CD4+CD25+Tregs express an increased LAG-3 and CTLA-4 in anterior chamber-associated immune deviation

  • Xuefei Zhu
  • Peizeng Yang
  • Hongyan Zhou
  • Bing Li
  • Xiangkun Huang
  • Qianli Meng
  • Li Wang
  • Aize Kijlstra
Laboratory Investigation



Regulatory CD4+CD25+ T cells have been proven to be essential for maintenance of peripheral tolerance and autoimmune diseases. ACAID is a model of immune privilege in the eye. Relatively little is known about the role and phenotype of these regulatory CD4+CD25+ T cells in ACAID.


Injection of OVA into the anterior chamber of BALB/C mice was performed to induce ACAID. The frequencies of splenic CD4+CD25+ Tregs and the expression of CTLA-4 and LAG-3 on these cells were determined by flow cytometry. Magnetic cell sorting was used to isolate CD4+CD25+ and CD4+CD25T cells. The function of CD4+CD25+ T cells was detected by in vitro immunosuppression assays and in vivo adoptive transfer.


ACAID was successfully induced following an i.c. injection of OVA. Frequencies of CD4+CD25+ and Tregs were significantly increased in ACAID mice as compared to those in controls. The CD4+CD25+ T cells stimulated with OVA in ACAID mice showed a stronger suppressive ability in vitro than those seen in non-ACAID mice. CD4+CD25+ T cells from ACAID mice, but not from non-ACAID mice, were able to suppress DTH responses in an antigen-specific manner following adoptive transfer. The frequencies of CTLA-4 or LAG-3 on Tregs in ACAID mice were higher as compared with those in naive mice.


Splenic CD4+CD25+Foxp3+T cells expressing CTLA4 and LAG3 play an important role in the induction of ACAID.


Regulatory T cells Immune regulation Foxp3 ACAID 


  1. 1.
    Ansari MJ, Salama AD, Chitnis T, Smith RN, Yagita H, Akiba H, Yamazaki T, Azuma M, Iwai H, Khoury SJ, Auchincloss H Jr, Sayegh MH (2003) The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 198:63–69PubMedCrossRefGoogle Scholar
  2. 2.
    Carreno BM, Collins M (2003) BTLA: a new inhibitory receptor with a B7-like ligand. Trends Immunol 24:524–527PubMedCrossRefGoogle Scholar
  3. 3.
    Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336PubMedCrossRefGoogle Scholar
  4. 4.
    Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY (2005) Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity 3:329–341CrossRefGoogle Scholar
  5. 5.
    Gangi E, Vasu C, Cheatem D, Prabhakar BS (2005) IL-10-producing CD4+CD25+ regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis. J Immunol 174:7006–7013PubMedGoogle Scholar
  6. 6.
    Graca L, Thompson S, Lin CY, Adams E, Cobbold SP, Waldmann H (2002) Both CD4+CD25+ and CD4+CD25 regulatory cells mediate dominant transplantation tolerance. J Immunol 168:5558–5565PubMedGoogle Scholar
  7. 7.
    Heuer JG, Zhang T, Zhao J, Ding C, Cramer M, Justen KL, Vonderfecht SL, Na S (2005) Adoptive transfer of in vitro-stimulated CD4+CD25+ regulatory T cells increases bacterial clearance and improves survival in polymicrobial sepsis. J Immunol 174:7141–7146PubMedGoogle Scholar
  8. 8.
    Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061PubMedCrossRefGoogle Scholar
  9. 9.
    Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, Hipkiss EL, Ravi S, Kowalski J, Levitsky HI, Powell JD, Pardoll DM, Drake CG, Vignali DA (2004) Role of LAG-3 in regulatory T cells. Immunity 10:503–513CrossRefGoogle Scholar
  10. 10.
    Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, Sakaguchi S (1999) Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol 162:5317–5326PubMedGoogle Scholar
  11. 11.
    Keino H, Takeuchi M, Kezuka T, Hattori T, Usui M, Taguchi O, Streilein JW, Stein-Streilein J (2006) Induction of eye-derived tolerance does not depend on naturally occurring CD4+CD25+ T regulatory cells. Invest Ophthalmol Vis Sci 47:1047–1055PubMedCrossRefGoogle Scholar
  12. 12.
    Kohm AP, Carpentier PA, Anger HA, Miller SD (2002) Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 169:4712–4716PubMedGoogle Scholar
  13. 13.
    Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders NJ, Powrie F (2003) CD4+CD25+TR cells suppress innate immune pathology through cytokine-dependent mechanisms. J Exp Med 197:111–119PubMedCrossRefGoogle Scholar
  14. 14.
    Meng QL, Yang PZ, Li B, Zhou HY, Huang YK, Zhu LX, Ren Y, Kijlstra A (2006) CD4+PD-1+T cells acting as regulatory cells during the induction of anterior chamber-associated immune deviation. Invest Ophthalmol Vis Sci 47:4444–4452PubMedCrossRefGoogle Scholar
  15. 15.
    Nakamura T, Terajewicz A, Stein-Streilein J (2005) Mechanisms of peripheral tolerance following intracameral inoculation are independent of IL-13 or STAT6. J Immunol 175:2643–2646PubMedGoogle Scholar
  16. 16.
    Niederkorn JY, Streilein JW (1983) Alloantigens placed into the anterior chamber of the eye induce specific suppression of delayed-type hypersensitivity but normal cytotoxic T lymphocyte and helper T lymphocyte responses. J Immunol 131:2670–2674PubMedGoogle Scholar
  17. 17.
    Niederkorn JY (2002) Immune privilege in the anterior chamber of the eye. Crit Rev Immunol 22:13–46PubMedGoogle Scholar
  18. 18.
    Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S (2002) Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 3:135–142PubMedCrossRefGoogle Scholar
  19. 19.
    Skelsey ME, Mayhew E, Niederkorn JY (2003) CD25+, interleukin-10-producing CD4+T cells are required for suppressor cell production and immune privilege in the anterior chamber of the eye. Immunology 110:18–29PubMedCrossRefGoogle Scholar
  20. 20.
    Streilein JW, Niederkorn JY (1985) Characterization of the suppressor cell(s) responsible for anterior chamber-associated immune deviation (ACAID) induced in BALB/c mice by P815 cells. J Immunol 134:1381–1387PubMedGoogle Scholar
  21. 21.
    Takahashi M, Ishimaru N, Yanagi K, Saegusa K, Haneji N, Shiota H, Hayashi Y (1999) Requirement for splenic CD4+T cells in the immune privilege of the anterior chamber of the eye. Clin Exp Immunol 116:231–237PubMedCrossRefGoogle Scholar
  22. 22.
    Teft WA, Kirchhof MG, Madrenas J (2006) A molecular perspective of CTLA-4 function. Annu Rev Immunol 24:65–97PubMedCrossRefGoogle Scholar
  23. 23.
    Totsuka T, Kanai T, Makita S, Fujii R, Nemoto Y, Oshima S et al (2005) Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells. Eur J Immunol 35:1773–1785PubMedCrossRefGoogle Scholar
  24. 24.
    Wilbanks GA, Streilein JW (1990) Characterization of suppressor cells in anterior chamber-associated immune deviation (ACAID) induced by soluble antigen. Evidence of two functionally and phenotypically distinct T-suppressor cell populations. Immunology 71:383–389PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Xuefei Zhu
    • 1
  • Peizeng Yang
    • 1
  • Hongyan Zhou
    • 1
  • Bing Li
    • 1
  • Xiangkun Huang
    • 1
  • Qianli Meng
    • 1
  • Li Wang
    • 1
  • Aize Kijlstra
    • 2
    • 3
  1. 1.State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Uveitis Study CenterSun Yat-sen UniversityGuangzhouPeople’s Republic of China
  2. 2.Animal Sciences Group van Wageningen UR Divisie VeehouderijLelystadThe Netherlands
  3. 3.Department Ophthalmology, Eye Research Institute MaastrichtUniversity of MaastrichtMaastrichtThe Netherlands

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