PD-L1 regulates tumor proliferation and T-cell function in NF2-associated meningiomas

Abstract

Introduction: Programmed death-ligand 1 (PD-L1) expression is an immune evasion mechanism that has been demonstrated in many tumors and is commonly associated with a poor prognosis. Over the years, anti-PD-L1 agents have gained attention as novel anticancer therapeutics that induce durable tumor regression in numerous malignancies. They may be a new treatment choice for neurofibromatosis type 2 (NF2) patients.

Aims: The aims of this study were to detect the expression of PD-L1 in NF2-associated meningiomas, explore the effect of PD-L1 downregulation on tumor cell characteristics and T-cell functions, and investigate the possible pathways that regulate PD-L1 expression to further dissect the possible mechanism of immune suppression in NF2 tumors and to provide new treatment options for NF2 patients.

Results: PD-L1 is heterogeneously expressed in NF2-associated meningiomas. After PD-L1 knockdown in NF2-associated meningioma cells, tumor cell proliferation was significantly inhibited, and the apoptosis rate was elevated. When T cells were cocultured with siPD-L1-transfected NF2-associated meningioma cells, the expression of CD69 on both CD4⁺ and CD8⁺ T cells was partly reversed, and the capacity of CD8⁺ T cells to kill siPD-L1-transfected tumor cells was partly restored. Results also showed that the PI3K-AKT-mTOR pathway regulates PD-L1 expression, and the mTOR inhibitor rapamycin rapidly and persistently suppresses PD-L1 expression. In vivo experimental results suggested that anti-PD-L1 antibody may have a synergetic effect with the mTOR inhibitor in reducing tumor cell proliferation and that reduced PD-L1 expression could contribute to antitumor efficacy.

Conclusions: Targeting PD-L1 could be helpful for restoring the function of tumor-infiltrating lymphocytes and inducing apoptosis to inhibit tumor proliferation in NF2-associated meningiomas. Dissecting the mechanisms of the PD-L1-driven tumorigenesis of NF2-associated meningioma will help to improve our understanding of the mechanisms underlying tumor progression and could facilitate further refinement of current therapies to improve the treatment of NF2 patients.

Keywords: NF2; PDL1; PI3K/AKT/mTOR; immunosuppression; meningioma.

FIGURE 1

Tumor‐infiltrating lymphocytes and PD‐L1 expression in NF2‐associated meningioma. (A) IHC staining of CD3‐, CD8‐, CD4‐ and CD20‐positive lymphocytes in NF2‐associated meningiomas. (B) IHC staining of PD–L1 expression in NF2‐associated meningiomas at 200×/400× magnification. (C) Western blotting results of PD‐L1 expression in NF2‐associated meningiomas.

FIGURE 2

The effect of PD‐L1 downregulation on NF2‐associated meningioma cell proliferation and apoptosis. (A) Top: Relative expression of PD‐L1 after transfection with siPD‐L1 for 72 h (unpaired t test, vs. NC, n = 3). Bottom: Western blotting results showed PD‐L1 expression after transfection with siPD‐L1 for 72 h. (B) The cell viability of NF2‐associated meningioma cells after transfection with siPD‐L1 for 72 h was determined using the CCK‐8 assay. (C) Top: Flow cytometric analysis of NF2‐associated meningioma cells after transfection with siPD‐L1 for 72 h using Annexin V‐FITC/7‐AAD staining. Bottom: Statistical data of apoptosis rates. *p < 0.05, **p < 0.01, ***p < 0.005 compared to the NC group.

FIGURE 3

The effect of PD‐L1 downregulation on T‐cell activation. NF2‐associated meningioma cells transfected with siPD‐L1 were added at 1:4 to normal CSC‐stimulated CD3⁺ T cells. (A) Flow cytometric analysis of CD4⁺CD69⁺ cell percentages in the coculture system. (B) Statistical analysis of the flow cytometric results shown in (A), n = 3. (C) Flow cytometric analysis of CD8⁺CD69⁺ cell percentages in the coculture system. (D) Statistical analysis of the flow cytometric results shown in (C), n = 3. *p < 0.05, **p < 0.01, ***p < 0.005 compared to the NC group.

FIGURE 4

The effect of PD‐L1 downregulation on T‐cell cytotoxicity. NF2‐associated meningioma cells transfected with siPD‐L1 were added at 1:4 to normal CSC‐stimulated CD8⁺ T cells, and cytotoxicity was analyzed. (A) Flow cytometric analysis of CFSE⁺7AAD⁺ cell percentages in the coculture system. (B) Statistical analysis of the flow cytometric results shown in (A), n = 3. (*p < 0.05, **p < 0.01).

FIGURE 5

Regulation of PD‐L1 expression in NF2‐associated meningiomas. (A) Western blot analysis showed upregulation of PD‐L1 expression in IOMM‐Lee cells after knockdown of NF2 expression. *p < 0.05 compared to the NC/518D group. (B) Western blot analysis showed that phosphorylation of NF2 led to increased PD‐L1 expression in IOMM^518D cells compared with IOMM^518A cells. **p < 0.01 compared to the 518D group. (C) Western blotting results showed that NF2‐associated meningioma tissues with higher P‐merlin expression also had higher PD‐L1 expression. (D) IOMM^NF2KD cells were treated with 50 μmol/L of a PI3K inhibitor (LY294002), 2 μmol/L of an mTOR inhibitor (rapamycin), or 5 μmol/L of an AKT inhibitor (MK‐2206 2HCL), and the phosphorylation events downstream of AKT/mTOR were monitored by Western blot analysis. (E) IOMM^518D cells were treated with 50 μmol/L of a PI3K inhibitor (LY294002), 2 μmol/L of an mTOR inhibitor (rapamycin), or 5 μmol/L of an AKT inhibitor (MK‐2206 2HCL), and the phosphorylation events downstream of AKT/mTOR were monitored by Western blot analysis.

FIGURE 6

The combination of rapamycin and anti‐PD‐L1 antibody inhibits NF2‐associated meningioma cell growth in vivo. (A) Figure of the animal experimental design and schedule. (B) Representative graph of tumor volume measured every 3 days from the beginning of treatment. Volume of the tumors excised from treated and control mice after the mice were sacrificed. (C) Pathologic analysis of H&E, Ki67 and PD‐L1 staining of the tumors excised from the treated and control mice after 21 days of treatment. (D) Western blot analysis of PD‐L1 expression in the tumors excised from treated and control mice after 21 days of treatment. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 compared to the CTRL group.