Article | Published:

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

Cellular & Molecular Immunologyvolume 16pages138153 (2019) | Download Citation



Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40−/− mice had significantly reduced numbers of CD25Foxp3low tTreg precursors and CD25+Foxp3+ mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Sakaguchi S. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 2000; 101: 455–458.

  2. 2

    Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4(+)CD25(+) regulatory T cells. Nat Immunol 2003; 4: 330–336.

  3. 3

    Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001; 27: 20–21.

  4. 4

    Lee HM, Bautista JL, Hsieh CS. Thymic and peripheral differentiation of regulatory T cells. Adv Immunol 2011; 112: 25–71.

  5. 5

    Jordan MS. Thymic selection of CD4(+)CD25(+) regulatory T cells induced by an agonist self-peptide (vol 2, pg 301, 2001). Nat Immunol 2001; 2: 468–468.

  6. 6

    Kieback E, Hilgenberg E, Stervbo U, Lampropoulou V, Shen P, Bunse M et al. Thymus-derived regulatory T cells are positively selected on natural self-antigen through cognate interactions of high functional avidity. Immunity 2016; 44: 1114–1126.

  7. 7

    Lio CW, Hsieh CS. A two-step process for thymic regulatory T cell development. Immunity 2008; 28: 100–111.

  8. 8

    Tai X, Erman B, Alag A, Mu J, Kimura M, Katz G et al. Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals. Immunity 2013; 38: 1116–1128.

  9. 9

    Mahmud SA, Manlove LS, Schmitz HM, Xing Y, Wang Y, Owen DL et al. Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells. Nat Immunol 2014; 15: 473–481.

  10. 10

    Lu FT, Yang W, Wang YH, Ma HD, Tang W, Yang JB et al. Thymic B cells promote thymus-derived regulatory T cell development and proliferation. J Autoimmun 2015; 61: 62–72.

  11. 11

    Nazzal D, Gradolatto A, Truffault F, Bismuth J, Berrih-Aknin S. Human thymus medullary epithelial cells promote regulatory T-cell generation by stimulating interleukin-2 production via ICOS ligand. Cell Death Dis 2014; 5: e1420.

  12. 12

    Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3(+) regulatory T cells in the human immune system. Nat Rev Immunol 2010; 10: 490–500.

  13. 13

    Caramalho I, Nunes-Cabaco H, Foxall RB, Sousa AE. Regulatory T-cell development in the human thymus. Front Immunol 2015; 6: 395.

  14. 14

    Nunes-Cabaco H, Caramalho I, Sepulveda N, Sousa AE. Differentiation of human thymic regulatory T cells at the double positive stage. Eur J Immunol 2011; 41: 3604–3614.

  15. 15

    Caramalho I, Nunes-Silva V, Pires AR, Mota C, Pinto AI, Nunes-Cabaco H et al. Human regulatory T-cell development is dictated by Interleukin-2 and -15 expressed in a non-overlapping pattern in the thymus. J Autoimmun 2015; 56: 98–110.

  16. 16

    Kumar P, Alharshawi K, Bhattacharya P, Marinelarena A, Haddad C, Sun Z et al. Soluble OX40L and JAG1 induce selective proliferation of functional regulatory T-cells independent of canonical TCR signaling. Sci Rep 2017; 7: 39751.

  17. 17

    Bhattacharya P, Gopisetty A, Ganesh BB, Sheng JR, Prabhakar BS. GM-CSF-induced, bone-marrow-derived dendritic cells can expand natural Tregs and induce adaptive Tregs by different mechanisms. J Leukoc Biol 2011; 89: 235–249.

  18. 18

    Gopisetty A, Bhattacharya P, Haddad C, Bruno JC Jr., Vasu C, Miele L et al. OX40L/Jagged1 cosignaling by GM-CSF-induced bone marrow-derived dendritic cells is required for the expansion of functional regulatory T cells. J Immunol 2013; 190: 5516–5525.

  19. 19

    Okamoto Y, Douek DC, McFarland RD, Koup RA. Effects of exogenous interleukin-7 on human thymus function. Blood 2002; 99: 2851–2858.

  20. 20

    Xing Y, Hogquist KA. Isolation, identification, and purification of murine thymic epithelial cells. J Vis Exp 2014, e51780 e-pub ahead of print Aug;10.3791/51780(90).

  21. 21

    Alharshawi K, Marinelarena A, Kumar P, El-Sayed O, Bhattacharya P, Sun Z et al. PKC- is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells. Sci Rep 2017; 7: 6594.

  22. 22

    Vail ME, Chaisson ML, Thompson J, Fausto N. Bcl-2 expression delays hepatocyte cell cycle progression during liver regeneration. Oncogene 2002; 21: 1548–1555.

  23. 23

    Zinkel S, Gross A, Yang E. BCL2 family in DNA damage and cell cycle control. Cell Death Differ. 2006; 13: 1351–1359.

  24. 24

    Cheng GY, Yu AX, Dee MJ, Malek TR. IL-2 R Signaling Is Essential for Functional Maturation of Regulatory T Cells during Thymic Development. Journal of Immunology 2013; 190: 1567–1575.

  25. 25

    Marie JC, Liggitt D, Rudensky AY. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-beta receptor. Immunity 2006; 25: 441–454.

  26. 26

    Chen W, Konkel JE. Development of thymic Foxp3(+) regulatory T cells: TGF-beta matters. Eur J Immunol 2015; 45: 958–965.

  27. 27

    Martin-Gayo E, Sierra-Filardi E, Corbi AL, Toribio ML. Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development. Blood 2010; 115: 5366–5375.

  28. 28

    Hsieh CS, Zheng Y, Liang YQ, Fontenot JD, Rudensky AY. An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat Immunol 2006; 7: 401–410.

  29. 29

    Bayer AL, Yu A, Malek TR. Function of the IL-2 R for thymic and peripheral CD4+CD25+ Foxp3+ T regulatory cells. J Immunol 2007; 178: 4062–4071.

  30. 30

    Paterson DJ, Jefferies WA, Green JR, Brandon MR, Corthesy P, Puklavec M et al. Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mol Immunol 1987; 24: 1281–1290.

  31. 31

    Mallett S, Fossum S, Barclay AN. Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes—a molecule related to nerve growth factor receptor. EMBO J 1990; 9: 1063–1068.

  32. 32

    Maddur MS, Sharma M, Hegde P, Stephen-Victor E, Pulendran B, Kaveri SV. Human B cells induce dendritic cell maturation and favour Th2 polarization by inducing OX-40 ligand. Nat Commun 2014; 5: 4092.

  33. 33

    So T, Song J, Sugie K, Altman A, Croft M. Signals from OX40 regulate nuclear factor of activated T cells c1 and T cell helper 2 lineage commitment. Proc Natl Acad Sci USA 2006; 103: 3740–3745.

  34. 34

    Kitamura N, Murata S, Ueki T, Mekata E, Reilly RT, Jaffee EM et al. OX40 costimulation can abrogate Foxp3(+) regulatory T cell-mediated suppression of antitumor immunity. Int J Cancer 2009; 125: 630–638.

  35. 35

    Alharshawi K, Marinelarena A, Kumar P, El-Sayed O, Bhattacharya P, Sun ZM et al. PKC-theta is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells. Sci Rep 2017; 7: 6594.

  36. 36

    Takeda I, Ine S, Killeen N, Ndhlovu LC, Murata K, Satomi S et al. Distinct roles for the OX40-OX40 ligand interaction in regulatory and nonregulatory T cells. J Immunol 2004; 172: 3580–3589.

  37. 37

    Piconese S, Pittoni P, Burocchi A, Gorzanelli A, Care A, Tripodo C et al. A non-redundant role for OX40 in the competitive fitness of Treg in response to IL-2. Eur J Immunol 2010; 40: 2902–2913.

  38. 38

    Sereti I, Gea-Banacloche J, Kan MY, Hallahan CW, Lane HC. Interleukin 2 leads to dose-dependent expression of the alpha chain of the IL-2 receptor on CD25-negative T lymphocytes in the absence of exogenous antigenic stimulation. Clin Immunol 2000; 97: 266–276.

  39. 39

    Chen Q, Kim YC, Laurence A, Punkosdy GA, Shevach EM. IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ T cells in vivo. J Immunol 2011; 186: 6329–6337.

  40. 40

    Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 2007; 5: e38.

  41. 41

    Koenecke C, Czeloth N, Bubke A, Schmitz S, Kissenpfennig A, Malissen B et al. Alloantigen-specific de novo-induced Foxp3+ Treg revert in vivo and do not protect from experimental GVHD. Eur J Immunol 2009; 39: 3091–3096.

  42. 42

    Okada M, Hibino S, Someya K, Yoshmura A. Regulation of regulatory T cells: epigenetics and plasticity. Adv Immunol 2014; 124: 249–273.

  43. 43

    Baeyens A, Saadoun D, Billiard F, Rouers A, Gregoire S. Zaragoza B, et al. Effector T cells boost regulatory T cell expansion by IL-2, TNF, OX40, and plasmacytoid dendritic cells depending on the immune context. J Immunol 2015; 194: 999–1010.

  44. 44

    Croft M, So T, Duan W, Soroosh P. The significance of OX40 and OX40L to T-cell biology and immune disease. Immunol Rev 2009; 229: 173–191.

  45. 45

    Croft M. Control of immunity by the TNFR-related molecule OX40 (CD134). Annu Rev Immunol 2010; 28: 57–78.

  46. 46

    So T, Croft M. Regulation of PI-3-kinase and Akt signaling in T lymphocytes and other cells by TNFR family molecules. Front Immunol 2013; 4: 139.

  47. 47

    Song J, So T, Croft M. Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival. J Immunol 2008; 180: 7240–7248.

  48. 48

    So T, Soroosh P, Eun SY, Altman A, Croft M. Antigen-independent signalosome of CARMA1, PKC theta, and TNF receptor-associated factor 2 (TRAF2) determines NF-kappa B signaling in T cells. Proc Natl Acad Sci USA 2011; 108: 2903–2908.

  49. 49

    Sauer S, Bruno L, Hertweck A, Finlay D, Leleu M, Spivakov M et al. T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR. Proc Natl Acad Sci USA. 2008; 105: 7797–7802.

  50. 50

    Huynh A, DuPage M, Priyadharshini B, Sage PT, Quiros J, Borges CM et al. Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability. Nat Immunol 2015; 16: 188–196.

  51. 51

    Vu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A et al. OX40 costimulation turns off Foxp3+ Tregs. Blood 2007; 110: 2501–2510.

  52. 52

    So T, Croft M. Cutting edge: OX40 inhibits TGF-beta- and antigen-driven conversion of naive CD4 T cells into CD25(+)Foxp3(+) T cells. J Immunol 2007; 179: 1427–1430.

  53. 53

    Nagar M, Jacob-Hirsch J, Vernitsky H, Berkun Y, Ben-Horin S, Amariglio N et al. TNF activates a NF-kappa B-regulated cellular program in human CD45RA(-) regulatory T Cells that modulates their suppressive function. J Immunol 2010; 184: 3570–3581.

  54. 54

    Ruby CE, Yates MA, Hirschhorn-Cymerman D, Chlebeck P, Wolchok JD, Houghton AN et al. Cutting edge: OX40 agonists can drive regulatory T cell expansion if the cytokine milieu is right. J Immunol 2009; 183: 4853–4857.

  55. 55

    Bresson D, Fousteri G, Manenkova Y, Croft M, von Herrath M. Antigen-specific prevention of type 1 diabetes in NOD mice is ameliorated by OX40 agonist treatment. J Autoimmun 2011; 37: 342–351.

  56. 56

    Haddad CS, Bhattacharya P, Alharshawi K, Marinelarena A, Kumar P, El-Sayed O et al. Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice. Autoimmunity 2016; 49: 298–311.

  57. 57

    Xiao X, Gong W, Demirci G, Liu W, Spoerl S, Chu X et al. New insights on OX40 in the control of T cell immunity and immune tolerance in vivo. J Immunol 2012; 188: 892–901.

  58. 58

    Guo Z, Wang X, Cheng D, Xia Z, Luan M, Zhang S. PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer. PLoS ONE 2014; 9: e89350.

  59. 59

    Shrimali RK, Ahmad S, Verma V, Zeng P, Ananth S, Gaur P et al. Concurrent PD-1 blockade negates the effects of OX40 agonist antibody in combination immunotherapy through inducing T-cell apoptosis. Cancer Immunol Res 2017; 5: 755–766.

  60. 60

    Montler R, Bell RB, Thalhofer C, Leidner R, Feng Z, Fox BA et al. OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer. Clin Transl Immunol 2016; 5: e70.

  61. 61

    Bell RB, Leidner RS, Crittenden MR, Curti BD, Feng Z, Montler R et al. OX40 signaling in head and neck squamous cell carcinoma: Overcoming immunosuppression in the tumor microenvironment. Oral Oncol 2016; 52: 1–10.

  62. 62

    von Boehmer H, Daniel C. Therapeutic opportunities for manipulating T-Reg cells in autoimmunity and cancer. Nat Rev Drug Discov 2013; 12: 51–63.

Download references


We thank the National Institutes of Health for grants #R01 AI107516-01A1 and #1R41AI125039-01. We thank the Juvenile Diabetes Research Foundation (JDRF) for grant #2-SRA-2016-245- S-B to Dr. Prabhakar. We thank the American Heart Association for offering a post-doctoral fellowship #15POST25090228 to PK.

Author information


  1. Department of Microbiology and Immunology, University of Illinois-College of Medicine, Chicago, IL, USA

    • Prabhakaran Kumar
    • , Alejandra Marinelarena
    • , Divya Raghunathan
    • , Vandhana K Ragothaman
    • , Shikha Saini
    • , Palash Bhattacharya
    • , Ajay V Maker
    •  & Bellur S Prabhakar
  2. Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

    • Alan L Epstein
  3. Department of Surgery, Division of Surgical Oncology, University of Illinois-College of Medicine, Chicago, IL, USA

    • Ajay V Maker


  1. Search for Prabhakaran Kumar in:

  2. Search for Alejandra Marinelarena in:

  3. Search for Divya Raghunathan in:

  4. Search for Vandhana K Ragothaman in:

  5. Search for Shikha Saini in:

  6. Search for Palash Bhattacharya in:

  7. Search for Jilao Fan in:

  8. Search for Alan L Epstein in:

  9. Search for Ajay V Maker in:

  10. Search for Bellur S Prabhakar in:

Conflict of interest

The authors declare no conflict of interest.

Corresponding author

Correspondence to Bellur S Prabhakar.

Electronic supplementary material

About this article

Publication history