KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma

Human NSCLC cell lines H460, H1299, H2228, H292 and H1993 were obtained from the American Type Culture Collection. Immortalized human lung bronchial epithelial cell (Beas-2B), EKVX, Beas-2B-vector, Beas-2B-KRAS-G12D and Beas-2B-KRAS-WT cells were generously provided by Prof. Liang Chen (National Institute of Biological Sciences, China). H358 was kindly provided by Prof. Mengfeng Li (Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, China). H358, H460 and EKVX are the KRAS-mutant NSCLC cell lines. H1299 is the N-RAS-mutant lung adenocarcinoma cell line. H2228 is the lung adenocarcinoma cell line with EML4-ALK fusion. H292 and H1993 are NSCLC cell lines with EGFR/ALK/KRAS wild-type (WT). Beas-2B-KRAS-G12D, Beas-2B-KRAS-WT and Beas-2B-vector are the Beas-2B cells stably transfected with KRAS G12D mutant, KRAS wild-type and control plasmid, respectively. H2228 and H358 were cultured in RPMI-1640 complete growth medium supplemented with 10% fetal bovine serum and antibiotics (10,000 U/ml penicillin and 10 μg/ml streptomycin). Other cell lines were grown in DMEM complete medium.

Western blot analysis was done as previously reported [15]. The primary antibodies for PD-L1 (E1L3N™), RAS (D2C1), mutant KRAS (G12D Mutant Specific) (D8H7), phospho-p44/42MAPK (ERK1⁄2) (Thr202⁄Tyr204), p44/42MAPK (ERK1/2), phosphor-AKT (Ser473), AKT and GAPDH were purchased from Cell Signaling Technology (CST). Quantitative real-time PCR experiments were performed as previous described [18].

Suspension cells (Beas-2B-vector, Beas-2B-KRAS-G12D and Beas-2B-KRAS-WT) were stained with PD-L1 (E1L3N, Rabbit mAb, PE Conjugated) or the corresponding isotype control (DA1E, Rabbit mAb IgG, PE Conjugated) (CST, Danvers, MA). The surface expression of PD-L1 were detected by flow cytometry and analyzed with FlowJo 7.6.1 software [15].

H358, H1993, Beas-2B-KRAS-G12D and Beas-2B-vector cells were fixed and blocked before addition of the primary antibodies including PD-L1 (E1L3N™, Rabbit mAb) or mutant KRAS (G12D mutant specific, D8H7, Rabbit mAb) at 4 °C overnight. Then cells were incubated with secondary antibody (Alexa Fluor 488 or 555 donkey anti-rabbit IgG [H+L], Life Technologies, LA) for 1 h. The detailed protocol was described in previous report [15].

KRAS siRNAs were purchased from Ribobio Corporation (Guangzhou, China). The target sequence of KRAS siRNA #1 and siRNA #2 are CGAATATGATCCAACAATA and CAAGAGGAGTACAGTGCAA, respectively. Beas-2B-KRAS-G12D and H358 cells were transiently transfected with KRAS siRNAs using Lipofectamine^® RNAiMAX_Reagent (Invitrogen) for 48 h. ERK1/2 inhibitor (SCH772984) and AKT1/2/3 inhibitor (MK-22062HCL) were purchased from Selleckchem (Houston, USA). Beas-2B-KRAS-G12D cells or H358 cells were exposed to climbing doses of ERK and AKT inhibitors for 72 h. The viability of the cells was tested with CCK8 kit (Cell Counting Kit-8, Dojindo.Co, Japan). Recombinant humanized anti-PD-1 antibody, Pembrolizumab (MK-3475, Keytruda) was from Merck Sharp & Dohme Corp (Whitehouse Station, NJ08889, USA).

Dendritic cells and cytokine-induced killer cells (DC-CIK) were kindly provided by Prof. Jianchuan Xia (Department of Biotherapy, Sun Yat-sen University Cancer Center, China) [19, 20]. The protocol of acquiring DC-CIK was described in our previous report [21]. Beas-2B-KRAS-G12D, Beas-2B-vector, H358 and EKVX cells were seeded into 12-well plates at a density of 1.0 × 10⁵ cells/well, respectively. The acquired DC-CIK were added into co-culture system with Beas-2B-KRAS-G12D, Beas-2B-vector,H358 or EKVX cells at the ratio of 1:1, respectively. Next, the DC-CIK/Beas-2B-KRAS-G12D, DC-CIK/H358 and DC-CIK/EKVX co-culture system were treated with mock, Pembrolizumab (500 μg/ml), ERK1/2 inhibitor (100 nM/L) or AKT inhibitor (1.0 μM/L), respectively. After 48 h, suspended DC/CIK cells were removed from the adherent Beas-2B-KRAS-G12D, H358 or EKVX cells in the cell culture plate with pipet. Then, after washing with PBS, suspended DC/CIK cells were stained with anti-human CD3 FITC antibody (OKT-3, 11-0037, Affymetrix eBioscience) for 15 min. Next, after washing, DC-CIK cells were stained with Annexin V-APC and 7-AAD for 15 min with Apoptosis Detection kit (KGA1023-1026, KeyGEN, Nanjing, China) [21]. The apoptotic cells of CD3+ T cells detected by flow cytometry (Beckman Gallios, Beckman-Coulter, Inc. USA) were defined as Annexin V-APC-positive cells (both 7-AAD-negative and -positive) from the gate of CD3-positive cells. The example of gating strategy is presented in supplementary Fig. 1.

The survival rates of KRAS-mutant tumor cells like H358 or EKVX cells were dynamically monitored in real time by the xCELLigence system (E-plate, Roche) which could exclude the interference of suspended DC-CIK. Firstly, 96-well E-plate with 50 μl of complete growth medium in each well was tested in the incubator to establish a background reading. Next, tumor cells (1.0 × 10⁴ cells/well) were seeded into 96-well E-plates for approximately 20 h followed by addition of DC-CIK (50 μl/well) into the E-plates at a DC-CIK: tumor cells ratio of 1:1. Finally, an additional 50 μl/well of the complete medium containing different drugs such as vehicle, Pembrolizumab (500 μg/ml), ERK1/2 inhibitor (100 nM/L) and Pembrolizumab (500 μg/ml) plus ERK1/2 inhibitor (100 nM/L) were added into the DC-CIK/H358 or DC-CIK/EKVX co-culture system, respectively. H358 cells alone were meanwhile treated with vehicle, Pembrolizumab (500 μg/ml) and ERK1/2 inhibitor (100 nM/L) as the control groups. Cell index values were monitored every 15 min from each well of E-plate and presented as the dynamic cell growth curves [21, 22].

Our study prospectively enrolled 216 newly diagnosed NSCLC patients who all underwent genomic analysis of EGFR, ALK and KRAS from April 2013 to December 2014 in Sun Yat-sen University Cancer Center (SYSUCC). This study was approved by the Institutional Review Board of SYSUCC and written informed consent was obtained before specimens were collected. The specimens were from surgical resection tissue or biopsies of the untreated patients. KRAS and EGFR mutation status were tested using real-time PCR. ALK rearrangements were detected by fluorescence in situ hybridization. Excluding the patients with EGFR mutation and ALK fusion, the remaining 69 patients were pathologically diagnosed as lung adenocarcinoma with EGFR/ALK wild-type. Among them, there were 19 patients harboring KRAS mutation. Patients’ baseline characteristics were collected including gender, age, smoking status, tumor differentiation and staging. Pathologic or clinical staging was determined according to the cancer staging manual (7th edition) of American Joint Committee on Cancer. Using “MatchIt” package of R programming language, baseline characteristics of patients were balanced matching between KRAS mutation group and EGFR/ALK/KRAS wild-type group by propensity matching score analysis [23]. Subsequently, statistic analysis has been carried out for 19 patients with KRAS mutation matched with 38 out of 50 patients with EGFR/ALK/KRAS wild-type. Finally, PD-L1 expression in the tissue of 57 patients after matching was detected by immunohistochemistry.

Immunohistochemical staining was performed using PD-L1 rabbit antibody (E1L3N™, CST; dilution 1:200) overnight at 4 °C. Immunoreactivity was detected using the DAKO ChemMateEnVision method according to the manufacturer’s instructions. Two pathologists blinded to patients’ information independently assessed expression of PD-L1. Semi-quantitative H score (H-SCORE) was determined by multiplying the percentage of positively stained cells by an intensity score (0, absent; 1, weak; 2, moderate; and 3, strong) and ranged 0–300.

The SPSS software (version 19.0) was used for statistical analysis. After matching with “MatchIt” package of R programming language, the differences of gender, smoking status, tumor differentiation, staging between KRAS mutation group and EGFR/ALK/KRAS wild-type group were examined by the Pearson Chi-square test and the difference of age between the two groups was examined by two independent samples’ t test. Wilcoxon rank-sum test was used to compare the H-SCORE of PD-L1 staining between KRAS mutation and EGFR/ALK/KRAS wild-type group. Representative results from three independent experiments were shown in this study. Numerical data were presented as the mean ± standard deviation of the mean (SD). The P values between two experimental groups were tested by two-tailed Student’s t test and P values less than 0.05 were considered significant.

In order to investigate the association between PD-L1 expression and KRAS mutation status, we conducted experiments in human lung adenocarcinoma cell lines and tissue. We found that the protein levels of PD-L1 in endogenous KRAS-mutant NSCLC cell lines (EKVX, H358, H460), EML4-ALK fusion cell line (H2228), and EGFR 19-exon deletion mutation cell line (H1650) were significantly higher than that in EGFR/ALK/KRAS wild-type cell lines (H292, H1993), N-RAS-mutant cell line (H1299), or lung bronchial epithelial cell line (Beas-2B cell) (Fig. 1a). Similar results of PD-L1 mRNA level were also observed in the above-mentioned cell lines (Fig. 1b). Sub-cellular localization of PD-L1 protein was detected in H358 and H1993 cells using immunofluorescence. PD-L1 showed stronger membrane localization in H358 cells (red fluorescence), compared with that in H1993 cells (Fig. 1c). Next, we detected PD-L1 expression on tumor cells and tumor-infiltrating immune cells in patients’ lung adenocarcinoma tissue by immunohistochemistry. Baseline characteristics of patients including gender, age, smoking history, tumor differentiation and stage were balanced matching between KRAS mutation group and EGFR/ALK/KRAS wild-type group by propensity matching score analysis [24] (Table 1). KRAS-mutant cases tended to have higher intensity of PD-L1 staining than EGFR/ALK/KRAS wild-type cases. PD-L1 immunoreactivity was detected mainly on the cytomembrane or slightly in the cytoplasm (or both) of tumor cells or tumor-infiltrating immune cells (Fig. 1e). The median of H-SCORE was significantly higher in KRAS mutation cases than in EGFR/ALK/KRAS wild-type cases (60 vs. 30, P = 0.042) (Fig. 1d). The results implied that PD-L1 expression was associated with KRAS mutation in lung adenocarcinoma.

Firstly, we found the expression of PD-L1 was significantly higher in Beas-2B-KRAS-G12D cells compared with Beas-2B-vector cells. However, the expression of PD-L1 in Beas-2B-KRAS-WT cell was not obviously increased compared with control cells. Likely, the phosphorylated expression of ERK1/2 and AKT were activated by mutant KRAS-G12D but not wild-type KRAS (Fig. 2a). Similarly, flow cytometric analysis showed the surface expression of PD-L1 in Beas-2B cells with KRAS G12D mutation was higher than that in Beas-2B cells with KRAS wild-type and vector control (Fig. 2b). The induction of PD-L1 by over-expression of exogenous KRAS was further confirmed at the mRNA level by PCR. The higher level of PD-L1 mRNA was observed in Beas-2B-KRAS-G12D cells than that in Beas-2B-vector and Beas-2B-KRAS-WT cells (Fig. 2c). Immunofluorescence staining also showed the association between KRAS and PD-L1 protein expression. Increased expression of KRAS and PD-L1 was observed on the cytomembrane of Beas-2B cells with KRAS G12D over-expression (Fig. 2d). In contrast, knocking down KRAS expression using two loci KRAS siRNAs decreased the expression level of Ras in Beas-2B-KRAS G12D cells. PD-L1 was also significantly down-regulated following KRAS knockdown (Fig. 2e). Similarly, in H358 cells, the protein expression and mRNA level of PD-L1 were both significantly down-regulated following the knockdown of KRAS by siRNAs (Fig. 2f, g). Taken together, our results demonstrated that over-expression of exogenous KRAS induced PD-L1 expression and knocking down KRAS reduced PD-L1 expression. PD-L1 expression level was positively correlated with KRAS mutation.

In order to investigate how KRAS regulate PD-L1 expression, firstly, we examined the two key downstream proteins of Ras pathway (p-ERK and p-AKT) by western blot. Figure 2a showed both p-ERK1/2 and p-AKT were activated by KRAS G12D mutation. Then, we used the inhibitors of ERK1/2 and AKT to block the downstream pathways of KRAS in Beas-2B cells with exogenous expression of KRAS G12D mutation and H358 cell with endogenous expression of KRAS G12C mutation. We found that ERK1/2 inhibitor (SCH772984) inhibited the p-ERK, which further resulted in the decrease of PD-L1 expression (Fig. 3a, c). However, AKT inhibitor (MK-2206 2HCL) could effectively suppress p-AKT, but did not alter the expression level of PD-L1 (Fig. 3b, d). To investigate whether PD-L1 is regulated through the mechanism of specific signaling pathway or the effect of cell viability, we performed the viability analysis of H358 and Beas-2B-KRAS G12D cells to find that the indicated concentration of ERK inhibitor and AKT inhibitor only weakly inhibited the viability of the two cells (Fig. 3e, f). Thus, PD-L1 regulated by ERK1/2 signaling is dependent on signaling pathway mechanism. Collectively, KRAS mutation induced PD-L1 expression through p-ERK1/2 signaling pathway but not p-AKT pathway.

PD-L1 could lead to apoptosis of T cells, and anti-PD-1 antibody could reverse this process. To investigate whether up-regulation of PD-L1 mediated by KRAS could lead to the apoptosis of T cells, we firstly constructed the stable cell line with KRAS G12D over-expression (Beas-2B-KRAS-G12D) (Fig. 4a). DC-CIK was prepared from peripheral blood donated by healthy volunteers and co-cultured with Beas-2B-KRAS-G12D cells or Beas-2B-vector cells at the ratio of 1:1. As shown in Fig. 4b, e, the apoptosis rates of CD3+ T cells co-cultured with Beas-2B-KRAS G12D cells were higher than that with Beas-2B-vector cells (21.40 ± 1.33 vs. 7.07 ± 0.09%, P = 0.0004). On the contrary, blockade of PD-1 on T cells with Pembrolizumab in Beas-2B-KRAS G12D/DC-CIK co-culture system reduced the apoptosis rates of CD3+ T cells to 8.00 ± 0.36% (P = 0.0006). Next, we used ERK inhibitor to investigate whether it could reverse the apoptosis of T cells through inhibiting the expression of PD-L1. As expected, the apoptosis rates of CD3+ T cells decreased from 21.40 ± 1.33 to 16.03 ± 0.27% (P = 0.0167). However, AKT inhibitor could not reduce the apoptosis rates of T cells compared with mock group (19.23 ± 0.52 vs. 21.40 ± 1.33%, P = 0.2037). Next, in H358/DC-CIK co-culture system, ERK inhibitor also reduced the apoptosis rates of CD3+ T cells from 28.30 ± 0.62 to 18.67 ± 0.30% (P = 0.0002, Fig. 4c, f). However, the apoptosis rates of CD3+ T cells in the AKT inhibitor group and mock group did not differ (26.10 ± 0.65 vs. 28.30 ± 0.62%, P = 0.0713). We also found that the apoptosis rates of CD3+ T cells decreased from 28.30 ± 0.62% in the mock group to 14.03 ± 0.84% in the Pembrolizumab group (P = 0.0002). Similarly, we chose EKVX cells (another KRAS-mutant NSCLC cell line) to further confirm that ERK inhibitor and Pembrolizumab could reduce the apoptosis rates of CD3+ T cells (25.47 ± 0.32 vs. 16.17 ± 0.61%, P = 0.0002 and 25.47 ± 0.32 vs. 12.60 ± 0.64%, P < 0.0001) in EKVX/DC-CIK co-culture system but not with AKT inhibitor (25.47 ± 0.32 vs. 23.83 ± 0.55%, P = 0.0616) (Fig. 4d, g). To understand whether the inhibitors and Pembrolizumab may directly affect the T cells, we tested the effects of the two inhibitors and Pembrolizumab on T cells alone. We found that Pembrolizumab (500 μg/ml), ERK1/2 inhibitor (100 nM/L) and AKT inhibitor (1 μM/L) could not influence the viability of DC-CIK and the apoptosis of CD3+ T cells (Supplementary Fig. 2).Taken together, our results demonstrated that KRAS-mediated up-regulation of PD-L1 could induce the apoptosis of CD3+ T cells through PD-1/PD-L1 axis while blocking PD-1 or inhibiting ERK pathway could alleviate the apoptosis of CD3+ T cells.

Further we examined the real-time survival signal of attached tumor cells after blockade of PD-1 by anti-PD-1 antibody or inhibiting PD-L1 by ERK inhibitor in tumor cells/DC-CIK co-culture system. The cell index represents the survival rate of attached tumor cells excluding the interference of suspended DC-CIK. When H358 cells alone was treated with Pembrolizumab (500 μg/ml), ERK inhibitor (100 nM/L) or vehicle, the cell index of H358 was not obviously changed. However, when H358 or EKVX cells, respectively, co-cultured with DC-CIK at the ratio of 1:1, the survival rates of H358 and EKVX cells decreased in the presence of Pembrolizumab (500 μg/ml), ERK inhibitor (100 nM/L) or both. But combined treatment of Pembrolizumab and ERK inhibitor did not show synergistic tumor-suppression effect (Fig. 5a, b). These results demonstrated that anti-PD-1 antibody or ERK inhibitor could recover the anti-tumor immunity of T cells and therefore decrease the survival rates of KRAS-mutant cells of lung adenocarcinoma in co-culture system.