Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Abstract

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud 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+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg 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+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg 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+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud 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