The immunoreceptor NKG2D promotes tumour growth in a model of hepatocellular carcinoma

Abstract

Inflammation is recognized as one of the drivers of cancer. Yet, the individual immune components that possess pro- and anti-tumorigenic functions in individual cancers remain largely unknown. NKG2D is a potent activating immunoreceptor that has emerged as an important player in inflammatory disorders besides its well-established function as tumour suppressor. Here, we provide genetic evidence of an unexpected tumour-promoting effect of NKG2D in a model of inflammation-driven liver cancer. Compared to NKG2D-deficient mice, NKG2D-sufficient mice display accelerated tumour growth associated with, an increased recruitment of memory CD8⁺T cells to the liver and exacerbated pro-inflammatory milieu. In addition, we show that NKG2D contributes to liver damage and consequent hepatocyte proliferation known to favour tumorigenesis. Thus, the NKG2D/NKG2D-ligand pathway provides an additional mechanism linking chronic inflammation to tumour development in hepatocellular carcinoma. Our findings expose the need to selectively target the types of cancer that could benefit from NKG2D-based immunotherapy.

Figure 1. DEN-treated Klrk1^+/+ mice display decreased survival and higher tumour burden in comparison to Klrk1^−/− mice.

HCC was induced by a single intraperitoneal injection of diethylnitrosamine (DEN) (25 mg kg⁻¹ body weight) at 14–21 days of age. (a) Kaplan-Meier representation of DEN-treated Klrk1^+/+ (n=30) and Klrk1^−/− (n=33) mice survival with significant differences determined by log-rank test (P=0.03). (b) Liver/body weight ratio was assessed in 15-month-old Klrk1^+/+, Klrk1^−/− mice treated with DEN and non-treated age-matched control mice (AMC). Statistical analysis was performed by unpaired Student's t-test. (c) Correlation between liver/body weight ratio and maximal tumour sizes in the liver of 15 months old Klrk1^+/+ mice injected with DEN (n=30) (P value <0.0001 from linear regression analysis). (d–e) Maximal tumour size, measured macroscopically by diameter and Tumour load (sum of tumour diameter for all tumours with diameters >5 mm) in 15-month-old Klrk1^+/+ and Klrk1^−/− mice treated with DEN. Graphs represent the mean±s.e.m. (n≥16). Statistical analysis was performed by unpaired Student's t-test. (f) Bar chart indicating the percentages of DEN-treated Klrk1^+/+ (n=30) and Klrk1^−/− (n=34) mice showing nodule, adenoma and carcinoma (HCC). HCC are further divided according to histological grading. (g) Representative H&E staining of normal AMC liver and of liver tumours showing HCC of grade 1, 2 and 3 –defined as follow: Grade 1: Focal lesion with mildly increased nuclear-cytoplasmic ratio and/or a small increase in the number of mitoses, plus mild architectural distortion. Grade 2: Focal lesion with moderately increased nuclear-cytoplasmic ratio and/or a moderate architectural distortion. Grade 3: Focal lesion with marked increase in nuclear-cytoplasmic ratio and/or a marked increase in mitotic activity plus marked architectural distortion and/or necrosis. Scale bar represents 100 μm. Statistical analyses were performed using unpaired Student's t-test. Statistically significant differences between groups are denoted as: *P≤0.05, **P≤0.01, ****P≤0.0001.

Figure 2. NKG2D favors the recruitment of CD8⁺ T cells to DEN-treated livers.

(a) Representative flow cytometry plots of CD8 versus CD4 staining (b) NK1.1 versus CD3 staining and (c) percentages of CD8⁺T cells (CD8⁺CD4⁻NK1.1⁻CD3⁺), (d) NK (NK1.1⁺CD3⁻) and (e) NKT (NK1.1^lowCD3^low) cells out of CD45⁺ cells, surrounding (S) and infiltrating tumours (T) in Klrk1^+/+ and Klrk1^−/− DEN-treated mice and AMC. (mean±s.e.m.) where dots represent individual mouse. Statistical analysis was performed by unpaired Student's t-test. (f) Absolute numbers of CD8⁺T cells, NK and NKT cells per gram of surrounding tissue (S) and tumour tissue (T) in Klrk1^+/+ and Klrk1^−/− DEN-treated, AMC and YC (n≥8 all groups). Graphs represent the mean±s.e.m. Statistical analysis was performed by Mann–Whitney test. (g) Absolute numbers of CD69⁺T cells per gram of surrounding tissue (S) and tumour tissue (T) in Klrk1^+/+ and Klrk1^−/− DEN-treated, AMC and YC (n≥5 all groups). Graphs represent the mean±s.e.m. Statistical analysis was performed by unpaired Student's t-test. (h) Quantification of CXCL9, CXCL10, CCL3 and CCL5 mRNA transcript in livers of DEN-treated mice (n≥20) and AMC (n≥8). Graphs represent the mean±s.e.m. Statistical analysis was performed by unpaired Student's t-test. Statistically significant differences between groups are denoted as: *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Figure 3. Hepatic CD8⁺T cells of DEN-treated mice have been chronically activated.

(a) Median fluorescence intensity of CD3 expression on CD8⁺T cells (n≥10) within tumors (T) and surrounding (S) liver tissue of Klrk1^+/+ and Klrk1^−/− DEN-treated mice and from AMC liver. (b) Representative flow cytometry plots of PD-1 expression ex vivo on CD8⁺T cells from DEN-treated Klrk1^+/+ mice and AMC mice (representative of n≥3). (c) Percentages of CD8⁺T cells expressing PD-1 from DEN-treated Klrk1^+/+ mice, age-matched control (AMC) mice and young controls (YC) (n≥3). Graphs represent the mean±s.e.m. (d) PD-1 positive cells per mm² on liver tissue sections from 15-month-old Klrk1^+/+ and Klrk1^−/− DEN-treated or AMC. Dots represent individual mice and show mean±s.e.m. Statistical analysis was performed by unpaired Student's t-test. (e,f) Relative expression of (e) Pd1 and (f) PD-L1 (CD274) mRNA transcripts, within tumours (T) and surrounding (S) liver tissue of Klrk1^+/+ and Klrk1^−/− DEN-treated mice, AMC and YC (n≥9). Graphs represent the mean±s.e.m.

Figure 4. Partial downregulation of NKG2D on CD8⁺T cells but not NK cells.

(a) Quantification of Klrk1 mRNA transcripts in livers of DEN-treated mice (n≥22), YC and AMC (n≥5). (b) Representative flow cytometry plots of NKG2D expression ex vivo on CD8⁺T cells from DEN-treated mice (representative of n≥6), AMC and YC mice (representative of n≥5). Graphs represent the mean±s.e.m. (c) Delta median fluorescence intensity (MFI) of NKG2D expression on CD8⁺T cells from DEN-treated mice, AMC and YC. Dots represent individual mice. (d) Histograms of NKG2D expression on NK cells (black lines) from DEN-treated mice (representative of n≥6) and AMC (representative of n≥4). Fluorescence-minus-one control is shown as grey filled histogram. Values in each quadrant indicate the mean±s.e.m. of NKG2D Delta MFI staining.

Figure 5. NKG2D ligands are expressed in DEN-treated livers.

(a) Representative immunohistochemical staining of RAE-1 (brown) on hepatocellular carcinoma tissue sections showing cell-surface staining of hepatocytes within tumours (T) and surrounding tissue (S) of Klrk1^+/+ (top left image) and Klrk1^−/− mice (middle left image) compared with isotype control staining (top and middle right image). RAE-1 staining of AMC (bottom left image) and 8-12 week old untreated young control mouse (YC) (bottom right image) are shown as indicated. A black dashed line demarcates boundary between tumour and surrounding tissue. DAB was used as revealing agent and eosin was used as a counter stain. Scale bar represents 100 μm. (b) Percentage area of RAE-1 positive hepatocytes on liver sections from 15-month-old Klrk1^+/+ and Klrk1^−/− DEN-treated, age-matched control mice (AMC) or young control mice (YC). Graphs represent the mean±s.e.m. Statistical analysis was performed by unpaired Student's t-test. (c) ELISA of soluble MULT1 from the sera of Klrk1^+/+ and Klrk1^−/− DEN-treated (n≥6), AMC, YC and ApoE^-/- mice (n≥4). Absorbance values normalised to isotype controls. Boxes represent 25th–75th percentiles, whiskers represent minimum to maximum. Statistical analysis was performed by Mann-Whitney test. (d) Quantification of MMP9, MMP14 and ADAM10 mRNA transcript in livers of DEN-treated mice (n≥17) and AMC (n≥9). Graphs represent the mean±s.e.m. Statistical analysis was performed by unpaired Student's t-test. Statistically significant differences between groups are denoted as: *P≤0.005 **P≤0.01, ***P≤0.001, ****P≤0.0001.

Figure 6. CD8⁺T cells are the main source of IFNγ in the tumour and surrounding milieu.

(a) Percentages of NK, NKT, CD8⁺T and CD4⁺T cells among CD45⁺IFN-γ⁺ cells isolated from the tumour and (b) surrounding tissue of DEN-treated Klrk1^+/+ and Klrk1^−/−mice, AMC and YC (n=2-8) upon 4 h in vitro stimulation with PMA & Ionomycin. Graphs represent the mean±s.e.m. (c) Representative flow plots and (d) average percentages (mean±s.e.m.) of CD8⁺T cells expressing PD-1 and/or IFNγ post in vitro stimulation as described for (a). (e) Percentages of CD8⁺T, (f) NK and (g) NKT cells expressing CD107a (n=2–7). Graphs represent the mean±s.e.m. (h) Percentages of CD107a⁺ cells among CD8⁺T cells expressing NKG2D (NKG2D⁺) or not (NKG2D⁻) in DEN-treated Klrk1^+/+ mice, AMC and YC. Dots represent populations from individual mice. Lines connect populations from the same mouse. (i) Relative expression of perforin and (j) granzyme B transcripts within tumour (T) and surrounding (S) tissue of Klrk1^+/+ and Klrk1^−/− DEN-treated mice (n≥19) and AMC liver (n≥9). Graphs represent the mean±s.e.m. Statistical analyses were performed by unpaired Student's t-test. Statistically significant differences between groups are denoted as: *P≤0.05 and **P≤0.01.

Figure 7. NKG2D exacerbates the local inflammation.

(a) Representative flow cytometry plots of Gr1 staining versus SSC-A from CD11b⁺ gated liver cells of Klrk1^+/+ and Klrk1^−/− DEN-treated mice (tumour and surrounding tissue) (representative of n≥19) and AMC (representative of n≥15). (b) Percentages of neutrophils (Ly6G⁺ or Gr1^hi) out of CD11b⁺ cells surrounding (S) and infiltrating tumours (T) of Klrk1^+/+ and Klrk1^−/− DEN-treated mice and from untreated AMC liver. Graphs represent the mean±s.e.m. (c) Representative flow cytometry plots of Ly6C versus SSC-A on CD11b⁺Ly6G^- F4/80⁺ cells from tumors and surrounding tissues of Klrk1^+/+ and Klrk1^−/− DEN-treated mice and untreated AMC liver are shown (representative of n≥15). (d) Ratio of inflammatory (Ly6C^hi or Gr1^intermediate) versus resident (Ly6C^lo or Gr1^lo) macrophages (CD11b⁺, F4/80⁺, Ly6G⁻ or Gr1^lo.int) from tumours (T) and surrounding (S) tissue of Klrk1^+/+ and Klrk1^−/− DEN-treated mice and untreated AMC. Graphs represent the mean±s.e.m. (e) Relative expression of CCR2 mRNA transcripts detected in tumours (T) and surrounding (S) tissue of Klrk1^+/+ and Klrk1^−/− DEN-treated mice (n≥19) and in AMC livers (n≥11). (f) Fold change in expression of IL-6, TNFα, IL-1β, IFN-γ, IL-15, IL-18, TGFβ, IL-10, IL-33 and IL-25 transcripts in tumours (bottom) and surrounding liver tissue (top) of DEN-treated Klrk1^+/+ over Klrk^−/− (n=9–22). Graphs represent the mean±s.e.m. All statistical analyses were performed by unpaired Student's t-test. Statistically significant differences between groups are denoted as: *P≤0.005, ***P≤0.001, ****P≤0.0001.

Figure 8. Hepatocyte cell death and proliferation induced by DEN treatment are increased in the presence of NKG2D.

(a) Serum alanine aminotransferase (ALT) levels in 15-month-old Klrk1^+/+ and Klrk1^−/− DEN-treated (n≥17) and untreated AMC (n≥5) mice. Graphs represent the mean±s.e.m. (b) Incidence of necrosis was detected by TUNEL staining on tissue sections from Klrk1^+/+ and Klrk1^−/− DEN-treated mice (n≥12). (c) Liver/body weight ratio of Klrk1^+/+mice showing no necrosis, necrosis or severe necrosis by TUNEL assay. Graphs represent the mean±s.e.m. (d) Relative expression of CDKN1A (p21) mRNA transcripts in livers of Klrk1^+/+ and Klrk1^−/− DEN-treated (n≥19) and AMC (n≥10). Graphs represent the mean±s.e.m. (e) Representative staining of proliferating hepatocytes by MCM4 (brown) with hematoxylin counterstained (blue). Black scale bar represents 100 μm. (f) MCM4 positive cells per mm² of liver tissue (n≥10). Graph represents the mean±s.e.m. Statistical analysis was performed by unpaired Student's t-test, where statistically significant differences between groups are denoted as: *P≤0.05, **P≤0.01, and ***P≤0.001, ****P≤0.0001.