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. 2003 May-Jun;26(3):190-201.
doi: 10.1097/00002371-200305000-00003.

Interleukin-2-independent proliferation of human melanoma-reactive T lymphocytes transduced with an exogenous IL-2 gene is stimulation dependent

Ke Liu  1 , Steven A Rosenberg
Affiliations

Interleukin-2-independent proliferation of human melanoma-reactive T lymphocytes transduced with an exogenous IL-2 gene is stimulation dependent

Ke Liu et al. J Immunother. 2003 May-Jun.

Abstract

A major obstacle limiting the efficacy of adoptive T-cell transfer (adoptive immunotherapy) to treat patients with cancer is the short survival of the transferred cells. These in vitro activated T cells depend on the growth factor, interleukin (IL)-2, and may undergo apoptosis in vivo when they are transferred. The authors previously reported that the need for an exogenous source of IL-2 could be abrogated in vitro by retrovirally transducing antitumor T lymphocytes with an exogenous IL-2 gene. Here they report that this growth of IL-2 transductants depended on restimulation of the T-cell receptor complex and appeared to be regulated at the transcriptional level of the transduced IL-2 gene. The transduced IL-2 transcript was barely detectable in IL-2-transductants just before they died without restimulation, and they expressed a low level of the CD25 molecule, the alpha chain of the IL-2 trimeric receptor complex. Melanoma-specific tumor-infiltrating lymphocytes (either bulk or CD8+ cells alone), when transduced with an IL-2 retroviral vector, could produce IL-2 upon tumor stimulation and proliferated after the destruction of autologous tumor cells in the absence of added IL-2. Control vector-transduced tumor-infiltrating lymphocytes failed to do so under the same conditions. These findings provide a foundation for the development of clinical efforts to adoptively transfer melanoma-specific tumor-infiltrating lymphocytes transduced with an IL-2 retroviral vector for the treatment of patients with metastatic melanoma to evaluate the fate and therapeutic effect of these IL-2 gene-modified antitumor T lymphocytes in vivo.

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Figures

FIG. 1
FIG. 1
IL-2YFP PBMC transductants self-regulated the transduced IL-2 gene in the absence of added IL-2 without further stimulation. Viable cells were counted by trypan blue exclusion and collected on days 8 (lane 1), 26 (lane 2), and 34 (lane 3) of the REP4 experiment without added IL-2, as described in the text. The RT-PCR was performed using primers for vector-derived (A) IL-2 gene and (B) beta-actin gene. (C) Genomic DNA PCR was performed using primers for retroviral genome. Different cycles of PCR were performed with the same pattern of results seen (data not shown). Shown here are photographs of 1% agarose gel electrophoresis of the PCR products after 29 cycles of amplification.
FIG. 2
FIG. 2
IL-2YFP PBMC transductants still expressed CD25 when they started to lose viability in the absence of exogenous IL-2. Viable cells were counted by trypan blue exclusion and taken on day 8 (A and B) and day 34 (C and D) of a REP4 experiment (B and D) without or (A and C) with 300 IU/mL added IL-2. Cells (5 × 105) were subjected to FACS analysis after staining with PE-conjugated antibodies. Shown here are histograms of anti-CD25–stained cells (thick line) superimposed over those of isotype control– stained cells (thin line).
FIG. 3
FIG. 3
The SBIL-2 vector-transduced human melanoma-specific TILs at frequencies greater than 73%. Bulk TIL1913 and TIL1941 were obtained and transduced with SBIL-2 and YFP retroviral vector, respectively, as described in Materials and Methods. Five days after transduction, the cells were subjected to FACS analysis and TIL 1913 cells were subjected to genomic DNA PCR. (A) Histograms of the percentage of YFP+ cells for YFP vector-transduced TILs (thick line) superimposed on the untransduced cells (thin line). (B) A schema for the genomic DNA PCR of the transduced TIL1913 cells. The forward primer indicated by a thin arrow and the reverse primer indicated by a thick arrow contained sequences from the exon 1 and exon 2 of the IL-2 gene, respectively. This pair of primers amplified a 311-bp fragment from the endogenous IL-2 gene containing the intron sequences, whereas the transduced retroviral IL-2 gene directed a synthesis of 221-bp fragment without intron sequences. (C, upper) A 2% agarose electrophoretic gel for the amplified PCR products using the schema illustrated in panel B. An equivalent amount of DNA representing 1,250 cells per sample was subjected to 29 cycles. No PCR cycle difference was seen in the pattern of amplified fragments (data not shown). Lane 1, untransduced cells; lanes 2 and 3, duplicate samples of the SBIL-2–transduced TIL1913 cells; lanes 4 and 5, duplicate samples of the control-vector YFP-transduced cells; M, DNA markers. The gel was subjected to Southern blotting and hybridization using a 32P-labeled oliogonucleotide containing the sequences of the exon 1 of the IL-2 gene (indicated by a small stippled box of panel B). (C, lower) A phosphoimage of the blot. The endogenous IL-2 gene band was indicated by a filled arrow, and the vector-derived IL-2 gene band was indicated by a solid arrow. After quantification of the signals by the phosphoimager, the ratio (percentage) shown at the bottom of the image was calculated by dividing the signal of the vector IL-2 gene band by that of the genomic DNA band. The fact that this ratio is greater than 100% in the SBIL-2–transduced cells implies that nearly all cells harbored one copy of the transduced IL-2 gene (a transduction efficiency of 100%), with some cells containing more than one copy of the retroviral sequences.
FIG. 4
FIG. 4
The SBIL-2–transduced TIL1913 cells proliferated after the destruction of the autologous tumor cells in the absence of added IL-2. Untransduced and transduced cells (1 × 105) were plated in duplicate 5 days after transduction in 100 μL medium in the wells of a 96-well flat-bottom plate, followed immediately by the addition of 1 × 105 autologous melanoma tumor cells previously irradiated at 100 Gy in 100 μL medium. Interleukin-2 was added to some wells at the indicated concentrations. Six days from the onset of the coculture at 37°C, 5% carbon dioxide, the cells in each well were transferred individually to wells of 48-well plates. (A) On day 8, each well was photographed (scale bar, 100 μm). Fourteen days after the onset of the experiment, viable cells were counted by trypan blue exclusion. Tumor-alone wells contained no viable cells. (B) The mean cell count ± SEM. The SBIL-2–transduced TIL1913 cells proliferated after the destruction of the autologous tumor cells in the absence of any exogenous IL-2. Untransduced or the control vector YFP-transduced cells failed to proliferate under the same condition.
FIG. 5
FIG. 5
SBIL-2–transduced TIL1913 cells produced intracellular IL-2 after additional expansion. Seven days after transduction, SBIL-2–transduced TIL cells were expanded in a REP protocol without IL-2 and YFP-transduced cells were expanded in the presence of 300 IU/mL IL-2. Fourteen days from the onset of this expansion, CD4+ cells were depleted using Dynal beads. The remaining CD8+ cells were washed with medium without IL-2 three times before replating onto the wells of a 24-well plate precoated with OKT3 without IL-2. After overnight stimulation in the presence of Golgi stop, cells were harvested and subjected to intracellular FACS with PE-conjugated antibodies. Shown here are histograms of cells stained for IL-2 superimposed over those of cells stained with an isotype-control antibody. Only SBIL-2–transduced TIL cells were positive for intracellular IL-2.
FIG. 6
FIG. 6
SBIL-2–transduced CD8+ TIL cells alone could destroy tumor cells and proliferate in the absence of added IL-2. Seven days after transduction, SBIL-2–transduced TIL1913 cells were expanded in a REP protocol without IL-2, and YFP-transduced cells were expanded in the presence of 300 IU/mL IL-2. Fourteen days from the onset of this expansion, CD4+ cells were depleted using Dynal beads. The remaining CD8+ cells and undepleted (bulk) cells were washed with medium without IL-2 three times before replating onto the wells of a 96-well plate containing preirradiated autologous tumor cells (100 Gy) without or with 300 IU/mL IL-2, as described in Figure 4. Six days from the onset of the coculture with tumor cells, each well was split one to two and 100 μL fresh medium with or without IL-2 was added. Twelve days after the onset of the coculture, photographs were taken of each well. (A) SBIL-2–transduced cells (scale bar, 100 μm). (B) YFP-transduced cells. Upper panels, no IL-2. Lower panels, +IL-2.
FIG. 6
FIG. 6
SBIL-2–transduced CD8+ TIL cells alone could destroy tumor cells and proliferate in the absence of added IL-2. Seven days after transduction, SBIL-2–transduced TIL1913 cells were expanded in a REP protocol without IL-2, and YFP-transduced cells were expanded in the presence of 300 IU/mL IL-2. Fourteen days from the onset of this expansion, CD4+ cells were depleted using Dynal beads. The remaining CD8+ cells and undepleted (bulk) cells were washed with medium without IL-2 three times before replating onto the wells of a 96-well plate containing preirradiated autologous tumor cells (100 Gy) without or with 300 IU/mL IL-2, as described in Figure 4. Six days from the onset of the coculture with tumor cells, each well was split one to two and 100 μL fresh medium with or without IL-2 was added. Twelve days after the onset of the coculture, photographs were taken of each well. (A) SBIL-2–transduced cells (scale bar, 100 μm). (B) YFP-transduced cells. Upper panels, no IL-2. Lower panels, +IL-2.
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