Radiotherapy enhances healthy PBMCs to suppress lung cancer in vitro
To investigate whether RT suppresses lung cancer and enhances immunity to eradicate tumors, the cell viability and colony formation were measured in the lung cancer A549 and LL/2 cells treated with X-ray irradiation. We found that radiotherapy by exposure of at least 10Gy and 4Gy, respectively, significantly suppressed A549 and LL/2 cell viability (Fig S1A) and colony formation (Fig S1B). We further found that healthy PBMCs isolated from a healthy volunteer suppressed 10Gy of RT-treated A549 cell viability compared to untreated A549 cells (Fig S1C). In addition to confirm the observation and exclude PBMCs-to-A549 suppression was mediated by allograft rejection, the experiment was repeated and validated in 4Gy of RT-treated LL/2 treated with syngeneic graft splenocytes isolated from a C57BL/6 mouse. The results revealed that splenocytes suppressed RT-treated LL/2 cell viability compared to untreated LL/2 (Fig S1C).
Radiotherapy increases IFNs and augments PBMCs for suppressing A549 in vitro
To investigate the detailed mechanism of RT of healthy PBMCs and splenocytes suppressing A549 and LL/2, respectively, the genes including IFNs and ISGs were detected using qPCR in the RT-treated tumor cells. We found that RT increased IFNα, IFNγ, CXCL9, and CXCL10 expression in A549 cells in a dose-dependent manner, whereas ISG15 was used as a radiotherapeutic marker (Fig. 1A). In addition, we confirmed the results in LL/2 cells (Fig. 1A). Moreover, we demonstrated that MSA-2, a STING agonist, significantly induced IFNα, IFNγ, and ISG15 expression in A549 cells (Fig. 1B), which revealed that RT induced IFN expression through the STING-mediated signaling pathway. Therefore, the additional IFNγ were incubated with parental A549 cells for 24 h, which were consequently re-incubated with a cultured medium containing healthy PBMCs for another 2 h, and CD8+ T cells were then isolated and analyzed using qPCR. We found that A549 pre-treated with 10 Gy of RT and 20 ng/mL of IFNγ significantly stimulated CD8+ T cells to increase cytotoxic markers GZMB, PRF1, and activation markers CD69, IFNγ compared to the untreated A549 group (Fig. 1C). In addition, A549 pre-treated with IFNs (20 ng/mL of IFNα and IFNγ mixture) for 24 h incubation increased PBMCs to suppress A549 colony formation (Fig. 1D). The results suggest that IFNs alter gene expression in A549 cells to activate CD8+ T cells in vitro.
Radiotherapy induces PD-L1 and ICAM-1 expression through autocrine IFNs stimulation in lung cancer
To investigate the mechanism that IFNs stimulated tumor recognition by healthy PBMCs in vitro, the differential gene expression was analyzed using a RANseq technique in A549 cells treated with 20 ng/mL of IFNγ for 2 h to discover the potential CD8+ T cells-recognized targets. We found 21 up-regulated (fold change > 2, p < 0.05) and 3 down-regulated gene expression (fold change < 2, p < 0.05) in the IFNγ-treated A549 compared to parental A549 cells (Table S2). The 9 up-regulated genes with fold change > 3, including CD274 (PD-L1), ICAM1, BATF2, IRF1, SOCS1, HAPLN3, TAP1, PSMB9 (LMP2), and MAFF, were selected for further analysis. To clarify the clinical significance of the selected genes, the Kaplan-Meier plotter (https://kmplot.com/analysis/) was used to analyze the correlation between the mRNA expression and survival probability in patients with lung cancer based on GEO, EGA, and TCGA databases. We found that CD274 (PD-L1), ICAM1, BATF2, SOCS1, HAPLN3, and MAFF overexpression were individually correlated with poor survival probability in patients with lung cancer (p < 0.05) (Fig. 2A). Consequently, qPCR was used to validate the 9 genes and another gene HLA-ABC expression in A549 cells treated with 20 ng/mL of IFNα and IFNγ. The results revealed that IFNα, IFNγ, and combined both significantly increased PD-L1, ICAM-1, IRF1, and TAP1 levels in A549 cells (Fig. 2B). Moreover, we found that there were increased levels of PD-L1, ICAM-1, IRF1, and TAP1 in the RT-treated A549 cells compared to untreated A549 (Fig. 2C). The RT (10Gy and 20Gy)-treated A549 medium was collected and added to parental A549 cells for 2 h incubation. The 4 genes, PD-L1, ICAM-1, IRF1, and TAP1, were then analyzed using qPCR, which indicated that 10Gy-treated A549 medium (10Gy-m) significantly induced the 4 gene expression in A549 cells (Fig. 2D). However, 20Gy-m caused less increase of PD-L1 and ICAM-1 (Fig. 2D) that may be less IFNγ expression in the 20Gy-treated A549 (Fig. 1A). In addition to confirm the observation, flow cytometry was used to validate the protein levels of PD-L1 and ICAM-1 in IFNs- and RT-treated A549 cells. We demonstrated that IFNα, IFNγ, and combined both significantly increased PD-L1 and ICAM-1 protein levels in A549 cells (Fig. 2E). Meanwhile, RT also increased PD-L1 and ICAM-1 protein levels in A549 cells in a dose-dependent manner (Fig. 2F). We further demonstrated that PD-L1 and ICAM-1 were positively correlated with each other for both mRNA and protein levels in patient with lung cancer based on the database in cBioPortal (https://www.cbioportal.org/) (Fig. 2G). To further clarify the correlation between genes and survival rate in lung cancer patients treated with immunotherapies, the Kaplan-Meier plotter (https://kmplot.com/analysis/) was used (restrict to anti-PD-1 therapy, N = 21). We found that high levels of the 4 genes, PD-L1, ICAM-1, IRF1, and TAP1 were correlated with better survival probability in lung cancer patients treated with immunotherapies (p = 0.17 for PD-L1, p = 0.28 for ICAM-1, p = 0.023 for IRF1, and p = 0.22 for TAP1, Fig. 2H).
IFNγ mediates PD-L1 and ICAM-1 expression through the JAK2-STAT3 signaling pathway in A549
To investigate the downstream transducers involved in IFNγ-mediated gene expression, IFNγ-downstream IFNGR1, IFNGR2, JAK1, JAK2, STAT1, and STAT3 were knocked down by short hairpin RNA (shRNA) (Fig S2A, S2C, and S2E), and qPCR was used to detected PD-L1, ICAM-1, IRF1 and TAP1 in A549 treated with 20 ng/mL of IFNγ for 2 h. We found that knockdown of IFNGR1 significantly suppressed IFNγ-mediated PD-L1, ICAM-1, IRF1, and TAP1 expression (Fig S2B). Knockdown of IFNGR2 significantly suppressed IFNγ-mediated PD-L1 and ICAM-1, but increased IRF1 and TAP1 (Fig S2B). In addition, knockdown of JAK1 reduced IFNγ-mediated ICAM-1 and IRF1; knockdown of JAK2 reduced IFNγ-mediated PD-L1 and ICAM-1 (Fig S2D). Knockdown of STAT3 significantly reduced IFNγ-mediated PD-L1 and ICAM-1, but knockdown of STAT1 increased PD-L1 and ICAM-1 (Fig S2F). The results are summarized in Fig S2G, indicating that IFNγ induced PD-L1 and ICAM-1 through the IFNGR1/2-JAK2-STAT3 axis.
Knockdown of PD-L1 increases activation of CD8+ T cells to suppress tumors
Pd-l1 was further knocked down in LL/2 cells, which was validated using qPCR (Fig. 3A) and flow cytometry (Fig. 3B). We found that knockdown of Pd-l1 significantly diminished splenocytes-mediated anti-LL/2 colony formation (Fig. 3C), whereas ODN1585, a class A oligodeoxynucleotides with unmethylated CpG dinucleotides binding to Toll-like receptor 9 in plasmacytoid dendritic cells (pDCs) [35], stimulated splenocytes-mediated anti-LL/2 activity (Fig. 3C). To validate the effect of PD-L1-knockdown tumors on CD8+ T cell activation, PD-L1 was further knocked down in A549 cells and validated using qPCR (Fig. 3D) and flow cytometry (Fig. 3E), which were incubated with healthy PBMCs for 12 h and 24 h, and CD8+ T cells were isolated consequently for detection of activation and exhaustion gene expression using qPCR. We noticed that knockdown of PD-L1 did not affect ICAM-1 expression in A549 cells (Fig. 3D) but stimulated CD8+ T cells to increase cytotoxic markers GZMB and PRF1 and activation marker CD69 after 12 h incubation (Fig. 3F). Particularly, knockdown of PD-L1 decreased PD-1 expression in CD8+ T cells (Fig. 3F). In addition to validate the observation, ELISA was used to detect granzyme B in the co-cultured supernatant. It validated that knockdown of PD-L1 in A549 stimulated healthy PBMCs to secrete higher granzyme B compared to A549shLuc (Fig. 3G).
Knockdown of ICAM-1 reduces PBMCs-mediated anti-A549 colony formation and CD8+ T cell activation
ICAM-1 is an adhesion molecule binding to LFA-1 (CD11/CD18) for T lymphocyte transmigration [36]. To investigate the role of ICAM-1 in regulating CD8+ T cells, ICAM-1 was knocked down in A549 cells that were further incubated with healthy PBMCs for functional and gene expression assays. Knockdown of ICAM-1 was validated using qPCR (Fig. 4A) and flow cytometry (Fig. 4B). Meanwhile, we noticed that knockdown of ICAM-1 resulted in the down-regulation of PD-L1 in A549 cells (Fig. 4A). Even so, we demonstrated that knockdown of ICAM-1 significantly reduced PBMCs-mediated anti-A549 colony formation (Fig. 4C). CD8+ T cells were consequently isolated and analyzed for activation, cytotoxic, and exhaustion gene expression. We found that knockdown of ICAM-1 significantly decreased A549-stimulated cytotoxic markers GZMB and PRF1 and activation marker CD69, in CD8+ T cells after 24 h incubation (Fig. 4D).
CXCR3 and its binding ligand CXCL10 decreased in patients with late-stage lung cancer, contributing to the reduction of tumor-mediated immune activation
To compare the difference between healthy volunteers and lung cancer patients for recognizing RT- or IFNγ-treated tumors, PD-1, a gene binding to PD-L1, CXCR3, a gene binding to CXCL10 for activating LFA-1, and LFA-1 levels were measured using flow cytometry. CD4+ T and CD8+ T were distinguished by the selection of anti-CD3-APC/anti-CD45-pacific blue with consequent anti-CD4-PE and anti-CD8-PE-cy5.5 staining (Fig. 5A). It indicated that CD8+ T levels were decreased in the patients with lung cancer compared to the healthy volunteers (p = 0.04, Table 1). Meanwhile, we found that CXCR3 and LFA-1 decreased in CD4+ T cells of patients with lung cancer (N = 6) compared to healthy volunteers (N = 14) (Fig. 5B). Meanwhile, only CXCR3 decreased in CD8+ T cells of patients with lung cancer (Fig. 5B). CXCR3 was positive correlated between CD4+ T and CD8+ T cells (R2 = 0.724, p < 0.001, Fig. 5C). In addition, we demonstrated that CD8+ T cells in a patient with lung cancer presented lower expression of cytotoxic marker PRF1 and activation marker CD69 in co-cultured with A549 for 24 h compared to a healthy volunteer (Fig. 5D). We further demonstrated that the CXCR3 ligand CXCL10 decreased in the PBMCs of patients with lung cancer compared to healthy volunteers (Fig. 5E). Moreover, non-CD8+ PBMCs, containing NKs, macrophages, DCs, and CD4+ T cells, presented lower A549-stimulated CXCL10 expression in a patient with lung cancer compared to a healthy volunteer after co-cultured with A549 for 24 h (Fig. 5F). Moreover, we found that CXCL10 significantly increased activation markers CD69 and IFNγ (Fig. 6A) and the genes involved in LFA-1 conformational activation (Fig. 6B), including Ca2+-binding calmoduline 1 (CALM1), calcineurin A (PPP3CA), and cytoskeletal talin-1 (TLN1) and kindlin-3 (FERMT3).[36, 37] The results suggested that CXCL10 triggered LFA-1 conformational activation in CD8+ T cells for further binding with ICAM-1.[38] Further to validate CXCR3 and LFA-1 involved in CD8+ T cell activation, their specific inhibitors were used in splenocytes-mediated anti-LL/2shPd-l1 colony formation (Fig. 3C). We demonstrated that SCH546738, an inhibitor of CXCR3, and A286982, an inhibitor of LFA-1, did not affect the cell viability of mouse splenocytes under 100 nM concentration (Fig. 6C) but suppressed splenocytes-mediated anti-LL/2 cell viability (Fig. 6D) and colony formation (Fig. 6E) after CD8+ T cells isolated, incubated with inhibitors for 4 h, and re-mixed with splenocytes. The results demonstrate that CXCR3 and LFA-1 in CD8+ T cells play significant roles in anti-tumor immunity.