Abstract
Heat-shock protein (HSP) GP96 is a well-known adjuvant in immunotherapy. It belongs to the HSP90 family. Our previous study demonstrated that DC pulsed with recombinant senescence marker protein 30 (SMP30) could induce cytotoxic T lymphocytes (CTLs) against liver cancer cells in vitro. In this study, SMP30 and GP96 were subcloned into lentiviruses and transfected into DCs from healthy donors. We included six groups: the GP96-SMP30 group, GP96 group, SMP30 group, DC group, empty vector control group, and hepatoma extracted protein group. We used ELISA to detect cytokines and flow cytometry to assess CD80 and CD86 on DCs and the effect of CTLs. Our vector design was considered successful and further studied. In the SMP30 group, DC expresses more CCR7 and CD86 than the control group; in the SMP30+GP96 group, DC express more CCR7, CD86, and CD80 than the control group. Transfected DCs secreted more TNF-α and interferon-β and induced more CTLs than control DCs. SMP30 + GP96 effectively stimulated the proliferation of T cells compared with control treatment (P < 0.01). We detected the cytokines TNF-α, TNF-β, IL-12, and IFN (α, β, and γ) via ELISA (Figure 5) and verified the killing effect via FCM. Four E : T ratios (0 : 1, 10 : 1, 20 : 1, and 40 : 1) were tested. The higher the ratio was, the better the effects were. We successfully constructed a liver cancer model and tested the CTL effect in each group. The GP96 + SMP30 group showed a better effect than the other groups. GP96 and SMP30 can stimulate DCs together and produce more potent antitumor effects. Our research may provide a new efficient way to improve the therapeutic effect of DC vaccines in liver cancer.
Highlights
Heat-shock protein (HSP) GP96 is a well-known adjuvant in immunotherapy
HSPpeptide complexes isolated from intact tumor cells or virusinfected cells or reconstituted by covalent cross-linking or fusion protein strategies are all capable of eliciting potent cytotoxic T lymphocytes (CTLs)
Human dendritic cells were cultured for 6 days and adjusted to 5 × 105 cells/mL in 24-well plates. en, supernatants from designated wells were harvested after 48 hours for quantification of cytokines, such as interleukin (IL)-12, IL-1, and tumor necrosis factor-α (TNF-α) or regulated on activation, normal T cell expressed and secreted (RANTES), using ELISA kits (R&D Systems, Minneapolis, MN)
Summary
Human peripheral blood monocyte-derived dendritic cells were collected, and rhGM-CSF and rhIL-4 were added every two days. E experiment included six groups: the DC group (negative control), empty vector group, GP96 + SMP30 group (mixed group), GP96 group, SMP30 group, and extracted protein group (positive control). E six groups of cells were harvested, and total protein was isolated and quantified. E protein-antibody complexes were detected using an ECL detection kit following the manufacturer’s protocol. En, supernatants from designated wells were harvested after 48 hours for quantification of cytokines, such as interleukin (IL)-12, IL-1, and tumor necrosis factor-α (TNF-α) or regulated on activation, normal T cell expressed and secreted (RANTES), using ELISA kits (R&D Systems, Minneapolis, MN) Human dendritic cells were cultured for 6 days and adjusted to 5 × 105 cells/mL in 24-well plates. en, supernatants from designated wells were harvested after 48 hours for quantification of cytokines, such as interleukin (IL)-12, IL-1, and tumor necrosis factor-α (TNF-α) or regulated on activation, normal T cell expressed and secreted (RANTES), using ELISA kits (R&D Systems, Minneapolis, MN)
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