Adjuvant Screen Identifies Synthetic DNA-Encoding Flt3L and CD80 Immunotherapeutics as Candidates for Enhancing Anti-tumor T Cell Responses
Amy Haseley ThorneKirsten N. MaloAshley J. WongTricia T NguyenNeil CoochCharles C. ReedJian YanKate E. BroderickTrevor R.F. SmithEmma L. MastellerLaurent Humeau
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Abstract:
Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.Keywords:
CD80
Cancer Immunotherapy
Objective:To investigate the effects of Jitai on the function of dendritic cells derived from patients with chronic hepatitis B(CHB).Methods:Dendritic cell(DC) derived from monocyte of the patients with chronic HBV infection and cultured with IL-4 /GMCSF were treated with different Jitai concentrations on 4 d.Cell morphology was monitored under light microscopy.Cell surface molecules including HLA-DR,CD80,CD83,CD1a were assayed by flow cytometry.T cell proliferation induced by DC was assayed by methyl thiazolyl tetrazolium(MTT).Results:The stimulatory capacity of Jitai(100 ug/mL) in the allogeneic mixed leukocyte reaction(All MLR) markedly enhanced compared with the control group(P0.05).The expressions of CD1a on DC treated with Jitai was significantly higher than those of DC untreated with Jitai(P0.05),so was the expression of CD80(P0.05).Conclusion:Jitai in certain concentration can improve the immunology function of DC derived from CHB.
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Objective To investigate the effects of house dust mites (HDM) with diverse concentrations on the expressions of co-stimulatory molecules of HDM major antigen gene Der p2 modified dendritic cell(DC). Methods Der p2 gene modified DC(Der p2-DC) and control DC(non-transfected DC)were treated 72 h by H DM with diverse concentrations respectively. The expressions of CD80,CD86 and MHC Ⅱ ,the surface co-stimulatory molecules on Der p2-DC and control DC, were detected by FACS.Results FACS analysis showed that positive expression percentages of CD80,CD86 and MHC Ⅱ on Der p2-DC treated with HDM had no significant change,compared with Der p2-DC treated without HDM.After treated with HDM, positive expression percentages of CD80, CD86 and MHC Ⅱ on control DC increased significantly. Conclusions Specific allergen pulsing could not increase the expressions of costimulatory molecules of allergen gene modified DC.
Key words:
Dendritic cell; Co-stimulatory molecule; Allergen; Immune tolerance
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Objective:To elucidate the effect of APS on inducing the cord blood monocyte in vitro into the dendritic cells(DCs).Methods:①The cord blood monocytes were isolated and obtained by lymphocyte isolation.two groups were divided;②Cultured in the RPMI-1640 culture with or DCs was identified by inverted optical microscope or scanning electron microscope.The phenotype of cultured 12 days DCs(CDla,CD80,CD86,and CD83)was identified by flowcytometry.Results:Cultured for 72 hours,the morphous of cell of the experiment group grew clustering and began to change from round to irregularity,appearing rough cell face and barb pustute.The longer cell cultured,the more obvious the dendritic structure is.The experiment group cell containing 100 mg/L APS cultured for 12 days had the most typical dendritic structure.the negative control group cell had no dendritic structure and became the macrophage when cultured for 12 days.The experiment group cell cultured for 10 days showed typical dendritic morphotype by SEM and significantly expressed the high level phenotype of DCs(CDla,CD80,CD86,and CD83)by flowcytometry after cultured for 12 days.Conclusion:APS and could induce the cord blood monocyte to committed differentiate into functional DC.
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Cord blood
Monocyte
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Dendritic cells (DCs) and cytokines play an important role in the tumor growth and recurrence.Sixty-six patients with superficial transitional cell carcinoma of the bladder (STCCB) and 38 healthy controls were studied to investigate the percentages of DC subsets, monocyte-derived DC (MoDC) function, and alterations of Th1 and Th2 cytokines. MoDCs were generated and three-color flow cytometry was used for determining the phenotype of MoDCs and DC subsets. The ability to stimulate autologous T cells was tested in mixed leukocyte reaction (MLR). The levels of various cytokines were measured using commercially available sandwich enzyme linked immunosorbent assay (ELISA) kit.The myeloid DC (mDC) counts, MoDC surface molecular expression, and stimulatory capacity to T cells were impaired in STCCB patients than in controls. The percentage of mDC and the expression of CD80, CD83, and CD86 were lower in patients showing recurrence. The serum levels of IL-2 and IFN-γ were found to be significantly lower while IL-4, IL-6, and IL-10 were significantly higher in STCCB patients than in controls. IL-6 was found to be significantly higher in recurrent patients.The impairment of mDC counts and MoDC function with imbalance of Th1/Th2 cytokines was closely associated with proliferation and recurrence of STCCB.
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ObjectiveTo investigate whether immune adjuvant can enhance the immunity of dendritic cell vaccine against murine breast cancer. Methods4 groups of mice with tumor are injected saline, immume adjuvant, dendritic cell (DC) vaccine and DC vaccine coupled with immune vaccine, respectively. Tumor volume and weight are measured 21 d later.ResultsThe tumor size in the DC vaccine coupled with immune vaccine group was significantly small compared with control group (P=0.001) and the DC vaccine group (P=0.047).ConclusionImmune adjuvant can enhance the immunity of dendritic cell vaccine against murine breast cancer.
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To determine whether gamma irradiation influences phenotype and function of human dendritic cells (DC) in vitro, dendritic cells were induced from the peripheral blood mononuclear cells of multiple sclerosis patients with RPMI 1640 medium containing recombinant human GM-CSF (rhGM-CSF, 800 U/ml) and recombinant human IL-4 (rhIL-4, 500 U/ml). Phenotypic changes were monitored by light microscopy. Lipopolysaccharide at a concentration of 5 micro g/ml was added into the cultures after 6 days of growth for DC complete maturation, and the cells were cultured for another 24 hours. The harvested DC on day 7 were divided equally into several parts. One part was used as non-irradiated DC (naive DC) while the other parts were irradiated by gamma ray at a dose of 25 Gy and 30 Gy respectively. Cell surface molecules were analyzed by flow cytometry. The capability of DC to stimulated autologous T cell proliferation were determined. The results showed that gamma irradiation reduced expression of CD86, CD80 and HLA-DR molecules on dendritic cells, especially CD86 molecules. Dendritic cells effectively stimulated autologous T cells proliferation while irradiated DC in all groups showed profound decrease of capability to promote T cells proliferation. It is concluded that gamma irradiation of dendritic cells not only influenced phenotype of DC but also altered their function as stimulator cells in mixed lymphocyte reaction.
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Objective To compare the phenotypes of bone marrow derived dendritic cell(BMDC)and DC2.4 cell stimulated with GST from Schistosoma japonicum.Methods Bone marrow cells were cultured in the presence of IL-4 and GM-CSF to induce dendritic cells.DC2.4 cells were cultured as routine.Both cells were stimulated with GST and the expressions of CD40,CD80 and CD86 on the cells'surface were analyzed by FACS,using PBS and lipopolysaccharide as controls. Results After stimulating with GST,the means of fluorescence intensity(MFI)for CD40,CD80 and CD86 on BMDC surface were 100.39,42.38 and 170.83,respectively.Compared with PBS control,the MFI of CD80 and CD86 on BMDC,but not CD40,enhanced significantly.The MFIs of CD40.CD80 and CD86 on DC2.4 loaded by GST were 23.73,72.13 and 59.58 respectively.Compared with PBS control,the expressions of CD40 and CD86 enhanced significantly after schistosome antigen stimulation.Conclusion The expressions of cell surface molecules after schistosome antigen GST stimulation were different between BMDC and DC2.4.
Key words:
Schistosoma japonicum; Dendritic cell; DC2.4; Phenotype
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Follicular dendritic cells
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