| Summary: | Background: The mechanisms responsible for the induction of human 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 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 using peripheral
T-lymphocytes from healthy human donors. CD4+ T-cells were anergized by
pre-stimulation with an anti-CD3 mAb followed by restimulation 72 hours later
with anti-CD3 with or without anti-CD28. Proliferation was measured by [3H]-
thymidine incorporation and IL-2 production using ELISA.
Results: CD4+ T-cells anergized with OKT3 displayed a marked reduction in
proliferation (P=0.0036) and IL-2 production (P<0.0001) compared with
controls. Simultaneous treatment with anti-CD28 prevented induction of anergy
measured either by proliferation (P=0.0002) or IL-2 production(P<0.0001). Co-incubation
with IL-10 reduced cellular proliferation in OKT3/CD28 pretreated
cells by 19% (P=n.s.) and reduced IL-2 production by 40% (P=0.0024).
Anergized T-cells demonstrated a reduced binding activity of the AP-1 protein
complex to the IL-2 gene promoter. Supershift experiments confirmed that the
individual binding of c-Fos, JunB and JunD, but not of FosB to the AP-1 region
of the IL-2 promoter was reduced in anergized cells when compared to
controls. Furthermore, there was a reduced Stat-binding at the SIE region of
the c-Fos promoter in anergized cells. Supershift experiments using specific
antibodies against Statl and Stat3 showed that binding of Statl, but not Stat3,
to the SIE region of the c-Fos promoter was diminished. Co-incubation of PBMC with OKT3/anti-CD28 and a blocking gp39 (CD40L) mAb resulted in a
significantly reduced proliferation rate (P=0.0003) and IL-2 production
(P=0.005). Co-incubation with anti-CD40L mAb also led to a marked reduction
of AP-1 binding to the IL-2 promoter similar to that observed in B7/CD28
abrogated cells.
Conclusions: T-cell anergy induced by OKT3 is characterized by reduced Tcell
proliferation and a profound decrease in IL-2 production accompanied by a
reduction in AP-1 binding to the IL-2 gene promoter, with selective reduction in
binding of the individual AP-1 components c-Fos, JunB and JunD. The
deficiency in binding of Stat l to the SIE region of the c-Fos promoter highlights
an involvement of the Jak-Stat pathways in the events of clonal anergy.
Furthermore, blockade of the CD40-CD40L pathway is able to achieve similar
anergizing effects as in cells where B7/CD28 costimulation is abrogated. This
highlights the importance of the CD40/CD40L pathway as a second
costimulatory pathway. It also provides insight into new mechanisms of clonal
anergy, in which co-blockade of both B7/CD28 and CD40/CD40L pathways
might lead to a more profound anergy induction and better graft survival. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate
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