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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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Role of p40 is conserved for the murine IL-12p70 production. Optimization of co-expression of the murine IL-12 subunits expressed alone (lanes 1 and 2, respectively) or together using the indicated plasmid ratios is shown. Cell-associated (top) and extracellular (middle) fractions were analyzed by a Western immunoblot assay and by a murine IL-12p70-specific ELISA (bottom). The mean of two independent plasmid preparations and the S.E. (error bars) are shown. The GFP values (mean ± S.E.) were 10.6 ± 0.4, 23.3 ± 1.2, 16.2 ± 0.1, 19.0 ± 0.1, and 8.1 ± 0.1 arbitrary units for lanes 1–5, respectively.
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Figure 2: Role of p40 is conserved for the murine IL-12p70 production. Optimization of co-expression of the murine IL-12 subunits expressed alone (lanes 1 and 2, respectively) or together using the indicated plasmid ratios is shown. Cell-associated (top) and extracellular (middle) fractions were analyzed by a Western immunoblot assay and by a murine IL-12p70-specific ELISA (bottom). The mean of two independent plasmid preparations and the S.E. (error bars) are shown. The GFP values (mean ± S.E.) were 10.6 ± 0.4, 23.3 ± 1.2, 16.2 ± 0.1, 19.0 ± 0.1, and 8.1 ± 0.1 arbitrary units for lanes 1–5, respectively.

Mentions: We also tested the production of the murine IL-12p70 heterodimer from RNA/codon-optimized coding sequences (Fig. 2) upon transfection in HEK293 cells using the same methods as described for Fig. 1B. The murine p35 subunit expressed alone remained cell-associated (Fig. 2, lane 1), whereas the murine p40 subunit was found both in the intra- and extracellular compartments (lane 2). Cotransfection of both subunits resulted in efficient export of the p35 subunit (lane 3), similar to their human counterparts. Production of the heterodimeric IL-12 was confirmed using the murine IL-12p70-specific ELISA (Fig. 2, bottom). Like for human IL-12 (Fig. 1, A and B), we found increased steady-state levels of the p35 subunit upon co-expression with p40 (Fig. 2, compare lanes 1 and 3). Cotransfection of higher amounts of the murine p40 plasmid (p35/p40 plasmid ratio 1:3; lane 4) further augmented both the p35 steady-state level and export, leading to the production of higher levels of murine IL-12p70. In contrast, excess of the p35 plasmid (lane 5) decreased the extracellular p40 and the IL-12p70 levels. Similar data were obtained upon transfection of the NIH3T3-derived murine PA317 cells (data not shown), indicating that this is a general property of the IL-12 subunits independent of the species of the producer cells. Thus, the studies of the human and murine subunits revealed a critical role of p40 in mediating stabilization and promoting trafficking and secretion of p35, suggesting that this is a conserved regulatory step in the biosynthesis of IL-12p70 heterodimer in mammals.


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 p40 is conserved for the murine IL-12p70 production. Optimization of co-expression of the murine IL-12 subunits expressed alone (lanes 1 and 2, respectively) or together using the indicated plasmid ratios is shown. Cell-associated (top) and extracellular (middle) fractions were analyzed by a Western immunoblot assay and by a murine IL-12p70-specific ELISA (bottom). The mean of two independent plasmid preparations and the S.E. (error bars) are shown. The GFP values (mean ± S.E.) were 10.6 ± 0.4, 23.3 ± 1.2, 16.2 ± 0.1, 19.0 ± 0.1, and 8.1 ± 0.1 arbitrary units for lanes 1–5, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585113&req=5

Figure 2: Role of p40 is conserved for the murine IL-12p70 production. Optimization of co-expression of the murine IL-12 subunits expressed alone (lanes 1 and 2, respectively) or together using the indicated plasmid ratios is shown. Cell-associated (top) and extracellular (middle) fractions were analyzed by a Western immunoblot assay and by a murine IL-12p70-specific ELISA (bottom). The mean of two independent plasmid preparations and the S.E. (error bars) are shown. The GFP values (mean ± S.E.) were 10.6 ± 0.4, 23.3 ± 1.2, 16.2 ± 0.1, 19.0 ± 0.1, and 8.1 ± 0.1 arbitrary units for lanes 1–5, respectively.
Mentions: We also tested the production of the murine IL-12p70 heterodimer from RNA/codon-optimized coding sequences (Fig. 2) upon transfection in HEK293 cells using the same methods as described for Fig. 1B. The murine p35 subunit expressed alone remained cell-associated (Fig. 2, lane 1), whereas the murine p40 subunit was found both in the intra- and extracellular compartments (lane 2). Cotransfection of both subunits resulted in efficient export of the p35 subunit (lane 3), similar to their human counterparts. Production of the heterodimeric IL-12 was confirmed using the murine IL-12p70-specific ELISA (Fig. 2, bottom). Like for human IL-12 (Fig. 1, A and B), we found increased steady-state levels of the p35 subunit upon co-expression with p40 (Fig. 2, compare lanes 1 and 3). Cotransfection of higher amounts of the murine p40 plasmid (p35/p40 plasmid ratio 1:3; lane 4) further augmented both the p35 steady-state level and export, leading to the production of higher levels of murine IL-12p70. In contrast, excess of the p35 plasmid (lane 5) decreased the extracellular p40 and the IL-12p70 levels. Similar data were obtained upon transfection of the NIH3T3-derived murine PA317 cells (data not shown), indicating that this is a general property of the IL-12 subunits independent of the species of the producer cells. Thus, the studies of the human and murine subunits revealed a critical role of p40 in mediating stabilization and promoting trafficking and secretion of p35, suggesting that this is a conserved regulatory step in the biosynthesis of IL-12p70 heterodimer in mammals.

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