*Purpose: Adoptive cell transfer of regulatory T cells (T_Reg) has shown great potential in animal models of auto- and alloimmunity. The translation of T_Reg therapies to the clinic, however, has been impeded by the short in vivo homeostasis of T_Regs. Here, we describe a novel drug-delivery platform which can be engineered on T_Regs to secrete interleukin (IL-)2 in response to TCR activation, and thus maintain T_Regs in sites of antigen encounter.

*Methods: CD4⁺CD25⁺FoxP3⁺ T_Regs were magnetically sorted from C57BL/6 (B6) splenocytes and conjugated with nanogel (NG) prior to adoptive transfer studies. NG particles were synthesized by reversibly crosslinking IL-2/Fc fusion (IL-2) protein with a bis-N-hydroxy succimide crosslinker. Further surface modification with monoclonal anti-CD45 and PEG-PLL ensured long-term surface retention.

*Results: In primary in vitro experiments we proved that IL-2 NG could be backpacked onto >90% of T_Regs and spur 4-fold greater expansion compared with unmodified (CT) T_Regs in a seven-day culture. Additionally, in a T_Reg suppression assay, we found the suppressive qualities of NG compared to CT T_Regs to be unaltered. To test the T_Reg homeostasis in vivo, we transplanted BALB/c skin onto B6.RAG1^-/- mice and injected an equal ratio of B6 T_Regs to CD8 T cells. Comparing CT T_Regs, CT T_Regs plus systemic IL-2, and NG T_Regs 7 days post adoptive transfer we found that only CT+IL-2 treatment improved peripheral T_Reg homeostasis. These mice, however, showed an unrepressed expansion of graft-resident CD8⁺CD69⁺ T cells. NG-treated mice, on the contrary, exhibited gross suppression of CD8 cells in the draining lymph nodes and skin graft. We later confirmed that solely NG T_Reg therapy could significantly prolong the mean skin graft survival (6 vs.18 days). We hypothesized that the NG platform was selectively benefitting antigen-specific T_Regs, and to test this, we performed OVA to B6.RAG1^-/- skin transplants and injected an equal ratio of OT-II T_Regs to OT-I CD8 T cells. For the NG T_Reg-treated mice we observed a resounding suppression of OT-I CD8 T cells in the skin graft and a 30-fold increase in the T_Reg:CD8 ratio. Finally, to assess if this platform could be translated to humans, we transplanted NSG mice with skin grafts from a healthy patient and transferred PBMCs and T_Regs from a healthy donor 7 days later. We found that 21 days post adoptive transfer, the human NG T_Reg treatment had efficiently attenuated CD8 T cell proliferation in the skin grafts, and while 5/6 (83%) of NG grafts remained healthy, only 3/6 (50%) of CT grafts and 1/4 (25%) of CT+IL-2 grafts did.

*Conclusions: Here, we describe a novel method for enhancing T_Reg transfer therapy through spatiotemporal provision of IL-2 to antigen-specific T_Regs. Importantly, our platform can be harnessed to complement CAR-T_Reg therapies.

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