Abstract
BackgroundCytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, it sefficacy is restricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells, we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, we tested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity, or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy.ResultsAfter stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice.ConclusionsOur findings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential to treat malignant tumors through in vivo adoptive therapy.
Highlights
Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors
The CIK cells had the CD3+CD56+ phenotype in the DC-CIK + Nb36 group with a total cells median percentage of 46.43%, which was significantly higher than the untreated DC-CIK group (33.46%, range 32.1–36.1%) and close to the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) mAb treated group (46.16%, range 42.8–49.42%) (Fig. 2B)
These results indicate that DC-CIK cells have a more efficient activation and expansion when CTLA-4 signaling is blocked
Summary
Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. It sefficacy is restricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In addition to dysfunctional stromal cells in the tumor microenvironment, there are various regulatory T-cells, myeloid-derived suppressor cells, and up-regulated tumor suppressor molecules such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) and programmed cell death protein-1 (PD-1) [5, 6] This complex tumor microenvironment is a hotbed for tumor development and leads to the exhaustion of infused DC-CIK cells, and is the main mechanism limiting the efficacy of DC-CIK adoptive therapy [7]
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