Immune Netw.  2013 Apr;13(2):63-69. 10.4110/in.2013.13.2.63.

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

• Lim H
• Do SA
• Park SM
• Kim YS

• KMID: 1431927
• DOI: http://doi.org/10.4110/in.2013.13.2.63

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-alpha. 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.

Keyword

Membrane-bound form; Interleukin-12; p35 subunit; p40 subunit; MethA fibrosarcoma

MeSH Terms

Animals
Antigen-Presenting Cells
Clone Cells
Corynebacterium
Fibrosarcoma
Interleukin-12
Interleukin-12 Subunit p35
Interleukin-12 Subunit p40
Macrophages
Mice
Rejection (Psychology)
T-Lymphocytes
T-Lymphocytes, Helper-Inducer
Tumor Necrosis Factor-alpha
Corynebacterium
Interleukin-12
Interleukin-12 Subunit p35
Interleukin-12 Subunit p40
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Expression of mbIL-12p35, mbIL-12p40, and MHC class I molecules on transfected MethA tumor cell clones. (A) The mock vector transfected, mbIL-12p35, and mbIL-12p40 expressing tumor clones were stained with anti-IL-12 antibody and analyzed by FACS, (B) Wild type MethA tumor cells, and mbIL-12p35 and mbIL-12 p40 expressing tumor clones were stained with anti-Ld antibody and analyzed by FACS.

• 

  Figure 2 The mbIL-12p35 and the mbIL-12p40 molecules on transfected tumor clones are not released. (A) The isolated mouse peritoneal macrophages were stained with specific antibody to CD11b and analyzed by FACS. (B) Cells (5×105 cells) of the wild type, mock vector transfected, mbIL-12p35, and mbIL-12p40 transfected clones were cultured for 48 hr in 2 ml normal medium, and then culture supernatants were analyzed for IL-12 by ELISA. As a positive control, culture supernatant from LPS (2µg/ml)-treated mouse peritoneal macrophages was used. ND, not detected.

• 

  Figure 3 The mbIL-12p35 and the mbIL-12p40 expressing tumor clones do not induce TNF-α production on macrophages. Mouse peritoneal macrophages (1×106 cells) were co-cultured with MMC-inactivated wild type MethA cells, mock vector transfected, mbIL-12p35, or mbIL-12p40 transfected clones (5×105 cells), respectively. After 24 hr, culture supernatants were harvested and measured for TNF-α level by TNF-α specific ELISA. Culture supernatant from LPS (2µg/ml)-treated mouse peritoneal macrophages was used as a positive control. ND, not detected.

• 

  Figure 4 Survival of T cell subset-depleted mice injected with mbIL-12p35 expressing tumor clone. To deplete CD4+ or CD8+ T cells in vivo, mice were injected with antibodies specific to CD4 (GK1.5, 200µg/time) or CD8 (53-6.72, 200µg/time) intraperitoneally on days -3, 0, 3, 7, respectively. The depletion of CD4+ or CD8+ T cells was confirmed by FACS analysis of peripheral blood cells from the mice (A). CD4+ or CD8+ T cell-depleted mice were injected with 5×105 cells of mbIL-12p35 expressing tumor clone subcutaneously, and survival (B) were monitored.

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