Alterations in transcription factor binding at the IL-2 promoter region in anergized human CD4+ T lymphocytes.
BACKGROUND
The mechanisms responsible for the induction of clonal anergy are not well understood. We have utilized an in vitro model of human T cell anergy to explore the perturbations in cell signaling at the level of interleukin (IL)-2 gene transcription and to define the contribution of other cytokines to this effect.


METHODS
An in vitro model of clonal anergy was established by using CD4+ T lymphocytes from healthy human donors. Cells were anergized by prestimulation with an anti-CD3 monoclonal antibody (mAb) followed by restimulation 72 hr later with anti-CD3 mAb with or without anti-CD28.


RESULTS
CD4+ T cells, anergized with anti-CD3 monoclonal antibody (OKT3) prestimulation, displayed a marked reduction in proliferation (P=0.0036) and IL-2 production (P<0.0001). Co-incubation with IL-10 reduced cellular proliferation in OKT3/CD28 pretreated cells by 19% (P=NS) and reduced IL-2 production by 40% (P=0.0024). Anergized T cells demonstrated a reduced binding activity of the AP-1 complex to the IL-2 promoter. Supershift experiments and Western blots confirmed that the binding of c-Fos, JunB, and JunD, but not of FosB, was reduced in anergized cells. At the sis-inducible element (SIE)-binding region of the c-Fos promoter, Stat3 binding was reduced.


CONCLUSIONS
T cell anergy, induced by prestimulation with OKT3, is characterized by reduced proliferation and a profound decrease in IL-2 production. Anergy can be prevented by co-incubation with anti-CD28 and partially re-established by IL-10. Anergy is accompanied by a reduction in AP-1 binding to the IL-2 promoter, with selective reduction in binding of c-Fos, JunB, and JunD. Defective binding for Stat3 at the c-Fos promoter suggests an involvement of the Jak-Stat pathway.
Heisel
O
O
Department of Medicine, Vancouver General Hospital, 910 West 10th Avenue, Vancouver, B.C., Canada.
Keown
P
P
D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D017635:Clonal Anergy; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D008213:Lymphocyte Activation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016753:Interleukin-10