Tumor Cell Clone Expressing the Membrane-bound Form of IL-12p35 Subunit Stimulates Antitumor Immune Responses Dominated by CD8⁺T Cells

Abstract

IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membrane-bound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-α. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the CD8+ T cell-depleted mice than in CD4+ T cell-depleted or normal mice. These results suggest that CD8+ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and CD4+ helper T cells.

Membrane-bound p35 Subunit of IL-12 on Tumor Cells is Functionally Equivalent to Membrane-bound Heterodimeric Single Chain IL-12 for Induction of Anti-tumor Immunity

“…Surprisingly, the tumor cell clone expressing only the membrane-bound form of p35 induced anti-tumor immunity (1718). Since endogenous IL-12 p40 is produced excessively in vivo (1920), we envisaged that mbIL-12 p35 on tumor cells might bind the p40 subunit to form a heterodimer.…”

The Relationship between the Antitumor Effect of the IL-12 Gene Therapy and the Expression of Th1 Cytokines in an HPV16-Positive Murine Tumor Model

“…We paid special attention to the differences in the Th1, Th2, and Th3 cytokines during treatment with IL-12. The shift from Th2-Th3 cytokines to Th1 cytokines suggests that IL-12 gene transfer in the tumor model promotes the cellular immune response and favors differentiation to Th1 among the tumor infiltrating cells [ 21 ]. The Th1 cytokine profile may induce effector cells as CD8+ T cells and NK-T cells that can function to eliminate tumor cells and promote tumor regression.…”

“…The antitumor effect of the IL-12 gene in several tumor models has been linked to activation of the cellular immune response, mainly activation of CD4+ T cells and CD8+ T cells [ 21 ]. Previously, we detected CD4 and CD8 mRNA in biopsies of tumor tissues from tumor models treated with the IL-12 gene.…”

The Nuclear Orphan Receptor NR4A1 is Involved in the Apoptotic Pathway Induced by LPS and Simvastatin in RAW 264.7 Macrophages

“…Fixed cells were labeled with 500µg/ml propidium iodide (PI) at 37℃ for 1 h. Genomic DNA was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) as previously described (26,27). All data were calculated and displayed using the Cell Quest analysis software provided by Becton Dickinson.…”

“…To analyze the fragmentation of genomic DNA in apoptotic cells, cells (1×10 6 cells/ml) were incubated with reagents for 12 h. The cells were harvested, washed with PBS, and fixed using 1 ml of ice-cold 70% ethanol at 4℃ overnight. Fixed cells were labeled with 500µg/ml propidium iodide (PI) at 37℃ for 1 h. Genomic DNA was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) as previously described ( 26 , 27 ). All data were calculated and displayed using the Cell Quest analysis software provided by Becton Dickinson.…”