ANALYSIS OF IL-2 TRANSCRIPTIONAL EVENTS IN ANERGIZED HUMAN T-CELLS

Abstract

263 Introduction: The mechanisms responsible for the induction and maintenance of clonal anergy are not well understood. We have established a human in vitro model of 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 the anti-CD3 monoclonal antibody OKT3 followed by restimulation 72 hours later with OKT3 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 (4,824 ± 461 cpm vs 134,134 ± 36505 cpm, p=0.0036).) and IL-2 production (<4 pg/ml vs 512 ± 49 pg/ml, p<0.0001) compared with controls. Simultaneous treatment with anti-CD28 prevented induction of anergy measured either by proliferation (80,639 ± 10652 cpm, p=0.0002) or IL-2 production (471 ± 22 pg/ml, 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), suggesting an additional effect of IL-10 in anergizing T-cells. Binding of the transcription factor AP-1 was measured by gel-shift using a non-radioactive method. Anergized T-cells demonstrated a clearly reduced binding activity of the AP-1 protein complex, as did fully stimulated (anti-CD3+anti-CD28) T-cells treated with anti-CD40L MoAb, suggesting that members of the Fos- and Jun-family play a crucial role in the signaling events leading to clonal anergy. Supershift experiments performed using specific antibodies against c-Fos, FosB, JunB and JunD confirmed that the binding of c-Fos, JunB and JunD, but not of FosB was reduced in anergized cells by comparison with controls. Conclusion: T-cell anergy induced by 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 re-established by IL-10 or anti-CD40L. Anergy is accompanied by a defect in AP-1 binding to the IL-2 gene promoter, with selective reduction in binding of c-Fos, JunB and JunD.