Calreticulin is an effective immunologic adjuvant to tumor-associated antigens

Abstract

As a key molecule involved in cell recognition, calreticulin (CRT) may be expressed on the surface of (pre-) apoptotic cells and provide the signal that is recognized by dendritic cells (DCs) or other antigen presenting cells (APCs), which results in phagocytosis. Within the APCs, tumor-associated antigens (TAAs) may be subsequently presented to T lymphocytes, which triggers a specific antitumor immune response. It has been hypothesized that CRT is able to act as the immunologic adjuvant and translocate itself and TAAs to the cell surface and induce a specific antitumor immune response. In the present study, CRT was demonstrated to translocate itself and mucin 1 (MUC1), a breast cancer antigen, to the surface of 4T1 cells and the MUC1-CRT-coated cells were able to induce apoptosis in a time-dependent manner. When DCs were infected with adenovirus containing MUC1-CRT, an increase in T cell proliferation and cytokine production was exhibited. These results suggest that CRT may act as an immunologic adjuvant with MUC1 and induce a strong immune response.

Keywords: breast cancer; calreticulin; dendritic cells; mucin 1; tumor-immunity.

Figure 1.

Detection of the subcellular localization of MUC1-CRT in 4T1 cells. Fluorescence microscopy was performed to elucidate the subcellular localization of MUCI-CRT in 4T1 cells. (A) pEGFP-CRT, (B) pEGFP-MUC1 and (C) pEGFP-MUC1-CRT were transiently transfected into 4T1 cells, treated with mitoxantrone (8 µg/ml) and cultured for 12 h prior to fluorescence microscopy analysis. Magnification, ×400. MUC1, mucin 1; CRT, calreticulin.

Figure 2.

Detection of CRT and MUC1 promoting a phagocytic signal on the cell surface of apoptotic 4T1 cells by DNA fragmentation analysis. DNA fragmentation was induced by mitoxantrone (10 µg/ml) for 24 h. Lane 1, 4T1 control cells; lane 2, 4T1 cells transiently transfected with pEGFP-MUC1; lane 3, 4T1 cells transiently transfected with pEGFP-CRT; lane 4, 4T1 cells transiently transfected with pEGFP-MUC1-CRT; M1 and M2, DNA markers. MUC1, mucin 1; CRT, calreticulin.

Figure 3.

Fluorescence microscopy analysis of the apoptotic behavior of CRT-MUC1-coated 4T1 cells. pEGFP-MUC1-CRT was transiently transfected into 4T1 cells and treated with (A) 0 µg/ml, (B) 2 µg/ml, (C) 4 µg/ml and (D) 8 µg/ml mitoxantrone for 12 h, or with (E) 0 µg/ml, (F) 2 µg/ml, (G) 4 µg/ml and (H) 8 µg/ml mitoxantrone for 24 h and the results were observed by fluorescence microscopy. Fluorescence microscopy was used to detect that the apoptotic behavior of CRT-MUC1-coated 4T1 cells treated with mitoxantrone was dose- and time-dependent. Magnification, ×400. MUC1, mucin 1; CRT, calreticulin.

Figure 4.

T cell proliferation induced by MUC1-CRT-infected dendritic cells. Splenocytes were incubated with adenovirus-infected dendritic cells and CD3⁺/CD4⁺/CD8⁺ T cells were detected by flow cytometry using anti-CD3-PerCP, anti-CD4-Pacific Blue and anti-CD8-Alexa Flour 700 antibodies. (A and B) Control, (C and D) pEGFP-MUC1 and (E and F) pEGFP-MUC1-CRT. A, C and E, CD3 T cells; B, D and F, CD4 and CD8 T cells. CD, cluster of differentiation; MUC1, mucin; PerCP, peridinin chlorophyll protein complex; CRT, calreticulin.

Figure 5.

IFN-γ and TNF-α production by MUC1-CRT-infected dendritic cells. Dendritic cells were infected with adenoviruses containing MUC1 and MUC1-CRT, respectively, and further co-incubated with lymphocytes. Non-treated cells were used as control, and cells treated with mock vehicle were used as Nco. Culture supernatants were used to analyze (A) IFN-γ and (B) TNF-α levels by ELISA assay. Data are presented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.005. INF-γ, interferon-γ; TNF-α, tumor necrosis factor-α; MUC1, mucin 1; CRT, calreticulin; Nco, null control.