P158 All-trans retinoic acid and rapamycin synergize with transforming growth factor-B to induce regulatory T cells but confer distinct in vivo migratory capacities

Introduction Regulatory T cells (Treg) play important roles in maintaining immune homeostasis and, for this reason, therapies based on Treg are promising candidates for the treatment of a variety of immune-mediated disorders. However, therapies based on naturally-occurring Treg face the challenge of obtaining sufficient numbers of these cells from peripheral blood. Inducing Treg from non-Treg has been suggested as a viable alternative. Different methods to induce Treg have been proposed, mainly involving use of TGFβ (TGFβ-iTreg). Although TGFβ alone is not sufficient to induce stable Treg, all-trans retinoic acid (RA) or rapamycin (Rapa) has been shown to synergize with TGFβ to induce stable Treg. While RA-induced Treg (RA-iTreg) have been well described in the literature, the phenotype, function and migratory characteristics of rapa-induced Treg (Rapa-iTreg) have yet to be elucidated. Methods iTreg were induced from mouse CD4 + CD25 − T cells by in vitro stimulation in presence of TGFβ alone or together with: RA, Rapa, or a combination thereof. Expression of Foxp3, Treg-associated markers, and chemokine receptors was assessed by flow cytometry. In vivo migration was determined by adoptive transfer of congenic iTreg followed by flow-mediated enumeration or by adoptive transfer of luciferase-expressing iTreg (Luc) and monitoring of the bioluminescent signal. Results Addition of RA, Rapa, or both increased the frequency of Foxp3 + cells over the proportion obtained with TGFβ alone. Interestingly, no significant differences were found in the expression of Treg-associated markers like CTLA-4, CD25, and GITR, although a significant increase in folate receptor-4 (FR4) was evident on Rapa- and RA+Rapa-iTreg. All 4 iTreg types were comparable in their ability to inhibit T cell activation in vitro. Major differences were evident in the surface expression of chemokine receptors and integrins: TGFβ-iTreg were CCR7 med CCR9 lo CD103 hi ; RA-iTreg were CCR7 med CCR9 hi CD103 hi ; Rapa-iTreg were CCR7 hi CCR9 lo CD103 lo ; finally, RA+Rapa-iTreg were characterized by heterogeneous composition with the two most abundant populations being CCR7 med CCR9 hi CD103 hi and CCR7 med CCR9 hi CD103 lo . This suggested different migratory properties for each population. Monitoring the intensity of bioluminescent signal from adoptively-transferred iTreg-Luc (3, 6, 8, and 10 days after transfer) confirmed that Rapa-iTreg had a greater tendency to accumulate in lymph nodes compared with TGFβ- and RA-iTreg, with the latter population mainly accumulating in the gut. These results were reproduced by enumeration of congenic adoptivelytransferred iTreg. Conclusion These results indicate that although RA and Rapa are considered interchangeable in their promotion of stable iTreg, they confer a different migratory potential onto these cells. It is conceivable that these differences in migratory capacities result in different abilities to control local immune responses in vivo , a hypothesis that we are currently testing using models of inflammatory bowel disease and other immune-mediated disorders.