Fig. 4. Nanogel-engineered T_regs prolong murine allograft survival better than conventional T_regs and differentially promote antigen-specific T_regs.

Allograft survival was studied using the same Rag1^−/− to BALB/c model as described in Figure 3, adoptively transferring 5.0×10⁵ C57BL/6 CD8α⁺ cells and 5.0×10⁵ C57BL/6 T_regs on day 3 post-transplant, without IL-2/Fc NG, with systemic IL-2/Fc, or with IL-2/Fc NG (n = 6 mice/group). (A–B) Experimental mouse survival model (A) and timeline (B). (C) Survival curves of each of the treatment groups. In vivo T cell responses were tracked by transplanting BALB/c skin grafts onto Rag1^−/− mice and adoptively transferring 5.0×10⁵ C57BL/6 CD8α⁺ cells transduced with Cypridina luciferase (CLuc) and 5.0×10⁵ C57BL/6 T_regs transduced with Gaussia luciferase (GLuc), either unmodified or decorated with IL-2/Fc NGs, on day 3 post-transplant (n = 3 biological replicates/condition, 1 representative experiment). (D) Experimental bioluminescent T cell tracking model and timeline. (E) In vivo imaging of CT and NG T_reg GLuc signals following intravenous coelenterazine administration. (F) In vivo imaging of CD8α⁺ T cell CLuc signals following intravenous Cypridina luciferin administration. (G) Ratios of T_reg-GLuc signal in the skin grafts compared with the whole animals, and fold changes of T_reg-to-CD8α (GLuc-to-CLuc) signal in the skin grafts compared with the whole animals. Finally, antigen-specific alloimmunity was assessed with an OVA-OT-1-OT-2 model. Rag1^−/− mice were transplanted with OVA allografts 3 days before the adoptive transfer of 5.0×10⁵ OT-1 CD8α⁺ T cells and 2.5×10⁵ OT-1 CD4⁺CD25⁺ T_regs and 2.5×10⁵ OT-2 CD4⁺CD25⁺ T_regs, left untouched or coupled IL-2/Fc NGs. On day 7, the mice were euthanized, and various tissues were analyzed by flow cytometry (n = 7 biological replicates/condition, 2 experiments). (H–I) Antigen-specific mouse model (H) and timeline (I). (J) Fold changes of the ratio of OT-2 T_regs to OT-1 CD8α in the distal-to-proximal draining sites. (K) Fold changes of the [³H]-Thymidine incorporation in mixed lymphocyte reactions of host Rag1^−/− splenocytes with irradiated donor OVA splenocytes. All fold changes were normalized against the CT condition. Throughout, data represent boxplots with median, interquartile range, minimum, maximum, and all individual data points of the denoted experimental groups. P values were calculated with log-rank Mantel-Cox tests, or with independent samples two-tailed Student t-tests, and non-parametric Kolmogorov-Smirnov tests were performed when the assumption of homoscedasticity could not be met.

CT, control; CLuc, Cypridina luciferase; GLuc, Gaussia luciferase; LNs, lymph nodes; MLR, mixed lymphocyte reactions; NG, nanogel.