Long noncoding RNA Pvt1 regulates the immunosuppression activity of granulocytic myeloid-derived suppressor cells in tumor-bearing mice

Lewis lung carcinoma (LLC) cells were obtained from the American Type Culture Collection. C57BL/6 mice (6–8 weeks, 18–22 g, male) were purchased from the Animal Research Center of Jiangsu University (Zhenjiang, China) and housed under specific-pathogen-free conditions. LLC cells (2 × 10⁶/mouse) were implanted in mice via s.c. injection to construct tumor models. All experimental protocols were approved by the Committee on the Use of Live Animals in Research and Teaching of Jiangsu University.

Spleen, tibia, femur and tumor tissues were harvested when mice were sacrificed. Spleen cells were lysed with ACK buffer. Bone marrow cells were immediately flushed from tibia and femur and then lysed with ACK buffer. Tumor tissues were removed, cut into small pieces (1–2 mm³), and digested in RPMI 1640 (Gibco, Carlsbad, CA) medium supplemented with 5% fetal calf serum (FBS), 0.5 mg/ml collagenase type V, 0.2 mg/ml hyaluronidase and 0.015 mg/ml DNase I (Sigma-Aldrich, St. Louis, MO) for 2 h at 37 °C. Single-cell suspensions derived from removed organs were obtained using a 70-μm cell strainer.

Murine G-MDSCs were isolated from the bone marrow, spleen and tumor tissues of LCC tumor-bearing mice using a mouse MDSC isolation kit (Miltenyi Biotec, Auburn, CA). To improve the purity of G-MDSCs harvested from tumor tissues, Ly6G-enriched G-MDSCs were subsequently isolated using flow cytometry (FCM). In addition, murine CD4⁺ T cells were isolated from spleens of wild-type (WT) C57BL/6 mice using monoclonal anti-mouse CD4 antibodies conjugated to MicroBeads (Miltenyi Biotec, Auburn, CA). The purity of G-MDSCs and CD4⁺ T cells obtained from the isolated cells was confirmed by FCM.

Single-cell suspensions were stained with relevant fluorochrome-conjugated mAbs: anti-mouse CD3, CD4, and CD8 antibodies from eBioscience (San Diego, CA) and anti-mouse CD11b, Gr-1, Ly6G and Ly6C antibodies from Biolegend (San Diego, CA). For detection of T helper 1 (Th1) cells and cytotoxic T lymphocytes (CTLs), single-cell suspensions from spleens, draining lymph nodes and tumor tissues were stimulated with 50 ng/mL PMA (Sigma-Aldrich, St. Louis, MO), 2 μg/mL ionomycin and 1 μg/mL monensin (eBioscience, San Diego, CA). After 5 h, the cells were stained with anti-CD3, anti-CD4, anti-CD8 mAbs; fixed; permeabilized; and stained with an anti-IFN-γ mAb according to instructions provided in an Intracellular Staining Kit (eBioscience, San Diego, CA). Flow cytometry was performed using FACSCalibur Flow Cytometer (Becton Dickinson).

Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA). Next, cDNA was synthesized with a Prime Script RT Reagent Kit (Takara, Osaka, Japan) according to the manufacturer’s instructions. Gene transcripts were quantified via real-time quantitative PCR performed with SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara, Osaka, Japan). The sequences for the primers used were as follows: Pvt1, 5′-ATCCACCCTCTTGTCTGATTTTCT-3′ (forward) and 5′-AATCCACAGGGTTCAGGAAGTC-3′ (reverse); c-myc, 5′-AGCGACTCTGAAGAGCAAG-3′ (forward) and 5′-ATGGAGATGAGCCCGACTC-3′ (reverse); β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (forward) and.

5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (reverse). Relative quantification of mRNA expression was calculated using the comparative threshold cycle (Ct) method.

Protein extracted from cells was lysed in radioimmunoprecipitation (RIPA) buffer, and cell debris was removed by centrifugation. The extracts were quantified using a protein assay and then boiled in SDS gel-loading buffer containing 10% β-mercaptoethanol. Proteins were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gels and then transferred onto immobilon PVDF membranes (Bio-Rad, Hercules, CA), which were probed with rabbit mAbs against mouse β-actin (CST, Danvers, MA), and c-myc and HIF-1α (Wanleibio, Co., Shenyang, China), and then incubated with HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK) followed by chemiluminescence detection (Champion Chemical, Whittier, CA).

G-MDSCs were plated in 48-well plates or 24-well plates (Costar, Corning, NY) with RPMI 1640 medium supplemented with 10% fetal calf serum and then transfected with 50 nM Pvt1 siRNA or the negative control (Ribobio Co., Guangzhou, China) using Entranster-R (Engreen Biosystem Co., Beijing, China) according to the manufacturer’s instructions. Six hours after transfection, 1 ng/mL GM-CSF was supplemented in culture system.

Arginase activity was detected using a quantitative colorimetric assay by employing a QuantiChrom Arginase Assay kit (BioAssay systems, Hayward, CA). Arginase activity was calculated according to the kit manufacturer’s instructions.

ROS produced by G-MDSCs was measured using the oxidation-sensitive dye 2′, 7′-dichlorofluorescin diacetate (Invitrogen, Carlsbad, CA). Cells were simultaneously cultured with 2.5 μM 2,7-dichlorofluorescin diacetate and 30 ng/mL PMA in PBS for 30 min. Then, ROS produced by G-MDSCs were detected by flow cytometry.

Tibia and femur from wild-type C57BL/6 mice were removed, and bone marrow cells were flushed from the bones. Erythrocytes were lysed with ACK for 5 min. To obtain BM-derived G-MDSCs, 1 × 10^{^6} bone marrow cells were plated in 24-well plates (Costar, Corning, NY) in 1 mL RPMI 1640 (Gibco, Carlsbad, CA) complete medium consisting of 10% fetal calf serum (FBS), 100 U/mL penicillin, 100 μg/ml streptomycin, 20 ng/mL IL-6 and 20 ng/mL GM-CSF (Peprotech, Rocky Hill, USA). After 3 days, the cells were collected and isolated using an MDSC Isolation Kit (Miltenyi Biotec, Auburn, CA) to acquire CD11b⁺ Ly6G⁺ G-MDSCs.

G-MDSCs were transfected with Pvt1 siRNA or the negative control. Responder cells (splenic CD4⁺ T cells) were cocultured with transfected G-MDSCs in U-bottomed 96-well plates (Costar, Corning, NY) in the presence of 10 μg/mL anti-CD3 mAb and 5 μg/mL anti-CD28 mAb (Biolegend, San Diego, CA) for 72 h and then pulsed with [³H]-thymidine (Pharmacia Biotech, Stockholm, Sweden, 1 μCi/well) for the last 16 h of culture. The capacity of G-MDSCs to suppress T cells was calculated according to the cpm value.

To detect the suppression function of G-MDSCs induced from bone marrow cells using IL-6 and GM-CSF treatment. 1 × 10⁷/mL splenic CD4⁺ T cells were stained with the fluorescent dye CFSE (5 μM, Invitrogen) at 37 °C for 10 min and keep protected from light. 5 times the original staining volume of RPMI 1640 (Gibco, Carlsbad, CA) complete medium consisting of 10% fetal calf serum were added, pellet cells were washed twice with RPMI 1640 medium. CFSE labeled CD4⁺ T cells were cocultured with BM-derived G-MDSCs in round-bottomed, 96-well plates (Costar, Corning, NY) in the presence of 10 μg/mL anti-CD3 mAb and 5 μg/mL anti-CD28 mAb (Biolegend, San Diego, CA) for 72 h. CD4⁺ T cell proliferation was measured by CFSE dilution using FACSCalibur.

A hypoxic environment was created by culturing G-MDSCs with AnaeroGen™ (Thermo Fisher Scientific, Massachusetts, USA) in a sealed box placed in an incubator at 37 °C (O₂ < 0.1, 5% CO₂). G-MDSCs cultured directly in the incubator chamber at 37 °C (20% O₂, 5% CO₂) were considered normoxia control cells.

Briefly, 1 × 10⁶ G-MDSCs isolated from tumor tissues and subsequently transfected with Pvt1 siRNA or the negative control mixed with 0.8 × 10⁶ LLC cells were implanted via s.c. injection into C57BL/6 mice, and tumor growth was monitored continuously. Tumor volume was calculated using the formula V = 1/2 × a² × b, where ‘a’ represents the smaller diameter and ‘b’ is the larger diameter. The Th1 cell and CTL proportion in spleens, draining lymph nodes (dLNs), and tumor tissues from the tumor-bearing mice were detected via FCM.

The data are expressed as the mean ± SD. The statistical significance of differences between groups was determined via t tests and ANOVA using SPSS 19.0 software. Data from all experiments were imported into GraphPad Prism 5.0 (GraphPad, San Diego, CA) to generate bar graphs. Differences were considered to be significant at a p level less than 0.05.

By comparing expression profile of lncRNAs between G-MDSCs isolated from tumor tissues of Lewis tumor-bearing (TB) mice and spleens from corresponding wild-type (WT) C57BL/6 mice using array-based lncRNA profiling, a large number of lncRNAs were found to be highly expressed in TB mice compared with WT mice. We screened out lncRNA Pvt1, which exhibited one of the 20 largest variations in the microarray (Fig. 1a). In addition, we confirmed the microarray results using qRT-PCR. In accordance with the array data, qRT-PCR analysis showed that Pvt1 expression was approximately 18 times higher in TB mice than in corresponding WT mice. To examine whether upregulation of Pvt1 in Lewis tumor-expanded G-MDSCs could be extrapolated to other tumors, murine CT26 colorectal tumors were established by implanting 1 × 10^{^6} CT26 cells via s.c. injection into BALB/c mice. An increase in the Pvt1 level in G-MDSCs from TB mice relative to that in G-MDSCs from corresponding WT mice was found (Additional file 1: Figure S1a).

It is widely known that MDSCs from both spleens and tumor tissues of TB mice effectively suppress the T cell response, but MDSCs from tumor tissues have a significantly stronger suppression effect. Therefore, we sorted G-MDSCs from bone marrow, spleens and tumor tissues of TB mice and analyzed cell purity using flow cytometry. As shown in Fig. 1b, the G-MDSC purity was higher than 90% in all samples, which met the requirements of subsequent experiments. The level of lncRNA Pvt1 in G-MDSCs from tumor tissues was clearly higher than that in G-MDSCs from spleens or bone marrow (Fig. 1c). All these data indicate that lncRNA Pvt1 is highly expressed in G-MDSCs from tumor tissues. To further determine the relationship between the suppressive activity of G-MDSCs and the Pvt1 level in G-MDSCs, we chose G-MDSCs from the bone marrow induced with IL-6 and GM-CSF for further study. Cytokine-induced G-MDSCs had a stronger inhibitory effect on CD4⁺T cell proliferation (Fig. 1d). At the same time, induced G-MDSCs had stronger Arg1 activity and higher ROS levels than G-MDSCs from fresh bone marrow (Fig. 1e and f). The level of Pvt1 was also upregulated in induced G-MDSCs (Fig. 1 g). Thus, the high level of Pvt1 might be related to a stronger immunosuppression effect of G-MDSCs.

As mentioned earlier, MDSCs are divided into two subgroups, of which G-MDSCs are the majority group. We also detected the expression of Pvt1 in G-MDSCs and M-MDSCs and found that the expression level of Pvt1 was not significantly different between G-MDSCs and M-MDSCs from the bone marrow, spleen and tumor tissues (Additional file 1: Figure S2).

As described previously, a high level of Pvt1 is associated with stronger immunosuppression induced by G-MDSCs. Therefore, we hypothesized that Pvt1 could regulate the function of G-MDSCs. To investigate the function of Pvt1 in G-MDSCs, G-MDSCs isolated from tumor tissues were transfected with Pvt1-specific small interfering RNA (si-Pvt1) to knockdown Pvt1 expression. Compared with the negative control, the expression of Pvt1 in G-MDSCs transfected with si-Pvt1 was downregulated, especially in cells transfected with si-Pvt1 003 (Fig. 2a). We chose si-Pvt1 003, rather than si-Pvt1 001 or si-Pvt1 002, for use in the following experiments. In addition, we found that G-MDSC-mediated T-cell suppression was weakened after Pvt1 knockdown (Fig. 2b). It has been reported that G-MDSCs suppress T-cell-induced antitumor immune responses through a large number of mechanisms, including through Arg1 and ROS. As shown in Fig. 2c and d, after transfection with si-Pvt1, Arg1 activity and ROS production were clearly decreased in G-MDSCs. Therefore, Pvt1 likely downregulates the immunosuppressive effect of G-MDSCs in vitro.

According to the regulatory mechanisms of lncRNA, intergenic lncRNA can regulate the expression of nearby genes. Pvt1 has been defined as an intergenic lncRNA in many diseases, and c-myc is adjacent to Pvt1 in the chromosome. As predicted by the gene microarray results (Fig. 3a), the mRNA level of c-myc was distinctly higher in G-MDSCs isolated from tumor tissues of TB mice than in G-MDSCs isolated from spleens of WT mice (Fig. 3b). In murine CT26 colorectal tumors, the same results were observed (Additional file 1: Figure S1b). In keeping with the Pvt1 findings, the c-myc level in G-MDSCs from tumor tissues was considerably higher than in the spleens and bone marrow from TB mice (Additional file 1: Figure S3a). Additionally, c-myc levels were increased in G-MDSCs induced from bone marrow cells with IL-6 and GM-CSF compared with G-MDSCs from fresh bone marrow (Additional file 1: Figure S3b). All these data suggested that the alteration in c-myc expression was in accordance with the change in Pvt1 levels; thus, c-myc might be a downstream target of Pvt1 in G-MDSCs. For further certification, we knocked down Pvt1 in G-MDSCs with si-Pvt1 and then measured the mRNA and protein levels of c-myc. As shown in Fig. 3c and d, both the mRNA and protein levels of c-myc were decreased in G-MDSCs transfected with si-Pvt1. The results indicate that c-myc is potentially a downstream target of Pvt1 in G-MDSCs, but whether c-myc participates in regulation of G-MDSC function through Pvt1 requires further study.

G-MDSCs isolated from tumor tissues of tumor-bearing C57BL/6 mice were transfected with si-Pvt1 in vitro. Then, 1 × 10^{^6} G-MDSCs transfected with si-Pvt1 mixed with 0.8 × 10^{^6} Lewis lung carcinoma (LLC) cells were implanted via s.c. injection into WT C57BL/6 mice. As shown in Fig. 4a, tumor growth was significantly reduced in mice injected with G-MDSCs transfected with si-Pvt1 (si-Pvt1 group) compared with mice injected with G-MDSCs transfected with the negative control (si-NC group). In the tumor microenvironment, MDSCs primarily suppress the antitumor response by inhibiting T cell responses. We investigated whether alteration of Pvt1 expression in G-MDSCs could regulate CTL and Th1 responses in vivo. Therefore, the proportions of CD8⁺ IFN-γ⁺ CTLs and CD4⁺ IFN-γ⁺ Th1 cells in draining lymph nodes (dLNs), spleens and tumor tissues were detected by flow cytometry. We found that the proportion of CTLs in dLNs from the si-Pvt1 group was much higher than that in dLNs from the si-NC group, and the proportions of CTLs and Th1 cells in tumor tissues exhibited a moderate increase in the si-Pvt1 group compared with the si-NC group (Fig. 4b and c).

As mentioned above, the expression of Pvt1 was higher in tumor-infiltrating G-MDSCs than in splenic G-MDSCs. We explored whether the tumor microenvironment might regulate the expression of Pvt1 in G-MDSCs, and which factor likely regulates the Pvt1 level. Considering the hypoxic conditions in the tumor microenvironment and that the primary molecular response to hypoxia is elicited through HIF-1α, we examined HIF-1α expression and found that it was higher in tumor-infiltrating G-MDSCs than in splenic G-MDSCs (Fig. 5a). We exposed splenic G-MDSCs to hypoxic conditions in vitro and measured HIF-1α expression to determine the success of the hypoxia treatment. Both the mRNA and protein levels of HIF-1α were upregulated in G-MDSCs under hypoxic conditions (Fig. 5b and c). Meanwhile, G-MDSCs exposed to hypoxic conditions expressed higher levels of Pvt1 than G-MDSCs under to normoxic conditions (Fig. 5d), which confirmed our hypothesis. In addition, consistent with the Pvt1 levels, the expression of c-myc in G-MDSCs was also upregulated under hypoxia (Additional file 1: Figure S4a). To adequately demonstrate whether HIF-1α regulates Pvt1 expression, we used a specific inhibitor of HIF-1α, YC-1, to block upregulation of HIF-1α under hypoxic conditions. When G-MDSCs were exposed to hypoxic conditions, we added YC-1 to inhibit HIF-1α expression. The results showed that HIF-1α expression was decreased (Fig. 5e and f). In addition, the upregulation of Pvt1 and c-myc under hypoxia was restored by treatment with the HIF-1α inhibitor (Fig. 5g and Additional file 1: Figure S4b). Taken together, these results suggest that the expression of Pvt1 in G-MDSCs might be regulated by HIF-1α under hypoxic stress.