Effects Of The Combined Administration Of Tumor/Gal And TLR Ligands On Function Of NKT Cells And DCs In Mice

Colorectal cancer is one of the most common malignant tumorsall over the world.Thecurrent treatment of surgery combined with radiotherapy and chemotherapycan not completely prevent its metastasis and recurrence. Butimmunotherapy which induces tumor-specific killing effect by improving the body’s immunity is expected to make up for thedefect of traditional treatment. Dendritic cells (DC) as the most effective professional antigen presenting cells play a key rollin immunotherapy. We previouslyconfirmed that as a preventive application, DC tumor vaccine significantly inhibited tumor growth, and the TLR ligand can effectively promote DC maturation; but DC tumor vaccine is not effective enough as a therapeutic application. Recent studies showed that NKT cells can enhance anti-tumor effect of DC, therefore, we assume that activate NKT cells to enhance the efficacyof tumor vaccine. As NKT cells can only recognize glycolipids presented by CD1d molecules, we firstly constructedCD1d molecules expressed MC38murine colon carcinoma cell lines, then loaded glycolipid on the cell lines to construct tumor/gal complex, next pulsed with TLR ligands as a new kind of tumor vaccine. We immunized mice via the tail vein injection with the new vaccine to observe its impact on function of NKT cells and DC.Part I.Preparation of Tumor/Gal complexObjectiveTo construct the CD1d expressingMC38murine colon carcinoma cell lines (CDld-MC38), verify its ability to combine with the glycolipida-GCstability, and the best conditionsin combination.Methods Transfected MC38cellswith lentivirus (GFP-FLAG-CD1d),screenedGFP+cells by flow cytometry; Verified the expression of CD1d molecules in GFP+cells by fluorescence microscopy, Western blot and flow cytometry; Investigatedwhether a-GC could combineCDld-MC38andthe optimal concentration and time in combinationby Binding experiment. ResultsObserved by a fluorescence microscope, number ofgreen fluorescence positive cells in CDld-MC38was (38.54±1.30)/FOV, which significantly higher than MC38cells (0±0)/FOV (P<0.001); Western blot showed that CD1d-MC38had a significant expression of FLAG proteins, while MC38hadn’t; CD1d-positive rate of MC38cells in flow cytometry was (0±0)%, contrast to CD1d-MC38cells (98.10±2.53)%(P<0.001);24h after CDld-MC38loaded a-GC (500ng/ml), CDld:a-GC binding rate was (12.11±2.15)%, which increased to (67.74±5.82)%after48h (P<0.01);24h after CD1d-MC38loaded a-GC (1μg/ml), CDld:a-GC binding rate was (21.07±3.48)%, which increased to (75.58±6.35)%after48h (P<0.01); there was no significant difference between the two rate in48h (P>0.05). Conclusion We successfully constructed the CDld expressing MC38murine colon carcinoma cell lines (CD1d-MC38); and it can be combined with a-GC stability.The optimal concentration and timeare1μg/ml and48h.Part Ⅱ.Effects of the combined administration of Tumor/Gal and TLR ligands (tumor vaccine) on function of NKT cells and DCs in MiceObjectiveTo study theeffects of administratethe tumor vaccine on function of NKT cells and DCs in Mice.Method C57BL/6mice were divided into five groups and injected PBS, a-GC, TLR9ligand, CD1d-MC38/a-GC complex and CD1d-MC38/a-GCcomplex+TLR9ligand by tail vein respectively:(1) Separating mononuclear cells from liver, detect CD69and CD40L on the surface andintracellular IFN-y of NKT cells by flow cytometry;(2) Separating lymphocytesfrom spleen, detect CD86and CD40on the surface of DCby flow cytometry;(3) Detect Thl/Th2cytokinesin serum by BD Cytometric Bead Array.Results The expression rate of CD69on NKT cells in the experimental group had no difference compared with a-GC group (P>0.05), but had significant difference compared with other three groups (P<0.01or P<0.05); the expression rate of CD40L on NKT cells in the experiment group was significant different from other four groups (P<0.01); the secretion rate oflFN-γ in NKT cells of the experimental group had no difference compared with a-GC group (P>0.05), but had significant difference compared with other three groups (P<0.01or P<0.05); the expression rate of CD86on DC in the experimental group had no difference compared with a-GC group (P>0.05), but had significant difference compared with other three groups (P<0.01); the expression rate of CD40on DC in the experiment group was significant different from other four groups (P<0.01); the IL-2concentration in the experiment group had significant difference compared with other four groups in each time point (P<0.01), and reached a peak after2hours; the TNF-α concentration in the experiment group had no difference compared with a-GC group(P>0.05), but had significant difference compared with other three groups in each time point (P<0.01), and reached a peak after2hours; the IFN-y concentration in the experimental group had significant difference compared with PBS, TLR9ligand and CDld-MC38/a-GC complex group (P<0.01or P<0.05); the IL-4concentration ina-GC group hadsignificant difference compared with PBS group (P<0.01), and reached a peak after2hours; theIL-4concentration in had difference compared with PBS only after2hours (P <0.05), but had no significant difference compared with other groups in each time potint (P>0.05).ConclusionThe tumor vaccinecan promote NKT cells and DC maturation, enhances their function and increase Thl cytokines (IL-2, TNF-α and IFN-γ) secretionin serum, but has no significant effect on secretion of Th2cytokine (IL-4).