Figure legends
Figure 1. Flow diagram of eligible study population. A total of 68 patients were selected with advanced solid tumors (including 42 lung cancers, 14 liver cancers, nine colon cancers and three renal carcinomas) who had experienced disease progression after at least one previous course of tumor-appropriate treatment for advanced or metastatic disease.
Figure 2. Naïve T cells from patients were related to CIK cell therapy response. (A) Cluster analysis of flow cytometry data acquired from patients before CIK cell therapy. The measured values of subsets of absolute blood cell numbers are displayed as heatmaps, which are colored per subset according to mean-centered and sigma-normalized data (Z-scores). The top-bars indicate the PD, SD or PR/CR of each individual. (B and C) Flow cytometry analysis of sorted human CD45RA+CCR7+CD62L+naïve CD8+ and CD4+ T cells acquired from patinaiveents before CIK cels therapy. Numbers indicate the percentage of cells in the gates. Graphs show PD vs SD vs PR/CR expression in the CD45RA+CCR7+CD62L+CD8+or CD4+ gate. (D, F, G, H) Percentages and absolute numbers of CD8+ (D and F) and CD4+(G and H) T cells in subsets of patients with different clinical prognoses before CIK cells therapy (obtained as described in panel C).
Figure 3. Light microscopic representation of CIK cells from patients with high and low numbers of naïve T cells on days 3, 5, 9 and 13 after amplification in vitro . (B) Quantification of proliferation of T cells after amplification in vitro . Cells were placed in 96-well plates (4,000 cells/well); the first bar represents the unstimulated control cells, the second bar represents stimulated T cells. (C) The frequency of PD-1+TIM3+CD8+cells in CIK cell expansions from patients with high and low numbers of naïve T cells at the indicated time points. (D) The frequency of Ki67+CD8+ cells in CIK cell expansions from patients with high and low numbers of naïve T cells at the indicated time points. (E) Cytolytic activity of non-activated CIK cells expanded from patients with high and low numbers of naïve T cells in response to their respective autologous tumor cells, which were obtained from two NSCLC patients. E:T ratio, effector cell to target cell ratio. (F, G, H) The frequency of PD-1+TIM3+CD8+cells at each time point from PD (F), SD (G) or PR/CR (H) patients before or after amplification in vitro . (I) The frequency of IFN-γ+CD3+CD56+cells before and after amplification in vitro from patients in different response categories.
Figure 4. The CIK cells derived from patients with different frequencies of naïve T cells drive the response to therapy. (A, B, C & D) The frequency of IL-2+CD3+CD56+(A), IFN-γ+CD3+CD56+(B), PD-1+CD3+CD56+ (C) and Ki67+CD3+CD56+cells (D) before and on days 5, 10, 15, 20 and 25 after CIK cell therapy in different response categories.
Figure 5. Patients with high numbers of naïve T cells show more potent and durable antitumor responses in vivo. (A) Best percent change in target lesion tumor burden from baseline. Data only includes patients with baseline target lesions and one or more post-baseline target lesion assessments with no missing values (n = 66). Maximum percent reductions in target lesion tumor burden from baseline across all tumor assessments before subsequent therapy were used. Positive change in tumor burden indicates tumor growth; negative change in tumor burden indicates tumor reduction. Horizontal lines denote 30% decrease and 20% increase. Not all reductions of ≥30% from baseline are partial responses (i.e., decrease in target lesion tumor burden but new or progressive nontarget lesions). (B) Percent change in target lesion tumor burden from baseline over time. Data only includes patients with baseline target lesions and one or more post-baseline target lesion assessments with no missing values (n = 66). Horizontal lines denote 30% decrease, 20% increase and no change. (C) MRI showing that a 43-year-old CRC patient with brain metastases who had a high naïve T count experienced a CR of the brain after eight cycles of CIK cell infusion (Top). Chest CT scans showing a 51-year-old NSCLC patient with high numbers of naïve T cells experienced PR of the lung after four cycles of CIK cell infusion (middle). Chest CT scans showing a 48-year-old NSCLC patient with a high naïve T cell count experienced PR of the lung after four cycles of CIK cell infusion (bottom); the arrows show regression of ascites. (D) Time to and duration of response. CR, complete response; DOR, duration of response. (E) Progression-free survival (PFS). Data for PFS are based on an August 2018 database lock. Symbols denote censored observations.