Article Figures & Data
Figures
- Figure 1.
Pmel-1 CD8+ T cells engineered to overproduce IL-12 possess enhanced functionality. A, the MSGV-1IL-12 retroviral vector (left), which encodes the p40 and p35 subunits of IL-12 linked by (Gly4Ser)3 flexible linker. SD, splice done; SA, splice acceptor; LTR, long terminal repeat; Ψ, packaging sequence. Pmel splenocytes were stimulated with 1 μmol/L hgp10025–33 for 2 d, transduced with the MSGV-1IL-12 retrovirus, and analyzed by flow cytometry 3 d after for IL-12 expression (right). B, the IL-12–transduced pmel-1 CD8+ cells in A were analyzed by intracellular staining without or with a 4-h secondary stimulation with PMA and ionomycin. C, quantification of the percentage of CD8+ IFN-γ+ cells following secondary stimulation in mock- versus IL-12–transduced pmel cells (n = 5; *, P < 0.05). All experiments are representative of at least two independent experiments.
- Figure 2.
Adoptive transfer of IL-12–engineered pmel-1 CD8+ T cells induces the regression of large, established tumors without exogenous IL-2 and vaccine. A, antitumor activity following the adoptive transfer of 1 × 105 (*), 5 × 104 (Ψ), or 1 × 104 (**) pmel-1IL-12-TD cells into sublethaly irradiated (5 Gy) mice bearing B16 tumors (n = 5) established for 14 d (*, Ψ, and **, P < 0.05, compared with no treatment) with no evidence of weight loss (right). B, mock- versus IL-12–transduced pmel-1 thy1.1+ CD8+ T cells (2.5 × 105) were transferred into sublethaly irradiated (5 Gy) mice (n = 3) and spleens were harvested on days 3, 7, and 14 after transfer and analyzed by flow cytometry for the percentage of transferred CD8+ thy1.1+ cells (left). B, right, quantification of the percentage of CD8+ thy1.1+ cells in spleens (* and **, P < 0.05, compared with mock). All experiments are representative of at least two independent experiments.
- Figure 3.
Treatment with IL-12–engineered CD8+ T cells leads to increased tumor infiltration of adoptively transferred cells stably expressing IL-12 and increased tumor infiltration by endogenous NK and CD8+ T cells. A, representative s.c. tumor samples (top) excised 7 d (n = 5) following the transfer of 5 × 105 mock- or IL-12–transduced pmel-1 CD8+ T cells into sublethaly irradiated (5 Gy) mice bearing B16 tumors established for 14 d. Corresponding H&E stains (bottom; ×100 magnification) for tumors in the top. B, mock- versus IL-12–transduced pmel-1 thy1.1+ CD8+ T cells (5 × 105) were transferred into sublethaly irradiated (5 Gy) mice (n = 3), and tumors were excised 7 d after transfer, mechanically disrupted, and enumerated by flow cytometry for infiltration of adoptively transferred thy1.1+ CD8+ cells per gram of tumor (left; *, P < 0.05, compared with control). B, right, IL-12 expression in tumor-infiltrating thy1.1+ CD8+ cells (gated on the CD8+ population). C, the tumor samples in B were also analyzed by flow cytometry for the number of endogenous thy1.1−, NK1.1+ cells (left; *, P < 0.05, compared with control) and thy1.1−, CD8+ cells (right; **, P < 0.10, compared with control) per gram of tumor. All flow cytometry plots gated on live propidium iodide–negative (PI−) populations. All experiments are representative of at least two independent experiments.
- Figure 4.
IL-12–engineered pmel-1 CD8+ T cells display enhanced antitumor responses compared with rIL-12 administered exogenously. A, antitumor activity following the adoptive transfer of 1 × 105 pmel-1IL-12-TD cells alone or in combination with 9 × 105 mock-transduced pmel-1 CD8+ T cells into sublethaly irradiated (5 Gy) mice bearing B16 tumors (n = 5) established for 14 d. B, treatment responses in sublethally irradiated (5 Gy) tumor-bearing mice (n = 5) following the transfer of 1 × 106 mock-transduced pmel-1 cells with exogenous rIL-12 daily administered i.p. for 3 d (0.25, 1, or 2 μg), 1 × 105 mock-transduced Tc1 cells (polarized with 3.33 ng/mL IL-12 ex vivo), or 1 × 105 pmel-1IL-12-TD cells cotransferred with 9 × 105 mock-transduced cells (*, P < 0.05, compared with all other treatment groups). All experiments are representative of at least two independent experiments.
- Figure 5.
Host irradiation (5 Gy) is required for antitumor immunity of adoptively transferred pmel-1IL-12-TD. A, tumor treatment of sublethally irradiated (5 Gy) or nonirradiated (0 Gy) WT B16 tumor-bearing mice (n = 5) treated with 1 × 105 pmel-1IL-12-TD cells. B, enumeration of thy1.1+ CD8+ T cells from isolated tumor samples and spleens (n = 4) 6 d after the adoptive transfer of 5 × 105 thy1.1+ pmel-1IL-12-TD into 0 Gy– or 5 Gy–treated hosts (left). Enumeration (n = 5) of thy1.1− CD4+ T cells in tumor samples (right; *, P < 0.05, compared with 0-Gy treatment). Flow cytometry of isolated thy1.1− CD4+ T cells for Foxp3 expression (right; gated on thy1.1−, CD4+ population). All flow cytometry samples gated on live PI− populations. C, antitumor responses following the transfer of 1 × 105 pmel-1IL-12-TD cells into nonablated (0 Gy) WT or TCRα−/− B16 tumor-bearing mice (n = 5). All experiments are representative of at least two independent experiments.
- Figure 6.
Tumor antigen–specific TCRs are critical for the therapeutic responses of IL-12–engineered T cells. A, cytofluorometric analysis of open-repertoire (WT) CD8+ T cells expressing IL-12 or pmel-1 TCR (Vβ13+ staining) individually or on the same cell following retroviral transduction. All plots gated on CD8+ cells. B, antitumor responses in sublethally irradiated (5 Gy) tumor-bearing WT mice (n = 5) treated with 1 × 106 single-transduced cells (expressing either the pmel-1 TCR or IL-12), a combination of both single-transduced populations (2 × 106 cells), or 1 × 106 CD8+ T cells coexpressing the pmel-1 TCR and IL-12 (*, P < 0.05, compared with all other treatments). C, enumeration of tumor, spleen, and draining lymph nodes (n = 5) for adoptively transferred open-repertoire CD8+ thy1.1+ T cells engineered to express IL-12 and/or the pmel-1 TCR as in A. *, P < 0.05, compared with mock. All flow cytometry samples gated on live PI− populations. All experiments are representative of at least two independent experiments.
Additional Files
Supplementary Data, Kerkar, et al.
Files in this Data Supplement:
Volume 70, Issue 17
