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Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.

Gaston DC, Odom CI, Li L, Markert JM, Roth JC, Cassady KA, Whitley RJ, Parker JN - PLoS ONE (2013)

Bottom Line: The purpose of these studies was to engineer an oHSV producing bioactive IL-15.Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV.These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham, Birmingham, Alabama, United States of America ; School of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

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Reduced viability of tumor cells following co-culture with NK cells and J100D-produced mIL-15/IL-15Rα.Enriched murine NK cells were co-cultured for 3 days with syngeneic murine glioma cells. The cells were co-cultured in the presence of supernatant obtained from cells that were mock infected, or infected with J100, or J100D as described in the Methods. Cells co-cultured with supernatant derived from J100D infection were cultured in the presence of 10ng/mL of the J100D-produced mIL-15/IL-15Rα complex. Percent viability was assayed by colorimetric conversion of the MTT reagent after 72 hours of co-culture. A) 4C8 glioma targets cultured at increasing effector:target ratios with syngeneic B6D2F1 enriched NK cells. Data represents average values with standard deviations from triplicate samples. B) GL261 and CT-2A glioma targets cultured at an effector:target ratio of 2:1 with syngeneic C57Bl/6 enriched NK cells. Data represents the average value from two independent experiments performed with triplicate samples. Error bars indicate standard deviation. * p < 0.05 for J100D measurements compared separately to mock and J100 measurements.
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pone-0081768-g005: Reduced viability of tumor cells following co-culture with NK cells and J100D-produced mIL-15/IL-15Rα.Enriched murine NK cells were co-cultured for 3 days with syngeneic murine glioma cells. The cells were co-cultured in the presence of supernatant obtained from cells that were mock infected, or infected with J100, or J100D as described in the Methods. Cells co-cultured with supernatant derived from J100D infection were cultured in the presence of 10ng/mL of the J100D-produced mIL-15/IL-15Rα complex. Percent viability was assayed by colorimetric conversion of the MTT reagent after 72 hours of co-culture. A) 4C8 glioma targets cultured at increasing effector:target ratios with syngeneic B6D2F1 enriched NK cells. Data represents average values with standard deviations from triplicate samples. B) GL261 and CT-2A glioma targets cultured at an effector:target ratio of 2:1 with syngeneic C57Bl/6 enriched NK cells. Data represents the average value from two independent experiments performed with triplicate samples. Error bars indicate standard deviation. * p < 0.05 for J100D measurements compared separately to mock and J100 measurements.

Mentions: 4C8 cells were cultured with enriched B6D2F1 NK cells at effector:target ratios of 2.5:1, 10:1, and 20:1 (Figure 5A). The percent of viable cells decreased inversely with the effector:target ratios in cells co-cultured with media obtained from J100D-infeced cells, whereas no viability reduction occurred in cells co-cultured with media obtained from mock or J100-infected cells. No reduction in viability was measured even at the 20:1 effector target ratio in cells co-cultured with media obtained from mock or J100-infected cells. GL261 and CT-2A cells were cultured with enriched C57Bl/6 NK cells only at an effector:target ratio of 2:1 (Figure 5B). The percent viable GL261 and CT-2A cells were significantly reduced in the presence of supernates containing J100D-produced mIL-15/IL-15Rα as compared to co-culture with supernates obtained from mock or J100 infected cells. No difference in the percent of viable GL261 or CT-2A cells was observed in co-culture with mock or J100 conditioned supernates.


Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.

Gaston DC, Odom CI, Li L, Markert JM, Roth JC, Cassady KA, Whitley RJ, Parker JN - PLoS ONE (2013)

Reduced viability of tumor cells following co-culture with NK cells and J100D-produced mIL-15/IL-15Rα.Enriched murine NK cells were co-cultured for 3 days with syngeneic murine glioma cells. The cells were co-cultured in the presence of supernatant obtained from cells that were mock infected, or infected with J100, or J100D as described in the Methods. Cells co-cultured with supernatant derived from J100D infection were cultured in the presence of 10ng/mL of the J100D-produced mIL-15/IL-15Rα complex. Percent viability was assayed by colorimetric conversion of the MTT reagent after 72 hours of co-culture. A) 4C8 glioma targets cultured at increasing effector:target ratios with syngeneic B6D2F1 enriched NK cells. Data represents average values with standard deviations from triplicate samples. B) GL261 and CT-2A glioma targets cultured at an effector:target ratio of 2:1 with syngeneic C57Bl/6 enriched NK cells. Data represents the average value from two independent experiments performed with triplicate samples. Error bars indicate standard deviation. * p < 0.05 for J100D measurements compared separately to mock and J100 measurements.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842420&req=5

pone-0081768-g005: Reduced viability of tumor cells following co-culture with NK cells and J100D-produced mIL-15/IL-15Rα.Enriched murine NK cells were co-cultured for 3 days with syngeneic murine glioma cells. The cells were co-cultured in the presence of supernatant obtained from cells that were mock infected, or infected with J100, or J100D as described in the Methods. Cells co-cultured with supernatant derived from J100D infection were cultured in the presence of 10ng/mL of the J100D-produced mIL-15/IL-15Rα complex. Percent viability was assayed by colorimetric conversion of the MTT reagent after 72 hours of co-culture. A) 4C8 glioma targets cultured at increasing effector:target ratios with syngeneic B6D2F1 enriched NK cells. Data represents average values with standard deviations from triplicate samples. B) GL261 and CT-2A glioma targets cultured at an effector:target ratio of 2:1 with syngeneic C57Bl/6 enriched NK cells. Data represents the average value from two independent experiments performed with triplicate samples. Error bars indicate standard deviation. * p < 0.05 for J100D measurements compared separately to mock and J100 measurements.
Mentions: 4C8 cells were cultured with enriched B6D2F1 NK cells at effector:target ratios of 2.5:1, 10:1, and 20:1 (Figure 5A). The percent of viable cells decreased inversely with the effector:target ratios in cells co-cultured with media obtained from J100D-infeced cells, whereas no viability reduction occurred in cells co-cultured with media obtained from mock or J100-infected cells. No reduction in viability was measured even at the 20:1 effector target ratio in cells co-cultured with media obtained from mock or J100-infected cells. GL261 and CT-2A cells were cultured with enriched C57Bl/6 NK cells only at an effector:target ratio of 2:1 (Figure 5B). The percent viable GL261 and CT-2A cells were significantly reduced in the presence of supernates containing J100D-produced mIL-15/IL-15Rα as compared to co-culture with supernates obtained from mock or J100 infected cells. No difference in the percent of viable GL261 or CT-2A cells was observed in co-culture with mock or J100 conditioned supernates.

Bottom Line: The purpose of these studies was to engineer an oHSV producing bioactive IL-15.Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV.These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham, Birmingham, Alabama, United States of America ; School of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

Show MeSH
Related in: MedlinePlus