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Abstract

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.