| BackgroundPhotodynamic therapy（PDT）is an effective treatment of superficial tumors in recent years.PDT can not only kill the tumor directly,but also induce the specific anti-tumor immune response.PDT acts on tumor tissue,resulting in immunogenicity death of some tumor cells and then the release of damage-associated molecular patterns（DAMPs）.Immature dendritic cell（DC）could recognize DAMPs and be activated into mature DC.Mature DC presents specific antigens to T cells,thus triggering specific immunity to tumor cells.Therefore,DC cells play a key role in the anti-tumor immune response induced by PDT,and DC maturation is the prerequisite for DC to play its function.It is important to study the factors and mechanisms that affect the maturation of DC in order to enhance the anti-tumor immune response induced by PDT.Protein 4.1R is our focus in the study of the factors and mechanisms affecting DC maturation induced by PDT.Protein 4.1R was first found in red blood cells and locates at the 4.1 band of erythrocyte membrane protein gel electrophoresis.As a80KD membrane skeleton protein,protein 4.1R connects the erythrocyte hemostatic protein-actin with transmembrane protein,which plays an important role in maintaining the structure,physiological function and mechanical stability of erythrocyte.Although the function of protein 4.1R in erythrocytes is clear,the role of protein 4.1R in nucleated cells has been unclear.Some studies have confirmed that protein 4.1R affected the occurrence and development of malignant tumors by participating in the formation of mitosis.Our previous studies have found that protein4.1N,which is very similar to protein 4.1R,was involved in the adhesion,migration and invasion of breast cancer cells.In addition,our study found that protein 4.1R affected the transmembrane transport of photosensitizer 5-ALA by interaction with transporter GAT1 and GAT2,and subsequent PDT anti-tumor effect.Our team also found that protein 4.1R affected the immune response by inhibiting the phosphorylation of T cell activating ligand（LAT）on the signal pathway mediated by T cell receptor.In view of the fact that protein 4.1R is directly involved in the signal activation of immune cells,and one of the mechanisms by which PDT kills tumor cells is to induce DC maturation and then anti-tumor immune response,it is reasonable to speculate that protein 4.1R may play an important role in the process of PDT induced DC maturation and affect the immune effect of PDT.However,the research on this aspect has not been reported yet.ObjectiveThe aim of this study is to investigate the effect of protein 4.1R on DC maturation and anti-melanoma immunity induced by PDT.This study can provide more theoretical basis for the study of the function of protein 4.1R and the mechanism of killing tumor cells by PDT.Methods1.Bone marrow protocells from normal and protein-deficient mice were induced into immature 4.1R+/+DC and 4.1R-/-DC respectively by cytokines;The purity of induced DC was detected by flow cytometry;PCR and Western blot techniques were used to detect the expression of protein 4.1R in two kinds of DC.2.Fluorescence spectrophotometer was used to measure the accumulation of PpⅨin the cells under different incubation concentration and incubation time,and to determine the best incubation concentration and incubation time of 5-ALA;The apoptosis of melanoma cell line B16 was detected by FITC Annexin V apoptosis kit,the expression of calcium reticulin on B16 cells was detected by flow cytometry and the secretion of HMGB1 in the supernatant was detected by ELISA,to determine the optimal dose of light to induce apoptosis of B16 cells.3.PDT treated B16 cells stimulated immature 4.1R+/+DC and 4.1R-/-DC into the mature;The expression of mature Marker CD80 and CD86 on the surface of mature 4.1R+/+DC and 4.1R-/-DC were detected by flow cytometry;The expression of IL-12p35,IFN-γgene in mature 4.1R+/+DC and 4.1R-/-DC was detected by fluorescence quantitative PCR;the difference of mature 4.1R+/+DC and 4.1R-/-DC morphology induced by PDT was observed under transmission electron microscope.4.Fluorescence quantitative PCR,flow cytometry and Western blot were used to detect the expression of PDT induced DC maturation related receptors in 4.1R+/+DC and 4.1R-/-DC.5.CD8+T cells were activated by 4.1R+/+DC and 4.1R-/-DC sensitized by PDT.The expression of Marker CD69 activated on the surface of CD8+T cells was detected by flow cytometry;the proliferation ability of CD8+T cells activated by two kinds of DCs was detected by CCK8 method;The cytotoxicity of CD8+T cells activated by two kinds of DCs to melanoma B16 was detected by LDH cytotoxicity kit.6.The lung metastasis model was established by injecting B16-F10 tail vein into C57BL/6J mice and then the mice were immunized with 4.1R+/+DC and 4.1R-/-DC sensitized by PDT,respectively.The concentration of IFN-γand IL-12 in the serum of model mice was detected by ELISA technique.The lung metastases of the two groups were counted and compared,and the survival curves of the mice in different treatment groups were calculated.Results1.The purity of 4.1R+/+DC and 4.1R-/-DC generated from bone-marrow progenitor cells and induced by cytokine GM-CSF and IL-4 were about 80%.The results of gene level and protein level test showed that protein 4.1 R was expressed in4.1R+/+DC,but not in 4.1R-/-DC.2.The apoptosis rate of B16 cells was the highest（28.45±3.04）%under the irradiation of 4mM 5-ALA 5 hours and 0.25J/cm2 doses in mice melanoma B16 cells.With the increase of apoptosis rate,the expression of calreticulin increased,up to（80.73±6.09）%,and the more HMGB1 was secreted in the supernatant,the highest was（7909.16±537.84）pg/ml.3.Mouse melanoma B16 was treated with optimal PDT and co-cultured with4.1R+/+DC and 4.1R-/-DC at 50:1 for 24 hours respectively.The expression ratio of CD80 on 4.1R+/+DC cell surface was（73.70±8.13）%,significantly higher than that of 4.1R-/-DC（47.33±14.50）%,P<0.01,and the expression of CD86 on 4.1R+/+DC（66.00±10.64）%was significantly higher than that on 4.1R-/-DC（42.57±5.60）%,P<0.01.The relative expression of IL-12p35 in 4.1R+/+DC（15.99±3.81）was higher than that in 4.1R-/-DC（9.19±0.83）,P<0.01;the relative expression of IFN-γin4.1R+/+DC（8.66±0.61）was higher than that in 4.1R-/-DC（5.33±0.95）,P<0.01.The dendrites formed on the surface of 4.1R+/+DC sensitized by PDT were more abundant than those on 4.1R-/-DC under transmission electron microscope.4.In the 8th hour of PDT sensitizing 4.1R+/+DC and 4.1R-/-DC,fluorescence quantitative PCR showed that the expression of TLR4 in 4.1R+/+DC was（3.92±0.68）,which was significantly higher than that of 4.1R-/-DC（2.97±0.34）,P<0.05;flow cytometry showed that the expression of TLR4 on 4.1R+/+DC membrane was significantly higher than that on 4.1R-/-DC membrane,（82.33±4.58）%vs（45.50±18.92）%,P<0.01;Western blot results showed that the total protein expression of TLR4 in 4.1R+/+DC was significantly higher than that in 4.1R-/-DC,（1.88±0.28）vs（0.61±0.14）,P<0.01.5.PDT sensitized 4.1R+/+DC and 4.1R-/-DC were co-cultured with 4.1R+/+CD8+T and 4.1R-/-CD8+T cell,respectively（mixed ratio 1:20 and co-culture time 72hours）.The proportion of CD69 expressed in 4.1R+/+DC-4.1R+/+CD8+T was（71.40±1.41）%,which was significantly higher than that in 4.1R-/-DC-4.1R+/+CD8+T（42.25±2.05）%,P<0.01.The expression of CD69 in 4.1R+/+DC-4.1R-/-CD8+T was（88.45±1.34）%significantly higher than that in 4.1R-/-DC-4.1R-/-CD8+T（59.05±1.77）%,P<0.01.These results suggested that the deletion of protein 4.1R on DC negatively regulates the activation of CD8+T cells stimulated by PDT-DC.6.PDT sensitized 4.1R+/+DC and 4.1R-/-DC were co-cultured with 4.1R+/+CD8+T and 4.1R-/-CD8+T cell,respectively（mixed ratio 1:20 and co-culture time 72hours）.The stimulation index of 4.1R+/+DC to 4.1R+/+CD8+T（3.73±0.50）was significantly higher than that of 4.1R-/-DC to 4.1R+/+CD8+T（1.75±0.31）,P<0.05.The stimulation index of 4.1R+/+DC to 4.1R-/-CD8+T was（5.23±0.33）,which was significantly higher than that of 4.1R-/-DC to 4.1R-/-CD8+T（2.30±0.39）,P<0.05.These results suggested that the deletion of protein 4.1R on DC negatively regulates the proliferation of CD8+T cells stimulated by PDT-DC.7.PDT sensitized 4.1R+/+DC and 4.1R-/-DC were co-cultured with 4.1R+/+CD8+T and 4.1R-/-CD8+T cell,respectively（mixed ratio 1:20 and co-culture time 72hours）.The cytotoxicity of 4.1R+/+DC-4.1R+/+CD8+T cells to B16 was significantly stronger than that of 4.1R-/-DC-4.1R+/+CD8+T cells under different target ratio and the killing rate of the two groups was（47.74±3.45）%vs（24.24±2.46）%（P<0.001）under the condition of the effective target ratio of 80:1.The cytotoxicity of 4.1R+/+DC-4.1R-/-CD8+T cells to B16 was significantly stronger than that of 4.1R-/-DC-4.1R-/-CD8+T cells under different target ratio and the killing rate of the two groups was（60.87±2.14）%vs（36.48±2.63）%（P<0.001）under the condition of the effective target ratio of 80:1.These results suggested that the deletion of protein 4.1R on DC negatively regulates the killing B16 of CD8+T cells mediated by PDT-DC.8.Serum concentration of IFN-γand IL-12 in mice immunized with PDT sensitized 4.1R+/+DC were（1501±415.2）pg/ml and（88.53±26.85）pg/ml,which were significantly higher than IFN-γ（1501±415.2）pg/ml and IL-12（42.57±11.12）pg/ml in 4.1R-/-DC immunized mice with lung metastasis model.The number of lung metastases in PDT sensitized 4.1R+/+DC immunized mice（43.40±15.08）was significantly lower than that in4.1R-/-DC immunized mice（91.30±20.37）,P<0.01.The survival time of mice immunized with PDT sensitized 4.1R+/+DC was significantly longer than that of PDT sensitized 4.1R-/-DC immunized mice,P<0.001.These results suggested that the deletion of protein 4.1R on DC down-regulates the inhibition of melanoma mediated by PDT induced mature DC.Conclusion1.Protein 4.1R is involved in the induction of DC maturation by PDT,and the deletion of protein 4.1R decreases in the ability of PDT to induce DC maturation,and then down-regulates the activation and proliferation of CD8+T cells mediated by DC;2.The deletion of protein 4.1R down-regulates the anti-melanoma effect mediated by PDT induced mature DC. |
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