Studies on naïve CD4+CD25+T cells inhibition of naïve CD4+CD25- T cells in mixed lymphocyte cultures

Bruce M Hall, Catherine M Robinson, Karren Michelle Plain, Nirupama Darshan Verma, Nicole M Carter, Rochelle A Boyd, Giang T Tran, Suzanne J Hodgkinson

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25 Citations (Scopus)


BACKGROUND: Naïve CD4+CD25+T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4+CD25+T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood.

METHOD: The alloreactivity of naïve CD4+CD25+, CD4+CD25(-) and unfractionated CD4+T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (LewisxPVG)F1 cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4+CD25+T cells.

RESULTS: Proliferation of CD4+CD25(-)T cells was significantly greater than unfractionated CD4+T cells to both allogeneic and syngeneic stimulator cells. CD4+CD25+T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3(-) cells. Admixing CD4+CD25+T cells with CD4+CD25(-)T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4+ T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-gamma, IL-10, TGF-beta and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4+CD25+ T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4+CD25(-)T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-gamma, IL-10, TGF-beta, IL-5 or iNOS did not prevent CD4+CD25+T cell's inhibition of CD4+CD25(-)T cell proliferation. Blocking IFN-gamma or iNOS enhanced CD4+CD25(-)T cell proliferation only in the absence of CD4+CD25+T cells. Depletion of CD4+CD25+T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators.

CONCLUSIONS: Two independent mechanisms that control proliferation of CD4+CD25(-)T cells in MLC were identified that naive CD4+CD25+T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4+CD25(-)T cells producing IFN-gamma to induce iNOS.

Original languageEnglish
Pages (from-to)291-301
Number of pages11
JournalTransplant Immunology
Issue number4
Publication statusPublished - Feb 2008
Externally publishedYes


  • Animals
  • Cell Differentiation
  • Cell Proliferation
  • Cell Separation
  • Coculture Techniques
  • Immune Tolerance
  • Interleukin-2 Receptor alpha Subunit
  • Lymphocyte Culture Test, Mixed
  • Rats
  • Rats, Inbred Lew
  • T-Lymphocyte Subsets
  • T-Lymphocytes, Regulatory
  • Comparative Study
  • Journal Article
  • Research Support, Non-U.S. Gov't


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