Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by immune-mediated destruction of hematopoietic stem cells. T cells from patients with AA overproduce IFN-γ, a cytokine that inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. In T cells, regulation of IFN-γ production occurs primarily at the level of transcription. A proximal site of the IFN-γ gene (−75 to −45bp of the IFN-γ promoter) is a binding site for different transcription factors including NFAT, AP-1, ATF, CREB, and T-bet. T-bet is a member of the T-box family of transcription factors, this family contains a highly conserved DNA binding domain, the T-box, that binds to a specific sequence in the promoter of different genes, including the IFN-γ promoter. T-bet is found in Th1 but not in Th2 cells and is the key regulator of Th1 development and function (

Rengarajan et al.,
Immunol Today
2000
;
21
:
479
). The inducible expression of T-bet is mediated in part by Itk kinase (
Sjabo et al.,
Science
2005
;
307
:
430
). In the present study, we examined T-bet protein levels in T cells from patients with AA. Samples from 17 of 20 patients examined (85%) by immunoblot showed increased T-bet protein levels in unstimulated T cells compared to normal controls (p = 0.0001). Normal controls showed undetectable T-bet protein levels in unstimulated T cells but T-bet expression was induced after 24 hrs of stimulation with PMA and ionomycin. In electrophoretical mobility shift assays, we observed increased T-bet binding to the proximal site of the IFN-γ promoter in T cells from patients with AA; no binding was detected in unstimulated T cells from healthy controls, but binding was present after stimulation for at least 24 hrs. T-bet protein levels correlated with disease activity. Patients with increased T-bet protein levels showed increased intracellular IFN-g levels compared to controls, as detected by flow cytometry (p<0.05). Patients that expressed increased T-bet protein levels also showed increased levels of the Itk kinase (p=0.02). We examined if other kinases that lie downstream of Itk in the signal transduction activation cascade in T cells affected the inducible expression of T-bet. In normal T cells, rottlerin, a PKC-theta (PKC-𝛉) inhibitor, decreased T-bet protein levels by 50%; in AA T cells, rottlerin also decreased T-bet protein levels and IFN-γ intracellular levels by 50%. Our results suggest that the increased IFN-γ levels observed in AA are the result of activation of transcription of the IFN-γ gene by the regulator T-bet. Blocking of transcription of the IFN-γ gene by kinase inhibitors might represent a therapuetic strategy for AA and other human T cell-mediated autoimmune diseases.

Topics:
acquired aplastic anemia, aplastic anemia, autoimmune diseases, bone marrow, hypocellular, cyclic amp-responsive dna-binding protein, cytokine, dna, electrophoretic mobility shift assay, flow cytometry, immunoblotting

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2005, The American Society of Hematology
2005
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