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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

Functional expression of IL-12 from a piggyBac transposon.A, HeLa cells were transfected with the pT-IL12-2A-venus transposon. DAPI (4',6-diamidino-2-phenylindole) stain was utilized to visualize cell nuclei (top) and immunofluorescence of the venus reporter gene was used to visualize protein expression (bottom). Shown is a representative of 3 independent experiments. Culture media from these cells was analyzed for mIL-12 production resulting in 31 ± 5pg/μl of mIL-12 (N = 3, ± SEM). B, B16 melanoma cells were stably transfected with pT-mIL12 in the presence of pCMV-m7pB. 5 X 105 B16 cells were implanted into mice on the hind quarter. The ability of mIL-12 expressing B16 cells to affect the growth of contralaterally implanted B16 cells was compared to that of untransfected cells. mIL-12 expressing B16 cells inhibited the contralateral growth of B16 cell in vivo.
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pone.0140744.g002: Functional expression of IL-12 from a piggyBac transposon.A, HeLa cells were transfected with the pT-IL12-2A-venus transposon. DAPI (4',6-diamidino-2-phenylindole) stain was utilized to visualize cell nuclei (top) and immunofluorescence of the venus reporter gene was used to visualize protein expression (bottom). Shown is a representative of 3 independent experiments. Culture media from these cells was analyzed for mIL-12 production resulting in 31 ± 5pg/μl of mIL-12 (N = 3, ± SEM). B, B16 melanoma cells were stably transfected with pT-mIL12 in the presence of pCMV-m7pB. 5 X 105 B16 cells were implanted into mice on the hind quarter. The ability of mIL-12 expressing B16 cells to affect the growth of contralaterally implanted B16 cells was compared to that of untransfected cells. mIL-12 expressing B16 cells inhibited the contralateral growth of B16 cell in vivo.

Mentions: The piggyBac vectors we used in our studies are shown in Fig 1. We engineered three piggyBac transposon vectors to express mIL-12 (p35/p40 IL-12): one followed by the venus fluorescent reporter after a 2A cleavage sequence, one followed by the Thy1.1 antigen, and one with mIL-12 alone. To validate IL-12 production, HeLa cells (1x106) were transiently transfected with pT-mIL12-2A-Venus and subjected to fluorescent microscopy for detection of the Venus reporter gene (Fig 2A). Media was collected prior to imaging at 24 hours post-transfection and mIL-12 concentration was measured using ELISA. Secreted mIL-12 was detected at a concentration of 31 ± 5pg/μl (N = 3, ± SEM) (S1 Fig). Therefore, piggyBac could be engineered to produce mIL-12 which could be secreted and detected.


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)

Functional expression of IL-12 from a piggyBac transposon.A, HeLa cells were transfected with the pT-IL12-2A-venus transposon. DAPI (4',6-diamidino-2-phenylindole) stain was utilized to visualize cell nuclei (top) and immunofluorescence of the venus reporter gene was used to visualize protein expression (bottom). Shown is a representative of 3 independent experiments. Culture media from these cells was analyzed for mIL-12 production resulting in 31 ± 5pg/μl of mIL-12 (N = 3, ± SEM). B, B16 melanoma cells were stably transfected with pT-mIL12 in the presence of pCMV-m7pB. 5 X 105 B16 cells were implanted into mice on the hind quarter. The ability of mIL-12 expressing B16 cells to affect the growth of contralaterally implanted B16 cells was compared to that of untransfected cells. mIL-12 expressing B16 cells inhibited the contralateral growth of B16 cell in vivo.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140744.g002: Functional expression of IL-12 from a piggyBac transposon.A, HeLa cells were transfected with the pT-IL12-2A-venus transposon. DAPI (4',6-diamidino-2-phenylindole) stain was utilized to visualize cell nuclei (top) and immunofluorescence of the venus reporter gene was used to visualize protein expression (bottom). Shown is a representative of 3 independent experiments. Culture media from these cells was analyzed for mIL-12 production resulting in 31 ± 5pg/μl of mIL-12 (N = 3, ± SEM). B, B16 melanoma cells were stably transfected with pT-mIL12 in the presence of pCMV-m7pB. 5 X 105 B16 cells were implanted into mice on the hind quarter. The ability of mIL-12 expressing B16 cells to affect the growth of contralaterally implanted B16 cells was compared to that of untransfected cells. mIL-12 expressing B16 cells inhibited the contralateral growth of B16 cell in vivo.
Mentions: The piggyBac vectors we used in our studies are shown in Fig 1. We engineered three piggyBac transposon vectors to express mIL-12 (p35/p40 IL-12): one followed by the venus fluorescent reporter after a 2A cleavage sequence, one followed by the Thy1.1 antigen, and one with mIL-12 alone. To validate IL-12 production, HeLa cells (1x106) were transiently transfected with pT-mIL12-2A-Venus and subjected to fluorescent microscopy for detection of the Venus reporter gene (Fig 2A). Media was collected prior to imaging at 24 hours post-transfection and mIL-12 concentration was measured using ELISA. Secreted mIL-12 was detected at a concentration of 31 ± 5pg/μl (N = 3, ± SEM) (S1 Fig). Therefore, piggyBac could be engineered to produce mIL-12 which could be secreted and detected.

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