Clinical trials of adoptively transferred regulatory T cells (T_Regs) have been hindered by the short lifespan of these transferred T_Regs. Systemic injection of low-dose interleukin (IL)-2 preferentially stimulates the high-affinity trimeric IL-2 receptor, abundantly expressed on T_Regs, leading to greater cellular expansion. However, cytotoxic CD8 T cells and NK cells are also significantly expanded by this strategy. Here, we describe novel engineered T_Regs conjugated with IL-2 nanogel (NG) particles before adoptive transfer. The NG's gradual secretion of IL-2 in vivo selectively promotes T_Reg viability, and is engineered by reversibly crosslinking carrier-free protein using a bis-N-hydroxy succinimide (NHS) crosslinker. Incorporation of monoclonal antibodies against the highly-expressed cell surface phosphatase CD45 acts as an anchor to retain the NG on T_Regs. Upon T_Reg activation, increased reduction activities lead to the progressive degradation of the crosslinker and sustained IL-2 release. We show that the coupling efficiency of NG to magnetically-isolated murine T_Regs is above 80% (n=5 experiments; ****P<0.0001), and that NG-conjugated T_Regs maintain their FoxP3 expression after coupling compared to the control (CT) T_Regs (99% vs. 92%, CT vs. NG; n=5 experiments; P=n.s.). To test T_Reg homeostasis in vivo, Immunodeficient RAG1^-/- mice received BALB/c skin transplants and were injected with 1:1 T_Regs:CD8 T cells from mismatched C57BL/6 donors to induce alloimmunity. Seven days post adoptive transfer, a two-fold increase in T_Regs was observed in the spleens of mice that received NG T_Regs compared to CT T_Regs (n=11/group; **P<0.01). Furthermore, contrary to when IL-2 is systemically injected, we saw a more than three-fold increase in T_Regs:CD8 and T_Regs:NK ratios in the NG-treated mice (n=11/group; *P<0.05). In a GvHD model, the adoptive transfer of NG T_Regs reduced disease progression compared to CT T_Regs. In summary, we show that the IL-2 NG T-cell conjugation strategy can efficiently and specifically improve the in vivo expansion of transferred T_Regs while sparing CD8 and NK T cells, thus improving the therapeutic efficacy of T_Regs and IL-2.

CITATION INFORMATION: Eskandari S., Bandeira Melo M., Assaker J., Mansouri A., Cai S., Al Dulaijan B., Mohamed M., Irvine D., Azzi J. Engineering Regulatory T Cells with Interleukin (IL-)2 Nanogel for Improved Alloimmune Suppression Am J Transplant. 2017;17 (suppl 3).

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