Characterization of ex vivo-expanded murine NK cells.

BM cells were cultured with IL-15 and IL-12 as described in Methods. (A) Analysis of NK cells (CD19-TCRαβ- TCRγδ-NK1.1+) among live expanded cells. Plots are representative of 20 independent cultures. (B)-(D) Expression of MHC-I-interacting activating and inhibitory NKRs, activation markers and anti-tumor effectors by the expanded NK cells. Representative flow histograms are presented for each molecule, and the compiled data from independent expansion cultures is indicated by the dot plots. (E) In vitro cytotoxicity of the expanded NK cells. CFSE-labeled EO771 or B16F10 cells (T, target) were co-cultured with expanded NK cells (E, effector) at the indicated E:T ratios. Tumor cell viability was analyzed by PI staining using FACS. One representative of three independent experiments is shown. Data represent mean ± SEM of triplicate wells. Statistical significance was determined by unpaired two-tailed Student’s t-test: *P<0.05, **P<0.01, ***P<0.001 relative to tumor only. (F) Tumor cells stimulate IFN-γ production by the expanded NK cells. Sorted NK cells were cultured alone (NK cells) or together with tumor cells (NK cells + EO771 and NK cells + B16F10) at a ratio of 1:1. Levels of intracellular IFN-γ of NK cells were determined by staining with anti-IFN-γ or isotype control antibody, and analyzed by FACS. The difference in mean fluorescence intensity (ΔMFI) was calculated by subtracting the MFI value of isotype control staining from that of anti-IFN-γ antibody staining. Representative plots of two independent experiments are shown.

Figure supplement 1. Expression of MHC-I molecules and Rae-1 by EO771 and B16F10 cells in vitro.

EO771 tumor resection and metastasis model for NK cell treatment.

(A) Positive correlation between primary tumor weight and lung metastasis. Pearson correlations between day 21 tumor weights and the number and total area of metastatic foci on the lung surface are shown. Each dot represents a mouse. (B) Survival of mice who received day 21 tumor resection, sham surgery, or no treatment. Mouse survival was analyzed by means of the Kaplan-Meier estimator and a Log-Rank test: ****P<0.0001. (C) Expanded NK cells express the chemokine receptors CXCR3, CCR5 and CXCR6. Representative flow plots of 10 independent cultures are shown. (D) Expression of CXCR3, CCR5 and CXCR6 ligands in the lung. Specific gene expression was determined by quantitative real-time PCR using lung RNA of day 21 tumor-resected mice collected on day 24 post tumor inoculation and normalized to the expression of cyclophilin a. Each symbol represents a mouse and means ± SEM are shown. (E) Expanded eGFP+ NK cells were transferred into day 21 EO771-resected mice on day 24, and quantified in the single cell suspensions of spleen and lung at the indicated times using FACS. Numbers of eGFP+ NK cells detected in each organ are shown. Each dot represents a mouse (means ± SEM).

Syngeneic NK cell therapy is effective in treating mice with low metastatic burden in a CD8+-T-cell-dependent manner.

(A) Day 21 tumor weight is inversely correlated with survival duration of NK-cell-treated tumor-resected mice. After tumor and sentinel LN resection on day 21, mice received either sorted NK cells (NK cell) or PBS (Control) at the indicated time-points. The correlation between day 21 tumor weight and survival time of mice was evaluated by Pearson correlation. Each dot represents a mouse. The results have been compiled from 5-8 independent experiments. The Control group is the same dataset as presented for the Resection group in Figure 2B. (B) NK cell therapy promotes long-term survival of mice who had light tumors on day 21. The mice described in (A) were separated into 95-400 mg and >400 mg day 21 tumor weight groups for survival analysis by means of the Kaplan-Meier estimator and a Log-Rank test: *P<0.05. (C) NK cell therapy induces tumor-specific protection. Surviving mice after NK cell therapy were rechallenged with EO771 (●, compiled from four independent experiments) or B16F10 cells (▴, compiled from two independent experiments) at 11-13 weeks post therapy, and then monitored for survival. Age-matched naïve mice were inoculated with EO771 (○, compiled from four independent experiments) or B16F10 cells (△, compiled from three independent experiments) as controls. Mouse survival, using 2000 mm3 tumor volume as a cut-off, was analyzed by means of the Kaplan-Meier estimator and a Log-Rank test: ****P<0.001. (D) Depletion of CD8+ cells abolished the efficacy of NK cell therapy. Mice were treated with indicated antibodies at day 19 post tumor inoculation, and then received resection followed by NK cell therapy. Mouse survival was analyzed by means of the Kaplan-Meier estimator and a Log-Rank test: ns, not significant; *P<0.05; ***P<0.001; ****P<0.0001. The data are compiled from 2-3 experiments. (E) T cells isolated from surviving mice post NK cell therapy and tumor re-challenge confer anti-tumor activity. Post NK cell therapy and re-challenge survivors were collected from three independent experiments. T cells and non-T cells isolated from each survivor were transferred separately into naïve recipient mice in a 1-donor-to-1-recipient manner, whereas other naïve mice that received no cells served as a control (compiled from two independent experiments). T cells and non-T cells were also isolated from age-matched naïve donors and transferred separately into naïve recipient mice, whereas other naïve mice that received no cells served as a control (compiled from two independent experiments). The recipient mice were then inoculated with EO771 cells one day after cell transfer. Mouse survival, using 2000 mm3 tumor volume as a cut-off, was analyzed by means of the Kaplan-Meier estimator and a Log-rank test: ns, not significant; **P<0.01; ***P<0.001.

Figure supplement 1. Effectiveness of in vivo depletion of CD4+ and CD8+ T cells by antibody (related to Figure 3D).

Syngeneic NK cell transfer modulates APC function of cDC in the metastatic lungs.

The experimental design is the same as depicted in the Fig. 3A schema except that NK cells (WT, Il10-/- or Ifng-/-) were transferred once on day 24 and lung immune cells were analyzed on day 25. (A) Gating strategies and characterization of cDC subsets in lung tissue. The dot plots show the gating of CCR7+LAMP3+MHC-IIhi, CCR7- LAMP3-MHC-IIhi and MHC-IIlo subsets of cDC1s and cDC2s. The histograms show the comparison of indicated molecules expressed by the three subsets of cDC1s and cDC2s. Negative controls are either fluorescence-minus-one (FMO) or single stain of another molecule. Data shown are from a representative PBS-treated mouse. (B) NK-cell transfer affects the activation of lung cDCs. The proportion of the three cDC subsets in cDC1s and cDC2s are shown. The expression of the indicated APC function molecules was analyzed as relative MFI, based on normalization against the mean MFI of the PBS group in each independent experiment. Data are compiled from two independent experiments and presented as mean ± SEM. Each symbol represents one mouse. Statistical significance was determined by unpaired two-tailed Student’s t-test: *P≤0.05, **P≤0.01, ***P≤0.001.

Syngeneic

NK cell transfer promotes T cell activation in metastatic lungs.

The experimental design is the same as Figure 4. The proportion of T cell subsets in CD45+ cells in lung tissue were analyzed (Figure 5-figure supplement 1). (A) Foxp3+CD4+, Foxp3-CD4+ and CD8+ T cells. (B) Foxp3-CD4+ T cell subsets. (C) PD1-Lag3-CD8+ subsets. (D) Total and KI67+ SCL, exhausted, and PEX CD8+ T cells. Data are compiled from two independent experiments and are presented as mean ± SEM. Each symbol represents one mouse. Statistical significance was determined by unpaired two-tailed Student’s t-test: *P≤0.05, **P≤0.01, ***P≤0.001.

Figure supplement 1. Analysis of T cells in lung tissue (related to Figure 5).

Syngeneic NK cell transfer affects migratory cDCs in mLN.

The experimental design is the same as Fig. 4. (A) The expression of CCR7 and LAMP3 by migratory and resident cDC1s and cDC2s. (B) NK cell transfer affected migratory cDC subsets. The proportion of migratory cDC1s and cDC2s in cDC are shown. The expression of the indicated APC function molecules was analyzed as relative MFI, based on normalization against the mean MFI of the PBS group in each independent experiment. Data are compiled from two independent experiments and presented as mean ± SEM. Each symbol represents one mouse. Statistical significance was determined by unpaired two-tailed Student’s t-test: *P≤0.05, **P≤0.01, ***P≤0.001. Figure supplement 1. Analysis of cDCs in mLN.

Ex vivo-expanded human NK cells exhibit anti-tumor activities in vitro.

(A) Analysis of expanded human NK cells. Live cells among total cells obtained from human PBMC culture (as described in Methods) were analyzed for the expression of indicated molecules by FACS. Representative plots of 33 healthy donors are shown. (B) Expanded human NK cells kill tumor cells in vitro. Sorted HLA-DR+ NK cells from two healthy donors were co-cultured with CFSE-labeled tumor cells at the indicated E:T ratio. Percentage of dead tumor cells represents the mean ± SEM of PI+ cells among CFSE+ tumor cells from triplicate wells. (C) Expanded human NK cells up-regulate IFN-γ production in response to tumor cells in vitro. Sorted HLA-DR+ NK cells from two healthy donors were co-cultured with tumor cells at a 1:1 ratio for 5 h. Levels of IFN-γ in NK cells were examined by intracellular staining and are calculated as the ΔMFI ± SEM between specific antibody-stained and isotype control antibody-stained samples. Statistical significance was determined by unpaired two-tailed Student’s t-test: *P<0.05, **P<0.01, ***P<0.001 relative to NK cells only.

Clinical outcomes of autologous NK cell therapy.

(A) Composition of the infused cells for each patient over six batches of cell preparation. Expression of (B) NKRs, CD16 and CXCR3 and (C) perforin and IFN-γ by the expanded HLA-DR+ NK cells over six batches of cell preparation. Each dot represents a batch of cell preparation. (D) Clinical responses and survival of the six patients. The blue and yellow blocks represent the durations of NK cell treatment and subsequent follow-up, respectively. The response of patients (SD, PD or PR) was evaluated by CT imaging according to RECIST 1.1. Sono symbol marks follow-up using ultrasound imaging. Colored dotted lines indicate the timeframes for additional medications.

Baseline characteristics of the patients enrolled in the phase I trial.