Figure 7.

RT +anti-CD25+anti-CD137 is sufficient to reprogram Tregs into cytokine-producing cytolytic effectors. (A) Experimental procedure for adoptive transfer by tail vein injection of GFP⁺ Tregs from non-tumor-bearing DEREG mice into MOC2 tumor-bearing RAG1^–/– mice followed by RT+anti-CD25+anti-CD137 treatment and harvesting of tumors for flow cytometric analysis. (B) Proportion of CD45⁺CD4⁺Foxp3⁺GFP⁺ Tregs expressing TNFα, IFNγ, or Perforin in MOC2 tumors from RAG1^–/– mice following adoptive transfer of Tregs from DEREG mice and treatment of RT+anti-CD25+anti-CD137 harvested 9 days post-RT versus RT alone controls (n=7 mice/group, two-tailed unpaired T test). (C) Mean fluorescence intensity of Eomesodermin in CD45⁺CD4⁺Foxp3⁺GFP⁺ Tregs in MOC2 tumors from RAG1^–/– mice following adoptive transfer of Tregs from DEREG mice and treatment of RT+anti-CD25+anti-CD137 harvested 9 days post-RT versus RT alone controls (n=7 mice/group, two-tailed unpaired t test). (D) Experimental procedure for Treg cytolytic assay using GFP⁺ Tregs harvested from tumor-bearing DEREG mice harvested 8 days post-RT with 10 Gy and MOC2 cells irradiated with 10 Gy 72 hours prior and stained with cell Tracker red. Cells were cultured in conditioned media from irradiated MOC2 cells with an 2:1 effector to target ratio for 4 hours at 37°C prior to detection of live cells by live/dead aqua viability dye. (E) Proportion of live MOC2 cells after incubation with Tregs harvested from tumor-bearing DEREG mice treated with IL-2 and anti-CD137 (n=4) compared with IL-2 and matching isotype control antibody (n=3). Each point represents technical replicates (p=0.0548, two-tailed unpaired t test). IFNγ, interferon γ; IL-2, interleukin 2; RT, radiation therapy; TNFα, tumor necrosis factor α; Treg, regulatory T cell.