Class A CpG oligodeoxynucleotide inhibits IFN-γ-induced signaling and apoptosis in lung cancer

Abstract

Background: Currently, anticancer immunotherapy based on PD-1/PD-L1 blockade with immune checkpoint inhibitors (ICIs) is being used as a standard therapy for non-small cell lung cancer (NSCLC). However, more effective treatments are required as these tumors are often resistant and refractory. Here, we aimed to determine the effects of immunomodulatory oligodeoxynucleotides (ODNs) in terms of the presence or absence of CpG motifs and the number of consecutive guanosines.

Methods: Western blots were used to measure the molecules which regulate the expression of PD-L1 in human lung cancer cell lines after incubation with several cytokines and ODNs. The expression of PD-L1 and β2-microglobulin (β2-MG) on A549 cells, and IFN-γ-induced apoptosis with ODNs were examined by flow cytometry. The relationship between IFN-γ receptor and ODN was analyzed by ELISA and immunofluorescence chemistry.

Results: Our results verified that A-CpG ODNs suppress the upregulation of IFN-γ-induced PD-L1 and β2-MG expression. In addition, we found that ODNs with six or more consecutive guanosines (ODNs with poly-G sequences) may competitively inhibit the IFN-γ receptor and abolish the effect of IFN-γ, thereby suppressing apoptosis and indoleamine 2,3-dioxygenase 1 expression in human lung cancer cells. The tumor microenvironment regulates whether this action will promote or suppress tumor immunity. Thus, in immunotherapy with CpG ODNs, it is essential to consider the effect of ODNs with poly-G sequences.

Conclusions: This study suggests that ODNs containing six or more consecutive guanosines may inhibit the binding of IFN-γ to IFN-γ receptor. However, it does not directly show that ODNs containing six or more consecutive guanosines competitively inhibit the IFN-γ receptor, and further studies are warranted to confirm this finding.

Key points: Significant findings of the study: Oligodeoxynucleotides with a contiguous sequence of six or more guanosines may competitively inhibit the IFN-γ receptor and abolish the action of IFN-γ. This may suppress IFN-γ-induced apoptosis and indoleamine-2,3-dioxygenase-1 expression in human lung cancer cells.

What this study adds: A-CpG and poly-G ODN may overcome tolerance if the cause of ICI tolerance is high IDO expression. However, IFN-γ also has the effect of suppressing apoptosis of cancer cells, and it is necessary to identify the cause of resistance.

Keywords: Apoptosis; CpG oligodeoxynucleotide; interferon-γ; non-small cell lung cancer; polyguanosine.

Figure 1

Effect of A‐CpG (D35) or B‐CpG (K3) ODN on the expression of PD‐L1 and β2‐ microglobulin in the A549 cell line. A549 cells were stimulated with or without recombinant human IFN‐γ (10 ng/mL) and/or the indicated ODNs (3 μM), and further cultured for 16 hours. PD‐L1 and β2‐microglobulin expression was detected using western blotting (a). D35A was constructed by replacing the consecutive guanosines in the 3′ tail of D35 with consecutive adenosines. ODN 1612, which contains the GpC motif instead of the CpG motif, was the control ODN for B‐CpG. The expression of PD‐L1 or B2‐MG in A549 cells after stimulation with IFN‐γ, A‐CpG (D35), or both was identified using flow cytometry (b), and immunofluorescence microscopy. Scale bar = 100 μm (c). () Iso type, () none, () D35, () IFN‐y and () IFN‐y+D35.

Figure 2

Effect of ODNs with consecutive guanosines on the IFN‐γ/JAK/STAT1 pathway. (a) IFN‐γ and the indicated ODNs containing several consecutive guanosines were added to A549 cells. JAK1, phospho‐JAK1, JAK2, phospho‐JAK2, STAT1, and phospho‐STAT1 levels were confirmed after 30 minutes, and PD‐L1 and β2‐microglobulin expression levels were confirmed after 16 hours using western blotting. Poly‐A ODN harbored pure consecutive adenosines. D122, which contains the GpC motif instead of CpG, was the control ODN for A‐CpG (D35). The D122G3 ODN contained three guanosines instead of six guanosines. The G6D122 ODN contained six guanosines in its 5′ head but not in its 3′ tail. (b) IFN‐γ and ODNs (poly‐G ODN, poly‐A ODN) were added to H226, H460, and H520 cells. Phospho‐STAT1 levels were confirmed after 30 minutes, and β2‐microglobulin expression levels were confirmed after 16 hours using western blotting.

Figure 3

Effect of ODNs with consecutive guanosines on binding of IFN‐γ to its IFN‐γ receptor. (a) Recombinant human IFN‐γ and the indicated ODNs were added to a plate coated with IFN‐γ capture antibody; IFN‐γ detection antibody and avidin‐HRP were subsequently added. The binding of IFN‐γ to the IFN‐γ capture antibody was determined from the absorbance value. Results are shown as the means + SD of two experiments. ns, not significant, **P < 0.01 (compared with IFN‐γ administration alone). (b) Subcellular localization of IFN‐γ receptor and poly‐G ODN in A549 cells. The localization of the IFN‐γ receptor was detected using GFP fluorescence (green), poly‐G ODN was conjugated with TAMRA (red), and cell nuclei were labeled with DAPI (blue) in A549 cells. Scale bar = 100 μm.

Figure 4

Effect of poly‐G oligodeoxynucleotide (ODN) on the JAK/STAT pathway regulated by IFN‐α or IFN‐β. (a) A549 cells were stimulated with recombinant human IFN‐α (800 U/mL) or IFN‐β (10 ng/mL) and/or poly‐G ODN (3 μM), and further cultured for 16 hours. PD‐L1 and β2‐microglobulin expression was assessed using western blotting. (b) A549 cells were treated as described above. PD‐L1 and β2‐microglobulin expression was determined using flow cytometry. () Iso type, () none, () IFN‐α and () IFN‐α+Poly‐G. () Iso type, () none, () IFN‐β and () IFN‐β+Poly‐G.

Figure 5

Effect of oligodeoxynucleotides (ODNs) with poly‐G sequences on induction of apoptosis and IDO by IFN‐γ. (a,b) A549 cells were incubated for 72 hours with IFN‐γ (200 ng/mL) and/or the indicated ODNs with consecutive guanosine sequences (3 μM). Apoptotic cells were detected as annexin V‐positive and 7AAD‐negative cells using flow cytometry. Results are shown as the means + SD of three independent experiments. ns, not significant; * P < 0.05 (compared to untreated). (c) A549 cells were treated with recombinant human IFN‐γ (50 ng/mL) and/or poly‐G ODN (3 μM), and further cultured for 16 hours. IDO expression was determined using western blotting. (d) A549 cells were treated as described above. IDO expression was assessed using immunofluorescence microscopy. Scale bar = 100 μm.