Ex Vivo Rapamycin Generates Th2 Cells with Enhanced Mitochondrial Function and Reduced Apoptosis Via Intrinsic and Extrinsic Pathways.

We previously showed that ex vivo rapamycin generates murine Th2 cells (Th2.R cells) with enhanced capacity to abrogate GVHD and graft rejection relative to control Th2 cells. Enhanced efficacy of Th2.R cells was associated with a 10-fold increase in Th2.R cell number in vivo relative to control Th2 cells. Experiments were performed to determine the contribution of increased proliferation or reduced cell death to this increased Th2 cell number. Upon adoptive transfer of CFSE-labeled Th2 or Th2.R cells after allogeneic BMT, we found that Th2.R cells were increased at each cell cycle number (1 through 6 cell divisions); increased Th2.R cell number post-BMT was attributable primarily to reduced cell death (relative to control Th2 cells, Hoechst red-positive cells reduced from 65%±2% to 39%±1%, p<.0001). Further experiments were performed to characterize the basis of the reduced apoptosis in Th2.R cells. Rapamycin-generated Th2.R cells were inhibited at the level of mTOR, as Western-blot analysis confirmed markedly reduced phosphorylation of 4EBP1 and S6 ribosomal protein. Ex vivo generated Th2.R and Th2 cells were compared for their apoptotic threshold via the extrinsic pathway (challenge with CD3, CD28 co-stimulation) and the intrinsic pathway (challenge with staurosporine [1 uM] or CCCP [100 uM]). Flow cytometric evaluation of cell death (PI staining), apoptosis (Annexin-V staining), and change in mitochondrial membrane polarization (DiOC6 staining) were tested at 6 and 24 hours after stimulation. Extrinsic pathway challenge through co-stimulation revealed that Th2.R cells had reduced AICD relative to control Th2 cells (PI-positive cells, reduced from 96%±0.2% to 66%±2%; p<.0001; n=12 experiments); furthermore, apoptotic CD4+Annexin-V+ cells were reduced from 21.3% ± 2.2% (Th2 cells) to 3.6% ± 0.5% (Th2.R cells) (p<.0001, n=12). This reduced extrinsic pathway apoptotic threshold in Th2.R cells was not attributable to modulation of the fas pathway, as Th2 and Th2.R cells had similar surface CD95 expression by flow cytometry and similar FLIP expression by Western-blot analysis. Rapamycin-generated Th2.R cells also had reduced apoptosis after challenge by staurosporine (annexin-positive cells, reduced from 79%±0.5% [Th2 cells] to 58%±1% [Th2.R cells]; p=.001, n= 4 experiments) or CCCP (annexin-positive cells, reduced from 67%±4% [Th2 cells] to 26%±1% [Th2.R cells]; p=.004, n= 2 experiments). After extrinsic pathway challenge, mitochondrial membrane gradient stability was enhanced in Th2.R cells, as the mean reduction in DiOC6 MFI was 83%±1% in Th2 cells and 35%±5% in Th2.R cells (p<.0001, n=8 experiments); in addition, the mitochondria-associated pro-apoptotic bcl-2 family gene members bid and bim were consistently reduced in Th2.R cells. In conclusion, rapamycin inhibition of mTOR during Th2 cell development generates Th2 cells with enhanced mitochondrial function and an associated reduction in apoptotic threshold via extrinsic and intrinsic apoptotic pathways that likely contributes to the enhanced in vivo efficacy of Th2.R cells for modulation of GVHD and graft rejection.