Blocking the PD-1/PD-L1 axis in dendritic cell-stimulated Cytokine-Induced Killer Cells with pembrolizumab enhances their therapeutic effects against hepatocellular carcinoma

Abstract

Immune checkpoint therapies for cancer, like the anti-programmed cell death 1 (PD-1) agent pembrolizumab, have gained considerable attention. However, the use of immune checkpoint inhibitors in the context of adoptive immunotherapy is poorly characterized. We investigated the therapeutic efficacy of dendritic cell-stimulated CIK (DC-CIK) cells pretreated with pembrolizumab against hepatocellular carcinoma (HCC) in cytotoxicity assay in vitro and in a nude mouse xenograft model. We used time-lapse imaging to investigate tumor killing. We also performed a survival analysis based on lymphocyte subpopulation-specific mRNA signatures using The Cancer Genome Atlas (TCGA) HCC cohort (n=371 patients). The results indicated that PD-1 inhibition increased the anti-tumor effects of DC-CIK cells over those of DC-CIK cells alone, resulting in a survival benefit importantly. Time-lapse imaging revealed that DC-CIK cells appeared to be more effective and aggressive after anti-PD-1 treatment than after culture in control conditions. The PD-1 inhibitor also induced more effective immune cell infiltration of the tumor. Our analysis of the TCGA HCC cohort confirmed that a genetic signature consistent with a high degree of intratumoral CD8+ T cell infiltration is associated with good prognosis. These results suggest that blockade of the PD-1/PD-L1 axis in DC-CIK cells with a PD-1 inhibitor prior to infusion is a promising therapeutic strategy against HCC.

Keywords: PD-1; cytokine-induced killer cells; dendritic cells; hepatocellular carcinoma; immunotherapy.

Figure 1

The phenotypic patterns of CIK cells and dendritic cells. (A-B) Representative flow cytometric analysis of the expression of CD3, CD56, and CD8 on CIK cells at days 7, 14, and 21. (C) The quantitative statistics of phenotypes of CIK cells. (D) Representative pictures of the generation and expansion of CIK cells. (E) Representative flow analysis of PD-1 expression on DC-CIK cells. (F) Analysis of divided cell using CFSE staining by flow cytometry. (G) Phenotype analysis of DCs.

Figure 2

Anti-PD-1 DC-CIK cells efficiently killed HCC cells. (A) PD-L1 expression was assessed in HCC cell lines. (B) DC-CIK cells exerting cytotoxicity against SK-Hep.1or HCCLM3 with or without PD-1 blockade. (C) Anti-PD-1 DC-CIK cells co-cultured with HCC cell lines at different Effect:Target ratio. (D) Time-lapse imaging (see supplemental video S1and S2) used to observe contact process between HCCLM3 cells and DC-CIK cells with or without antibody. Cancer cells were surrounded by DC-CIK cells.

Figure 3

PD-1 blockade promoted the antitumor activity of DC-CIK in vitro. (A-C) The apoptosis (A), proliferation (B) and migration (C) of HCCLM3 and SK-Hep.1 cells after incubation with anti-PD-1 DC-CIK cells/DC-CIK cells were evaluated by apoptosis assay, colony formation assay, transwell migration assay. (D) Cytokine secretion assay determined by ELISA. (E) Expression of perforin1 and granzyme B were detected in anti-PD-1 DC-CIK cells and DC-CIK cells.

Figure 4

Pembrolizumab (PD-1 inhibitor) combined with DC-CIK cells attenuates the growth of HCC cells in vivo. (A) Representative picture of xenograft tumors formed. (B) Tumor volume was periodically measured for each mouse and growth curves were plotted. (C) Tumors were weighted. (D) Survival curves using Kaplan-Meier analysis. (E) Representative H&E as well as Ki67, and PCNA IHC staining of primary tumor tissues are shown. (F) Infiltration of DC-CIK cells in tumor tissues was shown by immunohistochemistry (CD8 staining).

Figure 5

Increased prevalence of CD8+ t cell-associated genetic signatures correlates with good prognosis in HCC patients. (A-E) TCGA HCC patient cohort (n=371)was stratified into “high-expression” or “low-expression” groups for genes associated with CD8A/B, granzyme A/B, perforin 1, followed by Kaplan-Meier plotting of patient's OS.