NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype

Figures & data

Figure 1. Patients with PDAC have normal numbers of NK cells within blood but expression of CD16 is increased on the CD56dim subset

(A): Left panel: An example of dot plot from flow cytometry staining to gate the NK cells using CD3- CD56+ as the makers. Right panel: An example of dot plot to gate the four NK subsets according to CD56 and CD16 expression, CD56^dimCD16^bright, CD56^bright CD16^bright, CD56^dimCD16^negative and CD56^brightCD16^negative NK cells. (B): The percentage of CD3- CD56+ NK cells out of PBMCs were compared between PDAC patients and healthy donors using violin plot with each point represent a donor. (C): The composition of these four NK subsets in the bloods from PDAC patients and HDs were compared in bar chart. (D): Percentage of four NK subsets in the bloods from PDAC patients and HDs were compared individually using violin plot with each point represent a donor with significance was determined using Mann-Whitney testing, p< .01(**) and p<.0001(***).

Figure 2. Peripheral NK cells from PDAC patients express reduced levels of the activating receptors NKG2D and NKp30

The expression of differentiation markers including CD16, CD57, NKG2C(A) and activatory makers including DNAM-1. NKG2D, NKp30 (B) were compared between PDAC patients and HDs. The left panel of each marker is the histogram of representative examples to show the expression of each marker in PDAC patients and HDs. The comparison was carried out with percentage (middle panel) and mean florescence intensity (MFI) (right panel).The significance was determined using Mann-Whitney testing, p< .05(*), p< .01(**) and p< .001(***).

Figure 3. NK cells within the blood of PDAC patients have reduced cytotoxic function and express high levels of IL-10

(A and B): Whole PBMCs from PDAC patients and HDs were co-cultured with K562 cells at 1:1 ratio in the presence of Brefeldin A for overnight before the cells were stained for surface receptors, fixed permeabilised and stained with IFN-γ, IL-2, IL-10, TNF-α, IL-4 and IL-5 antibodies. The percentage of NK cells that produce each cytokine was compared between PDAC patients and HDs. Each plot represents one cytokine. (C) NK cells were enriched and co-cultured with CFSE labeled K562 cells for 16hrs. The cytotoxicity was calculated according to the relative cell count of the live populations of target cells using flow cytometry. Data shown are as violin plots with each dot represents adonor. The significance was determined using Mann-Whitney testing, p< .05(*) and p< .01(**).

Figure 4. NK cells are a minority population within PDAC tumors and show strong downregulation of CD16

(A): Examples of dot plot from flow cytometry staining to gate the NK cells using CD3- CD56+ as the makers with left panel represents PBMCs and right panel represents TILs from PDAC patient. (B) The percentage of CD3- CD56+ NK cells were compared between PBMCs and TILs from PDAC patients with each point represent a donor. (C) Examples of dot plots to gate the four NK subsets according to CD56 and CD16 expression, CD56^dimCD16^bright, CD56^bright CD16^bright, CD56^dimCD16^negative and CD56^brightCD16^negative NK cells. The left panel represents PBMCs and right panel represents TILs from PDAC patient. (D): The composition of these four NK subsets in PBMC and TILs from PDAC patients were compared in bar chart. (D): Percentage of four NK subsets in the PBMCs and TILs from PDAC patients were compared individually with each point represent a donor with significance was determined using Wilcoxon matched-pairs signed rank test, P< .01(**).

Figure 5. Expression of activatory receptors is reduced substantially on NK cells within the PDAC tumor

The expression of differentiation markers including CD16, CD57, NKG2C(A) and activatory makers including DNAM-1. NKG2D, NKp30 (B) were compared between PBMCs and TILs from PDAC patients. The left panel of each marker is the histogram of representative examples to show the expression of each marker in PBMCs and TILs from PDAC patients. The comparison was carried out with percentage (middle panel) and mean florescence intensity (MFI) (right panel).The significance was determined using Wilcoxon matched-pairs signed rank test, p< .05(*), P< .01(**), p< .001(***) and p< .0001(****).

Figure 6. The PDAC tumor microenvironment expresses high levels of NK cell ligands and tumors induce downregulation of CD16 and CD57 on NK cells

(A): Total RNA from tumor tissues and normal margins were extracted and and reverse-transcripted to cDNA before the Q-PCR was performed to calculate absolute quantity of copy numbers using standard curve. The copy numbers per reaction were identified for 18s, ULBP2, MICA and PVR. The expression of PVR, MICA/B and ULBP2 in PDAC was demonstrated using the immunohistochemistry staining contrasting to normal adjacent pancreatic tissue. (B): The PDAC tumor organoids were generated through 3D culture. These pictures are representative organoids culture from day2, day4 and day 10 after the setup of the organoids. Right panel is the histogram of the EpCAM staining with the organoids culture after they were matured. (C): After the organoids culture was matured, the co-culture with the autologous PBMCs were set up. The phenotype of NK cells from the co-culture were monitored at day 7 and These flow dot plots represent the expression pattern of CD16 and CD56 of NK cells (CD3- and CD56+) from one experiment(left panel: before the co-culture, middle panel: day 7 post co-culture and right panel: day14 post co-culture). (D) The percentage of CD16, CD57, DNAM-1, NKG2D and NKp30 on NK population was compared before the co-culture, D7 and D14 post co-culture. Expression of each marker (D7 andD14) is shown as relative percentage on NK cells cultured with organoids compared with NK cells under control medium. The data was shown as using violin plot with each point represent a donor with significance was determined using Mann-Whitney testing, p < .05(*).

Figure 7. The CD56^dim CD16^neg NK population within blood does not affect overall survival but is positively correlated with tumor recurrence

(A) Correlation between the percentage of total NK population in blood from PDAC patients with overall survival and tumor size was assessed. The line in the figure represents the linear regression line. In addition, the Spearman’s Rank correlation was used to test the significance and P value is indicated for each panel. (B) Kaplan-Meier curve (log-rank test) to compare the overall survival and disease free survival between two groups with different percentage of CD56^dim CD16^neg NK (cutoff point as median value 1%) from whole PBMCs. (C) Violin plot (Mann-Whitney test) to compare the percentage of CD56^dim CD16^neg NK cells in the patients who developed recurrent and who did not recurrent with significance was determined using Mann-Whitney testing, p < .05(*).

Table 1. Demographic, diagnostic and therapeutic characteristics of two groups of PDAC patients with defined on the basis of CD56^dim CD16^neg NK cell population (<1% and ≥ 1%)

CD56^dim CD16^neg NK cells	NK low (<1) n = 16	NK high (1+) n = 14	p
Age	67 (61–75)	75 (65–79)	0.525
BMI	27.3 (25.1–29)	26.9 (23.6 − 28.8)	0.856
Gender (male)	10 (63%)	6 (43%)	0.272
Smoker (yes)	3 (19%)	3 (21%)	1
Preoperative biliary drainage (Yes)	4 (25%)	2 (14%)	0.651
Jaundice at presentation (yes)	15 (94%)	13 (93%)	1
Vit D	25 (11.5–27.25)	31 (26–39)	0.9
CA19-9	109 (11–459)	625 (156–3036)	0.041
Neoadjuvant therapy (yes)	1 (6%)	1 (7%)	1
Resected at surgery (yes)	13 (81%)	13 (93%)	0.602
T stage 1 2 3	3 10 3	2 10 1	0.701
N Stage 0/1/2	2 7 5	3 7 3	0.775
Positive resection margin (yes)	7 (44%)	7 (50%)	1
Tumor size (mm)	25 (22.75–28.75)	25 (25–35)	0.223
Lymph node ratio	0.23 (0.13–0.53)	0.09 (0.04–0.43)	0.395
Comprehensive complication index	4.5 (0–17.9)	21 (0–26)	0.553
Adjuvant therapy (yes)	10 (62.5%)	12 (86%)	0.093
Palliative therapy (yes)	8 (50%)	5 (35%)	0.464

Table 2. Clinical survival of two groups of PDAC patients with defined on the basis of CD56^dim CD16^neg NK cell population (<1% and ≥ 1%)

CD56^dim CD16^neg NK cells	NK low (<1) n = 16	NK high (1+) n = 14	p
OS median (95% CI)	409 (383–434)	499 (276–718)	0.751
DSS median (95% CI)	405 (360–449)	487 (173–800)	0.853
DFS median (95% CI)	185 (17–352)	451 (164–737)	0.033

Availability of data and material

All data supporting the conclusion of this study have been included within the article. They can be requested through corresponding authors.