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. 2016 Apr 5;7(14):17579-90.
doi: 10.18632/oncotarget.8198.

Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes

Longhao Sun  1 , Hao Guo  1 , Ruoyu Jiang  1 , Li Lu  1 , Tong Liu  1 , Zhixiang Zhang  1 , Xianghui He  1
Affiliations

Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes

Longhao Sun et al. Oncotarget. .

Abstract

Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP(158-166)-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP(158-166) peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; adoptive immunotherapy; alpha-fetoprotein; artificial antigen-presenting cells; cytotoxic T lymphocytes; interleukin-15.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Expression of AFP158-166 peptide-HLA-A*02:01 complex, CD80, CD86, and IL-15 in DC, BA15, and BJAB cells
A. FCM revealed that there were not significant differences in the expression of HLA-A2, CD80, and CD86 among DC, BA15 and BJAB cells. B. qRT-PCR and ELISA showed that the mRNA and secreted protein levels of IL-15 in BA15 cells were significantly higher than those in DC and BJAB cells. C. HPLC showed that the eluting peak corresponding to the synthetic AFP158-166 peptide was found in acid-stripped BA15 cells but not in BJAB cells. Mass spectrometry also revealed that the molecular weight of the peptide in this eluting peak was the same as that of the synthetic peptide. Error bars indicate standard deviations. * indicates P < 0.05.
Figure 2
Figure 2. Stability of AFP158-166 peptide-HLA-A*02:01 complex, CD80, CD86, and IL-15 expression in BA15 cells after γ-ray irradiation
A. FCM revealed that the expression of HLA-A2, CD80, and CD86 were not significantly affected by different dosages of irradiation. B. ELISA showed that the secretion of IL-15 in BA15 cells decreased after exposure to 30 Gy of irradiation but was stable at lower dosages. C. HPLC showed that the eluting peak corresponding to the synthetic AFP158-166 peptide was found in acid-stripped BA15 cells both pre- and post-irradiation. Mass spectrometry revealed that the molecular weight of the peptide in this eluting peak was the same as that of the synthetic peptide. Error bars indicate standard deviations.
Figure 3
Figure 3. Inhibition of proliferation and induction of apoptosis of BA15 by γ-ray irradiation
After different dosages of irradiation, the cell viability and proliferation of BA15 cells were analyzed by MTT, cell counting, and CFSE assays. Apoptosis assays were performed every 3 days after irradiation. A. MTT assay indicated that the cell viability of BA15 cells decreased after exposure to 20 Gy and 30 Gy of irradiation. B. Cell counting indicated that the number of BA15 cells decreased after exposure to 20 Gy and 30 Gy of irradiation. C. CFSE labeling revealed that the proliferation of BA15 cells was completely inhibited after exposure to irradiation of 20 Gy and 30 Gy. D. The apoptosis assay revealed that all the cells in the 20-Gy and 30-Gy group were in apoptosis or dead 3 days after irradiation and that all the cells had died by day 12. There were fewer dead cells in the 20-Gy group than in the 30-Gy group at every time point. Error bars indicate standard deviations.
Figure 4
Figure 4. Activation and expansion of functional AFP158-166-specific CTLs with BA15 cells
CTLs isolated from HLA-A*02:01+ healthy donors were stimulated weekly by co-culturing with different APCs for 3 cycles. A. Cell counting showing CTL activation by different APCs and APC/lymphocyte ratios. BA15 cells activated CTLs at maximum efficiency at a ratio of 1:10. After 3 cycles of stimulation at this ratio, BA15 cells and DCs showed the same CTL activation efficiency. B. CFSE assay showing activation of CTLs by different APCs at different APC/lymphocyte ratios. BA15 cells had maximum CTL activation efficiency at a ratio of 1:10. C. AFP158-166 MHC Pentamer staining showed that CTLs generated by BA15 cells established a higher proportion of AFP158-166-specific CTL population than CTLs generated by peptide-pulsed DCs (6.7 ±0.4% vs. 4.5 ±0.3%, respectively, P < 0.05). D. Secretion of IFN-γ by antigen-specific CTLs activated by different APCs at different APC/lymphocyte ratios. A ratio of 1:10 displayed the highest levels of antigen-specific secretion of IFN-γ. The mean number of SFU per million was 810.1 (±58.3) in BA15-activated CTLs versus 660.4 (±54.2) in CTLs obtained with AFP-pulsed DCs. E. Comparison of the specific lysis rate of CTLs activated by different APCs toward HepG2. The CTLs activated by BA15 cells had significantly higher lysis rates toward HepG2 cells than did the CTLs obtained from AFP-pulsed DCs. F. Comparison of the specific lysis rate of AFP-positive (HepG2 and T2+AFP) and AFP-negative (SW480, MCF7, T2, and T2+HER2/neu) cells. AFP-positive cells were specifically lysed by antigen-specific CTLs stimulated with BA15 and peptide-pulsed DCs, but AFP-negative cells were not. E: T Ratio indicates effector to target ratio. Error bars indicate standard deviations. * indicates P < 0.05. NS: not significant.
Figure 5
Figure 5. Suppression of tumor growth in vivo by AFP158-166-specific CTLs generated by BA15 cells
HepG2 cells were used to establish xenograft tumors in NOD /SCID mice. Nonspecific activated CTLs, DCs, or BA15-induced AFP-specific CTLs (1 × 108 in 0.2 mL PBS) were transferred into mice of different groups via intravenous injection at days 0, 7, and 14. Black arrows indicate the treatment times. PBS-treated mice were used as a control group. A. Treatment with AFP-specific CTLs expanded by DCs and BA15 displayed significantly higher tumor-suppressive functions than treatment with nonspecific activated CTLs or PBS. CTLs expanded by BA15 were the most efficient of these groups. B. HE staining showed that there were significantly more tumor cells exhibiting morphological changes indicative of apoptosis and necrosis in the BA15 group than others. The infiltration of CD8+ CTLs in the BA15 group was also significantly higher than that in other groups (P < 0.01). Scale bar = 100 μm; magnification, 400 ×. Error bars indicate standard deviations. * indicates P < 0.05.
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