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Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

Galvan DL, O'Neil RT, Foster AE, Huye L, Bear A, Rooney CM, Wilson MH - PLoS ONE (2015)

Bottom Line: Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments.We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy.We next gene-modified OT-I cells to express mIL-12.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

No MeSH data available.


Related in: MedlinePlus

piggyBac transposon modification of mouse splenocytes.A, Splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1 using the Neon transfection system and transfection efficiency was evaluated via flow cytometry using antibodies directed against the Thy1.1 antigen at day 1 and day 7 post transfection. Shown is a representative of 3 independent experiments. B, mouse splenocytes could be cultured short term exhibiting cell growth. Shown is a representative of 3 independent experiments. Arrows indicate a split to 5 X 106 cells for continued growth.
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pone.0140744.g003: piggyBac transposon modification of mouse splenocytes.A, Splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1 using the Neon transfection system and transfection efficiency was evaluated via flow cytometry using antibodies directed against the Thy1.1 antigen at day 1 and day 7 post transfection. Shown is a representative of 3 independent experiments. B, mouse splenocytes could be cultured short term exhibiting cell growth. Shown is a representative of 3 independent experiments. Arrows indicate a split to 5 X 106 cells for continued growth.

Mentions: To determine the efficiency of piggyBac for the gene modification of murine T cells, we created a piggyBac vector expressing the luciferase reporter and Thy1.1 (Fig 1). Mouse splenocytes were transfected with pT-effLuc-Thy1.1 and pCMV-PB, then stimulated with concanavalin A, and transfection efficiency was quantitated by flow cytometry using an antibody to Thy1.1. From an initial transfection efficiency of 49% (± 5%, N = 3, SEM), 28% (± 4%, N = 3, SEM) of Thy1.1 and CD3 positive cells persisted on day 7 (Fig 3A). Stably transfected mouse T cells exhibited growth in short-term culture in vitro (Fig 3B).


Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

Galvan DL, O'Neil RT, Foster AE, Huye L, Bear A, Rooney CM, Wilson MH - PLoS ONE (2015)

piggyBac transposon modification of mouse splenocytes.A, Splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1 using the Neon transfection system and transfection efficiency was evaluated via flow cytometry using antibodies directed against the Thy1.1 antigen at day 1 and day 7 post transfection. Shown is a representative of 3 independent experiments. B, mouse splenocytes could be cultured short term exhibiting cell growth. Shown is a representative of 3 independent experiments. Arrows indicate a split to 5 X 106 cells for continued growth.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608718&req=5

pone.0140744.g003: piggyBac transposon modification of mouse splenocytes.A, Splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1 using the Neon transfection system and transfection efficiency was evaluated via flow cytometry using antibodies directed against the Thy1.1 antigen at day 1 and day 7 post transfection. Shown is a representative of 3 independent experiments. B, mouse splenocytes could be cultured short term exhibiting cell growth. Shown is a representative of 3 independent experiments. Arrows indicate a split to 5 X 106 cells for continued growth.
Mentions: To determine the efficiency of piggyBac for the gene modification of murine T cells, we created a piggyBac vector expressing the luciferase reporter and Thy1.1 (Fig 1). Mouse splenocytes were transfected with pT-effLuc-Thy1.1 and pCMV-PB, then stimulated with concanavalin A, and transfection efficiency was quantitated by flow cytometry using an antibody to Thy1.1. From an initial transfection efficiency of 49% (± 5%, N = 3, SEM), 28% (± 4%, N = 3, SEM) of Thy1.1 and CD3 positive cells persisted on day 7 (Fig 3A). Stably transfected mouse T cells exhibited growth in short-term culture in vitro (Fig 3B).

Bottom Line: Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments.We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy.We next gene-modified OT-I cells to express mIL-12.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

No MeSH data available.


Related in: MedlinePlus