Anergy is a state of immunologic tolerance in which T cells are viable but incapable of responding to antigenic stimulation. The authors found that anergic T cells selectively express small GTPase ADP-Ribosylation factor-6 (ARF). Anergic cells have sustained expression of ARF6 in the GTP-bound form which localizes at the plasma membrane. To address the role of ARF6 on TCR-mediated activation, freshly isolated peripheral blood human T cells were nucleofected with either empty vector, ARF6T27N mutant that remains in the GDP-bound state or ARF6Q67L mutant that remains in the GTP-bound state. Subsequently ERK1/2 (extracellular signal-regulated kinase1/2) was assessed by immunoblotting. ERKs, also known as MAP kinases, are conserved proteins that regulate cell growth, differentiation and survival. In a second experiment nucleofected cells were stimulated (CD3/CD28) and DNA synthesis was assessed by 3H-thymidine. The results revealed that forced expression of ARF6-GTP inhibited ERK1/2 activation and CD3/CD28 mediated proliferation.
Anergy is a state of immunologic tolerance in which T cells are viable but incapable of responding to antigenic stimulation. Recent data indicate that anergic cells have a distinct gene expression program that determines their unique function. In this study we show that anergic human T cells selectively express the small GTPase ADP-ribosylation factor-6 (ARF6), which is involved in membrane traffic and regulation of the cortical actin cytoskeleton. ARF6 was expressed in the GTP-bound form that localizes at the plasma membrane, resulting in a distinct morphologic appearance of anergic cells. Forced expression of ARF6-GTP in Jurkat T cells prevented TCR-mediated reorganization of cortical actin, extracellular signal-regulated kinase1/2 activation, and IL-2 transcription. Forced expression of ARF6-GTP in primary human T cells inhibited extracellular signal-regulated kinase1/2 activation and proliferative responses. Importantly, T cells with the distribution pattern of ARF6-GTP were detected in peripheral blood, suggesting that anergic T cells may constitutively exist in vivo.