The active ubiquitin E3 ligase GRAIL is crucial in the induction of CD4 T cell anergy. Here we show that GRAIL is associated with and regulated by two isoforms of the ubiquitin-specific protease otubain 1. In lethally irradiated mice reconstituted with bone marrow cells from T cell receptor-transgenic mice retrovirally transduced to express the genes encoding these proteases, otubain 1-expressing cells contained negligible amounts of endogenous GRAIL, proliferated well and produced large amounts of interleukin 2 after antigenic stimulation. In contrast, cells expressing the alternatively spliced isoform, otubain 1 alternative reading frame 1, contained large amounts of endogenous GRAIL and were functionally anergic, and they proliferated poorly and produced undetectable interleukin 2 when stimulated in a similar way. Thus, these two proteins have opposing epistatic functions in controlling the stability of GRAIL expression and the resultant anergy phenotype in T cells.
Nonstandard abbreviations used: antigen (Ag); bone marrow (BM); CG-rich motif (CpG); complete medium (CM); control oligonucleotide (ODN-CTR); effector/target (E/T); Fms-like thyrosine kinase 3 ligand (Flt3L); Langerhans cell (LC); macrophage inflammatory protein-3α (MIP-3α); mean fluorescence intensity (MFI); T cell receptor (TCR). Conflict of interest:The authors have declared that no conflict of interest exists. IntroductionThe ability of tumors to escape the immune system has been a major obstacle to the development of effective tumor immunotherapy. Both central and peripheral immune tolerance have been implicated in the failure of tumor-bearing hosts to mount an immune response to their tumors. Central tolerance may play a fundamental role in the lack of response against self-tumor-associated antigens (Ag's), while peripheral tolerance may explain the lack of response against tumor-specific Ag's. DCs are believed to play a critical role in antitumor immune responses. These cells are the most potent APCs known, uniquely capable of inducing immunity to newly introduced Ag's (1, 2). Normally, DCs reside as immature cells in peripheral tissues where they sample the environment by taking up and processing local Ag's. In the presence of certain toll-like receptor ligands, these cells not only take up and process Ag's but also undergo activation and maturation and then migrate to the draining LNs where they prime specific CD4 and CD8 T cells to these Ag's. The presence in a wide range of tumors of immature DCs that are unable to stimulate T cells (3-9) suggests a possible role for these cells in the failure of tumor-bearing hosts to mount an effective antitumor response. This view is supported by a recent study in melanoma patients that showed that tumor-associated DCs are present mostly at the periphery of tumors and express low levels of costimulatory molecules, while the majority of T cells infiltrating tumors have a naive phenotype (10). The presence of naive but not memory or effector T cells in tumors may be related to the failure of tumoral DCs to provide an adequate stimulus or possibly to the induction of T cell tolerance by the immature DCs. By contrast to tumoral DCs, in vitro-generated DCs can induce an effective T cell-mediated antitumor immune response in vivo (11), indicating that the T cells of tumor-bearing hosts are capable of recognizing and responding to tumor Ag's and suggesting again that the tumor milieu prevents tumoral DCs from inducing an effective immune response. Indeed, injection of immature Ag-pulsed DCs can induce a specific tolerogenic response, while similarly pulsed DCs, when matured, induced a typical Th1 immune response (12).Based on the recognition of the central role of DCs in initiating immune responses, a variety of strategies have been devised to use DCs to stimulate immunity against tumor Ag's. Most of these strategies rely on the activation and maturation of DCs ex vivo and their subsequent reinfusion to tumor-bearing recipients after a pulse with tumor Ag's expressed as...
Differential activation of CD4+ T-cell precursors in vivo leads to the development of effectors with unique patterns of lymphokine secretion. To investigate whether the differential pattern of lymphokine secretion is influenced by factors associated with either the display and/or recognition of the ligand, we have used a set of ligands with various class II binding affinities but unchanged T-cell specificity. The ligand that exhibited -10,000-fold higher binding to I-AU considerably increased the frequency of interferon y-producing but not interleukin (IL) 4-or IL-5-secreting cells in vivo. Using an established ligand-specific, CD4+ T-cell clone secreting only IL-4, we also demonstrated that stimulation with the highest affinity ligand resulted in interferon 'y production in vitro. In contrast, ligands that demonstrated relatively lower class II binding induced only IL-4 secretion. These data suggest that the major histocompatibility complex binding affinity of antigenic determinants, leading to differential interactions at the T cell-antigenpresenting cell interface, can be crucial for the differential development of cytokine patterns in T cells.
Acquisition of the anergy phenotype in T cells is blocked by inhibitors of protein synthesis and calcineurin activity, suggesting that anergic T cells may have a unique genetic program. Retroviral transduction of hemopoietic stem cells from TCR transgenic mice and subsequent reconstitution of syngeneic mice to express the E3 ubiquitin ligase, gene related to anergy in lymphocytes (GRAIL), or an enzymatically inactive form, H2N2 GRAIL, allowed analysis of the role of GRAIL in T cell anergy in vivo. Constitutive expression of GRAIL was sufficient to render naive CD4 T cells anergic, however, when the enzymatically inactive form H2N2 GRAIL was expressed, it functioned as a dominant negative of endogenous GRAIL and blocked the development of anergy. These data provide direct evidence that a biochemical pathway composed of GRAIL and/or GRAIL-interacting proteins is important in the development of the CD4 T cell anergic phenotype in vivo.
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