Immune Netw.  2016 Oct;16(5):305-310. 10.4110/in.2016.16.5.305.

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

• Kim HJ
• Park SM
• Lee H
• Kim YS

• KMID: 2355024
• DOI: http://doi.org/10.4110/in.2016.16.5.305

Abstract

In this study, we compared two different tumor cell vaccines for their induction of anti-tumor immunity; one was a tumor cell clone expressing a membrane-bound form of IL-12 p35 subunit (mbIL-12 p35 tumor clone), and the other was a tumor clone expressing heterodimeric IL-12 as a single chain (mb-scIL-12 tumor clone). The stimulatory effect of mb-scIL-12 on the proliferation of ConA-activated splenocytes was higher than that of mbIL-12 p35 in vitro. However, the stimulatory effect of mbIL-12 p35 was equivalent to that of recombinant soluble IL-12 (3 ng/ml). Interestingly, both tumor clones (mbIL-12 p35 and mb-scIL-12) showed similar tumorigenicity and induction of systemic anti-tumor immunity in vivo, suggesting that tumor cell expression of the membrane-bound p35 subunit is sufficient to induce anti-tumor immunity in our tumor vaccine model.

Keyword

Interleukin-12; Membrane-bound form; Cytokine gene therapy; Anti-tumor immunity

MeSH Terms

Clone Cells
In Vitro Techniques
Interleukin-12*
Vaccines
Interleukin-12
Vaccines

Figure

• 

  Figure 1 Structure of bioactive mbIL-12 p35 and mb-scIL-12 chimeric DNA, and expression in MethA tumor cells. (A) Structure of mbIL-12 p35 and mb-scIL-12 chimeric DNA. mbIL-12 p35 has a 225 bp TNF-α cDNA fragment encoding the cytoplasmic region (from –75 to –45), the transmembrane region (from –44 to –24), and the extracellular region (from –23 to –5) at the N-terminus, a three amino acid sequence spacer (GGI), and the entire sequence of IL-12 p35. In mb-scIL-12, the p35 region was replaced by the entire sequence of IL-12 p40 linked to the IL-12 p35 mature sequence by a flexible 15 amino acid spacer sequence (GGGGS3). (B) mRNA expression of mbIL-12 p35 and mbscIL-12 in each of the transfected MethA tumor cells.

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  Figure 2 Expression of chimeric IL-12 on the MethA cell surface and proliferation of ConA-activated murine splenocytes after co-culture with MethA clones expressing mbIL-12 p35 and mb-scIL-12. (A) Surface expression of chimeric IL-12 proteins on MethA tumor cells. Cells were stained with an IL-12 p35 specific antibody and analyzed by flow cytometry. A fluorescent-conjugated secondary antibody, without the primary antibody, was used as a control. (B) Splenocytes were treated with 2 µg/ml of ConA for 72 h to activate T cells and MethA cells stably transfected with a mock vector, mbIL-12 p35, or mb-scIL-12, and were treated with 50 µg/ml of MMC for 30 min. After treatment, 5×104 splenocytes and 1×104 cells from each tumor clone were co-cultured for 48 h. ConA-activated splenocytes were treated with 3 ng/ml rIL-12 as a positive control. Cells were then counted by trypan blue staining (*p <0.05; **p <0.01; ***p <0.001).

• 

  Figure 3 Tumor growth and survival of mice injected MethA tumor clones expressing membrane bound IL-12. BALB/c mice (n=5) were injected with 1×106 stable MethA transfectants (wild type, mbIL-12p35, or mb-scIL-12) subcutaneously on the right flank. (A) Tumor growth and (B) survival were monitored for 3 months. The tumor diameter was determined using calipers, and data represent the means±SD.

• 

  Figure 4 Re-challenge of surviving IL-12 MethA stable transfectants with wild type MethA tumor cells. Three months after the first injection of tumor cells, surviving mice were re-injected with 1×106 wild-type MethA cells, intraperitoneally. Age-matched untreated mice (n=5) were used as a control.

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