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The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

Jalah R, Rosati M, Ganneru B, Pilkington GR, Valentin A, Kulkarni V, Bergamaschi C, Chowdhury B, Zhang GM, Beach RK, Alicea C, Broderick KE, Sardesai NY, Pavlakis GN, Felber BK - J. Biol. Chem. (2013)

Bottom Line: We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit.Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences.Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.

ABSTRACT
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

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Related in: MedlinePlus

Role of IL-12p40 in the production and secretion of the IL-12p70 heterodimer. HEK293 cells were transfected with 100 ng of plasmids encoding the individual subunits alone, with a mixture of 100 ng each of the two plasmids together, or with a mixture of plasmids at the indicated ratios. Aliquots of the cell-associated and/or extracellular fraction were analyzed by Western immunoblot assays and by ELISA. The molecular mass standards (kDa) are indicated. Cotransfection of a GFP plasmid served as internal control. A, expression of the FLAG-tagged human IL-12p35 and IL-12p40 subunits alone (lanes 1 and 2) or together (lane 3) in the cell-associated (lanes 1–3) and extracellular (lanes 4–6) fraction. GFP values (mean ± S.E.) were 20.3 ± 6.0, 24.8 ± 2.4, and 17.5 ± 3.3 arbitrary units for lanes 1–3, respectively. A representative experiment is shown. B, optimization of co-expression of the untagged human IL-12p40 and IL-12p35 subunits expressed alone (lanes 1 and 2, respectively) or together using different DNA ratios as indicated (lanes 3–5). Analysis of the cell-associated (top) and extracellular fractions (middle) by Western immunoblot assays and by a human IL-12p70 specific ELISA (bottom) are shown. The mean of the values obtained from three independent and the S.E. (error bars) are shown. GFP values (mean ± S.E.) were 4.8 ± 0.7, 8.2 ± 0.9, 8.6 ± 2.1, 9.5 ± 1.5, and 8.4 ± 1.4 arbitrary units for lanes 1–5, respectively. C, IL-12p40 prefers the heterodimeric p70 form over homodimeric interactions. Shown is a comparison of extracellular samples from cells transfected with p40 alone (300 ng; lane 1) and p40 + p35 (300 ng of p40 plus 100 ng of p35; lane 2) under native (without β-mercaptoethanol, without SDS; top), non-reducing (without β-mercaptoethanol, plus SDS; middle), and reducing (treated with β-mercaptoethanol and with SDS; bottom) conditions. D, stability of human p35 and p40 subunits upon treatment with CHX. At 1 day posttransfection, the medium was replaced with fresh medium containing CHX (see “Experimental Procedures”). The cell-associated and extracellular fractions were collected at 0, 1, 2, and 4 h and analyzed on Western immunoblots. The bands were quantitated, and the accumulation of p35 and p40 when transfected alone or in combination was calculated over time by normalizing the value (sum of extracellular and cell-associated) at the start of the CHX treatment (time 0) as 100%. p35 (open circles), p40 alone (open squares), and a mixture of the p35 and p40 subunits (filled triangles) are shown. A combination of 2–3 independent experiments is shown.
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Figure 1: Role of IL-12p40 in the production and secretion of the IL-12p70 heterodimer. HEK293 cells were transfected with 100 ng of plasmids encoding the individual subunits alone, with a mixture of 100 ng each of the two plasmids together, or with a mixture of plasmids at the indicated ratios. Aliquots of the cell-associated and/or extracellular fraction were analyzed by Western immunoblot assays and by ELISA. The molecular mass standards (kDa) are indicated. Cotransfection of a GFP plasmid served as internal control. A, expression of the FLAG-tagged human IL-12p35 and IL-12p40 subunits alone (lanes 1 and 2) or together (lane 3) in the cell-associated (lanes 1–3) and extracellular (lanes 4–6) fraction. GFP values (mean ± S.E.) were 20.3 ± 6.0, 24.8 ± 2.4, and 17.5 ± 3.3 arbitrary units for lanes 1–3, respectively. A representative experiment is shown. B, optimization of co-expression of the untagged human IL-12p40 and IL-12p35 subunits expressed alone (lanes 1 and 2, respectively) or together using different DNA ratios as indicated (lanes 3–5). Analysis of the cell-associated (top) and extracellular fractions (middle) by Western immunoblot assays and by a human IL-12p70 specific ELISA (bottom) are shown. The mean of the values obtained from three independent and the S.E. (error bars) are shown. GFP values (mean ± S.E.) were 4.8 ± 0.7, 8.2 ± 0.9, 8.6 ± 2.1, 9.5 ± 1.5, and 8.4 ± 1.4 arbitrary units for lanes 1–5, respectively. C, IL-12p40 prefers the heterodimeric p70 form over homodimeric interactions. Shown is a comparison of extracellular samples from cells transfected with p40 alone (300 ng; lane 1) and p40 + p35 (300 ng of p40 plus 100 ng of p35; lane 2) under native (without β-mercaptoethanol, without SDS; top), non-reducing (without β-mercaptoethanol, plus SDS; middle), and reducing (treated with β-mercaptoethanol and with SDS; bottom) conditions. D, stability of human p35 and p40 subunits upon treatment with CHX. At 1 day posttransfection, the medium was replaced with fresh medium containing CHX (see “Experimental Procedures”). The cell-associated and extracellular fractions were collected at 0, 1, 2, and 4 h and analyzed on Western immunoblots. The bands were quantitated, and the accumulation of p35 and p40 when transfected alone or in combination was calculated over time by normalizing the value (sum of extracellular and cell-associated) at the start of the CHX treatment (time 0) as 100%. p35 (open circles), p40 alone (open squares), and a mixture of the p35 and p40 subunits (filled triangles) are shown. A combination of 2–3 independent experiments is shown.

Mentions: To dissect steps in the biosynthesis of the IL-12p70 heterodimer, its p35 and p40 subunits were expressed independently from the CMV promoter using the mammalian expression vector pCMVkan, bypassing their natural transcriptional control. In addition to their AU-rich 3′-UTRs (12–14), we noted that the native p35 and p40 coding sequences have a relatively low AU content of ∼50% and contain AU-rich segments, including AUUUA or AAUAA elements, which have been associated with low expression when present within the coding sequence, as we previously reported for HIV and SIV gag and env genes (25–27, 37), for IL-15 (30), and for the IL-15Rα (18, 19). To remove transcriptional and posttranscriptional impediments for IL-12p70 expression, the complete coding sequences of the human, macaque, or murine p35 and p40 were RNA- or codon-optimized (see “Experimental Procedures”). Production of optimized human p35 and p40 subunits was monitored upon transient transfection of HEK293 cells. Plasmids expressing FLAG-tagged human p35 and p40 were used to allow direct comparison of the production of respective subunits. Importantly, when expressed alone, the steady-state level of the p35 protein was much lower than that of p40 (Fig. 1A), both in the cell-associated and the extracellular fractions (compare lanes 1 and 4 versus lanes 2 and 5). A longer exposure was necessary to visualize both proteins; therefore, the p40 bands are overexposed. Most of the p35 subunit remained cell-associated, and the extracellular p35 migrated as diffuse bands due to different glycosylated forms (38, 39). In contrast, more p40 was found in the extracellular compartment. The steady-state protein levels and the localization changed when the two subunits were co-expressed in the same cell. We noted that the presence of p40 led to an overall increase in the total p35 protein level (Fig. 1A, compare lanes 1 and 4 with lanes 3 and 6) as well as to a shift from p35 to increased accumulation in the extracellular compartment, suggesting that p40 affected not only the trafficking but also the stability (supported by pulse-chase experiments with CHX; see below) of the p35 subunit (Fig. 1A, compare lanes 4 and 6).


The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

Jalah R, Rosati M, Ganneru B, Pilkington GR, Valentin A, Kulkarni V, Bergamaschi C, Chowdhury B, Zhang GM, Beach RK, Alicea C, Broderick KE, Sardesai NY, Pavlakis GN, Felber BK - J. Biol. Chem. (2013)

Role of IL-12p40 in the production and secretion of the IL-12p70 heterodimer. HEK293 cells were transfected with 100 ng of plasmids encoding the individual subunits alone, with a mixture of 100 ng each of the two plasmids together, or with a mixture of plasmids at the indicated ratios. Aliquots of the cell-associated and/or extracellular fraction were analyzed by Western immunoblot assays and by ELISA. The molecular mass standards (kDa) are indicated. Cotransfection of a GFP plasmid served as internal control. A, expression of the FLAG-tagged human IL-12p35 and IL-12p40 subunits alone (lanes 1 and 2) or together (lane 3) in the cell-associated (lanes 1–3) and extracellular (lanes 4–6) fraction. GFP values (mean ± S.E.) were 20.3 ± 6.0, 24.8 ± 2.4, and 17.5 ± 3.3 arbitrary units for lanes 1–3, respectively. A representative experiment is shown. B, optimization of co-expression of the untagged human IL-12p40 and IL-12p35 subunits expressed alone (lanes 1 and 2, respectively) or together using different DNA ratios as indicated (lanes 3–5). Analysis of the cell-associated (top) and extracellular fractions (middle) by Western immunoblot assays and by a human IL-12p70 specific ELISA (bottom) are shown. The mean of the values obtained from three independent and the S.E. (error bars) are shown. GFP values (mean ± S.E.) were 4.8 ± 0.7, 8.2 ± 0.9, 8.6 ± 2.1, 9.5 ± 1.5, and 8.4 ± 1.4 arbitrary units for lanes 1–5, respectively. C, IL-12p40 prefers the heterodimeric p70 form over homodimeric interactions. Shown is a comparison of extracellular samples from cells transfected with p40 alone (300 ng; lane 1) and p40 + p35 (300 ng of p40 plus 100 ng of p35; lane 2) under native (without β-mercaptoethanol, without SDS; top), non-reducing (without β-mercaptoethanol, plus SDS; middle), and reducing (treated with β-mercaptoethanol and with SDS; bottom) conditions. D, stability of human p35 and p40 subunits upon treatment with CHX. At 1 day posttransfection, the medium was replaced with fresh medium containing CHX (see “Experimental Procedures”). The cell-associated and extracellular fractions were collected at 0, 1, 2, and 4 h and analyzed on Western immunoblots. The bands were quantitated, and the accumulation of p35 and p40 when transfected alone or in combination was calculated over time by normalizing the value (sum of extracellular and cell-associated) at the start of the CHX treatment (time 0) as 100%. p35 (open circles), p40 alone (open squares), and a mixture of the p35 and p40 subunits (filled triangles) are shown. A combination of 2–3 independent experiments is shown.
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Figure 1: Role of IL-12p40 in the production and secretion of the IL-12p70 heterodimer. HEK293 cells were transfected with 100 ng of plasmids encoding the individual subunits alone, with a mixture of 100 ng each of the two plasmids together, or with a mixture of plasmids at the indicated ratios. Aliquots of the cell-associated and/or extracellular fraction were analyzed by Western immunoblot assays and by ELISA. The molecular mass standards (kDa) are indicated. Cotransfection of a GFP plasmid served as internal control. A, expression of the FLAG-tagged human IL-12p35 and IL-12p40 subunits alone (lanes 1 and 2) or together (lane 3) in the cell-associated (lanes 1–3) and extracellular (lanes 4–6) fraction. GFP values (mean ± S.E.) were 20.3 ± 6.0, 24.8 ± 2.4, and 17.5 ± 3.3 arbitrary units for lanes 1–3, respectively. A representative experiment is shown. B, optimization of co-expression of the untagged human IL-12p40 and IL-12p35 subunits expressed alone (lanes 1 and 2, respectively) or together using different DNA ratios as indicated (lanes 3–5). Analysis of the cell-associated (top) and extracellular fractions (middle) by Western immunoblot assays and by a human IL-12p70 specific ELISA (bottom) are shown. The mean of the values obtained from three independent and the S.E. (error bars) are shown. GFP values (mean ± S.E.) were 4.8 ± 0.7, 8.2 ± 0.9, 8.6 ± 2.1, 9.5 ± 1.5, and 8.4 ± 1.4 arbitrary units for lanes 1–5, respectively. C, IL-12p40 prefers the heterodimeric p70 form over homodimeric interactions. Shown is a comparison of extracellular samples from cells transfected with p40 alone (300 ng; lane 1) and p40 + p35 (300 ng of p40 plus 100 ng of p35; lane 2) under native (without β-mercaptoethanol, without SDS; top), non-reducing (without β-mercaptoethanol, plus SDS; middle), and reducing (treated with β-mercaptoethanol and with SDS; bottom) conditions. D, stability of human p35 and p40 subunits upon treatment with CHX. At 1 day posttransfection, the medium was replaced with fresh medium containing CHX (see “Experimental Procedures”). The cell-associated and extracellular fractions were collected at 0, 1, 2, and 4 h and analyzed on Western immunoblots. The bands were quantitated, and the accumulation of p35 and p40 when transfected alone or in combination was calculated over time by normalizing the value (sum of extracellular and cell-associated) at the start of the CHX treatment (time 0) as 100%. p35 (open circles), p40 alone (open squares), and a mixture of the p35 and p40 subunits (filled triangles) are shown. A combination of 2–3 independent experiments is shown.
Mentions: To dissect steps in the biosynthesis of the IL-12p70 heterodimer, its p35 and p40 subunits were expressed independently from the CMV promoter using the mammalian expression vector pCMVkan, bypassing their natural transcriptional control. In addition to their AU-rich 3′-UTRs (12–14), we noted that the native p35 and p40 coding sequences have a relatively low AU content of ∼50% and contain AU-rich segments, including AUUUA or AAUAA elements, which have been associated with low expression when present within the coding sequence, as we previously reported for HIV and SIV gag and env genes (25–27, 37), for IL-15 (30), and for the IL-15Rα (18, 19). To remove transcriptional and posttranscriptional impediments for IL-12p70 expression, the complete coding sequences of the human, macaque, or murine p35 and p40 were RNA- or codon-optimized (see “Experimental Procedures”). Production of optimized human p35 and p40 subunits was monitored upon transient transfection of HEK293 cells. Plasmids expressing FLAG-tagged human p35 and p40 were used to allow direct comparison of the production of respective subunits. Importantly, when expressed alone, the steady-state level of the p35 protein was much lower than that of p40 (Fig. 1A), both in the cell-associated and the extracellular fractions (compare lanes 1 and 4 versus lanes 2 and 5). A longer exposure was necessary to visualize both proteins; therefore, the p40 bands are overexposed. Most of the p35 subunit remained cell-associated, and the extracellular p35 migrated as diffuse bands due to different glycosylated forms (38, 39). In contrast, more p40 was found in the extracellular compartment. The steady-state protein levels and the localization changed when the two subunits were co-expressed in the same cell. We noted that the presence of p40 led to an overall increase in the total p35 protein level (Fig. 1A, compare lanes 1 and 4 with lanes 3 and 6) as well as to a shift from p35 to increased accumulation in the extracellular compartment, suggesting that p40 affected not only the trafficking but also the stability (supported by pulse-chase experiments with CHX; see below) of the p35 subunit (Fig. 1A, compare lanes 4 and 6).

Bottom Line: We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit.Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences.Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.

ABSTRACT
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

Show MeSH
Related in: MedlinePlus