Figure 1.

Design of IL-12 and TCR vectors and in vitro validation.

(a) Diagrams representing the molecular structure of the retroviral vectors. IRES; internal ribosome entry site, LTR; long terminal repeat, GFP; green fluorescent protein, NFAT; nuclear factor activated T cells. (b) Anti-CD3/CD28-activated splenocytes were transduced with the Tet-IL-12 construct (Tet-IL-12) or vector control (VC) construct containing GFP only (Tet-GFP), and treated with Dox (1 µg/ml) overnight or left untreated. Representative flow cytometry plots showing the expression of Q8 and GFP in transduced T cells demonstrating the transduction efficacy and the level of induction in the presence and absence of Dox. Representative histogram overlay showing intracellular IL-12 staining in GFP-positive (induced) and GFP-negative (non-induced) cells after 4hrs treatment with BFA. The experiments were done at least 3 times with similar results. (c) Anti-CD3/CD28-activated splenocytes were transduced with NFAT-IL-12 construct or mock-transduced and analyzed by flow cytometry for GFP expression the following day. (d) Representative flow cytometry plots depicting intracellular IFNγ and TNFα staining of T cells transduced with NP-specific F5-TCR and either Tet-IL-12 (T_TCR+iIL-12) or Tet-GFP vector control (T_TCR+iGFP), and stimulated with EL4 (control) or EL4-NP tumor cells expressing the cognate antigen for 4hrs in the presence and absence of Dox. Dot plots show live-gated TCR-expressing cells (CD19⁺). Data shown represents at least 3 independent experiments. (e) Measurements of IL-12 secretion in culture supernatant of transduced T cells by enzyme-linked immunosorbent assay (ELISA). Graph shows mean ± SEM of duplicate values from two experiments. (f) Mean of body weight measurements over time post transfer of 5 × 10⁵ T_cIL-12 or T_mock transduced cells into sublethally irradiated (4Gy) recipient mice; baseline is 100%. n = 3 mice per group. (g)Mean of body weight measurements over time of mice receiving 5 × 10⁵ Tet-IL-12 or NFAT-IL-12 transduced T cells. Mice received Tet-IL-12 transduced T cells were split into two cohorts: one received Dox (2mg/ml) in drinking water (+ Dox) and the other cohort left untreated (-Dox). n = 5 mice per group. (h) Kinetics of transient IL-12 induction in vivo. C57BL/6 mice (Thy1.2⁺) were sublethally irradiated (4Gy) and injected intravenously with 5 × 10⁵ Tet-IL-12 transduced T cells (Thy1.1⁺). On day 4 post T cell transfer, mice were split into two groups, one group received Dox-containing water (2mg/ml) for 3 consecutive days and the other group left untreated. Blood samples were obtained at 24hrs, 48hrs and 72hrs following Dox administration, and 24hrs following Dox withdrawal. Representative flow cytometry plots showing the levels of GFP expression. Cells were pre-gated on PI- singlet Thy1.1+ lymphocytes. n = 4 mice (-Dox); n = 6 mice (+ Dox) (top). Mean of body weight measurements over time showing lack of toxicity with temporal Dox induction post transfer of 5 × 10⁵ Tet-IL-12 transduced cells into sublethally irradiated (4Gy) recipient mice. Mice received Tet-IL-12 transduced T cells were split into two cohorts: one received Dox (2mg/ml) in drinking water (+ Dox) for 3 days and the other cohort left untreated (-Dox). Graph shows mean ± SEM. n = 5 mice per group (bottom).