CD4+CD25+Foxp3+ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4+ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.

The natural CD4+CD25+ T regulatory (Treg) lymphocyte has emerged as a critical cell for controlling immune responses to self, foreign proteins, and pathogens. Identified initially by the constitutive expression of CD4 and CD25, natural Tregs suppress a variety of immune cells and responses, including CD4+CD25- proliferation and IL-2 production, and CD8 cell proliferation, IFNgamma production and CTL activity. Although natural Tregs require activation with specific antigen to attain their suppressive phenotype, once activated they execute inhibition in an antigen specific as well as non-specific (bystander) fashion. Treg suppression depends on IL-2, CD25, and cell:cell contact. The use of live cell imaging in vivo and in vitro to visualize the dynamic cell:cell interactions involving natural Tregs as well as the CD4+CD25+ Treg inhibitory hybridoma RD6 has refined the mechanistic models of contact dependent Treg suppression.

Regulatory T cell plays an important role in controlling immunity to self and foreign antigens. CD4+CD25+ require T cell receptor and IL-2 receptor activation for in vivo / in vitro expansion and function, but the source of IL-2 remains unknown. In identifying the important paracrine source of IL-2 for Treg cells, we concentrated on the dendritic cell as the paracrine source, because in vitro imaging studies demonstrated fine localization of CD25 at the membrane interface between RD6 and dendritic cells. We used ELISPOT assay to detect IL-2 production by the freshly isolated splenic and bone marrow dendritic cells Dendritic cells secrete a small fraction of IL-2 early in culture, CpG-B enhances the IL-2 production of DCs. Coculture with regulatory cells reduces the level of detectable IL-2.We examined the direct effect of dendritic cell IL-2 on Treg function, CD4+CD25+ cells suppressed CD4+CD25- effectors cells when stimulated with WT dendritic cells, but not when stimulated with IL-2KO DCs. Anti-CD25 abrogates the function of both Treg and RD6 co-cultured with WT DCs, suggesting that CD25 is required for delivery of IL-2 into the Tregs for processing and the addition of high doses of exogenous IL-2 can restore the inhibitory function of Treg and RD6 cells co-cultured with IL-2 -/- dendritic cells. These data suggest that DC IL-2 is essential for Treg function.

The CD4+CD25+ regulatory T cell plays an important role in controlling immunity to self and foreign antigens. CD4+CD25+ require T cell receptor and IL-2 receptor activation for in vivo development/ expansion, in vitro expansion and for in vitro function, but the source of IL-2 remains unknown. we used the DO11.10 transgenic mouse model, whose T cell receptor shows allogeneic cross-reactivity to B6. Purified dendritic cells from WT or IL-2 −/− B6 were labeled with cell tracker green and cultured overnight with natural CD4+CD25+ Tregs from DO11.10 transgenic mice, the RD6 inhibitory hybridoma, or the control RD6N non-inhibitory hybridoma. DCs were FACS sorted then used to stimulate fresh CD4+CD25− DO11.10 effectors T cells in an IL-2 ELISPOT assay. CD4+CD25+ cells suppressed CD4+CD25− effectors cells up to 90% when stimulated with WT dendritic cells, but only 29% when stimulated with IL-2 −/− DCs. Similar results were obtained using the RD6 inhibitory hybridoma> RD6 suppressed a mean of 97% with WT dendritic cells and only 25% with IL-2 −/− dendritic cells. The result supports a model that Tregs require signal 3 activation for the APC to gain inhibitory function.