NKG2D upregulation sensitizes tumors to combined anti-PD1 and anti-VEGF therapy and prevents hearing loss

Domain: neuroscience
Published: 2026-02-11
Editorial outcome: major_revision

Abstract
NF2-related schwannomatosis (NF2-SWN) is a debilitating condition, characterized by bilateral vestibular schwannomas (VSs) that progressively cause irreversible sensorineural hearing loss. Current management relies on surgery or radiotherapy, while bevacizumab (αVEGF) is used off-label, with variable and often transient efficacy. Effective therapies that durably suppress tumor growth and preserve hearing are urgently needed. Although immune checkpoint inhibitors have transformed cancer treatment, their efficacy in non-malignant tumors such as VS remains unclear. Here, we evaluate combined anti-PD1 (αPD1) and αVEGF therapy in two syngeneic, immune-competent VS models. Combination treatment significantly outperforms either monotherapy, inhibiting tumor growth and preventing hearing loss. Mechanistically, αVEGF enhances αPD1 efficacy by normalizing tumor vasculature, improving drug delivery and immune cell infiltration, and promoting cytotoxicity of T and NK cells via NKG2D upregulation. Combined treatment effectively controls tumor growth that progresses despite anti-VEGF therapy. These findings support αPD1 and αVEGF combination therapy as a promising strategy for NF2-SWN.

Introduction
NF2-related schwannomatosis (NF2-SWN) is a hereditary neoplastic syndrome resulting from germline pathogenic variants in the NF2 tumor suppressor gene. It has an incidence rate of about 1 in 61,000 individuals, with nearly complete penetrance at close to 100%1,2. Patients with NF2-SWN are characterized by bilateral vestibular schwannomas (VS) that arise from cranial nerve VIII. The progressive growth of VSs can lead to sensorineural hearing loss (SNHL), significant morbidity, and greatly affect the quality of life of patients3. In some cases, large VSs can put pressure on the brainstem, leading to severe complications and even fatality4. Current treatments for progressive VSs include surgery and radiotherapy, both of which carry the risk of further nerve damage and may result in profound deafness, chronic dizziness, and paralysis of the facial and other cranial nerves5–7. Bevacizumab, a humanized monoclonal antibody that neutralizes vascular endothelial growth factor-A (VEGF-A), is approved in the UK for the treatment of NF2-SWN and has documented benefits in 30-40% of patients, with improvement in hearing or tumor shrinkage7–9. However, not all patients respond to bevacizumab and the effect is not always durable. There remains a significant unmet medical need for effective treatments that can halt VS growth and prevent SNHL associated with these tumors.

Immune checkpoint inhibitors (ICI), including monoclonal antibodies targeting programmed cell death protein-1 (PD-1) and its ligand 1 (PD-L1), work by blocking immune inhibitory signals, thereby enhancing T cell activation and function. This process leads to improved antitumoral immunity and better prognostic outcomes10,11. FDA has approved ICIs for a variety of cancers, either as standalone treatments or in conjunction with other therapies such as chemotherapy12. However, their therapeutic potential in non-malignant tumors, such as schwannomas, has yet to be investigated. VS tumors contain a significant population of T cells13. However, many of these infiltrating CD4+ and CD8+ T cells express PD-114 and display a transcriptome signature indicative of CD8+ T cell senescence15. Additionally, PD-1 and its ligand PD-L1 are expressed in VS tumors13,16. Despite these findings, there has been limited research on the therapeutic potential of ICIs in VS. Notably, an αPD1 antibody was shown to modestly slow subcutaneous mouse schwannoma growth17, and αPD1 salvage therapy resulted in tumor growth inhibition in a VS patient with recurrent tumors18. These insights indicate that ICIs could represent a promising treatment approach for VS.

In this study, we aim to address two questions: 1) Can αVEGF-induced vessel normalization enhance intratumoral delivery of αPD1 drug and immune effector cells, thus augmenting the ICI efficacy in VS? and 2) Can αPD1 serve as an effective alternative for patients unresponsive to or unable to tolerate bevacizumab? Using schwannoma mouse models, we achieve several key findings: i) we conduct the first comprehensive investigation of the immune checkpoint inhibitor αPD1 as a potential treatment for non-malignant VS, characterizing its effects on tumor growth; (ii) we evaluate the impact of αPD1 on hearing preservation, which has not been previously examined. Importantly, we show that combining αVEGF with αPD1 significantly enhances the efficacy of either monotherapy. Specifically, iii) αVEGF improves drug delivery and promotes intratumoral infiltration of immune effector cells, thereby enhancing αPD1 efficacy; and iv) combined αPD1 with αVEGF treatment effectively controls tumor growth that progresses despite αVEGF treatment. Our long-term goal is to develop therapies for NF2-SWN that both suppress VS tumor growth and preserve hearing. These findings provide a strong foundation for αPD1 with αVEGF combination therapy for patients with NF2-SWN.

Results
αVEGF treatment normalizes tumor vasculature and increases αPD1 drug delivery and intratumoral infiltration of immune effector cells in the mouse schwannoma model
To address our first question of whether αVEGF-induced vessel normalization enhances the intratumoral delivery of ICI drugs and immune effector cells, thereby augmenting ICI efficacy in VS, we evaluated how αVEGF treatment affects tumor vasculature in the cerebellopontine angle (CPA) Nf2−/− schwannoma model. αVEGF treatment did not significantly reduce microvessel density (MVD; Fig. 1A-B), but it did significantly increase the fraction of pericyte-covered vessels, indicating that the treated tumor blood vessels are structurally similar to normal vessels (Fig. 1C). Next, to determine whether the structural normalization of tumor vessels leads to normalized vessel perfusion, we measured the fraction of perfused vessels (Fig. 1D). In concert with the blood vessel structural normalization, αVEGF treatment increased the percentage of perfused vessels (Fig. 1E). These data indicate that αVEGF treatment normalized the schwannoma vasculature.

To determine whether anti-VEGF-mediated vascular normalization enhances αPD1 antibody delivery, the αPD1 antibody was conjugated with FITC and administered i.p. to control IgG- or αVEGF-treated mice. Twenty-four hours later, tumor-bearing brains were collected, and the distribution of fluorescently labeled αPD1 antibody was assessed by confocal microscopy (Fig. 1F). We observed significant angiogenesis (CD31+, cyan) at the peritumoral margin, and the FITC-labeled αPD1 antibody (magenta) predominantly accumulated within the tumor, with minimal signal in the adjacent brain tissue (Fig. 1G). In αVEGF-treated tumors, intratumoral green fluorescence intensity was significantly higher than in control IgG-treated tumors (Fig. 1H). Image analysis confirmed that antibody delivery improved following αVEGF treatment (Fig. 1I).

To determine whether αVEGF-mediated vascular normalization increases the intratumoral infiltration of immune effector cells, we analyze the intratumoral immune cell population using flow cytometry (Supplementary Fig. 1). We found that αVEGF treatment significantly increased the number of tumor-infiltrating cytotoxic CD8+ T cells and NK cells, as well as increased the ratio of CD8+ T cells over immune suppressive Treg (CD8+/Treg) (Fig. 1J).

Combined αVEGF treatment enhances αPD1 efficacy in the mouse schwannoma models
The αVEGF-induced increased delivery of αPD1 antibody and immune effector cells provides the rationale to test if αVEGF treatment could enhance αPD1 efficacy. We treated mice bearing Nf2−/− schwannomas in both the sciatic nerve and CPA models with: i) control IgG, ii) αPD1, iii) αVEGF, or iv) αPD1 + αVEGF (Fig. 2A). In both models, all treatments - including monotherapies and the combination therapy - were well tolerated, with no significant body weight loss observed in any treatment group (Supplementary Fig. 2A). In the sciatic nerve model, combined αVEGF treatment enhanced the antitumor effect of αPD1 and significantly delayed tumor growth compared to αPD1 or αVEGF monotherapies (Fig. 2B). This enhanced efficacy was also evident in the SC4 schwannoma model (Supplementary Fig. 2B). In the CPA Nf2−/− schwannoma model, combined αVEGF treatment significantly prolonged animal survival compared to either monotherapy – with 65% of mice surviving ≥100 days in the combination group, compared to 20% of long-survivor mice in the αPD1 group and 25% of long-survivor mice in the αVEGF group (Fig. 2C).

In a separate cohort of Nf2−/− sciatic nerve tumor-bearing mice, all treatments were discontinued after administering three doses. Remarkably, tumor growth remained suppressed even after treatment cessation, indicating a durable antitumor effect that persisted beyond the active treatment phase (Supplementary Fig. 2C-2D).

To examine the effects of immunotherapy on hearing function, we first evaluated potential ototoxicity from αPD1 treatment in non-tumor-bearing mice by measuring ABR, which represents the summed activity of the auditory nerve and central auditory nuclei. In non-tumor-bearing mice, three doses of αPD1 treatment did not alter the ABR threshold in mice 21 days post-treatment (Supplementary Fig. 2E–2F), demonstrating that αPD1 does not cause acute ototoxicity in mice. In mice bearing Nf2−/− tumors in the CPA region, αVEGF treatment prevented tumor-induced hearing loss (blue line), reproducing the hearing benefit from bevacizumab treatment observed in patients with NF2-SWN. αPD1 monotherapy (red line) and its combination with αVEGF treatment (pink line), restored the ABR threshold to normal levels observed in non-tumor-bearing mice. No treatment demonstrated superiority over the others (Fig. 2D).

αVEGF-enhanced αPD1 efficacy is mediated by CD8+ T cells and NK cells
To investigate the mechanisms of how αVEGF enhanced αPD1 efficacy, we first examined the CPA tumor proliferation and apoptosis. In the combination group, we observed fewer proliferating tumor cells (PCNA+) and more apoptotic tumor cells (TUNEL+) compared to those in the control or monotherapy groups (Supplementary Fig. 3A-3B).

Next, using flow cytometry to profile intratumoral immune cell populations, we found that both αPD1 and αVEGF monotherapies significantly increased the number of tumor-infiltrating cytotoxic CD8+ T and NK cells, reduced the number of immunosuppressive MDSCs, compared with the control group. Compared with αPD1 monotherapy, the combination of αVEGF and αPD1 further enhanced the infiltration of effector CD8+ T cells and NK cells, while reducing MDSCs (Fig. 3A). Tumor secretome analysis revealed that anti-PD1 treatment elevated the levels of IFN-γ and TNF-α - cytokines known to synergistically activate T cells and produced by activated T cells. Combined αVEGF and αPD1 treatment further increased IFN-γ and TNF-α production compared with αPD1 monotherapy (Fig. 3B). These findings suggest that, beyond recruiting more CD8+ T and NK cells to the tumor, αVEGF treatment may also enhance the activation of these immune effector cells. Consistent with this, flow cytometry analysis showed that αVEGF treatment significantly increased the proportion of CD8+ T cells and NK cells expressing granzyme B and perforin - key mediators of cytotoxicity in these effector cells (Fig. 3C and Supplementary Fig. 4).

To investigate if αVEGF-driven infiltration and activation of CD8+ T cells and NK cells are essential for the enhanced antitumor efficacy of the combination therapy, we depleted mice of CD8+ T cells or NK cells prior to treatment (Fig. 3D). αCD8 antibodies effectively deplete CD8+ T cells, and αNK1.1 antibodies specifically reduce NK cells, as confirmed by flow cytometry (Fig. 3E). CD8+ T cell depletion (pink line) completely abolished the therapeutic benefit of the combination therapy, whereas NK cell depletion (blue line) significantly reduced the antitumor response (Fig. 3F).

NKG2D upregulation enhances T and NK cell cytotoxicity, sensitizes tumors to combined αPD1 and αVEGF therapy
To investigate whether αVEGF treatment regulates the cytotoxic function of NK cells and CD8+ T cells, from control- and αVEGF-treated mice, we isolated: i) tumor-associated CD8+ T cells, and ii) spleen NK cells, given the low number of tumor-infiltrating NK cells. CD8+ T cells were co-cultured with calcein AM-labeled Nf2−/− tumor cells at an effector-to-target ratio (E:T) of 10:1. Similarly, NK cells were co-cultured with calcein AM-labeled Nf2−/− tumor cells or YAC-1 cells, which are sensitive to NK cell-mediated cytotoxicity, at the same E:T ratio. CD8+ T cells and NK cells isolated from αVEGF-treated mice exhibited significantly enhanced cytotoxicity against both Nf2−/− tumor cells and YAC-1 target cells compared to effector cells isolated from control IgG-treated mice (Fig. 3G).

We next investigated whether systematic αVEGF treatment activates CD8+ T cells and NK cells. In Nf2−/− tumors, αVEGF treatment significantly increased the expression of natural killer group 2D (Nkg2d) (Fig. 3H). NKG2D is an activating receptor expressed on NK cells, CD8+ T cells, and activated CD4+ T cells, which promotes NK cell cytotoxicity19 and augments T cell receptor-mediated activation of CD8+ T cells20. In mice, NKG2D binds to its ligands RAE-1 and H60A21,22. αVEGF treatment also significantly increased the expression of Rae-1 and H60a, but did not affect the expression of the inhibitory receptor Nkg2a (Fig. 3H).

Lastly, to determine whether NKG2D signaling is required for the enhanced antitumor efficacy of the combination therapy, we blocked NKG2D signaling using a neutralizing antibody. Nf2−/− tumor-bearing mice were treated with i) control IgG, ii) αVEGF, iii) αVEGF+αNKG2D neutralizing antibody, iv) αVEGF+αPD1, or v) αVEGF+αPD1+αNKG2D (Fig. 3I). Blockade of NKG2D signaling markedly attenuated the anti-tumor response to both αVEGF monotherapy and combination treatment (Fig. 3J), indicating that NKG2D signaling is critical for the therapeutic benefit from the αVEGF treatment.

scRNA-Seq analysis reveals elevated cytotoxic profiles of CD8+ T and NK cells in VS from an NF2-SWN patient treated with bevacizumab
To profile the T cells and NK cells in VSs with or without bevacizumab treatment, we dissociated fresh VS tissues from NF2-SWN patients who were either bevacizumab-naïve or treated with bevacizumab for about 10 years. Single-cell transcriptomic profiling was performed using the 10X Genomics platform. Within the VS tissues, cell clusters were partitioned into major cell types, including Schwann cells, macrophages, lymphocytes, fibroblasts, and other stromal cells (Chang LS, et al., manuscript in preparation). We further subclustered the lymphocyte compartment and identified distinct lymphocyte-derived cell states, including CD8+ cytotoxic T cells, early naïve T cells, CD8+ PD-1+ T cells, regulatory T cells, and NK cells in naïve VSs (Fig. 4A).

In NK cells and CD8+ T cells from naïve VSs, we observed elevated RNA expression of i) KLRC1, which encodes NKG2A, a major inhibitory receptor expressed on NK cells and CD8+ T cells23,24; ii) KLRD1, which encodes CD94, forming a heterodimer with NKG2A; and iii) HLA-E, the ligand for NKG2A:CD94 heterodimer, which sends a strong inhibitory signal regulating the cytotoxic activity of NK and CD8+ T cells (Fig. 4B)25,26.

In bevacizumab-treated VS, compared to naïve VS, we observed changes in RNA expression of CD8+ T cell and NK cell cytotoxicity markers: i) reduced expression of inhibitory receptors on NK cells and T cells, including KIR2DL3, KIR2DL2, KIR3DL1, KIR3DL227,28, ii) reduced expression of immune checkpoint proteins and receptors, including CD96, PVRIG, HLA-E29–31, iii) increased expression of molecules mediating NK cell and CD8+ T cell cytotoxicity and recruitment, including Granzyme B (GZMB), the key mediator of32, C-C motif chemokine ligand 4 (CCL4), chemokine recruiting NK cells and T cells33, CLEC2B and SH2D1B which activates NK cells34,35, and iv) NFkB pathway genes, including REL, NFkB1, and NFkB2, which play a crucial role in regulating the cytotoxic activity of both NK cells and T cells36 (Fig. 4C-4D and Supplementary Fig. 5). These scRNASeq findings indicate that bevacizumab treatment enhances the cytotoxic function of CD8+ T and NK cells.

αPD1 treatment can control the growth of tumors that progress despite αVEGF treatment in Schwannoma models
We next experimented to address our second question: can αPD1 serve as an effective alternative for patients who are unresponsive to or unable to tolerate bevacizumab? We implanted mouse Nf2−/− cells in both the sciatic nerve and CPA models and treated the mice with three doses of αVEGF. Then, the mice were randomized into three groups, receiving: i) continued αVEGF monotherapy, ii) combined αPD1 with αVEGF treatments, or iii) switched to αPD1 monotherapy, with αVEGF discontinued (Fig. 5A).

In the sciatic nerve model, both switching to αPD1 monotherapy and combining αPD1 with αVEGF significantly delayed tumor growth compared to αVEGF alone, with the combination therapy demonstrating the greatest efficacy (Fig. 5B). In the CPA model, while switching to αPD1 modestly prolonged animal survival, discontinuing αVEGF completely abolished the hearing benefit (red line). Combining αPD1 with αVEGF treatment led to the most significant survival benefit and further prevented tumor-induced hearing loss compared to αVEGF alone (pink line) (Fig. 5C, D).

Flow cytometry analysis revealed that both switching to αPD1 and combining αPD1 with αVEGF significantly increased intratumoral CD8+ T cells and NK cells while reducing immune suppressive MDSCs compared to αVEGF monotherapy (Fig. 5E). These findings suggest that αPD1 antibody can serve as an effective alternative or be combined with αVEGF in treating VS. Furthermore, the αPD1 and αVEGF combination provides superior efficacy in tumor control and hearing preservation.

Methods
Cell lines
Mouse Nf2−/− Schwann cells were grown in Schwann cell medium containing 10% fetal bovine serum (FBS), which includes Schwann cell growth supplement (SCGS, ScienCell)54. Mouse SC4 Schwannoma cells (gift from Dr. Vijaya Ramesh, Massachusetts General Hospital, MGH) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Corning) containing 10% FBS46,55.

Animal models
Nf2−/− and SC4 tumor cells were inoculated into immunocompetent C57/FVB mice. Both male and female mice (1:1 ratio), aged 8-12 weeks, were used to ensure sufficient statistical power and to examine potential sex-related differences. Given that schwannomas develop in patients’ vestibular and peripheral nerves, we employed two mouse models by injecting tumor cells into the cerebellopontine angle or into the sciatic nerve.

Cerebellopontine angle (CPA) model. To simulate the intracranial microenvironment of VSs, tumor cells were injected into the CPA region of the right hemisphere56,57. Each mouse received an implantation of 1 µl of tumor cells suspension containing 2,500 cells.

Sciatic nerve (SN) schwannoma model. To mimic the microenvironment associated with peripheral schwannomas, we injected schwannoma cells into the sciatic nerve of the mice46. Tumor cell suspension (5×104 cells in 3 µl) was slowly injected over the course of 45-60 seconds under the sciatic nerve sheath to avoid any leakage.

All mice were housed in the Massachusetts General Hospital (MGH) specific pathogen–free animal facility under a 12h light/12h dark cycle, at an ambient temperature of 20–24°C and relative humidity of 30–70%, with ad libitum access to food and water. All experiments were conducted in accordance with the approved animal protocol, and no experimental endpoints exceeded those specified in the protocol.

The experimental endpoint for the CPA model is the onset of neurological symptoms in mice. The experimental endpoint for the SN model is when the tumor grows to ~1 cm in diameter. At the experiment endpoint, mice were euthanasized by i.p. administration of penobarbital at an overdose dose (150-200 mg/kg).

Treatment protocols
In the SN model, tumor diameter was measured by caliper, and treatment began once the tumor reached a diameter of 3 mm. In mice with CPA tumors, we started treatment when the blood concentration of the Gaussia luciferase (Gluc) reporter gene expression reached 1×104 RLU (relative luminescence unit). For αPD1 treatment, the αPD1 antibody or isotype IgG control (200 µg/mouse, BioXCell) was delivered i.p. in 4 doses every 3 days58. For αVEGF treatment, the αVEGF antibody B20.4.4 (B20, 2.5 mg/kg, Genentech), which neutralizes both human and mouse VEGF, was administered i.p. once every week46. Detailed antibody information is provided in Supplementary Table 1.

Measurement of tumor growth
To track tumor growth in the CPA model, both tumor cell lines were transfected with lentivirus carrying the secreted Gluc reporter gene, and plasma Gluc was measured as previously described57,59–61. In brief, 13 µl of whole blood was collected from the tail vein and mixed with 5 µl of 50 mM EDTA to prevent clotting. The blood sample was then transferred to a 96-well plate, where Gluc activity was assessed using a GloMax 96 Microplate Luminometer (Promega). The luminometer was configured to automatically inject 100 µl of 100 mM coelenterazine (Nanolight) in PBS, with photon counts recorded for 10 s. The tumor size in the sciatic nerve was measured with calipers every 3 days until the tumors reached a diameter of 1 cm.

Audiometric testing in mice
Auditory brainstem responses (ABRs) were assessed as previously outlined57. In brief, the animals were anesthetized by intraperitoneal injection of ketamine (0.1 mg/g) and xylazine (0.02 mg/g). All mice were verified by microscopic examination of the tympanic membrane and the middle ear to have well-aerated middle ears and no signs of otitis media, and all animals showed. ABRs were collected using subdermal needle electrodes positioned with the positive electrode placed on the inferior aspect of the ipsilateral pinna, the negative electrode at the vertex, and the ground electrode at the base of the tail. The responses were amplified 10,000-fold, filtered within the 0.3–3.0 kHz range, and averaged over 512 repetitions for frequency and sound levels. Data acquisition was achieved using custom LabVIEW software operating on a PXI chassis from National Instruments Corp. For each frequency tested, the auditory threshold was determined as the lowest stimulus level at which consistent peaks could be visually identified. If no auditory threshold was detected, a value of 85 dB was recorded, representing 5 dB above the highest level tested.

Flow cytometry analysis of tumor-infiltrating immune cells
To analyze the immune cells that infiltrate the tumor, tumor lysates were dissociated into single cells in digestion buffer (collagenase P 0.2 mg/ml, dispase II 0.8 mg/ml, DNase 0.1 mg/ml, RPMI with 1% FBS) at 37°C. Every 5-8 min, tubes were agitated, and then the contents were allowed to resettle. Next, the supernatant was transferred to the collecting buffer (RPMI with 1% FBS and 2 mM EDTA). After 45-60 min, the tumor tissues were completely dissociated and centrifuged at 4°C, at 1200 rpm for 5 min. After discarding the supernatant, the Fc block was added to the pellet (1:500) and kept on ice for 15 min. After blocking, tumor cells were stained for flow cytometry by a BD flow cytometer using antibodies against CD45 (1:200), CD4 (1:200), CD8 (1:200), NK1.1 (1:200), Gr1 for MDSC (1:200, for myeloid-derived suppressor cells [MDSC]), CD11b (1:200), Granzyme B (1:100), perforin (1:100) (Supplementary Table 1).

Single-cell RNA-Sequencing (scRNA-seq)
Human VSs were obtained from NF2-SWN patients who provided informed consent, including those who received bevacizumab treatment, in accordance with the Human Subjects protocols approved by the Institutional Review Boards of The Ohio State University and Massachusetts General Hospital. Fresh surgically-removed VS tissues were placed in DMEM or saline and transported to the lab. To prepare single-cell suspension, each tumor was minced and then digested with collagenase I (2 mg/ml) and dispase (1 U/ml), followed by filtering through a 40-µm cell strainer as previously described62. Dissociated cells were cryopreserved in 10% dimethyl sulfoxide-containing DMEM plus 10% FBS until processing on the 10X Genomics’ Chromium Single Cell platform and sequencing on a NovaSeq 6000 System (Illumina) at a depth of approximately 400 million reads per sample. For computational analysis, we used Cellranger v5.0.1 to align reads to the hg19 human reference sequence. For each sample dataset, unsupervised clustering was performed using the R package Seurat (version 4) (https://www.satijalab.org/seurat; https://www.github.com/satijalab/seurat)63. Following normalization using Seurat’s NormalizeData function, highly variable genes were identified and used for principal components analysis. Then, we performed clustering using graph-based clustering and visualized using Uniform Manifold Approximation and Projection (UMAP) with Seurat function RunUMAP. To identify the cell type in tumor sample datasets, we input marker gene lists generated by the Seurat FindMarkers function in Toppgene to identify the top cell type makers or cell identities. Differentially expressed genes (DEGs) in a given cell type compared with all other cell types were determined with the FindAllMarkers function in the Seurat package.

Quantitative RT-PCR
Changes in the mRNA levels were quantified with qPCR, using SYBR Green-based protocol64. All qPCR analyses were performed on a Stratagene MX3000 qPCR System with the operating MXPro qPCR software (Stratagene)65.

Western Blot
Thirty micrograms of protein lysates per sample were separated on 10% SDS-polyacrylamide gels66. Membranes were probed with antibodies against perforin (1:500) and granzyme B (1:500) (both from Cell Signaling Technology), or β-actin (1:5000, Sigma) as a loading control67.

ELISA
Plasma or protein samples obtained from snap-frozen tumors were diluted to a concentration of 2 µg/µl protein levels were measured using mouse multiplex enzyme-linked immunosorbent assay plates following the manufacturer’s guidelines (Meso-Scale Discovery). Each sample was tested in triplicate64,68.

In vitro cytotoxicity assay
To isolate CD8+ T cells from treated tumors and NK cells from the mouse spleen, tumor tissues and the spleens were placed on a petri dish containing pre-cooled phosphate-buffered saline (PBS) supplemented with 2% FBS. Tumor tissues and the spleens were minced into small pieces and filtered through a 70 µm-pore cell strainer positioned above a 50-ml conical tube (Thermo Fisher Scientific). The petri dish and the strainer were rinsed with pre-cooled PBS supplemented with 2% FBS. Erythrocytes were lysed with ammonium chloride potassium lysing buffer. Subsequently, single cell suspensions were transferred through another 70 µm-pore cell strainer into a new 50-ml conical tube. Cell numbers were counted by trypan blue staining. CD8+ T cells were purified using a MojoSort Mouse CD8 T cell isolation kit (BioLegend), and NK cell fractions were harvested using the NK Cell Isolation Kits and MACS columns (Miltenyi Biotech) following the manufacturer’s instructions. Experiments were done when the purity of CD8+ T cells and NK cells was above 90%, as determined by flow cytometry. Cultured mouse lymphoma YAC-1 cells or schwannoma tumor cells were used as the target cells and labeled with calcein AM (10 nM for 1×106 cells/ml, Becton Dickson). After overnight incubation, 1×104 cells/well were seeded in a U-bottom 96-well plate (100 µl/well), protected from light. The effector cells were added in triplicate at the effector:target (E:T) cell ratio of 10:1. Target cells only were plated for baseline fluorescent reading, and target cells in lysis buffer (containing 1% NP40) were included as maximum cell lysis. After incubation for 6 hours, the supernatants were collected, and fluorescent intensity was measured using a fluorescent plate reader. Cytolysis was assessed by measuring the release of calcein AM from target cells and calculated as: effector cell cytotoxicity (%) = Sample OD - Baseline OD / Maximum OD - Baseline OD × 100% as described previously69.

Histological staining
To evaluate tumor cell proliferation and apoptosis, sections of tumor tissues were stained with proliferating cell nuclear antigen (anti-PCNA, 1:1000; Abcam) and TUNEL (ApopTag®; Millipore). Microvessel density (MVD, detected by anti-CD31, 1:200; Millipore) and myofibroblast infiltration (detected by anti-α-smooth muscle actin-Cy3™, 1:200; Sigma-Aldrich) were determined in frozen sections (8 µm). To evaluate the percentage of perfused vessels, 50 µl of FITC-lectin (2 mg/kg) was injected i.v. ten minutes before sacrifice to identify perfused vessels. Then, tumors were harvested and embedded in frozen sections (20 µm). Endothelial cell labeling with FITC-lectin demonstrates perfused blood vessels, and CD31+ staining displays all blood vessels in solid tumors. The percentage of FITC-lectin+/CD31+ vessels was quantified using Image J software. To fluorescently label the αPD1 antibody, Antibody Labeling Kit – [FITC (Fluorescein) F10240; Thermo Fisher Scientific] was used following the manufacturer’s protocol. The labeled αPD1 antibody was processed through a purification column to remove the unbound dye. Four specimens of normal peripheral nerves were obtained post mortem and used as controls. Appropriate positive and negative controls were used for all stains68,70. Acquisition was performed on an Olympus IX81 confocal microscope with Fluoviewer FV10-ASW 4.2 software. FITC channel shown in magenta in the figures. Histological evaluation using digital quantitative image analysis was performed using ImageJ. Positive staining in 20 random fields/slides was quantified via automated built-in functions based on fluorescent pixel intensity after establishing a threshold to exclude background staining.

Statistical analyses
Differences in sciatic nerve tumor growth were analyzed using repeated measures two-way ANOVA. Survival curves were generated using the Kaplan-Meier method. Kaplan-Meier survival curves were analyzed by the Log-rank (Mantel-Cox) test. ABR thresholds were analyzed with a linear mixed-effects model. Flow cytometry, ELISA, qPCR, and cytotoxicity studies were analyzed using Student’s t test and Mann-Whitney U test, as appropriate. All statistical analyses were carried out using GraphPad Prism Software version 9.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Ethics approval
The study was approved by MGH’s Institutional Animal Care and Use Committee (Protocol #2016N00004).

Data availability
The raw data generated in this study are provided in the Supplementary Information/Source Data file. The RNA Seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession code GSE315579. All data and the supplementary materials from this study are included in this manuscript and are available after publication upon request from the corresponding author. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study highlights the synergistic effects of combined αVEGF and αPD1 treatment in an in vivo schwannoma model. The\nauthors demonstrate that αVEGF enhances the anti-tumour efficacy of αPD1, and the combination therapy significantly\ndelayed tumour growth, reduced the number of proliferating cells, and increased apoptotic cell numbers, leading to improved\nsurvival in mice compared to monotherapies. The mechanisms underlying αVEGF’s action are elucidated, including tumour\nvasculature normalisation, which enhances αPD1 delivery. Additionally, the αVEGF treatment promotes increased\ninfiltration and activation of cytotoxic T cells and NK cells via upregulation of Natural Killer Group 2 Member D (NKG2D).\nImportantly the authors revealed that αPD1 treatment in combination with αVEGF, decrease tumour growth of tumours that\ndid not respond to αVEGF.\nThese findings are a novel contribution to schwannoma research and hold promising potential for future clinical trials\ninvolving NF2 patients. αVEGF (bevacizumab) is currently used as a treatment for vestibular schwannomas in NF2 patients,\ndemonstrating partial effectiveness in some patients. Furthermore, several FDA-approved αPD1 inhibitors, which have been\npreviously tested for other conditions, could be repurposed for use in combination therapy with αVEGF in NF2 patients.\nSimilar co-treatment studies have been conducted in lung carcinoma mouse models, yielding comparable outcomes\n(doi/full/10.2217/imt-2021-0196). Several clinical trials testing αVEGF and αPD1 combination treatment are currently\nongoing. Toxicity reports should be thoroughly evaluated before initiating any clinical trials involving NF2 patients.\nThe results are consistent with the conclusions and claims; however, there are several aspects that require further attention:\nFigure 1. Title: B20 should be referred to αVEGF. This also should be addressed throughout the manuscript.\n1G: No DAPI staining is visible. Could you clarify which type of microscope was used in the study? The manuscript does not\nprovide any details regarding the microscopy techniques employed.\n1I: Flow cytometry data should be presented using histograms or scatter plots, at least a few representative examples from\nthe selected datasets.\n3A and G: Same comment as in 1I.\n5A-C: Was the effect seen in mice where the switch from αVEGF to αPD1 was done, due to αVEGF pre-treatment?\n5E: Same comment as in 1I.\nFigure S2. What do the authors mean by 'enhance tumour control'? This statement should be more specific.\nPictures in Figure S2 A are PCNA staining not TUNEL.\nPictures in FigureS2 B are TUNEL staining not PCNA.\nImage quantifications: Are these really flow cytometry data?\nThe legends for all figures should be more detailed and informative.\nAdditional questions:\n1. Will tumours regrow when the treatment is discontinued? This would be important to investigate.\n2. Were any adverse events observed in the mice?", "reviewer_2": "This manuscript claims that combined treatment with anti-PD1 significantly improves the therapeutic effects of anti-VEGF on\nthe prevention of hearing loss as well as tumor growth in their unique model of vestibular schwannomas (VSs) linked to\nNF2-related schwannomatosis (NF2-SWN). As a mechanism of action, they claims that anti-VEGF normalizes tumor\nvasculature for drug delivery and immune cell infiltration, and by activating antitumor cytotoxicity of T and NK cells via\nNKG2D upregulation, leading to enhanced anti-PD1 efficacy and tumor controls. Overall, the topic is of interest in the context\nof newly suggested combination of immune checkpoint inhibitors such as anti-PD1 with anti-VEGF as a promising\ntherapeutic strategy to treat VS with currently no FDA-approved medications. However, the conclusion of this manuscript is\nnot sufficiently supported by the results provided and requires more experimentation to support the mechanism of action\nclaimed and the rationale of combined anti-VEGF and anti-PD1 therapy in VS.\nMajor comments\nTo assess the anti-PD1 drug delivery, the authors fluorescently labeled anti-PD1 antibody with Alexa Fluor 647 labeling kit\nin histological staining section of Methods (p. 8). This raises the question if the labeled antibody was functionally intact for\nthe binding and blocking of PD1 receptor on target cells. In addition, as they described the labeling of anti-PD antibody with\na green fluorescent tag in the Results section (p. 10) and FITC-labeled anti-PD1 antibody (green) in the Figure 1 legend (p.\n21), I am wondering if they independently tested two different labeling of anti-PD1 otherwise it is a simple mistake.\np. 12: To profile the T cells and NK cells in VSs with or without bevacizumab treatment, the authors performed scRNA-seq\nanalysis by employing a single VS case treated with bevacizumab for 10 years in comparison to three VS cases without the\ntreatment. Given the heterogeneity of patient response to certain biologics, the authors should include more VS cases in\ntheir analysis to draw more convincing conclusions.\np. 13: The authors argued that CD8+ T cells and NK cells are in a suppressed functional state in the VS tumor\nmicroenvironment simply based on the results from scRNA-seq analysis for NKG2A, CD94, HLA-E. This should be\nsupported by the functional assessment of isolated CD8+ T cells or NK cells from VS tissues with or without bevacizumab\ntreatment.\np. 15: The approval of bevacizumab in UK along with its therapeutic benefit and limitation is repeatedly described in the\nIntroduction and Discussion, which should be removed and replaced with more insightful discussion based on additional\nmechanistic studies.\nFor statistical analyses, they simply used Student’s t-test or Mann-Whitney test even for the comparison of multiple groups,\ntime-dependent curves between two groups (may be better with ANOVA), or Kaplan-Meier survival analysis (may be better with log-rank test). In this regard, they should consult the statistician in the Institute and provide the rationale for each\nanalysis. In addition, they should indicate which statistical analysis was used for specific data in the legends along with the\npresentation of exact p-value instead of P<0.01.\nThey authors used simple bar graphs for the presentation of the results performed with multiple mice, which should be\npresented with each dot indicating individual mice to strengthen their results.\nFor the presentation of flow cytometry analysis of various immune cells, the authors only used bar graphs for each immune\ncell type in Figure 1I, 3A, 3G, and 5E. In this regard, they are required to provide representative figures and gating strategy in\nthe main figure or supplementary figures.\nIn in vivo experiments, they commented the use of both male and female mice (1:1 ratio) to ensure sufficient statistical power\nand to examine any potential sex-bias. Given their use of 6 or 8 mice in each experiment, I am wondering if they used\nadequate numbers of mice.\nIn figure 3C and 3D, to claim the activation of CD8+ T cells and NK cells, they performed Western blot analysis for Perforin\nand Granzyme B proteins and qRT-PCR analysis for mRNA expression of NKG2D and its ligands using tumor lysates.\nTo support their claim, they are required to perform flow cytometric analysis for assessing proteins levels and scRNA-seq for\ngene expression in a per-cell basis. Moreover, given their claim of NKG2D upregulation as a major mechanism of action,\nthey are required to test the functional effect of NKG2D upregulation such as blockade of NKG2D receptor.\nIn figure 3E, it seems curious that anti-VEGF treatment enhanced the cytotoxicity of NK cells in the spleen compared to that\nof CD8+ T cells in the tumor tissues. In this regard, I am wondering if anti-VEGF treatment had a systemic effect on the\ncytotoxicity of NK cells and CD8+ T cells.\nIn figure 4 of scRNA-seq analysis, the authors presented the limited set of receptors (NKG2A, CD94, HLA-E) and molecules\n(GZMB, CCL4, REL, RELB, BFKB1/2). In this regard, they are required to provide more comprehensive data linked to the\nactivation of CD8+ T cells and NK cells (e.g., more receptors including activation markers, immune checkpoint receptors,\nand etc.)\nMinor comments\nProvide detailed information on Materials and Methods\n- Preparation of tumor lysates for flow cytometry (p. 7), dissociated cells for scRNA-seq (p. 7), and CD8+ T cell isolation from\ntreated tumors for in vitro cytotoxicity assay (p. 8).\nProvide detailed information (clone, catalogue number, etc) of antibodies including anti-PD1 and isotype IgG control,\nperforin and granzyme B\nCorrect the following errors or typos\np. 8: “Diluted to a concentration of 2 ug/ul inflammatory cytokine levels were measured using ….” in ELISA section\np. 15: “in preventing preserving hearing….”\np. 16: “This This study….” In Funding section\nIn figure 1 legend, the statement of “Representative images of …. In:” appears to cover the data (F), (G), and (H). If the case,\nthe authors are required to indicated the caption (F-H) in front of the statement.", "reviewer_3": "In their manuscript, Lu et al. demonstrate that combining aPD1 and aVEGF therapy enhances anti-tumor efficacy in\nsyngeneic non-malignant vestibular schwannoma (VS) models and prevents hearing loss. In more detail, aVEGF-induced\nnormalization of tumor blood vessels improved delivery of aPD1 antibodies in preclinical models and is associated with an\nincreased tumor infiltration of effector immune cells in murine and patient VS tissue. Combined aVEGF/aPD1 treatment was\neffective also after failure of aVEGF monotherapy.\nBy preclinical development of an effective treatment regimen for non-malignant VS which takes into account not only tumor\ngrowth but also hearing function, the authors address a timely topic in the field of neurooncology and may impact future VS\nmanagement. The study is very well presented and clearly written. The results are supported by a broad methodology, and\nthe inclusion of human post-treatment tissue even strengthens the impact of the study in terms of clinical translation.\nMinor comments:\n- Please depict individual values and means (+-SEM) in all bar plots.\n- Please comment on tolerability and potential side effects beyond ototoxicity."}, "author_response": "POINT-BY-POINT RESPONSE TO REVIEWERS’ COMMENTS\nNKG2D Upregulation Enhances T and NK Cell Cytotoxicity, Sensitizes Tumors to Combined αPD1 and\nαVEGF Therapy, and Contributes to Hearing Loss Prevention in Vestibular Schwannoma Model\nCorresponding Author: Lei Xu\nWe thank the reviewers for their helpful comments. We have revised our manuscript to address all of their\ncomments. Our responses are provided below in blue, and the changes made in the revised manuscript are\nshown in red.\nReviewer #1\nThe study highlights the synergistic effects of combined αVEGF and αPD1 treatment in an in vivo schwannoma\nmodel. The authors demonstrate that αVEGF enhances the anti-tumour efficacy of αPD1, and the combination\ntherapy significantly delayed tumour growth, reduced the number of proliferating cells, and increased apoptotic\ncell numbers, leading to improved survival in mice compared to monotherapies. The mechanisms underlying\nαVEGF’s action are elucidated, including tumour vasculature normalisation, which enhances αPD1 delivery.\nAdditionally, the αVEGF treatment promotes increased infiltration and activation of cytotoxic T cells and NK\ncells via upregulation of Natural Killer Group 2 Member D (NKG2D). Importantly the authors revealed that\nαPD1 treatment in combination with αVEGF, decrease tumour growth of tumours that did not respond to\nαVEGF.\nThese findings are a novel contribution to schwannoma research and hold promising potential for future clinical\ntrials involving NF2 patients. αVEGF (bevacizumab) is currently used as a treatment for vestibular\nschwannomas in NF2 patients, demonstrating partial effectiveness in some patients. Furthermore, several FDA-\napproved αPD1 inhibitors, which have been previously tested for other conditions, could be repurposed for use\nin combination therapy with αVEGF in NF2 patients. Similar co-treatment studies have been conducted in lung\ncarcinoma mouse models, yielding comparable outcomes (doi/full/10.2217/imt-2021-0196). Several clinical\ntrials testing αVEGF and αPD1 combination treatment are currently ongoing. Toxicity reports should be\nthoroughly evaluated before initiating any clinical trials involving NF2 patients. The results are consistent with\nthe conclusions and claims; however, there are several aspects that require further attention.\nResponse summary: We thank the reviewer for the thorough and positive review of our work. We appreciate\nthe recognition of the novelty and translational potential of our findings. In response to the reviewer’s\ncomments, we have:\ni) Addressed all points in detail,\nii) Performed new experiment investigating whether tumors grow after discontinuation of therapy\n(Supplemental Fig. 2A),\niii) Performed new experiment assessing body weight loss and tolerability (Supplemental Fig. 2C-2D).\niv) Added references to αVEGF and αPD1 combination treatment preclinical studies and clinical trials,\n1. Figure 1. Title: B20 should be referred to αVEGF. This also should be addressed throughout the manuscript.\nReply: As suggested by the reviewer, we have replaced all ‘B20’ with ‘aVEGF’ throughout the manuscript,\nincluding the main text, figures, and figure legends.\n2. 1G: No DAPI staining is visible. Could you clarify which type of microscope was used in the study? The\nmanuscript does not provide any details regarding the microscopy techniques employed.\nReply: We apologize for the lack of clarity in the previous version. The image shown in Figure 1H (previously\nFig 1G) was acquired using confocal microscopy. In this experiment, we visualized the distribution of anti-PD1\nantibody conjugated to a green fluorescent tag to assess antibody delivery. To better visualize the green\nfluorescence signal, we omitted the DAPI channel, as the strong nuclear staining could obscure the green signal.\nTo address the reviewer’s concern, we have now added a detailed description of confocal microscopy methods\nin the Materials and Methods section (p9).\n3. 1I: Flow cytometry data should be presented using histograms or scatter plots, at least a few representative\nexamples from the selected datasets.\nReply: We appreciate the reviewer’s helpful suggestion. Following the recommendation, we have replaced Fig.\n1I flow cytometry data with scatter plots in the revised Figure 1J, and we included representative gating\nstrategies in Supplemental Figure 1 and Supplemental Figure 4.\n4. 3A: Same comment as in 1I.\nReply: As suggested by the reviewer, we have replaced the Fig. 3A flow cytometry data with scatter plots in the\nrevised Figure 3A.\n5. 5A-C: Was the effect seen in mice where the switch from αVEGF to αPD1 was done, due to αVEGF pre-\ntreatment?\nReply: We thank the reviewer for this insightful question. The enhanced tumor control observed after switching\nfrom aVEGF to aPD1 likely reflects a sustained effect of aVEGF-induced vascular normalization. As shown in\nFigure 1C, aVEGF treatment increases pericyte coverage, making tumor vessels structurally similar to normal\nvasculature. Correspondingly, Figure 1D-E demonstrated that this structural normalization leads to normalized\nvessel perfusion, which facilitates intratumoral immune cell infiltration (Figure 1J), thereby creating a more\nimmunostimulatory microenvironment that can potentiate aPD1-mediated cytotoxicity. We have added a\ndiscussion of this temporal relationship between aVEGF pre-treatment and aPD1 efficacy in the Discussion\nsection (p16).\n6. 5E: Same comment as in 1I.\nReply: As suggested by the reviewer, we have replaced the old Fig. 5E flow cytometry data with scatter plots in\nthe revised Figure 5E.\n7. Figure S2. What do the authors mean by 'enhance tumour control'? This statement should be more specific.\nReply: We have revised the title of Figure S3 (previously Figure S2) to read: ‘Combined aVEGF and aPD1\ntreatment reduces proliferating and increases apoptotic tumor cells compared to control and monotherapies in\nNf2-/- model’.\n8. Pictures in Figure S2 A are PCNA staining not TUNEL. Pictures in FigureS2 B are TUNEL staining not\nPCNA. Image quantifications: Are these really flow cytometry data?\nReply: We apologize for the labeling error. The text has been corrected to indicate that Figure S3A (previously\nFigure S2A) shows PCNA staining, and Figure S3B shows TUNEL staining. We also clarified in the figure\nlegend that the quantification data were obtained from image analysis, not flow cytometry. See Result section\np11.\n9. The legends for all figures should be more detailed and informative.\nReply: We have revised all figure legends to provide more detailed information about the experiments.\nAdditional questions:\n1. Will tumours regrow when the treatment is discontinued? This would be important to investigate.\nReply: As suggested by the reviewer, we conducted a new experiment to address this question. In a separate\ncohort of Nf2-/- sciatic nerve tumor-bearing mice, all treatments were discontinued after three doses.\nRemarkably, tumor growth remained suppressed even after treatment cessation, indicating a durable antitumor\neffect that persisted beyond the active treatment phase. Please see Result (p11) and Supplemental Figure 2C-\nD.\n2. Were any adverse events observed in the mice?\nReply: In both sciatic nerve and CPA models, all treatments - including monotherapies and the combination\ntherapy - were well tolerated, with no significant body weight loss observed in any treatment group. This new\ndata has now been added to Result (p10-11) and Supplemental Figure 2A. We also showed that aPD1 does not\naffect hearing function in non-tumor bearing mice, indicating that aPD1does not cause acute ototoxicity in this\nmouse model. These results were included in Supplemental Figure 2E-F.\nReviewer #2:\nThis manuscript claims that combined treatment with anti-PD1 significantly improves the therapeutic effects\nof anti-VEGF on the prevention of hearing loss as well as tumor growth in their unique model of vestibular\nschwannomas (VSs) linked to NF2-related schwannomatosis (NF2-SWN). As a mechanism of action, they\nclaims that anti-VEGF normalizes tumor vasculature for drug delivery and immune cell infiltration, and by\nactivating antitumor cytotoxicity of T and NK cells via NKG2D upregulation, leading to enhanced anti-PD1\nefficacy and tumor controls. Overall, the topic is of interest in the context of newly suggested combination of\nimmune checkpoint inhibitors such as anti-PD1 with anti-VEGF as a promising therapeutic strategy to treat\nVS with currently no FDA-approved medications. However, the conclusion of this manuscript is not\nsufficiently supported by the results provided and requires more experimentation to support the mechanism\nof action claimed and the rationale of combined anti-VEGF and anti-PD1 therapy in VS.\nResponse summary: We thank the reviewer for their thoughtful and encouraging comments regarding the\nsignificance of our study and the potential of combining αPD1 with αVEGF as a therapeutic strategy for\nvestibular schwannomas. We appreciate the reviewer’s suggestion that additional data are needed to further\nsubstantiate the proposed mechanism of action. In response to the reviewer’s comments, we have:\ni) Addressed all points in detail,\nii) Performed new flow cytometry experiment assessing CD8 T cell and NK cell activation (Figure\n3C).\niii) Performed new animal experiment using NKG2D blockade to investigate if αVEGF enhances αPD1\nefficacy via promoting NKG2D-mediated cytotoxic (Figures 3I-3J).\niv) Expanded scRNASeq analysis to profile T cell and NK cell activation landscape (Figure 4 and\nSupplemental Figure 5)\nMajor comment:\n1. To assess the anti-PD1 drug delivery, the authors fluorescently labeled anti-PD1 antibody with Alexa Fluor\n647 labeling kit in histological staining section of Methods (p. 8). This raises the question if the labeled\nantibody was functionally intact for the binding and blocking of PD1 receptor on target cells.\nReply: We thank the reviewer for this important point. Our earlier description may not have been sufficiently\nclear. The fluorescently labeled αPD1 antibody was not used for treatment, it was used exclusively to assess\nantibody delivery following αVEGF-induced vascular normalization.\nIn this experiment, mice bearing Nf2-/- schwannomas in the CPA were treated with either control IgG or αVEGF\nfor three weeks. Then, FITC-conjugated αPD1 was administered intraperitoneally. Twenty-four hours later,\ntumor-bearing brains were collected and for vascular staining and confocal imaging to evaluate the distribution\nof the fluorescent αPD1 antibody.\nTo improve clarity, we have revised the Results section (p10) and added a schematic illustration in the new\nFigure 1F to clarify this experimental design.\nIn addition, as they described the labeling of anti-PD antibody with a green fluorescent tag in the Results\nsection (p. 10) and FITC-labeled anti-PD1 antibody (green) in the Figure 1 legend (p. 21), I am wondering if\nthey independently tested two different labeling of anti-PD1 otherwise it is a simple mistake.\nReply: We have corrected the text to ensure a consistent description of the fluorescent labeling throughout the\nmanuscript. The αPD1 antibody was labeled with FITC (Fluorescein), which emits green fluorescence\n(Antibody Labeling Kit, Cat#F10240, Thermo Fisher Scientific). This information has been added to\nthe Methods section (p9).\n2. p. 12: To profile the T cells and NK cells in VSs with or without bevacizumab treatment, the authors\nperformed scRNA-seq analysis by employing a single VS case treated with bevacizumab for 10 years in\ncomparison to three VS cases without the treatment. Given the heterogeneity of patient response to certain\nbiologics, the authors should include more VS cases in their analysis to draw more convincing conclusions.\nReply: We agree with the reviewer that additional VS cases would strengthen the analysis and better represent\npatient heterogeneity in response to bevacizumab. However, VS is a rare disease, and surgical samples from\npatients treated long-term with bevacizumab are extremely limited, as surgery is generally avoided whenever\npossible due to the substantial risks of further nerve damage, causing hearing loss, facial paralysis, and chronic\ndizziness. We hope the reviewer agrees with us that obtaining more VS cases is challenging. We have now\nacknowledged this limitation and emphasized the importance of expanding the scRNA-seq analysis in future\nstudies as more patient samples become available, please see Discussion section (p17).\n3. p. 13: The authors argued that CD8+ T cells and NK cells are in a suppressed functional state in the VS\ntumor microenvironment simply based on the results from scRNA-seq analysis for NKG2A, CD94, HLA-E.\nThis should be supported by the functional assessment of isolated CD8+ T cells or NK cells from VS tissues\nwith or without bevacizumab treatment.\nReply: We agree with the reviewer that scRNASeq data alone cannot provide direct functional evidence of\nCD8+ T cell and NK cell activity. To address this point, we evaluated the functional status of CD8 T cells and\nNK cells in our mouse model with and without anti-VEGF treatment (Figures 3C, 3G), which supports our\nconclusions regarding their activation state.\nWe fully recognize that direct functional assays on human VS-infiltrating lymphocytes would provide the most\ndefinitive evidence. However, VS is a rare disease, and isolating sufficient numbers of patient CD8 T cells and\nNK cells is highly challenging. To avoid overinterpretation of our human data, we have removed the original\nstatement regarding patient CD8 T cell and NK cell function from the Results section and revised the text to\nmore accurately reflect these limitations.\n4. p. 15: The approval of bevacizumab in UK along with its therapeutic benefit and limitation is repeatedly\ndescribed in the Introduction and Discussion, which should be removed and replaced with more insightful\ndiscussion based on additional mechanistic studies.\nReply: As suggested by the reviewer, we have removed the redundant discussion of bevacizumab’s approval\nand therapeutic limitations from the Introduction and revised the Discussion to focus on the mechanistic\ninsights gained from our study and their potential clinical implications (page 16-17).\n5. For statistical analyses, they simply used Student’s t-test or Mann-Whitney test even for the comparison of\nmultiple groups, time-dependent curves between two groups (may be better with ANOVA), or Kaplan-Meier\nsurvival analysis (may be better with log-rank test). In this regard, they should consult the statistician in the\nInstitute and provide the rationale for each analysis. In addition, they should indicate which statistical analysis\nwas used for specific data in the legends along with the presentation of exact p-value instead of P<0.01.\nReply: As suggested by the reviewer, we consulted our biostatistician, Dr. Alona Muzikansky (a co-author on\nthe manuscript), and re-performed all statistical analyses accordingly. We have specified the statistical methods\nused for each dataset in the corresponding figure legends and reported exact p-values.\n6. They authors used simple bar graphs for the presentation of the results performed with multiple mice, which\nshould be presented with each dot indicating individual mice to strengthen their results.\nReply: As suggested by the reviewer, we have revised all figures with bar graphs and replaced them with new\ndot blots, displaying individual data points for each mouse to better illustrate data distribution.\n7. For the presentation of flow cytometry analysis of various immune cells, the authors only used bar graphs\nfor each immune cell type in Figure 1I, 3A, 3G, and 5E. In this regard, they are required to provide\nrepresentative figures and gating strategy in the main figure or supplementary figures.\nReply: As suggested by the reviewer, we have updated the flow cytometry data with dot plots in the revised\nFigures 1, 3, and 5. We included representative gating strategies in Supplemental Figure 1 and Supplemental Figure 4.\n8. In in vivo experiments, they commented the use of both male and female mice (1:1 ratio) to ensure\nsufficient statistical power and to examine any potential sex-bias. Given their use of 6 or 8 mice in each\nexperiment, I am wondering if they used adequate numbers of mice.\nReply: We appreciate the reviewer’s comment. All in vivo data shown in the figures are representative of at\nleast three independent experiments. We have updated the N values in the figure legends to accurately reflect\nthe total number of mice used across all experiments, which demonstrates that the sample sizes were adequate\nfor the analyses performed.\n9. In figure 3C and 3D, to claim the activation of CD8+ T cells and NK cells, they performed Western blot\nanalysis for Perforin and Granzyme B proteins and qRT-PCR analysis for mRNA expression of NKG2D and its\nligands using tumor lysates. To support their claim, they are required to perform flow cytometric analysis for\nassessing proteins levels and scRNA-seq for gene expression in a per-cell basis.\nReply: As suggested by the reviewer, we performed new flow cytometry analyses to assess Granzyme B and\nPerforin expression in CD8+ T cells and NK cells. The previous Western blot data have been replaced with these\nnew flow cytometry results, now presented in the revised Figure 3C, p28.\n10. Moreover, given their claim of NKG2D upregulation as a major mechanism of action, they are required to\ntest the functional effect of NKG2D upregulation such as blockade of NKG2D receptor.\nReply: Following the reviewer’s suggestion, we performed additional experiments using an NKG2D\nneutralizing antibody to evaluate the functional role of NKG2D signaling in mediating the therapeutic benefit of\nthe combination therapy.\nOur new data showed that blockade of NKG2D markedly attenuates the antitumor effects of both anti-VEGF\nmonotherapy and the combination treatment, confirming the critical role of NKG2D in this mechanism.\n11. In figure 3E, it seems curious that anti-VEGF treatment enhanced the cytotoxicity of NK cells in the spleen\ncompared to that of CD8+ T cells in the tumor tissues. In this regard, I am wondering if anti-VEGF treatment\nhad a systemic effect on the cytotoxicity of NK cells and CD8+ T cells.\nReply: We thank the reviewer for this insightful comment. The enhanced cytotoxicity of NK cells observed in\nthe spleen following anti-VEGF treatment likely reflects a systemic immune-modulatory effect of VEGF\nblockade. VEGF is known to suppress immune cell maturation and cytotoxic function; thus, its inhibition can\nrestore NK cell activity in peripheral lymphoid organs. The activation of tumor-associated CD8+ T cells is\nmore dependent on tumor-specific antigen presentation within the tumor microenvironment. Future studies are\nneeded to further investigate the systemic versus local immune effects of anti-VEGF treatment.\n12. In figure 4 of scRNA-seq analysis, the authors presented the limited set of receptors (NKG2A, CD94, HLA-\nE) and molecules (GZMB, CCL4, REL, RELB, BFKB1/2). In this regard, they are required to provide more\ncomprehensive data linked to the activation of CD8+ T cells and NK cells (e.g., more receptors including\nactivation markers, immune checkpoint receptors, and etc.)\nReply: As suggested by the reviewer, we have expanded our scRNA-seq analysis to comprehensively profile\nthe landscape of T cell and NK cell activation, including co-stimulatory and co-inhibitory receptors, cytokines\nand chemokines, immune checkpoint molecules, and transcription factors, in naïve and bevacizumab-treated\nNF2-SWN patient samples.\nReviewer #3:\nIn their manuscript, Lu et al. demonstrate that combining aPD1 and aVEGF therapy enhances anti-tumor\nefficacy in syngeneic non-malignant vestibular schwannoma (VS) models and prevents hearing loss. In more\ndetail, aVEGF-induced normalization of tumor blood vessels improved delivery of aPD1 antibodies in\npreclinical models and is associated with an increased tumor infiltration of effector immune cells in murine and\npatient VS tissue. Combined aVEGF/aPD1 treatment was effective also after failure of aVEGF monotherapy.\nResponse summary: We sincerely thank the reviewer for the positive and encouraging comments on the\nsignificance and clarity of our study. We appreciate the recognition of our preclinical approach, which integrates\nboth tumor control and hearing preservation, as well as the translational relevance strengthened by the inclusion\nof human post-treatment VS samples. In response to the reviewer’s comments, we have addressed all points in\ndetail and add new data on tolerability assessment (Supplemental Fig. 2A).\nMinor comments:\n- Please depict individual values and means (+-SEM) in all bar plots.\nReply: As suggested by the reviewer, all bar graphs have been revised to dot blots, displaying individual data\npoints across all Figures.\n- Please comment on tolerability and potential side effects beyond ototoxicity.\nReply: In both sciatic nerve and CPA models, all treatments - including monotherapies and the combination\ntherapy - were well tolerated, with no significant body weight loss observed in any treatment group. These data\nhave now been added to the new Supplemental Figure 2A."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed all my concerns and performed the additional experiments requested. After\nreviewing their responses, including those to other reviewers, I have no outstanding issues. I am happy to recommend this\nmanuscript for publication. The work is novel, relevant to vestibular schwannoma treatment, and shows potential for clinical\napplication.\nThe statistical evaluation has been corrected using appropriate analytical methods. The methodology is sound, and the data\ninterpretation accurately reflects the results.", "reviewer_2": "The authors addressed my concerns to a reasonable extent although some key issues related to the inclusion of more VS\ncases and their functional assessment was discussed as the limitation of the study. Given a rarity of VS case treated long-\nterm with bevacizumab but a conceptual advancement of VS treatment with the combination of anti-VEGF with immune\ncheckpoint inhibitors such as anti-PD1, I recommend the publication of the study in Nature Communications.", "reviewer_3": "The authors have sufficiently addressed the previous points raised. I believe that the manuscript in its current form is an\nimportant contribution to the brain tumor literature and recommend accepting the paper for publication."}, "author_response": "POINT-BY-POINT RESPONSE TO REVIEWERS’ COMMENTS\nNKG2D Upregulation Enhances T and NK Cell Cytotoxicity, Sensitizes Tumors to Combined αPD1 and\nαVEGF Therapy, and Contributes to Hearing Loss Prevention in Vestibular Schwannoma Model\nCorresponding Author: Lei Xu\nReviewer #1:\nThe authors have adequately addressed all my concerns and performed the additional experiments requested.\nAfter reviewing their responses, including those to other reviewers, I have no outstanding issues. I am happy to\nrecommend this manuscript for publication. The work is novel, relevant to vestibular schwannoma treatment,\nand shows potential for clinical application.\nThe statistical evaluation has been corrected using appropriate analytical methods. The methodology is sound,\nand the data interpretation accurately reflects the results.\nResponse:\nWe sincerely thank the reviewer for their careful re-evaluation of our manuscript and for their positive\nassessment. We are grateful for the reviewer’s recognition of the novelty, methodological rigor, and translational\nrelevance of our work, as well as for acknowledging the additional experiments and corrected statistical\nanalyses. We appreciate the reviewer’s support for publication.\nReviewer #2:\nThe authors addressed my concerns to a reasonable extent although some key issues related to the inclusion of\nmore VS cases and their functional assessment was discussed as the limitation of the study. Given a rarity of VS\ncase treated long-term with bevacizumab but a conceptual advancement of VS treatment with the combination\nof anti-VEGF with immune checkpoint inhibitors such as anti-PD1, I recommend the publication of the study in\nNature Communications.\nResponse:\nWe thank the reviewer for their thoughtful and constructive evaluation of our study. We appreciate the\nreviewer’s understanding of the limitations imposed by the rarity of vestibular schwannoma cases treated long-\nterm with bevacizumab, and we have explicitly discussed these constraints in the revised manuscript. We are\ngrateful for the reviewer’s recognition of the conceptual advancement represented by combining anti-VEGF\ntherapy with immune checkpoint inhibition and for their recommendation to publish our work in Nature\nCommunications.\nReviewer #3:\nThe authors have sufficiently addressed the previous points raised. I believe that the manuscript in its current\nform is an important contribution to the brain tumor literature and recommend accepting the paper for\npublication.\nResponse:\nWe sincerely thank the reviewer for their careful reassessment of the revised manuscript and for their positive\nfeedback. We are grateful for the reviewer’s recognition of the significance of this work and their\nrecommendation for publication."}]

Decision letter