Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4(+) CD25(high)Foxp3(+) regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-gamma chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.
Methodology/principal findings: CD4(+)CD25(+) and CD4(+)CD25(neg) T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1-100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4(+)CD25(high) and CD4(+)CD25(neg) T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4(+)CD25(neg) or CD8(+)CD25(neg) T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4(+)CD25(+) T cells in the presence of 1-100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4(+)CD25(high)Foxp3(+) cells and were resistant to apoptosis, while CD4(+)CD25(neg) T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4(+)CD25(neg) cells. Activated Treg+/-RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway.
Conclusions/significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.