Rapamycin-resistant Proliferation of CD8+ T Cells Correlates with p27 Down-regulation and bcl-xL Induction, and Is Prevented by an Inhibitor of Phosphoinositide 3-Kinase Activity

Abstract

Rapamycin inhibits the proliferation of many mammalian cell types, including lymphocytes, making the compound useful as an immunosuppressant. Rapamycin has also been a useful tool for studying signaling mechanisms regulating cellular proliferation. However, the effects of rapamycin remain poorly understood, and the precise mechanisms of clinical action remain elusive. Previously, we found that, depending on the strength of the signal delivered to the T cell via both the T cell receptor and the costimulatory molecule CD28, CD8؉ T cells are capable of rapamycin-resistant proliferation. Here, we have further elucidated the mechanism of rapamycin-resistant proliferation of human CD8؉ T cells. Under conditions where rapamycin inhibited proliferation, p27 kip1 down-regulation was prevented, whereas under conditions resulting in rapamycin-resistant proliferation, p27 kip1 was down-regulated. Further, T cell receptor/CD28-dependent induction of bcl-x L expression was not inhibited by rapamycin, which correlated with both rapamycin-resistant proliferation and increased cell survival. Moreover, an inhibitor of phosphoinositide 3-kinase activity was able to eliminate rapamycin-resistant proliferation of freshly isolated CD8؉ human cells, strongly suggesting that phosphoinositide 3-kinase activity was required for the rapamycin-resistant proliferation of CD8؉ T cells. The selective immunosuppressive effect of rapamycin in human CD8؉ T cell populations could be predictive of a selective effect allowing cytotoxic responses during microbial infections where there are strong strengths of signals associated with high affinity T cell receptors and strong costimulatory second signals. In contrast, the weaker autoimmune and perhaps allogeneic responses can be selectively inhibited by the actions of rapamycin.Rapamycin is an immunosuppressant that functions by inhibiting entry into the cell cycle. This inhibition of proliferation by rapamycin occurs at a later stage of cellular activation than the inhibition by cyclosporin A and FK506, which inhibit calcineurin or calcineurin-dependent transcriptional activation of lymphokine genes (Ref. 1; reviewed in Ref. 2). It is thought that rapamycin primarily inhibits growth factor signaling rather than growth factor synthesis (3). The potent immunosuppressive activity of rapamycin has led to its wide clinical use in solid organ transplantation (reviewed in Ref. 4). More recent studies demonstrated successful islet cell transplantation in patients with type I diabetes treated with low dose rapamycin in combination with humanized anti-IL-2 1 receptor monoclonal antibody (mAb) and FK506, which prevented autoimmune destruction of islet cells (5). However, the clinical mechanism of action of rapamycin in suppressing immune responses is not well defined.Although it is clear that entry into the cell cycle is generally inhibited by rapamycin, we recently made the surprising observation that increasing the strength of signal delivered to the T cell via both the T cell receptor (TcR) and the costimula...

Ex Vivo Rapamycin Generates Apoptosis-Resistant Donor Th2 Cells That Persist In Vivo and Prevent Hemopoietic Stem Cell Graft Rejection

“…Our finding that rapamycin-generated Th2 cells manifest a broad apoptosis-resistant phenotype may not have been predicted from the numerous studies that associate mTOR inhibition with increased T cell apoptosis (49 -53), but is consistent with knowledge that rapamycin-resistant human CD8 ϩ T cells have an increase of antiapoptotic Bcl-x L (24) and that rapamycin resistance in murine T cells is mediated through the Pim-1 and Pim-2 kinases that confer antiapoptotic, growth, and proliferative effects (44,45). The Th2.R cells used in our study were rapamycin resistant, as they expanded in high-dose rapamycin in the setting of reduced phosphorylation of molecules dependent upon mTOR kinase activity, 4EBP1 and S6 ribosomal protein.…”

“…Relative to control Th2 cells, we found that Th2.R cells expressed similar levels of Pim-1 (six of six cases) and Pim-2 (six of six cases). As a third possibility, we evaluated Th2.R cell expression of the cell cycle inhibitor p27, which is down-regulated in rapamycin-resistant human CD8 ϩ T cells (24). Consistent with this previous observation, we found that Th2.R cells had reduced expression of p27 in six of six cases.…”

“…Our observation that Pim-1 and Pim-2 were equally expressed in Th2 and Th2.R cells suggests that these kinases contribute to the capacity of Th2.R cells to expand in the continued presence of rapamycin. However, the mechanism of resistance in our system is likely multifactorial, because Th2.R cells also had: 1) reduced expression of the cell cycle inhibitor p27, which is a previously described pathway of rapamycin resistance in human CD8 ϩ T cells (24) and 2) reduced expression of the active form of the T cell inhibitor GSK3␤. Additional experiments will be required to determine the relative contributions of pim kinase function, cell cycle inhibitor down-regulation, and modulation of the GSK3 pathway in the observed Th2.R cell rapamycin resistance.…”

“…23). Furthermore, rapamycin-resistant human CD8 ϩ T cells have increased in vitro survival that may be mediated through increased expression of antiapoptotic Bcl-x L (24). Given this information, the second goal of our project was to perform serial T cell tracking studies to compare the in vivo persistence of Th2/Tc2.R cells to control Th2/Tc2 cells in the graft rejection model and to further characterize any observed antiapoptotic property of rapamycin-generated T cells.…”

Intracellular Signals of T Cell Costimulation

“…In particular, one tyrosine (Y170 in mouse CD28, Y173 in human CD28) that is important in PI3K activation permits CD28 to recruit SH2-containing signaling molecules, including PI3K, Grb2, and Gads that control the regulation of bcl-xL (21,74). However, whether or not this regulation was dependent on mammalian target of rapamycin pathway is still controversial (13,75). CD28 also promotes expression of bcl-2, bfl-1 (A1), and myeloid cell leukemia sequence 1 (mcl-1) to regulate T cell survival (76)(77)(78).…”