T cells given full stimulation (Signal 1 + 2) in the presence of rapamycin are rendered anergic () (Powell et al., 1999
). Rapamycin inhibits TORC1 activity by blocking the interaction between mTOR and raptor. To confirm this in our system, T cells were incubated in serum-free conditions in the presence of rapamycin or the Hsp90 inhibitor 17-AAG (a derivative of geldanamycin, as a negative control) for 3 hours, and then given TCR and costimulation for 3 hours. Immunoprecipitation (IP) of mTOR demonstrates the mTOR-raptor interaction is inhibited by rapamycin but not by 17-AAG ().
Hsp90 is a binding partner of the TORC1 component raptor
Since rapamycin promotes anergy by disrupting TORC1 signaling, we were interested in finding novel binding proteins for raptor that might be involved in regulating T cell function. To do this, we utilized a proteomic strategy involving mass spectrometry (). Raptor was immunoprecipitated from lysates of either resting or hyper-activated Jurkat T cells, separated by SDS-PAGE and silver stained. Protein bands were identified that were differentially bound to raptor in the lysate from stimulated versus unstimulated cells. One band identified near 90 kDa was excised, digested with trypsin, extracted and analyzed by nanospray LCMS/MS. The raw LCMS/MS data was analyzed using the MASCOT search engine against the NCBInr human database and two peptide sequences were identified and matched to Hsp90, a chaperone protein necessary for the correct folding of many protein “clients” (Bishop et al., 2007
To confirm these results, we sought to demonstrate raptor-Hsp90 interaction in primary T cells. Primary 5C.C7 T cells were stimulated with peptide and expanded in IL-2 to create previously activated Th1 cells. The cells were either rested or stimulated in serum free media for 3 hours. In the stimulated T cells, IP of raptor leads to concomitant precipitation of Hsp90 (). Likewise, IP of Hsp90 from lysates of activated T cells leads to the concomitant precipitation of raptor. Thus, the TORC1 adaptor protein raptor binds to Hsp90 upon T cell activation.
Hsp90 is a chaperone that plays a critical role in the proper folding of a variety of proteins key to cellular survival. Using a similar proteomic approach, Ohji and colleagues also observed an interaction between raptor and Hsp90 in HEK293 cells (Ohji et al., 2006
). To further determine the role of the Hsp90-raptor interaction in T cells we employed several inhibitors of Hsp90: 17-AAG and radicicol, as well as CCT018159, a newly reported pyrazole Hsp90 inhibitor. A.E7 T cells were stimulated in the presence of rapamycin or the Hsp90 inhibitors for 16 h. As expected, rapamycin had no effect on raptor expression, but stimulation during Hsp90 blockade markedly decreased raptor protein levels, consistent with the concept that raptor is an Hsp90 client (). T cell activation leads to increased mTOR signaling as determined by the phosphorylation of the TORC1 substrate S6K1 (Zheng et al., 2007
). Consistent with previous reports (Ohji et al., 2006
) in HEK293 cells, in T cells, Hsp90 inhibitors, like rapamycin, have the ability to inhibit TORC1 activity ().
Hsp90 inhibition in T cells leads to the development of an anergic state
Next we examined the functional consequences of Hsp90 inhibition in T cells. A.E7 T cells were stimulated with anti-CD3 alone or with anti-CD3 and anti-CD28 in the presence of rapamycin or Hsp90 inhibitors. The cells were washed and rested without drug. Upon initial stimulation, all T cells given costimulation produced equivalent amounts of IL-2 (). Such an observation suggests that Hsp90 activity is not required for T cell activation. After five days, the cells were rechallenged with anti-CD3 and anti-CD28. Importantly, immunoblot analysis performed prior to rechallenging the cells revealed that these cells have fully regained expression of raptor protein (). That is by pharmacologic means, we were able to successfully knock down raptor expression during the initial encounter with antigen. On the other hand, during the rechallenge, five days later, raptor levels returned to normal. Upon rechallenge, cells initially treated with rapamycin or Hsp90 inhibitor display a marked decrease in IL-2 production. (). The inability to produce IL-2 upon full rechallenge is the functional hallmark of T cell clonal anergy(Schwartz, 2003
). To further confirm this anergic phenotype, we interrogated the cells for IFN-γ production as well as their ability to proliferate. As expected, cells receiving Signal 1 alone or Signal 1 + 2 with rapamycin produced less IFN-γ and proliferated less upon rechallenge. In addition, those cells initially receiving Hsp90 inhibition even in the context of costimulation also produced less IFN-γ and proliferated less (). These data demonstrate that similar to Signal 1 alone and Signal 1+2+rapamcyin, Signal 1+2 in the presence of Hsp90 blockade induced T cell anergy. That is, five days later upon rechallenge without drug present, the cells failed to proliferate or produce IFN-γ and IL-2.
Our group and others have been interested in understanding the role of mTOR in regulating T cell activation and tolerance (Powell and Zheng, 2006
). In this report we demonstrate that T cell activation results in the increased binding between Hsp90 and the TORC1 component raptor. Raptor acts as an mTOR scaffolding protein facilitating the phosphorylation of mTOR substrates (Guertin et al., 2006
). We performed our proteomic approach with the intention of identifying raptor binding proteins and potential TORC1 substrates upon T cell activation. However, our data suggest that raptor is in fact a client of Hsp90 rather than Hsp90 being a substrate of mTOR. Consistent with this conclusion is the observation that the Hsp90 inhibition led to a decrease in raptor protein levels. In transformed HEK293 cells, raptor is a client of Hsp90 and that its inhibition results in a decrease in TORC1 signaling(Ohji et al., 2006
). Our data show that not only does this hold true in non-transformed primary T cells, but, functionally, inhibition of Hsp90 results in anergy induction. We cannot completely rule out off-target effects of pharmacologic inhibition of Hsp90 in our system. However, our approach employed inhibitors with different mechanisms of action and facilitated the rapid return of raptor expression. Whereas knockdown of Hsp90 via RNAi might be more specific, it also may be more
problematic, as a prolonged decrease in expression of such a vital protein could show increases in non-raptor-related events. In our system, we can control the timing of Hsp90 inhibition and remove blockade from culture enabling us to study the long term downstream consequences of TCR engagement in the absence of raptor even after raptor levels are returned to normal. In this fashion, we have defined Hsp90 inhibitors as novel pharmacologic inducers of anergy. Interestingly, Hsp90 inhibitors are currently being evaluated as anti-neoplastic agents (Bishop et al., 2007
). Our data suggest that while such agents might not acutely inhibit T cell function (), they may induce anergy in Th1 cells. As such Hsp90 inhibitors might be incorporated into immunosuppressive regimens to treat autoimmune disease or prevent transplant rejection through promotion of T cell tolerance.