ludwig Cancer Center at Memorial Sloan-Kettering Cancer Center (A.Y.R.), and the Howard Hughes Medical Institute (A.Y.R.).

Author information

Authors and Affiliations



Z.L. designed and conducted most of the experiments, data analysis, and drafted the manuscript; M.Y.G. performed the analysis of DC phenotype and provided assistance with histo-cytometry studies; N.V.P. conducted 2P intravital imaging experiments; A.G.L. and A.Y.R. created Tracwt/wtFoxp3eGFP-cre-ERT2 and Tracfl/flFoxp3eGFP-cre-ERT2 mice, generated tissue samples from tamoxifen treatment of these mice, and discussed data interpretation; R.N.G. helped design experiments, interpret data, and with input from all other authors, develop the final version of the manuscript.

Corresponding author

Correspondence to Ronald N. Germain.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Role of IL-2 in induction of pSTAT5 in Treg cells.

a, Representative flow data showing pSTAT5 staining of Treg cells from lymphoid organs as indicated after treatment with isotype antibody or IL-2-neutralizing antibody (1 mg per mouse) for 24 h. The numbers in the quadrants indicate the percentages of cells in each quadrant. pLN, popliteal lymph node; iLN, inguinal lymph node; mLN, mesenteric lymph node. b, Quantification of pSTAT5+ Treg cells (n = 3 for each treatment). Results are representative of two independent experiments. c, Detection of pSTAT5+ Treg cells in wild-type, IL-2-knockout and IL-15-knockout mice. The numbers in the quadrants indicate the percentages of cells in each quadrant. Results are representative of two independent experiments. ***P < 0.001, as calculated by two-tailed Student’s t-test.

Extended Data Figure 2 Spatial correlation between IL-2+ cells and pSTAT5+ Treg clusters.

a, A positive control showing immunofluorescence staining of IL-2 in situ, and the comparison of IL-2 detection sensitivity between flow cytometry and histo-cytometry. OVA323-339 peptide-loaded splenic dendritic cells (DC, cyan) were stained with CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) and injected into the footpad, and Rag2−/− TCR transgenic OT-II CD4+ T cells (green) stained with CMFDA (5-chloromethylfluorescein diacetate) were transferred 18 h post-transfer of dendritic cells. For each recipient, 16 h after T cell transfer, one draining popliteal lymph node was isolated for IL-2 (red) immunofluorescence staining, while the contralateral lymph node was isolated and dissociated into single cells for intracellular IL-2 staining ex vivo. Each dot in the middle panel of the upper row indicates a lymph node (n = 3, 3 lymph nodes total from 3 mice for each analysis). *P < 0.05 as calculated by two-tailed Student’s t-test. b, Immunofluorescence staining of IL-2 and pSTAT5 in lymph nodes from FOXP3–eGFP reporter mice. Upper row indicates an IL-2+ cell associated with clustered Treg cells among a group of pSTAT5+ Treg cells. Lower row indicates an unassociated IL-2+ cell surrounded by a group of pSTAT5+ Treg cells. c, Quantification of IL-2+ cells associated or unassociated with Treg clusters. Data are pooled from seven lymph nodes.

Extended Data Figure 3 Phenotypic characterization of IL-2-producing cells in the steady state in Il2WT/GFP heterozygous mice.

a, Representative flow data showing the phenotypes of cells from inguinal lymph nodes of wild-type (WT) or Il2WT/GFP heterozygous mice. b, Quantification of the percentages of CD4+ and CD8+ cells within GFP+CD3+ population from different lymphoid organs (n = 3, mean ± s.e.m.). iLN, inguinal lymph node; mLN, mesenteric lymph node.

Extended Data Figure 4 Histo-cytometry analysis of dendritic cell subsets associated with Treg clusters.

a, Immunofluorescence staining of an inguinal lymph node section from FOXP3–eGFP mice (upper row) and Histo-cytometry analysis of dendritic cell (DC) subsets (lower row). Surfaces for each identified dendritic cell subset are shown in Fig. 2b. In brief, a new dendritic cell channel (CD11c+MHC-II+CD3B220) was generated and used to gate on dendritic cell surface markers, as well as to create dendritic cell surfaces for subset analysis and visualization. b, Gating strategy for the phenotypic characterization of dendritic cell subsets associated with pSTAT5+ or pSTAT5 Treg clusters.

Extended Data Figure 5 CD86 expression on dendritic cells associated with Treg clusters.

a, Representative images showing immunofluorescence staining of CD86 in lymph nodes from FOXP3–eGFP reporter mice. b, Histo-cytometry analysis of CD11c, MHC-II, and CD86 expression for dendritic cells (DCs) associated or unassociated with Treg clusters. c, d, Quantification of percentage of CD86high (c) and CD86 mean fluorescence intensity (MFI; d) in different dendritic cell subsets. Each dot indicates a section. n = 6, 6 sections total from 3 inguinal lymph nodes from 3 mice. ***P < 0.001, as calculated by two-tailed Student’s t-test (c) or one-way ANOVA with Tukey’s post hoc test (d). ns, not significant.

Extended Data Figure 6 The correlation between CD73/CTLA4 expression on Treg cells and their distance to the centre of Treg clusters.

ad, Regression analysis of CD73 (a, b) and CTLA4 (c, d) based on images shown in Fig. 3e, g. MFI, mean fluorescence intensity. Each circle in a and c or each dot in b and d indicates a Treg cell. The red lines in a and c indicate the trend lines. The red lines in b and d indicate the mean. ***P < 0.001 and *P < 0.05 as calculated by one-way ANOVA with Tukey’s post hoc test.

Extended Data Figure 7 Role of TCR signalling in high expression of CD73 and CTLA4 in Treg cells.

a, c, Immunofluorescence staining of CD73 (a) and CTLA4 (c) in mesenteric lymph node sections from TracWT/WTFoxp3eGFP-cre-ERT2 and Tracfl/flFoxp3eGFP-cre-ERT2 mice 7 days after tamoxifen treatment. CD73 and CTLA4 signals were gated on eGFP+ Treg cells. Images are representative of lymph node sections from two mice for each group. b, d, Histo-cytometry analysis of CD73 and CTLA4 mean fluorescence intensity (MFI) in indicated cell populations from the sections shown in a and c. Error bars represent mean ± s.d. The dots in the yellow rectangles in the left panels indicate CD73high or CTLA4high Treg cells. The right panels in b and d show the distribution pattern of the gated wild-type CD73high and CTLA4high Treg cells (in left panels) in sections. Arrows indicate CD73high and CTLA4high Treg cells in representative clusters. ***P < 0.001 as calculated by one-way ANOVA with Tukey’s post hoc test.

Supplementary information

Visualization of a pSTAT5+ Treg cluster in three dimensions.

Immunofluorescence staining of a 350-μm thick inguinal lymph node section from Foxp3-eGFP mice. Green indicates eGFP+ Treg cells, red indicates pSTAT5 signals, and cyan indicates B220+ cells. (MOV 9495 kb)

Two-photon intravital imaging of interaction between dendritic cells and wild-type (WT) or IL-2-/- 5C.C7 transgenic Rag2-/- T cells.

WT or IL-2-/- 5C.C7 transgenic Rag2-/- T cells (green) were each co-transferred with polyclonal CD4+ T cells (blue) into distinct WT host mice, 18 h post-transfer of PCC peptide loaded splenic dendritic cells (red). Imaging was performed 24 h after T cell transfer. (MP4 3802 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, Z., Gerner, M., Van Panhuys, N. et al. Immune homeostasis enforced by co-localized effector and regulatory T cells. Nature 528, 225–230 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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