Investigations of the functional states of dendritic cells under different conditioned microenvironments by Fourier transformed infrared spectroscopy

Abstract

Background: Dendritic cells are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. The dendritic cell-based vaccination against cancer has been clinically achieved promising successes. But there are still many challenges in its clinical application, especially for how to identify the functional states.

Methods: The CD14+ monocytes were isolated from human peripheral blood after plastic adherence and purified to approximately 98% with cocktail immunomagnetic beads. The immature dendritic cells and mature dendritic cells were induced by traditional protocols. The resulting dendritic cells were cocultured with normal cells and cancer cells. The functional state of dendritic cells including immature dendritic cells (imDCs) and mature dendritic cells (mDCs) under different conditioned microenvironments were investigated by Fourier transformed infrared spectroscopy (FTIR) and molecular biological methods.

Results: The results of Fourier transformed infrared spectroscopy showed that the gene transcription activity and energy states of dendritic cells were specifically suppressed by tumor cells (P < 0.05 or 0.01). The expression levels of NF-kappa B (NF-κB) in dendritic cells were also specifically inhibited by tumor-derived factors (P < 0.05 or 0.01). Moreover, the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were closely correlated with the expression levels of NF-κB (R2:0.69 and R2:0.81, respectively).

Conclusion: Our results confirmed that the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were positively correlated with the expression levels of NF-κB, suggesting that Fourier transformed infrared spectroscopy technology could be clinically applied to identify the functional states of dendritic cell when performing dendritic cell-based vaccination. It's significant for the simplification and standardization of dendritic cell-based vaccination clinical preparation protocols.

Figure 1

The schematic diagram of infrared spectrum of the contents of lipids and proteins in cells. Cells were adjusted to 2 × 10⁶/ml and washed twice with 0.9% NaCl in 1000 RPM centrifugation for 6 min. The supernatant was removed by centrifugation. The cells were transferred to the CaF₂ crystals at 37°C and left to stand for about 10 min. The water in the cell suspension was evaporated, until the formation of 2 ~ 3 mm film in the window. The crystals were fixed in the sample holder and covered with another CaF₂ crystal. To measure the background spectrum of a blank group, before each sample measurement by Infrared Spectrometer (ENXUS-470 FT-IR), blank control with 0.9% NaCl was used in the detection of infrared absorption spectra. The parameters of measurement were the scanning range of 400 ~ 4000 cm^-1, the resolution of 8 cm^-1, scanning the stack up to 256 times. The data analyses were performed using OMNIC6.0 software. All the spectra were subtracted blank control, and Fourier self-deconvolution, broadband = 56.4, sensitivity enhancement factor = 2.6, in deconvolution spectrum.

Figure 2

The expression levels of RelB in DCs under different conditioned microenvironments. Cells were lysed with RIPA buffer (20 mM sodium phosphate, pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 5 mM EDTA, 200 μM phenymethylsulfonyl fluoride, 1 μg/ml aprotinin, 5 μg/ml leupeptin, 1 μg/ml pepstatin and 500 μM Na₃VO₄). The protein extracts were electrophoresed on 12% ~ 14% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Invitrogen, USA). After blocking with 5% BSA in 0.1% Tween 20 in PBS, membranes were probed with primary antibodies. Anti-RelB and anti-β-actin antibodies (Sigma) were diluted in blocking buffer and incubated with the blots overnight at 4°C. The bound primary antibodies were probed with a 1:2000 diluted secondary antibody (goat anti-human IgG-HRP antibody) and visualized by the ECL chemiluminescence system (Amersham, USA). The gray values of proteins were measured by Image J (1.45). The expression levels of proteins were normalized to those of corresponding β-actin. Compared with DCs: ^*P < 0.05 or ^**P < 0.01.

Figure 3

Analyses of linear regression of the activities gene transcription and the expression levels of NF-κB. A. imDCs (R²: 0.69), B. mDCs (R²: 0.81).