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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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The coexpression of human IL-12p35 and p40 alters intracellular localization.A, HLtat cells were transfected with p35-FLAG (A–D), p40-FLAG (E–H), and the combinations of p35-FLAG and p40 (I–L) or p35 and p40-FLAG (M–P) and fixed. FLAG-p35 and FLAG-p40 were visualized with anti-FLAG primary followed by Alexa-Fluor 488 secondary antibody (A, E, I, and M). The TGN was visualized with anti-TGN46 primary antibody followed by Alexa-Fluor 594 secondary antibody (B, F, J, and N). The nuclei were visualized with DAPI (C, G, K, and O). The images were merged to show similar localization within the TGN (D, H, L, and P). B, increase of glycosylated p35 in the presence of p40. The supernatants and cell lysates from HEK293 cells transfected with plasmids expressing the individual p35 or p40 subunits either alone or together were treated with Endo F or Endo H or left untreated. The samples were analyzed by Western immunoblot assay using the human IL-12p70 antibody. *, Endo H-resistant forms of p35 in the cell-associated fraction.
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Figure 3: The coexpression of human IL-12p35 and p40 alters intracellular localization.A, HLtat cells were transfected with p35-FLAG (A–D), p40-FLAG (E–H), and the combinations of p35-FLAG and p40 (I–L) or p35 and p40-FLAG (M–P) and fixed. FLAG-p35 and FLAG-p40 were visualized with anti-FLAG primary followed by Alexa-Fluor 488 secondary antibody (A, E, I, and M). The TGN was visualized with anti-TGN46 primary antibody followed by Alexa-Fluor 594 secondary antibody (B, F, J, and N). The nuclei were visualized with DAPI (C, G, K, and O). The images were merged to show similar localization within the TGN (D, H, L, and P). B, increase of glycosylated p35 in the presence of p40. The supernatants and cell lysates from HEK293 cells transfected with plasmids expressing the individual p35 or p40 subunits either alone or together were treated with Endo F or Endo H or left untreated. The samples were analyzed by Western immunoblot assay using the human IL-12p70 antibody. *, Endo H-resistant forms of p35 in the cell-associated fraction.

Mentions: To study the effect of p40 on p35, the intracellular localization of p35 was investigated. Cells transfected with plasmids expressing the FLAG-tagged subunits, alone or in combination, were examined by fluorescent microscopy (Fig. 3A). The p35-FLAG (Fig. 3A) was found in punctate foci in the cytoplasm when expressed alone. Several cellular markers with known localization, such as calreticulin for the ER, Rab5 for early endosomes, and Rab11 and transferrin for recycling endosomes, were tested, but no colocalization with p35-FLAG was found, indicating that p35 did not accumulate appreciably in the ER. The p40-FLAG was also found in punctate foci in the cytoplasm (Fig. 3E) and did not colocalize with the above listed markers, also indicating no appreciable accumulation in the ER. When present alone, a minority of the p35-FLAG (∼10%) and p40-FLAG (∼14%) colocalized with TGN46, a marker for the trans-Golgi network (TGN), a system of membranes responsible for the sorting of proteins. Cotransfection of plasmids expressing the FLAG-tagged subunit with its untagged partner led to a significant change in the localization of the p35 and p40 subunits to the perinuclear area (Fig. 3, I and M, respectively), compatible with Golgi localization. Staining of the cells with TGN46 demonstrated that the FLAG-tagged p35 (∼91% of cells; Fig. 3L) and p40 (∼89% of cells; Fig. 3P), respectively, significantly co-localized with TGN46, indicating more efficient association with the trans-Golgi network when expressed together. Accumulation at the Golgi is only found in the presence of the two partners and is critical for the export of the heterodimer, supporting the notion that this colocalization reflects a transient accumulation in the Golgi. Together, these data demonstrate that p40 increased the stability of p35 (Fig. 1D) and promoted the intracellular trafficking of the p35 subunit through the Golgi export pathway (Fig. 2A).


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)

The coexpression of human IL-12p35 and p40 alters intracellular localization.A, HLtat cells were transfected with p35-FLAG (A–D), p40-FLAG (E–H), and the combinations of p35-FLAG and p40 (I–L) or p35 and p40-FLAG (M–P) and fixed. FLAG-p35 and FLAG-p40 were visualized with anti-FLAG primary followed by Alexa-Fluor 488 secondary antibody (A, E, I, and M). The TGN was visualized with anti-TGN46 primary antibody followed by Alexa-Fluor 594 secondary antibody (B, F, J, and N). The nuclei were visualized with DAPI (C, G, K, and O). The images were merged to show similar localization within the TGN (D, H, L, and P). B, increase of glycosylated p35 in the presence of p40. The supernatants and cell lysates from HEK293 cells transfected with plasmids expressing the individual p35 or p40 subunits either alone or together were treated with Endo F or Endo H or left untreated. The samples were analyzed by Western immunoblot assay using the human IL-12p70 antibody. *, Endo H-resistant forms of p35 in the cell-associated fraction.
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Related In: Results  -  Collection

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Figure 3: The coexpression of human IL-12p35 and p40 alters intracellular localization.A, HLtat cells were transfected with p35-FLAG (A–D), p40-FLAG (E–H), and the combinations of p35-FLAG and p40 (I–L) or p35 and p40-FLAG (M–P) and fixed. FLAG-p35 and FLAG-p40 were visualized with anti-FLAG primary followed by Alexa-Fluor 488 secondary antibody (A, E, I, and M). The TGN was visualized with anti-TGN46 primary antibody followed by Alexa-Fluor 594 secondary antibody (B, F, J, and N). The nuclei were visualized with DAPI (C, G, K, and O). The images were merged to show similar localization within the TGN (D, H, L, and P). B, increase of glycosylated p35 in the presence of p40. The supernatants and cell lysates from HEK293 cells transfected with plasmids expressing the individual p35 or p40 subunits either alone or together were treated with Endo F or Endo H or left untreated. The samples were analyzed by Western immunoblot assay using the human IL-12p70 antibody. *, Endo H-resistant forms of p35 in the cell-associated fraction.
Mentions: To study the effect of p40 on p35, the intracellular localization of p35 was investigated. Cells transfected with plasmids expressing the FLAG-tagged subunits, alone or in combination, were examined by fluorescent microscopy (Fig. 3A). The p35-FLAG (Fig. 3A) was found in punctate foci in the cytoplasm when expressed alone. Several cellular markers with known localization, such as calreticulin for the ER, Rab5 for early endosomes, and Rab11 and transferrin for recycling endosomes, were tested, but no colocalization with p35-FLAG was found, indicating that p35 did not accumulate appreciably in the ER. The p40-FLAG was also found in punctate foci in the cytoplasm (Fig. 3E) and did not colocalize with the above listed markers, also indicating no appreciable accumulation in the ER. When present alone, a minority of the p35-FLAG (∼10%) and p40-FLAG (∼14%) colocalized with TGN46, a marker for the trans-Golgi network (TGN), a system of membranes responsible for the sorting of proteins. Cotransfection of plasmids expressing the FLAG-tagged subunit with its untagged partner led to a significant change in the localization of the p35 and p40 subunits to the perinuclear area (Fig. 3, I and M, respectively), compatible with Golgi localization. Staining of the cells with TGN46 demonstrated that the FLAG-tagged p35 (∼91% of cells; Fig. 3L) and p40 (∼89% of cells; Fig. 3P), respectively, significantly co-localized with TGN46, indicating more efficient association with the trans-Golgi network when expressed together. Accumulation at the Golgi is only found in the presence of the two partners and is critical for the export of the heterodimer, supporting the notion that this colocalization reflects a transient accumulation in the Golgi. Together, these data demonstrate that p40 increased the stability of p35 (Fig. 1D) and promoted the intracellular trafficking of the p35 subunit through the Golgi export pathway (Fig. 2A).

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