Sunitinib indirectly enhanced anti-tumor cytotoxicity of cytokine-induced killer cells and CD3⁺CD56⁺ subset through the co-culturing dendritic cells

Abstract

Cytokine-induced killer (CIK) cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs). We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6) in DCs at the expense of Th2 inducing phenotype (IL-13) and regulatory phenotype (PD-L1, IDO). Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet) and the downregulation of the Th2 signature (GATA-3) and the Th17 marker (RORC) on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.

Figure 1. The cytotoxic activity against HubCCA1 cell line after the priming with sunitinib-pretreated DCs.

The CIK cells (A) at 1.0×10⁵ cells/well from each condition were incubated with the attached HubCCA1 cells (5,000 cells/well) for 4 h before the PI assay. The CIK cell preparations comprised untreated condition, direct sunitinib treatment, macrophage co-culture, sunitinib-treated macrophage co-culture, iDC co-culture, and sunitinib-treated iDC co-culture, mDC co-culture, and sunitinib-treated mDC co-culture. The isolated CD3⁺CD56⁺ cells (B) were studied in similar fashion. These included untreated CD3⁺CD56⁺ cells, direct sunitinib treatment, mDC co-culture, and sunitinib-treated mDC co-culture. * and ** designate data with significant different from those of the untreated CIK cells at the same effector to target (E:T) ratio with p<0.05 and <0.01 respectively.

Figure 2. The real-time RT-PCR analysis in macrophages (Φ), iDC and mDC after sunitinib exposure.

These cells were evaluated for the expression of IL-12 (A), IFN-γ (B), IL-6 (C), IL-13 (D), IL-10 (E), PD-L1 (F), IDO (G), and IL-23 (H). The expression levels of these genes were normalized with those of their respective untreated mDCs.

Figure 3. The FACS analysis for the maturity of macrophages, iDCs, and mDCs after sunitinib exposure.

The markers for the maturity of DCs (A) are CD80, CD83 and CD86. The macrophage markers (B) are CD14 and CD40. The selected immunosuppressive molecules (C) are IDO and IL-10.

Figure 4. The FACS analysis for the alteration in the proportion of subpopulations in CIK cells.

The studied subpopulations included CD3⁺CD56⁺(A), Th17 (RORC⁺IL-17⁺, B) and Treg (CD4⁺CD25⁺Foxp3⁺, C) subsets. The corresponding dot plot analysis demonstrated the gating of each subset. The CIK cells were either exposed to sunitinib directly, primed with mDCs or primed with sunitinib-pretreated DCs.

Figure 5. The real-time RT-PCR analysis for the polarization of CD3⁺CD56⁺ subset after different treatments.

The CD3⁺CD56⁺ subset that had been directly exposed to sunitinib or co-cultured with sunitinib-pretreated mDCs were analyzed for the expression of IFN-γ, T-bet, IL-4, GATA-3, RORC, STAT3, and IDO.

Figure 6. The cytotoxic activity of all conditions of CD3⁺CD56⁺ cells could be neutralized with αIFN-γ treatment.

The CD3⁺CD56⁺ cells from each condition were incubated with the attached HubCCA1 cells (5,000 cells/well) for 4 h before the PI assay. These conditions included the untreated CD3⁺CD56⁺ cells, αIFN-γ treatment to CD3⁺CD56⁺ cells, CD3⁺CD56⁺ cells primed with mDC, αIFN-γ treatment to CD3⁺CD56⁺ cells primed with mDC, CD3⁺CD56⁺ cells primed with sunitinib-pretreated mDC, and αIFN-γ treatment to CD3⁺CD56⁺ cells primed with sunitinib-pretreated mDC. * designates conditions that provided statistically difference after αIFN-γ treatment at the same E:T ratio with p<0.05.