Interplay between mTOR and STAT5 signaling modulates the balance between regulatory and effective T cells

Immune response outcome, inflammation or tolerance, often depends on the balance between regulatory T cells (Tregs) and effective T cells (Teffs). Rapamycin (Rapa) has been reported to selectively expand Tregs and promote de novo generation of foxp3(+) Tregs, suggesting its potential role in inducing tolerance. But the mechanism by which Rapa regulating the Treg-Teff balance is yet to be understood.

Mouse CD4(+)CD25(-) Teffs and CD4(+)CD25(+) Tregs are sorted by MACS. These T cell subsets were labeled with CFSE and cultured with anti-CD3/CD28 Ab±IL-2 for 6 days. Two rounds of stimulation of 3 days each were performed. Rapa or Jak Inhibitor I was added to the culture when indicated. Cells were harvested after each round of stimulation. CFSE dilution, FOXP3, miR-155 expression and the signaling via the mTOR and STAT5 pathways were determined. And miR-155-mimic and miR-155-antagomir were transfected into purified CD4(+) T cells respectively to detect miR-155 role in regulating STAT5 signaling.

Firstly, we confirmed that the effect of Rapa on proliferating T cells is time-dependent: it reduces both Teffs and Tregs proliferation at early stage, but selectively promotes Tregs proliferation after second round of stimulation. Then we found there is direct interaction between mTOR and STAT5 signaling, and this interaction explained the time-dependent effect of Rapa and may participate in deciding Teff-Treg balance: mTOR inhibition up-regulated the expression of phos-STAT5 in both proliferating Tregs and Teffs via miR-155. And foxp3 is the down streaming target of phos-STAT5, thus Rapa could maintain expanded Tregs function and promote de novo generation of foxp3(+) Tregs. However, the phos-4E-BP1 expression pattern is different in proliferating Tregs and Teffs. 4E-BP1 is the common target of mTOR and STAT5 signaling, and plays a key role in cell proliferation. Rapa inhibits phos-4E-BP1 expression in both Tregs and Teffs at early stage of proliferation, but selectively raises its expression in Tregs after second round of stimulation. This may explains why Rapa inhibits Teffs growth, but delays Tregs proliferation.

Together, these findings indicate that the dynamic interaction between mTOR and STAT5 signaling modulates the reciprocal differentiation of the effective and regulatory T cells, and differently affect their proliferation activity. This provides a new insight of how Treg-Teff balance is regulated.