GRAIL is expressed in human and mouse naive CD4 T cells, and its degradation following TCR/CD3 engagement and costimulation is required for proliferation. These data demonstrate a role for GRAIL in controlling naive CD4 T cell activation and proliferation in addition to GRAIL's role in the induction and maintenance of anergy. As demonstrated in these studies, not only TCR/CD3 engagement and CD28 costimulation are required for full activation, IL-2 production and IL-2R signaling are also necessary to allow proliferation. Phosphorylation of Akt following IL-2R engagement drives mTOR activation leading to Otub1 protein expression, degradation of GRAIL, and T cell proliferation. These data demonstrate a pathway of GRAIL regulation that links critical components of CD4 T cell stimulation to CD4 T cell proliferation. Interference in this pathway highlights the potential importance of this pathway in peripheral T cell tolerance and may suggest new targets for immunotherapeutics ().
FIGURE 9 The CD28 costimulation, IL-2 signaling, and mTOR pathway regulate Otub1 and GRAIL expression, controlling proliferation in primary naive CD4 T cells. CTLA4-Ig, anti-IL-2, and Rapamycin regulation of Otub1 and GRAIL expression controls naive CD4 T cell (more ...)
Our results link TCR/CD3 engagement and CD28 costimulation with IL-2 production and IL-2R signaling to activation of mTOR kinase that is required for activation induced proliferation of human and mouse naive CD4 T cells. Our studies highlight the importance of IL-2R signaling in sustaining mTOR activation during naive CD4 T cell activation. However, we also found that at early times (10 min to 1 h) following activation by CD3 and CD28 signaling, Akt is phosphorylated even in the presence of anti-IL-2 Abs, resulting in mTOR activation independent of IL-2R signaling (our unpublished observations) in agreement with previous reports (36
). This discrepancy is resolved by differentiating the IL-2R signaling requirement at different time points following naive CD4 T cell activation. At later times (24–72 h) following activation, IL-2R signaling was required for sustained mTOR activity as anti-IL-2 Abs blocked phosphorylation of Akt and mTOR activation at these later time points (), resulting in the sustained presence of GRAIL () and decreased proliferation ().
Previous studies have implicated S6K1 regulation by mTOR in CD4 T cell activation (39
), identifying a role for this pathway in directing mTOR-dependent protein translation. In this study, we demonstrate that naive CD4 T cells also regulate 4E-BP1 through the mTOR-dependent pathway via phosphorylation on Thr37/46
. Phosphorylation of 4E-BP1 leads to its dissociation from eIF4E, allowing active eIF4E to bind eIF4G during translation initiation complex formation (46
). A functional consequence attributed to active eIF4E is preferential translation of specific mRNAs normally translated into protein at low or absent rates (47
). The phosphorylation of 4E-BP1, and subsequent activation of eIF4E, may allow protein translation of a subset of mRNAs important for T cell activation. We propose that Otub1
mRNA is under such regulation as its protein expression does not appear to be mediated through changes in mRNA transcript levels yet is sensitive to mTOR inhibition. The therapeutic effects of rapamycin in the inhibition of CD4 T cell activation and proliferation may be due not only to decreased overall protein translation but also to prevention of translation of a subset of mRNAs critical for successful activation.
This study is the first demonstration that endogenous GRAIL protein regulation in primary human and mouse naive CD4 T cells plays an important role in controlling T cell activation and proliferation. In mice, GRAIL expression can be traced to Qa-2+
CD4 single-positive thymocytes poised for export to the periphery; thus, GRAIL expression may be an important component of peripheral tolerance in naive CD4 T cells, in addition to its role in CD4 T cell anergy. Qa-2+
CD4 single-positive thymocytes, but not earlier stage thymocytes, respond to TCR ligation in a manner similar to peripheral CD4 T cells (49
). The observations of GRAIL expression in Qa-2+
CD4 single-positive thymocytes and expression in peripheral naive CD4 T cells suggest a possible role for GRAIL in CD4 T cell tolerance to TCR self-peptide/MHC encountered during the transition from the thymus to the peripheral environment. TCR engagement of self selecting-peptide/MHC needs to remain a nonresponsive event for the naive CD4 T cell, and yet TCR engagement is necessary for maintaining their survival and keeping them poised for potential activation by non-self (50
). When foreign Ag is presented as non-self-peptide in the context of MHC class II, the increased affinity/avidity of the TCR engagement, as well as the presence of danger-induced APC costimulatory signals following B7-CD28 ligation, breaks the quiescent state of the naive CD4 T cell that these data suggest is maintained by GRAIL. IL-2 signals through the IL-2R on CD4 T cells via mTOR to ensure GRAIL degradation to allow proliferation. Thus, maintenance of GRAIL serves to preserve quiescence of naive CD4 T cells and its down-regulation is required to allow proliferation.
Anergic CD4 T cells express multiple E3 ubiquitin ligases, suggesting possible unique roles in maintaining cellular nonresponsiveness (7
). The differential expression of these E3 ubiquitin ligases in primary CD4 T cells during quiescence and activation may provide insights into further elucidation of their functions in periperhal T cell tolerance. We found that while GRAIL was present in naive quiescent CD4 T cells and down-regulated upon activation, by contrast, Cbl-b was expressed at low levels in naive quiescent CD4 T cells and up-regulated upon activation (data not shown). Another group has recently also reported on the observed Cbl-b up-regulation upon activation in primary CD4 T cells (56
). Their findings suggest that Cbl-b acts to limit CD4 T cell proliferation following TCR and CD28 activation through Cbl-b ubiquitination and degradation of phospholipase Cγ
) and PI3K (58
). Cbl-b decrease of PI3K expression diminishes downstream phosphorylation of ERK and Akt (56
). We proposed that GRAIL and Cbl-b both serve to counteract CD4 T cell activation, however, at different stages. GRAIL, by maintaining quiescence in the absence of CD28 costimulation, and Cbl-b, by dampening proliferation of activated cells. GRAIL and Cbl-b may be mechanistically linked through Cbl-b down-regulation of Akt phosphorylation. A decrease in Akt phosphorylation would decrease mTOR activation, abrogating Otub1 protein expression and thus resulting in the re-expression of GRAIL and inhibition of cell proliferation. In this regard, we found that human and mouse naive CD4 T cells activated and subsequently rested eventually diminished their levels of phosphorylated Akt, S6K1, and 4E-BP1 (data not shown). The return of these cells to a nonproliferating quiescent state was correlated with the re-expression of GRAIL. Reactivation by TCR/CD3 and CD28 stimulation again led to Otub1 protein expression and down-regulation of GRAIL before cell proliferation (data not shown). An investigation into this proposed interrelationship between GRAIL and Cbl-b control of CD4 T cell proliferation would help characterize their overlapping and distinct roles.
NOD mice serve as a murine model of human type 1 diabetes with increasing incidence of hyperglycemia with age (60
). The disease process is thought to occur initially through autoimmune T cell activation, possibly in the pancreatic lymph node, followed by inflammation of the islets of langerhans (insulitis) that, at ~12 wk of age, leads to islet β
-cell destruction and resultant hyperglycemia (61
). In search of genes differentially expressed during disease initiation and progression, we examined pancreatic lymph nodes from NOD and disease-resistant NOD.B10 (H-2b
) congenic mice. We conducted genome-wide analyses of gene expression using microarrays comparing NOD vs NOD.B10 pancreatic lymph node RNA (22
). At certain ages, including 12 wk, GRAIL
expression was decreased in pancreatic lymph nodes of NOD mice compared with NOD.B10 mice (supplemental Fig. 7A
This differential GRAIL
expression was verified by quantitative PCR of pancreatic lymph node RNA samples from multiple 12-wk-old NOD and NOD.B10 mice (supplemental Fig. 7B
Our findings suggest a potential peripheral tolerance role for GRAIL on naive CD4 T cells in vivo, which might be lost during NOD disease pathogenesis. In a study of primate HIV infection, GRAIL was up-regulated in anergic CD4 T cells isolated from disease-susceptible SIV-infected rhesus macaques, whereas SIV-resistant sooty mangabey primates showed no increase in GRAIL (62
). A role for GRAIL in human disease was recently demonstrated in patients successfully treated for ulcerative colitis: patients in remission expressed higher levels of GRAIL in CD4 T cells vs patients with ongoing disease or normal controls (63
) These studies and the findings reported above suggest that regulation of GRAIL and Otub1 may play an important role in peripheral tolerance.