Direct cytotoxicity by Trp1 cells as a result of OX40 engagement during CTX-induced lymphopenia is Eomes dependent. (A) C57BL/6 wild-type or Rag1^−/− mice (6–10/group) were inoculated intradermally in the flank with B16 cells. After 3 wk, mice were injected with CTX, followed the next day by OX86 (or IgG as control) and Trp1 cells. Tumors were measured periodically. Graphs represent tumor area of individual mice over time for each treatment. (B) C57BL/6 mice (10 mice/group) were injected subcutaneously with B16 in Matrigel. 3 wk after tumor challenge, CTX was injected. The next day, mice were injected with Trp1 cells and OX86 (or IgG). On day 14 after treatment, Trp1 cells were purified by FACS from splenocytes and were used for in vitro killing assays using B16 cells as targets at a 10:1 effector to target ratio. Means of three individual wells and SEM are shown. *, P < 0.05. (C) Single cell suspensions of TDLNs and spleens from mice that were treated, as in B (8 mice/group), were stimulated overnight with Trp1 peptide in the presence of monensin and anti–CD107a-FITC. The next day, the samples were stained for CD45.1 (Trp1 cells) and other phenotypic markers and analyzed by flow cytometry. Plots show CD107a MFIs of individual mice gated on ViD⁻ CD45.1⁺ CD4⁺ Foxp3⁻. *, P < 0.005; **, P < 0.05. Error bars represent SEM. (D) Single cell suspensions of TDLNs and spleens from mice that were treated as in B (4–5 mice/group) were stained and analyzed by flow cytometry for GrzB and other phenotypic markers on day 14 after treatment. Representative plots are shown for GrzB versus CD45.1. Events were pregated on ViD⁻, CD4⁺, and Foxp3⁻. (E) C57BL/6 mice (7–10/group) were treated as in B and bled on day 14 after treatment. PBMCs were stained for GrzB and Eomes and analyzed by flow cytometry. Events were pregated on ViD⁻, CD45.1⁺, CD4⁺, and Foxp3⁻. Representative plots are shown of GrzB versus Eomes. (F) C57BL/6 mice (4–5/group) were injected subcutaneously with B16 in Matrigel. 3 wk after tumor challenge, CTX was injected. The next day, mice were injected with Trp1 cells and OX86, DTA-1, FGK45, 9D9, 10F.9G2, or IgG. Two additional doses of 9D9 and 10F.9G2 were given every 3 d. On day 14 after treatment, single cell suspensions were stained for Eomes and other phenotypic markers and analyzed by flow cytometry. Events were pregated on ViD⁻, CD45.1⁺, CD4⁺, and Foxp3⁻. Representative plots are shown of GrzB versus Eomes. (G) In vitro activated Trp1 cells were transduced with Eomes shRNA-GFP retrovirus or GFP retrovirus as control. Transduced cells were FACS sorted on GFP^high gate 5 d later. Cells were stained intracellularly for Eomes, GrzB, and T-bet and analyzed by flow cytometry. Representative histograms are shown. (H) In vitro cytotoxicity assay with transduced Trp1 cells sorted on GFP^high gate. B16 cells were used as targets at a 10:1 effector to target ratio. Bars represent the means of duplicate wells and the SEM is shown. (I) C57BL/6 Rag1^−/− mice (6/group) were inoculated intradermally in the flank with B16 cells. 3 wk later, CTX was injected followed the next day by the transfer of 70,000 transduced Trp1 cells (sorted on GFP^high) and injection of OX86. Tumor area was periodically monitored. Each point represents the mean tumor area and error bars are SEM. *, P < 0.05. All experiments were repeated at least twice with similar results.