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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

IL-12 transfected OT-I cells produce IL-12 and produce IFNγ when co-cultured with B16/OVA cells.A, OT-I splenocytes were transfected with pT-eGFP (control) or pT-mIL12 and co-cultured with B16 or B16/OVA cells. Flow cytometry confirmed the presence of eGFP expressing CD8 positive OT-I cells at the end of the co-culture. Shown is a representative of 3 independent experiments. B, cytometric bead analysis was used to measure mIL-12 (au, arbitrary units) in the media derived from the co-culture. *, p<0.05 comparing mIL-12 groups to eGFP. C, cytometric bead analysis was used to measure INFg production from transfected OT-I cells in the presence of B16 or B16/OVA cells. *, p<0.05 comparing B16/OVA groups to B16 (without OVA).
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pone.0140744.g005: IL-12 transfected OT-I cells produce IL-12 and produce IFNγ when co-cultured with B16/OVA cells.A, OT-I splenocytes were transfected with pT-eGFP (control) or pT-mIL12 and co-cultured with B16 or B16/OVA cells. Flow cytometry confirmed the presence of eGFP expressing CD8 positive OT-I cells at the end of the co-culture. Shown is a representative of 3 independent experiments. B, cytometric bead analysis was used to measure mIL-12 (au, arbitrary units) in the media derived from the co-culture. *, p<0.05 comparing mIL-12 groups to eGFP. C, cytometric bead analysis was used to measure INFg production from transfected OT-I cells in the presence of B16 or B16/OVA cells. *, p<0.05 comparing B16/OVA groups to B16 (without OVA).

Mentions: We performed co-culture experiments of gene modified OT-I cells with B16 cells to confirm mIL-12 expression from and antigen-specificity of the OT-I cells. OT-I cells were transfected with either pT-eGFP (control vector) or pT-mIL12 to produce mIL-12. Transfected OT-I splenocytes were then co-cultured with B16 or B16/OVA cells (Fig 5). Flow cytometry confirmed that 25 ± 3% of CD8 positive OT-I cells expressed eGFP (N = 3, ± STD) at the end of the co-culture (Fig 5A). Cytometric bead array analysis of media from the co-culture revealed increased mIL-12 (3.2 ± 0.9 fold when co-cultured with B16 and 2.5 ± 0.3 with B16/OVA; N = 3 ± STD) in pT-IL12 transfected splenocytes compared to eGFP controls (Fig 5B). To confirm antigen specificity, cytometric bead analysis of media from the co-culture for interferon-γ (INFγ) revealed increased INFγ (8.9 ± 3.3 fold for eGFP and 9.5 ± 4.4 fold for mIL-12 transfected splenocytes; N = 3 ± STD) when OT-I splenocytes were co-cultured with B16/OVA compared to B16 without OVA (Fig 5C). These results demonstrate mIL-12 expression from the pT-mIL12 vector from OT-I splenocytes in the presence of B16 or B16/OVA. Additionally, OT-I splenocytes increased INFγ production only when co-cultured with B16/OVA and this is unaffected by mIL-12 production from the OT-I splenocytes.


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)

IL-12 transfected OT-I cells produce IL-12 and produce IFNγ when co-cultured with B16/OVA cells.A, OT-I splenocytes were transfected with pT-eGFP (control) or pT-mIL12 and co-cultured with B16 or B16/OVA cells. Flow cytometry confirmed the presence of eGFP expressing CD8 positive OT-I cells at the end of the co-culture. Shown is a representative of 3 independent experiments. B, cytometric bead analysis was used to measure mIL-12 (au, arbitrary units) in the media derived from the co-culture. *, p<0.05 comparing mIL-12 groups to eGFP. C, cytometric bead analysis was used to measure INFg production from transfected OT-I cells in the presence of B16 or B16/OVA cells. *, p<0.05 comparing B16/OVA groups to B16 (without OVA).
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pone.0140744.g005: IL-12 transfected OT-I cells produce IL-12 and produce IFNγ when co-cultured with B16/OVA cells.A, OT-I splenocytes were transfected with pT-eGFP (control) or pT-mIL12 and co-cultured with B16 or B16/OVA cells. Flow cytometry confirmed the presence of eGFP expressing CD8 positive OT-I cells at the end of the co-culture. Shown is a representative of 3 independent experiments. B, cytometric bead analysis was used to measure mIL-12 (au, arbitrary units) in the media derived from the co-culture. *, p<0.05 comparing mIL-12 groups to eGFP. C, cytometric bead analysis was used to measure INFg production from transfected OT-I cells in the presence of B16 or B16/OVA cells. *, p<0.05 comparing B16/OVA groups to B16 (without OVA).
Mentions: We performed co-culture experiments of gene modified OT-I cells with B16 cells to confirm mIL-12 expression from and antigen-specificity of the OT-I cells. OT-I cells were transfected with either pT-eGFP (control vector) or pT-mIL12 to produce mIL-12. Transfected OT-I splenocytes were then co-cultured with B16 or B16/OVA cells (Fig 5). Flow cytometry confirmed that 25 ± 3% of CD8 positive OT-I cells expressed eGFP (N = 3, ± STD) at the end of the co-culture (Fig 5A). Cytometric bead array analysis of media from the co-culture revealed increased mIL-12 (3.2 ± 0.9 fold when co-cultured with B16 and 2.5 ± 0.3 with B16/OVA; N = 3 ± STD) in pT-IL12 transfected splenocytes compared to eGFP controls (Fig 5B). To confirm antigen specificity, cytometric bead analysis of media from the co-culture for interferon-γ (INFγ) revealed increased INFγ (8.9 ± 3.3 fold for eGFP and 9.5 ± 4.4 fold for mIL-12 transfected splenocytes; N = 3 ± STD) when OT-I splenocytes were co-cultured with B16/OVA compared to B16 without OVA (Fig 5C). These results demonstrate mIL-12 expression from the pT-mIL12 vector from OT-I splenocytes in the presence of B16 or B16/OVA. Additionally, OT-I splenocytes increased INFγ production only when co-cultured with B16/OVA and this is unaffected by mIL-12 production from the OT-I splenocytes.

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