Introduction: Currently, Treg therapy focusses on adoptive transfer of ex vivo expanded natural Tregs (nTregs). Ex vivo expansion requires time-consuming and costly protocols due to low frequency of nTregs in human blood. Thus, induced Tregs (iTregs) provide an attractive avenue for Treg therapy. The combination of TGF-β, retinoic acid and rapamycin has been shown to generate highly suppressive human iTregs under various sources of TCR stimulation, even though, successful induction and stability were not observed. Here, we investigated the impact of rapamycin concentration and TCR stimulation strength on human iTreg differentiation and stability.
Method: iTregs were differentiated from naïve conventional T-cells (Tconv) using IL-2, TGF-β, retinoic acid and rapamycin concentrations (0, 1, 10 and 100 ng/mL; iTreg-0, iTreg-1, iTreg-10 and iTreg-100 respectively) at 1:1 α-CD3/CD28 bead to cell ratio. Additionally, iTregs were differentiated using IL-2, TGF-β, retinoic acid and 100 ng/mL of rapamycin at 1:10 bead to cell ratio (iTreg-1:10). Naïve nTregs and Tconvs were stimulated as controls. Initial differentiation and stability upon re-stimulation was assessed by evaluating CD25 and FOXP3 expression and measuring suppressor function.
Results and Discussion: nTreg, iTreg-0, iTreg-1, iTreg-10 and iTreg-100 exhibited >85% CD25+FOXP3+. No differences were shown with FOXP3 MFI among cell types while CD25 MFI of iTreg-10 and iTreg-100 were significantly higher than for other cell types. iTreg-1, iTreg-10 and iTreg-10 were highly suppressive, while iTreg-0 showed significantly lower suppressive capacity than nTregs. Upon re-stimulation, iTreg-1, iTreg-10 and iTreg-100 remained >85% CD25+FOXP3+, while %CD25+FOXP3+ of iTreg-0 significantly reduced to 66%. FOXP3 and CD25 MFI of iTreg-0 were significantly lower than nTreg. CD25 MFI of iTreg-0 was also significantly lower than iTreg-10 and iTreg-100. iTreg-1, iTreg-10 and iTreg-100 remained highly suppressive upon re-stimulation. Moreover, iTreg-1:10 showed <55% CD25+FOXP3+ with significant differences compared with nTreg and iTreg-100. FOXP3 and CD25 MFI of iTreg-1:10 were significantly lower than nTreg and iTreg-100. Nonetheless, iTreg-1:10 was highly suppressive with no significant differences compared with nTreg and iTreg-100. Upon re-stimulation, iTreg-1:10 showed slight increase in %CD25+FOXP3+ to <65%, although still significantly lower than nTreg and iTreg-100. Interestingly, no significant differences were shown with FOXP3 MFI while CD25 MFI of iTreg-1:10 was still significantly lower than nTreg and iTreg-100. iTreg-1:10 remained highly suppressive upon re-stimulation.
Conclusion: Here, we demonstrated that rapamycin is required for generation of stable human iTregs. Higher concentrations of rapamycin generate iTregs with greater expression of CD25. Furthermore, we showed that the strength of TCR stimulation is important for successful differentiation and stability of iTregs.