Dual phagocytosis-checkpoint blockade revitalizes immune surveillance in mouse models of glioblastoma

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Macrophage-mediated phagocytosis of tumor cells elicits potent antitumor immunity. Nonetheless, sole-blockade of the anti-phagocytosis molecule CD47 has yielded insufficient therapeutic outcomes. Here, we report that glioblastoma (GBM) cells expressed abundant levels of phagocytosis checkpoint CD24. We further show that dual blockade of CD24 and CD47 synergistically enhances the pro-phagocytic activity of macrophages, thereby improving tumor antigen cross-presentation and activating the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS-STING) pathway. This innate immune activation facilitates T cell infiltration into tumors and sensitizes tumors to anti-PD1 therapy, improving survival outcomes in murine GBM models, including immunosuppressive tumors reflecting human GBM-like features. Thus, our results indicate that dual-phagocytosis checkpoint blockade offers a promising therapeutic avenue to potentiate cancer immunotherapy.

Introduction
The innate immune system acts as the frontline defense against infections and cellular malignancies.1 Antigen-presenting cells (APC), such as macrophages, dendritic cells (DC), and monocytes, specialize in phagocytosis to facilitate malignant cell clearance and present tumor-derived antigens.2,3 This process bridges innate and adaptive immunity, priming T cells and activating subsequent adaptive immune responses.4,5 However, tumor cells evade phagocytosis by upregulating phagocytosis checkpoints.3,6 Among these, CD47 has been intensively studied to overcome innate immune evasion.7-13 Although blockade of CD47 with monoclonal antibodies has demonstrated clinical potential in various preclinical human cancer models, the therapeutic efficacy of anti-CD47 monotherapy varies across different tumor models.14,15 CD24, known to interact with Siglec-10 to suppress inflammatory responses, has emerged as a novel phagocytosis checkpoint.16 Both genetic ablation and antibody-mediated blockade of CD24 have been shown to promote phagocytosis and elicit macrophage-dependent immune responses.16

Here, we show that CD24 is predominantly expressed in glioma cells, whereas Siglec-10 is specifically expressed in myeloid cells, suggesting that CD24 is a promising therapeutic target for glioblastoma (GBM). Combined blockade of CD24 and CD47 synergistically promotes GBM cell phagocytosis and antigen presentation, accompanied by activation of the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS-STING) pathway in APCs and downstream adaptive immune activation. This strategy effectively remodels the cold tumor microenvironment and restores responsiveness to conventional PD1 blockade in an immunosuppressive tumor model. Together, our findings highlight the potential of targeting multiple phagocytosis checkpoints as a strategy to overcome immune resistance and improve cancer immunotherapy.

Results
CD24–Siglec-10 axis is selectively expressed on glioma and myeloid cells
To identify key regulators of phagocytosis resistance in GBM, we analyzed previously published single-cell RNA sequencing data obtained from 18 patients with glioma (Fig. 1a).17 We observed selective and prominent expression of CD24 within glioma cells, with its receptor Siglec-10 was predominantly expressed on myeloid cell populations (Fig. 1b). By contrast, CD47 and its cognate receptor SIRPα demonstrated relatively widespread expression across various cell types, lacking specificity to glioma and myeloid compartments (Fig. 1b). Similarly, other well-known 'don’t eat me' signals, including PD1–PDL1 and B2M–LILRB1 axes, as well as the 'eat me' signal SLAMF7, exhibited non-selective enrichment patterns across diverse cellular subsets (Fig. 1c).18-20 In addition, protein-level expression of CD24 and CD47 was confirmed with immunohistochemistry in human GBM patient tissues (Fig. 1d). Given the selective expression of the CD24–Siglec-10 axis, therapeutic targeting of CD24 could specifically enhance macrophage-mediated glioma phagocytosis.

Next, we evaluated the expression of CD47 and CD24 on various mouse and human GBM cell lines (Fig. 1e, f, Suppl. Figs. 1, 2). CD47 was highly expressed across all tested cell lines. CD24 expression was detected on most of the tested GBM cell lines except for the astrocyte-like CT-2A and U138 cells.21,22 In contrast, higher CD24 expression was observed in proneural-like SB28 cells and epithelial-like U87 cells.23,24

Dual blockade of CD24 and CD47 synergistically improves glioma cell phagocytosis by macrophages
To assess the therapeutic potential of CD24 and CD47 blockade, we evaluated phagocytic activity using bone marrow-derived macrophages (BMDM) and mouse glioma cells treated with varying antibody concentrations (Fig. 2a, b). CD24 blockade induced a dose-dependent increase of phagocytosis in CD24-expressing GL261 and SB28 cells, whereas no effect was seen in CD24-negative CT-2A cells (Fig. 2a). CD47 blockade also enhanced phagocytosis in a dose-dependent fashion across all glioma cell lines tested, although slightly higher antibody concentrations were required compared with CD24 blockade (Fig. 2b). Both anti-CD47 clones MIAP301 and MIAP410 showed comparable phagocytic activity when combined with anti-CD24 (Suppl. Fig. 3), and MIAP301 was used for subsequent experiments. To evaluate synergy between CD24 and CD47 blockade, various antibody concentration ratios were tested (Suppl. Fig. 4). The highest numerical value of phagocytosis was observed at a CD24:CD47 ratio of 5:3 in GL261 cells and 1:3 in SB28 cells; however, both ratios demonstrated no significant advantage over the simpler 1:1 ratio, which was selected for subsequent studies. By using this 1:1 ratio, we observed that dual blockade significantly enhanced macrophage-mediated phagocytosis as compared with mono-treatments in all cell lines tested except for the CD24-negative cell line CT-2A (Fig. 2c, d, Suppl. Fig. 5). In addition, treatment with soluble CD24-Fc neutralized the effect of anti-CD24, demonstrating that the observed increase was dependent on CD24 engagement (Suppl. Fig. 6). Conversely, CD24 transfection in CT-2A cells conferred sensitivity to the dual blockade, further validating CD24-dependent responsiveness (Suppl. Fig. 7). To distinguish between phagocytosis from trogocytosis, GBM cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and PKH26 to stain the intracellular and membrane compartments, respectively. Antibody treatments increased the proportion of double-positive macrophages fully engulfing tumor cells, indicating that antibody treatment increased bona fide phagocytosis (Suppl. Fig. 8). In human GBM cell lines, synergy was less pronounced than in mouse models, but the combination treatment still improved phagocytosis in CD24-positive T98G cells (Suppl. Fig. 9). Notably, no significant enhancement in phagocytosis was observed in bone marrow-derived dendritic cells (BMDC), likely reflecting the predominant macrophage-specific expression of SIRPα and Siglec-10 (Suppl. Fig. 10).

Macrophages process and present tumor-derived antigens after phagocytosis to facilitate crucial crosstalk between innate and adaptive immunity, consequently leading to T cell priming. To explore whether enhanced phagocytosis by dual blockade could improve downstream immune responses, we co-cultured macrophages with cytoplasmic ovalbumin (cOVA)-expressing GBM cells, followed by exposure to ovalbumin (OVA)-specific CD4+ (OT-II) and CD8+ (OT-I) T cells. Dual blockade markedly enhanced OVA peptide presentation on macrophages co-cultured with CD24-positive cell lines, whereas mono-blockade conditions produced only modest increases (Fig. 2e, f, Suppl. Fig. 11). Furthermore, dual blockade consistently resulted in significant and robust enhancement of both CD4+ and CD8+ T cell proliferation compared with IgG and mono-treated groups (Fig. 2g, h, Suppl. Figs. 12, 13).

Phagocytosis-mediated lysis of tumor cells releases double-stranded DNA into the macrophage cytosol, initiating cGAS-dependent synthesis of cyclic GMP-AMP. This, in turn, triggers STING activation and downstream type I interferon (IFN) signaling, resulting in the secretion of pro-inflammatory cytokines that promotes T cell priming.25-31 In line with this mechanism, dual blockade induced significant STING and interferon regulatory factor 3 (IRF3) phosphorylation in macrophages co-cultured with GL261 cells, as assessed by immunoblotting (Fig. 2i, Suppl. Fig. 14), whereas this effect was absent in CT-2A co-cultures (Suppl. Figs. 15, 16). Confocal microscopy further demonstrated nuclear translocation of phosphorylated IRF3 (pIRF3) in macrophages co-cultured with GL261 cells but not CT-2A cells (Fig. 2j). Activation of the cGAS-STING pathway was observed between 6 and 24 hours and declined by 48 hours (Suppl. Figs. 17, 18). NF-κB, another key effector of the cGAS–STING pathway, was likewise activated upon dual blockade, as evidenced by increased phosphorylation of IKKα/β, IκBα, and p65 only in GL261 co-cultures (Fig. 2i, Suppl. Figs. 14-16).32 Consistent with these findings, combination treatment led to substantially higher IFNβ and TNF mRNA expression relative to anti-CD47 monotherapy in GL261 co-cultures. By contrast, in CT-2A co-cultures, the combination treatment did not further enhance these cytokine transcripts beyond levels observed with anti-CD47 alone (Suppl. Fig. 19).

Dual blockade-mediated phagocytosis fosters an antitumor immune environment
To evaluate the in vivo therapeutic efficacy of dual blockade–mediated phagocytosis observed in vitro, we tested orthotopic mouse GBM models, including the GL261 and CT-2A cell lines. Intratumoral administration of optimized dosages (Suppl. Fig. 20) led to significantly improved survival outcomes in mice treated with anti-CD24 antibodies alone or in combination with anti-CD47 blockade, with the combination therapy demonstrating notably greater effectiveness relative to anti-CD24 monotherapy (Fig. 3a, Suppl. Fig. 21). However, in the CD24-negative CT-2A model, anti-CD24 treatment did not produce any antitumor efficacy, and all treatment groups exhibited comparable survival outcomes (Fig. 3b).

To elucidate cGAS-STING pathway activation after dual blockade-enhanced phagocytosis, we visualized the nuclear translocation of pIRF3. Consistent with the survival outcomes, anti-CD24 monotherapy moderately increased nuclear pIRF3, but the combination therapy resulted in a greater increase (Fig. 3c). Given that the cGAS-STING pathway is a critical signaling cascade known to initiate macrophage activation, we next assessed the expression of ionized calcium-binding adaptor molecule 1 (Iba1), a representative macrophage activation marker. In line with our prior findings, abundant numbers of Iba1-positive macrophages were identified within tumors treated with dual blockade (Fig. 3d, e). Moreover, dual blockade synergistically skewed the intratumoral macrophage population toward an M1-like phenotype, as evidenced by a reduced CD206+/CD86+ ratio (Fig. 3h). In addition to macrophage activation, a marked increase in CD4+ and CD8+ T cell infiltration was noted exclusively in the combination treatment group, underscoring that dual blockade-mediated phagocytosis fosters an antitumor immune environment (Fig. 3d, f, g). However, the CD24low CT-2A model did not exhibit a notable increase in immune cell infiltration, except for a moderate increase in Iba1-expressing macrophages in the anti-CD47 antibody-treated groups (Fig. 3e-g, Suppl. Fig. 22). Additionally, within T helper cell population, pro-tumoral Th2 cells were remarkably reduced following antibody treatments, while anti-tumoral Th1 cells remained unchanged, with no synergistic effects observed in the dual blockade compared to mono-treatments (Suppl. Fig. 23).

This immune modulation extended beyond the tumor site to the systemic circulation, as evidenced by increased antigen-specific CD8+ T cells in spleen in both OVA and endogenous GL261 antigen models (Fig. 3i, Suppl. Fig. 24), as well as elevated serum levels of IFNα, IFN, and tumor necrosis factor (TNF) (Fig. 3i, Suppl. Figs. 25, 26). Together, these findings illustrate the capability of dual blockade therapy to stimulate both innate and adaptive antitumor immune responses in the GL261 murine GBM model.

Antitumor efficacy of dual blockade depends on cGAS-STING activation in host macrophages and the presence of CD8+ T cells
Given the established role of dual blockade in activating intratumoral macrophages via the cGAS-STING pathway, we sought to delineate the necessity of this signaling cascade in host macrophages for effective antitumor responses. To this end, we initially isolated BMDMs from STING knockout (KO) mice and co-cultured them with GL261 cells, observing markedly diminished phosphorylation of key signaling molecules within the cGAS-STING and NF-κB pathways after dual blockade treatment (Fig. 4a, Suppl. Fig. 27). Furthermore, nuclear translocation of pIRF3 and pNF-κB was substantially impaired in STING KO BMDMs, indicating disrupted pathway activation (Fig. 4b). In line with these findings, the survival advantage conferred by dual blockade observed in wild-type mice was significantly reduced in STING KO mice, underscoring the crucial involvement of macrophage cGAS-STING signaling in mediating antitumor efficacy (Fig. 4c). Flow cytometric analysis further confirmed significantly reduced levels of pIRF3 within the intratumoral CD11b+ macrophage populations of STING KO mice (Fig. 4d). Immunostaining corroborated these observations, demonstrating dual blockade–induced elevation of F4/80+ and Iba1+ populations and nuclear translocation of pIRF3 in wild-type mice, which were absent in STING KO mice (Fig. 4e, f).

Mounting an effective antitumor immune response necessitates the activation of adaptive as well as innate immunity. As type I IFN bridges these immune responses, dual blockade showed markedly diminished survival benefits in Ifnar1 KO mice (Fig. 4g). To investigate the contribution of CD8+ T cells in the antitumor effects mediated by dual blockade, we depleted CD8+ T cells through intraperitoneal administrations of an anti-CD8 antibody (Fig. 4h). This depletion notably attenuated the therapeutic efficacy of dual blockade, leading to decreased survival outcomes (Fig. 4i). Analysis of intratumoral immune cell populations demonstrated a significant reduction in IFNγ+CD8+ T cells upon CD8+ T cell depletion, whereas regulatory T cell (CD4+FoxP3+CD25+) populations remained unaltered (Fig. 4j).

Dual blockade sensitizes tumors to PD1 blockade
Although activation of innate immune responses can enhance T cell infiltration, it may simultaneously induce adaptive immune resistance through increased expression of PD1 and PDL1 within the tumor microenvironment.33-36 To examine whether such adaptive immune resistance was triggered by dual blockade, we assessed PD1 and PDL1 expression levels in GL261 tumors after phagocytosis checkpoint blockade. Notably, increased expression of PD1 and PDL1 was solely associated with dual blockade treatment (Fig. 5a, b). These effects were dependent on type I interferon signaling, as IFNAR1 blockade significantly abrogated the upregulation of PD1 and PDL1 following dual blockade (Suppl. Fig. 28). We subsequently explored the therapeutic potential of combining anti-PD1 antibody with dual blockade to overcome this resistance. Remarkably, the triple combination therapy significantly enhanced survival outcomes in GL261-bearing mice relative to either dual blockade or anti-PD1 monotherapy alone (Fig. 5c). Although the other treatment groups showed no significant changes, the triple combination uniquely reduced the population of myeloid-derived suppressor cells (MDSC; CD11b+Gr1+) (Fig. 5d). In addition, this combination therapy enhanced the infiltration of IFNγ+CD8+ T cells, whereas regulatory T cell populations remained unchanged (Fig. 5e). Immunostaining images also indicated increased infiltration and activation of effector T cells, evidenced by elevated expressions of IFNγ and granzyme B (Fig. 5f).

To ascertain whether triple combination therapy could establish long-term tumor-specific immune memory, surviving mice previously treated with the triple combination were rechallenged with GL261 tumor cells. As expected, all naïve (unimmunized) mice succumbed by day 25, whereas rechallenged mice exhibited prolonged survival up to 60 days (Fig. 5g). Visualizing with in vivo imaging system confirmed rapid tumor progression in naïve mice, but no detectable signals were present in rechallenged mice, indicating effective tumor-specific immune memory capable of preventing tumor engraftment (Fig. 5h, i).

To further validate the broader applicability of this combination therapy, we investigated its efficacy in immunosuppressive GBM models, including SB28 and QPP8. The SB28 model is characterized by low intrinsic MHC-I expression and minimal immunogenicity, closely mimicking human GBM phenotypes.23,37 The QPP8 model harbors recurrent human GBM mutations, including Qki, PTEN, and p53, providing translational similarity to clinical immunotherapy scenarios.38-40 Dual phagocytosis checkpoint blockade significantly prolonged survival in both models (Fig. 5j, Suppl. Fig. 29). Moreover, triple combination therapy further improved survival duration in the SB28 model compared with IgG-treated controls, extending median survival time from 23 to 35 days (Fig. 5j). Magnetic resonance imaging further substantiated these outcomes, demonstrating significant suppression of tumor growth after triple combination therapy (Fig. 5k, l, Suppl. Figs. 30, 31).

Methods
Mice
Age-matched male and female C57BL/6 (bred at MD Anderson Cancer Center), C57BL/6-Ifnar1tm1.2Ees/J (The Jackson Laboratory, USA, #028288), and C57BL/6J-Sting1gt/J mice (The Jackson Laboratory, USA, #017537) were used for tumor engraftment and growth studies. For T cell isolation, C57BL/6-Tg(TcraTcrb)1100Mjb/J (The Jackson Laboratory, USA, #003831) and C57BL/6-Tg(TcraTcrb)425Cbn/J mice (The Jackson Laboratory, USA, #004194) were used. All animals were maintained in a specific pathogen-free animal facility under controlled conditions (22°C, 50% relative humidity, 12 h light/dark cycle) with access to standard diet and water. Experimental and control mice were co-housed under identical conditions. All procedures were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. In the orthotopic glioblastoma model, direct measurement of tumor size is not practical and endpoints were therefore determined based on the clinical condition of mice. Humane endpoints for euthanasia included signs of distress such as labored breathing, abnormal movement, hypothermia, hunched posture, or body weight loss of greater than 20% of the baseline. Mice were monitored three times per week for clinical signs starting two weeks after the tumor inoculation. Mice were euthanized by CO inhalation followed by cervical dislocation. This study was conducted in accordance with all relevant ethical regulations. All experimental procedures were approved by and carried out in compliance with the institutional policies of The University of Texas MD Anderson Cancer Center (no. 00002163).

Cell lines
GL261 (ACC 802) and SB28 (ACC 880) cell lines were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). CT-2A (SCC194) and U87 (89081402-1VL) cell lines were purchased from Millipore Sigma Aldrich. THP-1 (TIB-202), U138 (HTB-16), and T98G (CRL-1690) cell lines were purchased from the American Type Culture Collection (ATCC). QPP8 cells were kindly provided by Dr. Michael A. Curran.

Cell cultures
CT-2A, GL261, and SB28 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. U87, U138, T98G, and THP-1 cells were cultured in Roswell Park Memorial Institute 1640 medium containing 10% heat-inactivated FBS and 1% penicillin-streptomycin. QPP8 cells were cultured in NeuroCult Basal Medium (Mouse & Rat) (Stemcell Technologies, #05700) supplemented with 10% NeuroCult Proliferation Supplement (Stemcell Technologies, #05701) and 1% penicillin-streptomycin. All cell lines were cultured at 37°C in a humidified incubator with 5% CO except for the SB28 cells, which required 10% CO .

Monocytes were isolated from the femurs and tibias of C57BL/6 or C57BL/6J-Sting1gt/J mice according to previously established protocols.43 The surrounding skeletal muscles were removed, and bone marrow was flushed out with PBS and filtered through cell strainer to obtain monocytes. For BMDM differentiation, monocytes were cultured in DMEM supplemented with 10% FBS, 1% sodium pyruvate, 1% penicillin-streptomycin, and 10 ng/mL macrophage-colony-stimulating factor. For differentiation into dendritic cells (BMDC), monocytes were cultured in DMEM with 10% FBS, 1% sodium pyruvate, 1% penicillin-streptomycin, 20 ng/mL granulocyte-macrophage colony-stimulating factor, and 10 ng/mL IL-4. THP-1 macrophage differentiation was achieved by treating THP-1 cells with 200 nM phorbol 12-myristate 13-acetate for 3 days, followed by an additional 2 days of incubation with fresh medium to generate human macrophage-like cells.

cOVA-expressing CT-2A, GL261, and SB28 tumor cells were generated by transfection with pLenti-cOVA plasmid according to previously published protocols.44 Lentivirus was generated in 293T cells by using Lipofectamine 3000 and ViraPower packaging mix (ThermoFisher). Tumor cells were plated at 106 cells in 60 mm culture dishes and 10 µg/mL polybrene (EMD Millipore) and lentivirus were added dropwise. These cells were selected and maintained in DMEM complete medium supplemented with 0.5 mg/mL G418 sulfate.

CD24-expressing CT-2A cells were generated by transfection with Lipofectamine 3000 following the manufacturer's protocol. Phagocytosis assays were conducted 2 days after transfection.

Human single-cell RNA sequencing data
Expression of "Eat me" and "Don't eat me" signals in GBM patient cells was analyzed by using publicly available single-cell RNA sequencing data (GEO accession: GSE182109), comprising 201,986 cells from 18 glioma patients (1 astrocytoma, 1 oligodendroglioma, 11 newly diagnosed GBM, and 5 recurrent GBM).17 Data visualizations are available via the Single Cell Portal: https://singlecell.broadinstitute.org/single_cell/study/SCP1985/single-cell-analysis-of-human-glioma-and-immune-cells-identifies-s100a4-as-an-immunotherapy-target-gse182109.

Phagocytosis assay
Tumor cells (1.0 × 105) labeled with CFSE were treated with antibodies (IgG control, anti-CD24, anti-CD47, or a combination) for 15 min, followed by co-culture with 1.0 × 105 macrophages or BMDCs labeled with eFluor670. For CD24-Fc competition assay, GBM cells were treated with CD24-Fc for 15 min before antibody treatment. Co-cultures were maintained for 2 hours at 37°C in ultra-low attachment 24-well plates, harvested, stained with 4′,6-diamidino-2-phenylindole (DAPI), and analyzed with an Attune NxT Flow Cytometer (ThermoFisher). The phagocytic index was defined as the percentage of CFSE+ tumor cells within eFluor670+ phagocytes. For concentration-response experiments, relative phagocytic index was determined by normalizing the phagocytosis ratio of each treatment condition to that of the IgG control within the same cell line.

Distinguishing phagocytosis and trogocytosis
GBM cells (GL261 or SB28) were dual-labeled with CFSE and PKH26 according to the manufacturer’s instructions. Labeled tumor cells (1.0 × 105) were treated with antibodies and co-cultured with eFlour670-labeled macrophages. For confocal microscopy, cells were incubated in µ-Slide 8 Well chambers (Ibidi, #80826) for 2 hours and visualized using an LSM880 confocal microscope (Zeiss). For flow cytometry, co-cultures were maintained for 2 hours in ultra-low attachment 24-well plates, stained with DAPI, and analyzed with an Attune NxT Flow Cytometer (ThermoFisher). Phagocytosis and trogocytosis were defined as PKH26+CFSE+ and PKH26+CFSE- populations, respectively, within eFlour670+ macrophage populations. The detailed gating strategy is provided in Suppl. Fig. 32.

Antigen presentation
GBM-cOVA cells were treated as described in the phagocytosis assay and then co-cultured with BMDMs in 24-well cell culture plates for 24 hours. After incubation, cells were detached with trypsin, stained with the corresponding antibodies, and resuspended in phosphate-buffered saine (PBS) containing 2% FBS and 300 nM DAPI. Antigen presentation was quantified with an Attune NxT Flow Cytometer (ThermoFisher), measuring the mean fluorescence intensity of the SIINFEKL–H2Kb complex on CD11b+F4/80+ BMDMs.

Visualizing phagocytosis and antigen presentation by confocal laser scanning microscopy
For visualizing phagocytosis, tumor cells were treated as described in the phagocytosis assay and co-cultured with eFluor670-labeled BMDMs in µ-Slide 8 Well chambers (Ibidi, #80826) for 4 hours. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde solution. To visualize antigen presentation, cells were co-cultured for 24 hours, followed by washing and fixation. Fixed cells were then stained with SIINFEKL-H2kb and F4/80 antibodies overnight at 4°C and images obtained with an LSM880 confocal microscope (Zeiss).

T cell proliferation
Spleens harvested from OT-I (CD8) or OT-II (CD4) transgenic mice were washed with cold PBS, mechanically dissociated, and filtered through a 70-μm cell strainer. Naïve T cells were isolated using a Naïve CD4+ or CD8+ T Cell Isolation Kit (Miltenyi Biotec, #130-104-453 for CD4+ and #130-093-244 for CD8+). GBM-cOVA cells and BMDMs were treated and co-cultured as described for antigen presentation, then incubated with CFSE-labeled naïve T cells for an additional 72 hours. Cells were harvested, stained with the corresponding antibodies, and resuspended in PBS containing 2% FBS and 300 nM DAPI. T cell proliferation was evaluated using an Attune NxT Flow Cytometer (ThermoFisher), calculating the percentage of CFSEmed cells within CD3+CD4+ or CD3+CD8+ T cell populations.

Western blotting
Tumor cells (1.5 × 107 cells) treated with different antibody combinations (20 μg/mL) were co-cultured with BMDMs (1.5 × 107 cells) in 150-mm dishes for 24 hours, except for kinetic studies in which co-cultures were assessed at 6, 12, 24, and 48 hours. After co-culture, macrophages were isolated using CD45 MicroBeads (Miltenyi Biotec, #130-052-301) and lysed in RIPA buffer containing protease inhibitor (1%) and phosphatase inhibitor cocktail (1%). Protein concentration was normalized to 2 mg/mL using a BCA kit. Protein samples were separated by gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in TBS containing 2% bovine serum albumin (BSA) and 0.1% Tween 20 for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C, rinsed four times, and subsequently incubated with secondary antibodies. The blot signals were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, #RPN2232) and visualized with a ChemiDoc MP imaging system (BioRad). Antibodies include: GAPDH (Cell Signaling, #2118, 1:2000), STING (Cell Signaling, #50494, 1:1000), IRF-3 (Cell Signaling, #4302, 1:1000), pSTINGSer366 (ThermoFisher, PA5-105674, 1:1000), pIRF-3Ser396 (ThermoFisher, MA5-14947, 1:1000), NF-κB p65 (Cell Signaling, #8242, 1:1000), IκBα (Cell Signaling, #4814, 1:1000), IKKα (Cell Signaling, #2682, 1:1000), IKKβ (Cell Signaling, #8943, 1:1000), pNF-κB p65Ser536 (Cell Signaling, #3033, 1:1000), pIκBαSer32 (Cell Signaling, #2859, 1:1000), pIKKα/βSer176/180 (Cell Signaling, #2697, 1:1000). For densitometric analysis, band intensities were quantified using ImageJ software. Phosphorylated protein levels were normalized to their corresponding total protein levels, and the resulting values were further normalized to the IgG-treated control group. A full list of the antibodies used for Western Blot analysis is provided in Supplementary Table 1.

Immunofluorescence
Tumor cells (1.0 × 105) treated with different antibody combinations (10 μg/mL) were co-cultured with BMDMs (1.0 × 105) in µ-Slide 8-well chambers (Ibidi, #80826) for 24 hours. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde, blocked and permeabilized using PBS containing 2% BSA, anti-CD16/32 antibody (10 μg/mL), and 0.3% Triton X-100. Cells were subsequently stained with primary antibodies against CD11b and p-IRF3Ser396 overnight, followed by secondary antibodies. Immunofluorescence images were acquired using an LSM880 confocal microscope (Zeiss).

Quantitative PCR with reverse transcription
Tumor cells and BMDMs were co-cultured, and BMDMs were isolated as described for western blot. RNA samples were extracted using a PureLink RNA Mini kit (ThermoFisher, #12183025) according to the manufacturer’s protocol. Isolated RNA was reverse transcribed into complementary DNA using High Capacity cDNA Reverse Transcription Kit (ThermoFisher, #4368814). Quantitative reverse transcription PCR (qRT-PCR) was conducted using TaqMan Fast Advanced PCR Master Mix (ThermoFisher, #4444557) with corresponding primers (7500 FAST real-time PCR system, Applied Biosystems). The ΔCt values were calculated by Ct target - Ct GAPDH, and -ΔΔCt values were obtained by ΔCt cont - ΔCt treated. Relative mRNA levels were determined as 2-ΔΔCt.

Tumor implantation
For flow cytometry and immunohistochemistry analyses, only female mice were used. For survival experiments, both male and female were used. All experiments were performed using age-matched mice (6-12 weeks old).

CT2A-Luc-mCherry, GL261-Luc-mCherry, SB28, or QPP8 tumors were inoculated into the caudate nucleus via a guide screw system. Mice were anesthetized, and analgesia was provided with buprenorphine in polymer (0.1 mg/kg) immediately before surgery and 48 hours post-surgery. After disinfecting the surgical site, a 3-mm midline scalp incision was made to expose the skull. A 2-mm burr hole was drilled at coordinates 2 mm lateral and 2 mm anterior to Bregma, and a guide screw was inserted. Seven days later, tumor cells (1 × 105 cells for CT-2A-Luc-mCherry and GL261-Luc-mCherry, 1 × 104 cells for SB28, and 2 × 105 cells for QPP8) in 5 μL of PBS were infused slowly at 0.2 μL/min through the guide screw. The mice were allowed to recover from anesthesia on a heating pad. Engrafted tumors were visualized using an IVIS Spectrum Imaging System (IVIS Lumina XR, PerkinElmer), and mice were randomized into four treatment groups.

Tumor inhibition
For the GL261 and CT-2A models, MIAP-301 (anti-CD47, 40 μg) and M1/69 (anti-CD24, 40 μg) antibodies were administered intracranially on days 7, 14, and 21 post-implantation, and anti-PD-1 antibody (100 μg) was injected intraperitoneally on days 8, 15, and 22. In the SB28 model, anti-CD47 and anti-CD24 antibodies (80 μg each) were administered intraperitoneally on days 5, 7, and 9, while anti-PD-1 antibody (200 μg) was given intraperitoneally on days 10, 12, and 14. For the QPP8 model, anti-CD47 and anti-CD24 antibody cocktails (80 μg each) were administered intraperitoneally on days 5, 7, and 9. Tumors were harvested one day following the final treatment for flow cytometry and immunohistochemical analyses. Survival outcomes were assessed by Kaplan–Meier survival analysis, and statistical significance was evaluated using a log-rank (Mantel–Cox) test.

CD8+ T cell depletion
C57/BL6 mice received anti-CD8α antibody (300 μg) intraperitoneally every 4 days, starting 2 days before treatment was begun. To confirm CD8+ T cell depletion, splenocytes were isolated and analyzed by flow cytometry.

Ifnar1 blockade
C57/BL6 mice were administered anti-Ifnar1 antibody intraperitoneally, with an initial dose of 500 μg per mouse, followed by maintenance doses of 250 μg per mouse every 3 days, beginning 2 days prior to treatment initiation.

Immunohistochemical analyses
For human samples, primary GBM tumor specimens were obtained from patients with pathologically confirmed diagnoses under the University of Arizona. All samples were de-identified, and written informed consent was obtained from each patient after approval by the Institutional Review Board of the University of Arizona (IRB number: 1507974522). At the time of tissue collection, all patients were treatment naïve. For mouse samples, brain tissues were collected 1 day after the final treatment, fixed in 4% paraformaldehyde for 24 hours, and then placed in 30% sucrose solution until completely submerged. Fixed tissues were embedded in Tissue-Tek optimal cutting temperature compound, frozen on dry ice, and stored at –80°C. Frozen blocks were sectioned into 10-μm slices and mounted on adhesive glass slides. Slides were blocked with 10% goat serum solution for 1 h, with 0.3% Triton X-100 added for intracellular staining. Blocked slides were incubated with primary antibodies overnight at 4°C, washed three times, and subsequently incubated with fluorescent secondary antibodies overnight at 4°C. After three additional washes, slides were stained with 300 nM DAPI solution, mounted with coverslips, and visualized with an LSM880 microscope (Carl Zeiss). Image processing was performed with Zeiss Zen software.

Flow cytometry in vivo
Tumors and spleens were harvested 1 day after the final treatment. Tumors were digested at 37°C for 30 min in a solution containing 300 U/mL collagenase D and 15 U/mL DNase I. The dissociated tumor cells were passed through a 70-μm cell strainer, and leukocytes were enriched by using CD45 or CD11b microbeads (Miltenyi Biotec, #130-052-301 for CD45 and #130-097-142 for CD11b) as required by the assay. Splenocytes were obtained by grinding the spleens and then lysing red blood cells. The isolated cells were blocked with 2% FBS solution containing anti-CD16/CD32 antibody for 10 minutes to minimize nonspecific Fc receptor binding. Subsequently, the cells were stained with fluorescence-labeled antibodies for 30 minutes at 4°C and analyzed on an Attune NxT Flow Cytometer (ThermoFisher) or Cytek Aurora (Cytek Bioscience). Fluorescence-labeled antibodies included: CD3 (clone 145-2C11, BioLegend, 100330, 1:100), CD4 (clone RM4-5, BioLegend, 100510 or 100552, 1:100), CD8 (clone 53-6.7, BioLegend, 100722 or 100744, 1:100), IFN-γ (clone XMG1.2, BioLegend, 505808, 1:50), CD25 (clone PC61, BioLegend, 102012, 1:50), FoxP3 (clone MF-14, BioLegend, 126404, 1:200), F4/80 (clone BM8, BioLegend, 123116 or 123137, 1:100), CD11b (clone M1/70, BioLegend, 101226, 101259, or 101208, 1:100), pIRF-3Ser396 (clone PSH06-75, ThermoFisher, MA5-57852, 1:1000), CD11c (clone N418, BioLegend, 117318, 1:100), Gr-1 (clone RB6-8C5, BioLegend, 108406 or 108457, 1:50), CD45 (clone 30-F11, Biolegend, 103114, 1:100), Thy1.2 (clone 30-H12, Biolegend, 105327, 1:100), T-bet (clone 4B10, Biolegend, 644814, 1:50), GATA3 (clone 16E10A23, Biolegend, 653814, 1:50), CD86 (clone GL-1, Biolegend, 105008, 1:100), CD206 (clone C068C2, Biolegend, 141703, 1:100), H-2Db Tetramer-AALLNKLYA (MHC Tetramer Production Facility in Balyor College of Medicine), and H-2Kb OVA Tetramer-SIINFEKL (MBL Life Science, MBL-TS-5001-4C, 1:10). Detailed gating strategies are provided in the Suppl. Figs. 33-38. A full list of the antibodies used for Flow cytometry analysis is provided in Supplementary Table 2.

Magnetic resonance imaging
Tumor growth was monitored by using a 7 Tesla (T) small animal MRI scanner (Bruker Biospin, Billerica, MA) at the MD Anderson Small Animal Imaging Facility. Mice were anesthetized with 2% isoflurane during imaging. To detect and measure tumors, T2-weighted axial and coronal MR images were captured. Axial and coronal images were acquired by using a rapid acquisition with relaxation enhancement (RARE) sequence with the following parameters: repetition time (TR) of 3000 ms and echo time (TE) of 57 ms. For coronal images, slices were 0.75 mm thick with a field of view (FOV) of 30 × 40 mm and a matrix resolution of 256 × 192 pixels, yielding an in-plane resolution of approximately 0.156 µm. Axial images were similarly acquired with 0.75 mm thickness, a FOV of 30 × 22.5 mm, and the same matrix resolution, resulting in an in-plane resolution of approximately 0.117 µm. Tumor regions were manually delineated on each MRI slice in a blinded fashion with ImageJ, and tumor volumes were calculated by summing the delineated areas in mm2 × 1 mm slice intervals.

Statistics and reproducibility
Statistical analyses were done with GraphPad Prism 10, and data are presented as mean ± SD. Statistical significance was determined with unpaired two-tailed Student’s t tests or one-way analysis of variance.

Data availability
Publicly available single-cell RNA sequencing data were obtained from GEO under accession code GSE182109 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE182109]. All data are included in the Supplementary Information or available from the authors, as are unique reagents used in this Article. The raw numbers for charts and graphs are available in the Source Data file whenever possible.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript Dual phagocytosis-checkpoint blockade revitalizes immune surveillance in glioblastoma by Ha et. al attempts to discover additional phagocytosis checkpoint inhibitors expressed on glioblastoma cells. Phagocytosis checkpoint inhibitors (primarily anti CD47 antibodies) have shown great promise in animal models against many different cancers and are currently being evaluated in clinical trials. phagocytosis inhibitor. A comprehensive investigation of “don’t eat me” vs “eat me” signal genes on GBM and associated myeloid cells in sc RNA-Seq databases has identified CD24 as a potential candidate. The authors proceed to validate CD24 using function blocking antibodies in combination with anti-CD47. Representative confocal microscopy images seem to show an increase GBM cells (green) phagocytosed by macrophages (red). Furthermore, this enhanced phagocytosis translated into increased antigen presentation as CD4+ CD8+ T cells are activated in vitro. These observations are consistent with what is seen in-vivo. Enhanced phagocytosis with both dual antibody increases activation of the cGAS/STING pathway which is known to promote antigen presentation and T cell activation. Indeed this pathway is responsible for the effect as it is not seen in STING KO mice. Finally, the authors observe that dual antibody treatment results in an increase in PDL/PDL1 signaling in tumors. Upregulation of the PD1 pathway is exploited by treating animals with a triple antibody combination (aCD47, aCD24, aPD1) which does modestly enhance survival.\nOverall this is a very compelling study which highlights the interaction between different immune checkpoint pathways and the importance of innate-adaptive immune communication for immunotherapy. The data is thorough and presented clearly. However there are several issues which should be addressed.\nOne of the main concerns is whether in fact what is observed is truly phagocytosis. It has become apparent that macrophages can pick up cellular debris or engage in “trogocytosis” (cellular gnawing) which is not complete phagocytosis of the tumor cell target.\nThe GL261 model is well established and is known to involve a substantial degree of macrophage infiltration. The of tumor associated macrophages in Figure 3 in the GL261 tumor model seems extremely low compared with other studies using this same cell line in C57BL/6 mice (for two examples see Coniglio Mol Mel 2012 May 9;18(1):519-27. doi: 10.2119/molmed.2011.00217, Xu J Natl Cancer Inst. 2014 Jun 28;106(8):dju162. doi: 10.1093/jnci/dju162. Print 2014 Aug.)\nAre these images representative of the entire tumor or just specific regions?\nIt would be of great interest to know the polarity of the macrophages associated with the tumor under the different treatment conditions. (i.e. M1, M2)\nAlso vital for this study would be to know the status of the recruited CD4 t cells within the tumor (i.e. Th1 or Th2). The presence of CD8+ effector T cells has been well established here, but there is controversy regarding the involvement of CD4 Th cells in tumor progression. Profiling these specific cells in this study could help clarify this issue.\nThe study does an excellent job with various cell lines and matched controls (such as the CD24 negative CT-2A cell line). However some verification of protein expression in patient samples would be helpful.", "reviewer_2": "Overall Summary\nThis study demonstrates that glioblastoma (GBM) cells express high levels of CD24, a phagocytosis checkpoint, alongside CD47. Dual blockade of CD24 and CD47 enhances macrophage-mediated phagocytosis, activates the cGAS–STING pathway, and promotes antigen presentation, leading to robust T cell responses. In murine GBM models, this dual blockade improves survival and sensitizes tumors to anti-PD1 therapy. Mechanistic experiments confirm the necessity of STING signaling and CD8+ T cells for therapeutic efficacy. The triple combination of CD24, CD47, and PD1 blockade induces durable immune memory, offering a promising strategy to overcome immune resistance in immunosuppressive GBM.\nThis manuscript builds incrementally on the authors’ prior work (PMID: 32198351), extending their exploration of phagocytosis checkpoint blockade in glioblastoma by adding CD24 to the previously studied CD47 axis. While the dual blockade strategy enhances macrophage activation and T cell priming via cGAS–STING signaling, the mechanistic novelty is low. The study is well-executed with strong in vivo data, but translational relevance is limited by murine models and intracranial antibody delivery. The work would benefit from deeper mechanistic dissection of CD24’s role and more clinically relevant delivery strategies. Nonetheless, it reinforces the therapeutic potential of innate-adaptive immune synergy.\nMajor Concerns\n1. Biological Replication and Statistical Validity\n• Lack of Independent Replicates: Many experiments seem to rely on technical replicates from a single biological experiment. For instance, the tumor growth curves (Fig. 4) and macrophage phagocytosis assays (Fig. 2C–D) appear to be derived from one animal cohort or cell passage, lacking independent biological replication. Results are reported with SEMs that obscure intra-experimental variability.\n• Statistical Tests: The authors frequently apply uncorrected multiple t-tests. For multi-group comparisons, ANOVA with post-hoc testing would be more appropriate.\n2. Lack of Appropriate Controls\n• Rescue Experiments Missing: While inhibition of CD24 is shown to affect phagocytosis, rescue with a non-targetable construct (e.g., siRNA-resistant cDNA or inactive mutant) is not performed. This is critical to prove specificity.\n• No Null Tumor Model: The tumor models used all express [target antigen]. The authors do not test whether their strategy fails in antigen-negative tumors, which would serve as a critical specificity control.\n3. Mechanistic Redundancy and Oversights\n• The proposed mechanism (cGAS-STING) has already been described in earlier papers by the same senior author (e.g., PMID 32198351) and is an incremental extension rather than a fundamentally new insight.\n• No demonstration of direct causality: The study infers a mechanism from correlations and phenotypic observations, but does not directly link the pathway activation to the functional outcome (e.g., chromatin accessibility, transcription factor binding, or downstream effector knockout).\n• No temporal analysis: The authors do not assess whether changes in gene expression precede phenotypic outcomes (e.g., phagocytosis, tumor shrinkage). Time-course analysis would help establish causality.\nAdditional Critiques\n1. Line 68… The assumption that SB28 may be a progenitor like cell lines and the references cited are incorrect\n2. Line 84: Misleading sentence, how is it optimal if there's no significant advantage\n5:3 and 1:3 are vastly different ratios, why is there such a difference to achieve an optimal phagocytosis\n3. How is their \"relative\" phagocytic index calculated.\n4. Line 98: This is an insufficient experiment\n1. Increase of ova presentation on macrophages makes sense and is a logical assay\n2. Using ova specific CD4 and CD8 cells does nothing to prove the crosstalk between innate and adaptive immunity. We need to see an in vivo assay demonstrating increased ova-specific CD4 and CD8 in response to ova expressing tumor cells.\n5. In Fig 2, while the combination treatment leads to a modest increase in phagocytosis it seems to increase antigen presentation in a significant way. Please explain\n6. The cell line QPP8 appears to be most susceptible to phagocytosis however its has not been used in any of the subsequent studies.\n7. Line 101: This experiment is insufficient to show cross-presentation as it's simply showing that activation of macrophages can cause secretion of cytokines that promote CD4, CD8 activity. The real issue that needs is addressing is whether these macrophages can prime an adaptive immune response by presenting neo-antigens from the ingested tumor cells\n8. Results from Fig 2i are unclear whether they are looking at signal from macrophages and tumor cells. It might be better to carry out phospho-flow to characterize what is happening to the macrophages post phagocytosis.\n9. Line 153: A rescue experiment of using wt-macrophages in Sting-KO mice is required to nail down their hypothesis.", "reviewer_3": "This manuscript by Ha and colleagues addresses a critical roadblock in glioblastoma immunotherapy. To date immunotherapy approaches have failed in GBM, and activation of phagocytosis in immunosuppressed macrophage environment has not been possible. The authors have noted the highly GBM-associated checkpoint CD24 on GBM cells, and found that Ab inhibition of CD24, in conjunction with CD47 inhibition can synergistically induce innate and adaptive anti tumor responses in mouse and human GBM cells. Triple inhibition with addition of anti-PD-L1 (which is induced by anti-CD24) resulted in even further anti-GBM phagocytic activity by tumor associated macrophages and T-cells, and even anti-tumor memory. These results open a novel potential avenue for combination checkpoint inhibitor immunotherapy for human GBM.\nOverall the work is substantiated by carefully planned experiments. I see no significant flaws in experimental design.\nMinor comments:\nFig. 2D: very difficult to see arrows pointing out phagocytosis. Please enlarge.\nFig. 2F: Please provide arrows to identify cells undergoing phagocytosis.\nGeneral comment: Although beyond the scope of this project, could the authors briefly discuss current clinical trials with anti-CD24 and potential utility for GBM...off target risks, etc?"}, "author_response": "We sincerely thank the Reviewers for their thoughtful evaluation of our manuscript and for the constructive comments that have greatly improved the clarity and impact of our work. We have carefully addressed all points raised in the reviews in a detailed, point-by-point manner. Our responses are provided below, with our revisions and answers highlighted in blue for ease of reference.\nReviewer #1 (Remarks to the Author):\nThe manuscript Dual phagocytosis-checkpoint blockade revitalizes immune surveillance in glioblastoma by Ha et. al attempts to discover additional phagocytosis checkpoint inhibitors expressed on glioblastoma cells. Phagocytosis checkpoint inhibitors (primarily anti CD47 antibodies) have shown great promise in animal models against many different cancers and are currently being evaluated in clinical trials. phagocytosis inhibitor. A comprehensive investigation of “don’t eat me” vs “eat me” signal genes on GBM and associated myeloid cells in sc RNA-Seq databases has identified CD24 as a potential candidate. The authors proceed to validate CD24 using function blocking antibodies in combination with anti-CD47. Representative confocal microscopy images seem to show an increase GBM cells (green) phagocytosed by macrophages (red). Furthermore, this enhanced phagocytosis translated into increased antigen presentation as CD4+ CD8+ T cells are activated in vitro. These observations are consistent with what is seen in-vivo. Enhanced phagocytosis with both dual antibody increases activation of the cGAS/STING pathway which is known to promote antigen presentation and T cell activation. Indeed this pathway is responsible for the effect as it is not seen in STING KO mice. Finally, the authors observe that dual antibody treatment results in an increase in PDL/PDL1 signaling in tumors. Upregulation of the PD1 pathway is exploited by treating animals with a triple antibody combination (aCD47, aCD24, aPD1) which does modestly enhance survival.\nOverall this is a very compelling study which highlights the interaction between different immune checkpoint pathways and the importance of innate-adaptive immune communication for immunotherapy. The data is thorough and presented clearly. However there are several issues which should be addressed.\nOne of the main concerns is whether in fact what is observed is truly phagocytosis. It has become apparent that macrophages can pick up cellular debris or engage in “trogocytosis” (cellular gnawing) which is not complete phagocytosis of the tumor cell target.\nResponse: We thank the reviewer for this insightful comment regarding the distinction between phagocytosis and trogocytosis. To directly address this concern, we performed additional experiments designed to discriminate between the two processes. Glioblastoma (GBM) cells were dual-labeled with CFSE (intracellular) and PKH26 (cell-membrane) dyes and co-cultured with eFluor 660-labeled bone-marrow-derived macrophages (BMDMs) for two hours, followed by visualization by confocal microscopy (Supplementary Fig. S6a). In this system, phagocytosis was defined as macrophage uptake of both CFSE and PKH26 signals (double-positive), whereas trogocytosis was defined as uptake of PKH26 signal alone without CFSE. This dual-color strategy allowed us to clearly differentiate partial membrane nibbling from complete cellular engulfment.\nIn GL261 co-cultures, control IgG treatment predominantly yielded PKH26-positive puncta within macrophagesm, consistent with trogocytosis (blue arrows), whereas treatment with anti-CD24 or anti-CD47 antibodies increased the proportion of macrophages fully enclosing tumor cells, indicative of phagocytosis (yellow arrows). The combination treatment produced a synergistic enhancement of phagocytosis. Notably, trogocytosis levels remained relatively constant across treatment groups, whereas phagocytosis markedly increased following antibody treatment.\nA similar pattern was observed in SB28 cells: IgG-treated controls exhibited primarily trogocytosis with minimal phagocytosis, while antibody treatment promoted both processes, again showing synergy under dual blockade. The relative increase in trogocytosis seen in SB28 cells may reflect their larger size, which could favor partial membrane uptake events.\nThese microscopy results were corroborated by flow-cytometric quantification (Supplementary Fig. S6b), which showed trends consistent with our imaging data. Together, these findings demonstrate that while antibody treatments can modestly influence trogocytosis, they predominantly and significantly promote bona fide phagocytosis of GBM cells by macrophages.\nSuppl. Fig. S6\nThe GL261 model is well established and is known to involve a substantial degree of macrophage infiltration. The of tumor associated macrophages in Figure 3 in the GL261 tumor model seems extremely low compared with other studies using this same cell line in C57BL/6 mice (for two examples see Coniglio Mol Mel 2012 May 9;18(1):519-27. doi: 10.2119/molmed.2011.00217, Xu J Natl Cancer Inst. 2014 Jun 28;106(8):dju162. doi: 10.1093/jnci/dju162. Print 2014 Aug.) Are these images representative of the entire tumor or just specific regions?\nResponse: We thank the reviewer for this important observation regarding Iba1 expression and macrophage infiltration in the GL261 model. As noted, some reports describe substantial macrophage infiltration in GL261 tumors; however, the degree of Iba1 positivity varies considerably across studies, reflecting both biological heterogeneity within the GL261 model and methodological differences among laboratories. For example, the study cited by the reviewer (Coniglio et al., Mol Med, 2012) reported relatively high Iba1 expression, whereas other publications, such as Simoes et al., eLife, 2025 (doi: 10.7554/eLife.100570.3) and our previous work (von Roemeling et al., Nat Commun, 2020; doi: 10.1038/s41467-020-15129-8), document lower levels of macrophage infiltration.\nIn our original submission, the representative Iba1 IHC images depicted a relatively limited field of view, which may have contributed to the impression of underrepresentation. In the revised manuscript, we have replaced these panels with images encompassing a broader tumor area (Fig. 3d). The updated figures more accurately capture the overall macrophage distribution within GL261 tumors, while confirming that the general level of Iba1 expression in our model remains consistent with that observed in prior studies reporting moderate macrophage infiltration.\nFig. 3d\nIt would be of great interest to know the polarity of the macrophages associated with the tumor under the different treatment conditions. (i.e. M1, M2)\nResponse: We thank the reviewer for this valuable suggestion. As recommended, we analyzed the polarization status of tumor-associated macrophages in the GL261 model under the IgG, anti-CD24, anti-CD47, and combination treatment conditions. We used CD86 as a marker for M1 macrophages and CD206 as a marker for M2 macrophages, and we quantified macrophage polarity by calculating the CD206 /CD86 ratio (Fig. 3h).\nIn IgG-treated tumors, this⁺ ratio w⁺as the highest, indicating a predominance of M2-polarized macrophages. Both anti-CD24 and anti-CD47 monotherapies reduced the CD206 /CD86 ratio, consistent with a shift toward an M1 phenotype. Importantly, the combination treatment produced the lowest CD206 /CD86 ratio, reflecting a pronounced ⁺ ⁺repolarization of macrophages toward a proinflammatory, M1-like state within the tumor ⁺ ⁺microenvironment.\nFig. 3h\nAlso vital for this study would be to know the status of the recruited CD4 t cells within the tumor (i.e. Th1 or Th2). The presence of CD8+ effector T cells has been well established here, but there is controversy regarding the involvement of CD4 Th cells in tumor progression. Profiling these specific cells in this study could help clarify this issue.\nResponse: We thank the reviewer for this valuable suggestion. In accordance with the recommendation, we examined the polarization of intratumoral CD4 T cells under the different treatment conditions. Using T-bet as a Th1 marker and GATA3 as a Th2 marker, we quantified ⁺their relative proportions within the CD4 Foxp3 population (Supplementary Fig. S22).\nOur analysis revealed that the pr⁺oportio⁻n of Th1 cells remained largely unchanged across treatment groups, whereas Th2 cells were significantly reduced following antibody treatment. This decrease was observed in both monotherapy groups and in the combination treatment, with comparable magnitude.\nGiven the established anti-tumoral role of Th1 and pro-tumoral role of Th2 subsets, these findings suggest that antibody treatment shifts the CD4 T cell compartment toward a more anti-tumor phenotype, primarily through selective reduction of Th2 cells. However, no ⁺synergistic enhancement was observed under dual blockade compared with monotherapies.\nSuppl. Fig. S22\nThe study does an excellent job with various cell lines and matched controls (such as the CD24 negative CT-2A cell line). However some verification of protein expression in patient samples would be helpful.\nResponse: We thank the reviewer for this thoughtful suggestion. As recommended, we performed immunohistochemical (IHC) staining of human glioblastoma patient samples to evaluate protein-level expression of CD24 and CD47 within tumor tissues (Fig. 1d). Consistent with our scRNA-seq analyses, both CD24 and CD47 were found to be highly expressed in tumor regions, thereby validating the translational relevance of our findings in patient specimens.\nFig. 1d\nReviewer #2 (Remarks to the Author):\nOverall Summary\nThis study demonstrates that glioblastoma (GBM) cells express high levels of CD24, a phagocytosis checkpoint, alongside CD47. Dual blockade of CD24 and CD47 enhances macrophage-mediated phagocytosis, activates the cGAS–STING pathway, and promotes antigen presentation, leading to robust T cell responses. In murine GBM models, this dual blockade improves survival and sensitizes tumors to anti-PD1 therapy. Mechanistic experiments confirm the necessity of STING signaling and CD8+ T cells for therapeutic efficacy. The triple combination of CD24, CD47, and PD1 blockade induces durable immune memory, offering a promising strategy to overcome immune resistance in immunosuppressive GBM.\nThis manuscript builds incrementally on the authors’ prior work (PMID: 32198351), extending their exploration of phagocytosis checkpoint blockade in glioblastoma by adding CD24 to the previously studied CD47 axis. While the dual blockade strategy enhances macrophage activation and T cell priming via cGAS–STING signaling, the mechanistic novelty is low. The study is well-executed with strong in vivo data, but translational relevance is limited by murine models and intracranial antibody delivery.\nResponse: We thank the reviewer for the thoughtful and constructive comments on our study. We appreciate the opportunity to clarify both the mechanistic novelty and the translational significance of our work. While our previous publication (PMID: 32198351) established the role of CD47 blockade in promoting macrophage-mediated phagocytosis and cGAS-STING-dependent immune activation in glioblastoma, the current study represents the first investigation of dual phagocytosis-checkpoint blockade targeting both CD24 and CD47 in GBM. Through integrative scRNA-seq analyses and functional validation, we identified CD24 as a previously unrecognized, functionally relevant phagocytosis checkpoint highly expressed on glioblastoma cells.\nImportantly, we demonstrate that CD24 and CD47 regulate distinct but convergent macrophage-tumor interaction pathways: CD47 blockade primarily relieves SIRPα-mediated inhibitory signaling, whereas CD24 blockade disrupts Siglec-10-mediated sialoside recognition. Although both interventions ultimately converge on cGAS-STING pathway activation, our data reveal that dual blockade elicits a qualitatively distinct and synergistic immune response, characterized by enhanced phagocytosis, increased antigen presentation, and robust T cell priming. This dual mechanism of action, integrating two independent inhibitory axes to cooperatively amplify innate and adaptive immune communication, represents a novel conceptual advance beyond CD47 monotherapy. Moreover, we identify adaptive PD-L1 upregulation as a feedback response to combined CD24/CD47 inhibition, providing a mechanistic rationale for a triple-checkpoint combination (anti-CD24, anti-CD47, anti-PD1) that achieves durable immune memory and long-term survival in vivo.\nWe also appreciate the reviewer’s comments regarding the translational aspects of the work. While intracranial delivery was used for certain experiments initially to test the efficacy of combined therapy, we further validated our findings through systemic intraperitoneal administration in the SB28 model, which closely recapitulates molecular and immunological features of human GBM. The demonstration of therapeutic efficacy following systemic treatment in this model strengthens the translational relevance of our findings. We fully acknowledge that efficient antibody penetration into intracranial tumors remains a general challenge in GBM research; however, our results provide a strong mechanistic foundation for future studies aimed at developing clinically feasible delivery strategies and rational combination regimens to overcome this barrier.\nThe work would benefit from deeper mechanistic dissection of CD24’s role and more clinically relevant delivery strategies. Nonetheless, it reinforces the therapeutic potential of innate-adaptive immune synergy.\nMajor Concerns\n1. Biological Replication and Statistical Validity\n• Lack of Independent Replicates: Many experiments seem to rely on technical replicates from a single biological experiment. For instance, the tumor growth curves (Fig. 4) and macrophage phagocytosis assays (Fig. 2C–D) appear to be derived from one animal cohort or cell passage, lacking independent biological replication. Results are reported with SEMs that obscure intra-experimental variability.\nResponse: We thank the reviewer for this important observation regarding biological replication and statistical rigor. As recommended, we performed additional independent biological replicates to strengthen the reproducibility and robustness of our findings. Specifically, the in vitro macrophage phagocytosis assays using bone marrow-derived macrophages (BMDMs) from a single donor mouse were repeated with three additional biological replicates (Figs. 2a-c, e, g, and h; Supplementary Figs. S3 and S9). These experiments consistently reproduced the trends observed in the original datasets.\nFor the in vivo survival studies, all experiments were conducted with at least five biological replicates per group, ensuring sufficient statistical power. In instances where fewer than five replicates were used (e.g., in certain flow cytometry-based immune profiling experiments), we increased reliability by performing two independent experimental cohorts using distinct batches of animals and reagents. Furthermore, in line with the reviewer’s recommendation, we have updated all figure representations to display standard deviation (SD) rather than standard error of the mean (SEM) to more accurately reflect biological variability across replicates.\nThese additional biological replicates and revisions strengthen the statistical validity and reproducibility of our findings across both in vitro and in vivo datasets.\n• Statistical Tests: The authors frequently apply uncorrected multiple t-tests. For multi-group comparisons, ANOVA with post-hoc testing would be more appropriate.\nResponse: We thank the reviewer for this helpful comment regarding statistical analysis. In accordance with the recommendation, we have reanalyzed all multi-group datasets using one-way or two-way ANOVA followed by appropriate post-hoc multiple comparison tests instead of uncorrected t-tests. The resulting updated p-values are now reflected in the revised figures and figure legends, ensuring that all reported statistical results meet the appropriate standards for multiple-group comparisons.\n2. Lack of Appropriate Controls\n• Rescue Experiments Missing: While inhibition of CD24 is shown to affect phagocytosis, rescue with a non-targetable construct (e.g., siRNA-resistant cDNA or inactive mutant) is not performed. This is critical to prove specificity.\nResponse: We thank the reviewer for this insightful comment regarding the need for rescue experiments to confirm specificity. While a genetic rescue approach using an siRNA-resistant CD24 construct or an inactive mutant would conceptually validate the target, we opted for a functional rescue strategy using soluble CD24 protein (CD24-Fc) to directly assess antibody specificity. In this assay, soluble CD24 competitively inhibited the binding of anti-CD24 antibody to cell-surface CD24, thereby serving as a biologically relevant rescue model (Supplementary Fig. S5).\nIn both CD24-positive glioma cell lines (GL261 and SB28), addition of soluble CD24 did not alter phagocytosis in the IgG or anti-CD47 groups, but it significantly reduced phagocytosis in the anti-CD24 monotherapy and dual-blockade groups. These results confirm that the enhanced phagocytic activity observed upon anti-CD24 treatment is specific to CD24 inhibition by the anti-CD24 antibody, thereby addressing the reviewer’s concern regarding target specificity.\nSuppl. Fig. S5\n• No Null Tumor Model: The tumor models used all express [target antigen]. The authors do not test whether their strategy fails in antigen-negative tumors, which would serve as a critical specificity control.\nResponse: We thank the reviewer for this important comment regarding the need for an antigen-negative control model to confirm target specificity. To address this point, we included the CT-2A glioma model, which is CD24-negative, as a null comparator in our study. In vitro phagocytosis assays using CT-2A cells confirmed the absence of response to anti-CD24 treatment, in clear contrast to the enhanced phagocytosis observed in CD24-positive GL261 and SB28 cells.\nWe further evaluated immune cell infiltration by IHC in CT-2A tumors and, in this revision, have added survival studies under all treatment conditions (Fig. 3b). As expected, anti-CD24 monotherapy did not confer any antitumor efficacy, and survival outcomes across all treatment groups were comparable. These results provide direct evidence that the therapeutic effect of anti-CD24, alone or in combination, is specific to tumors expressing CD24, thereby strengthening the mechanistic and specificity conclusions of our study.\nFig. 3b\n3. Mechanistic Redundancy and Oversights\n• The proposed mechanism (cGAS-STING) has already been described in earlier papers by the same senior author (e.g., PMID 32198351) and is an incremental extension rather than a fundamentally new insight.\nResponse: We thank the reviewer for this thoughtful comment and the opportunity to clarify the mechanistic and conceptual advances of our study. While the cGAS-STING pathway was indeed implicated in our prior work involving CD47 blockade, the present study provides the first demonstration of this pathway’s cooperative activation under dual phagocytosis-checkpoint blockade targeting CD24 and CD47, which has not been previously investigated in glioblastoma.\nMechanistically, we show that CD24 and CD47 regulate distinct yet convergent inhibitory axes, the Siglec-10-sialoside and SIRPα pathways, that together suppress macrophage activation. Their simultaneous blockade elicits synergistic activation of the cGAS-STING cascade, driving enhanced phagocytosis, antigen presentation, and T cell priming. Using STING- and Ifnar1-knockout models, we directly establish that this pathway is essential for the observed synergy.\nImportantly, although our combination leverages a previously characterized signaling pathway, this does not diminish its novelty or impact. Many transformative immunotherapies, such as combinations targeting the PD-1/PD-L1 axis or exploiting DNA damage repair pathways, derive their significance not from discovering entirely new mechanisms, but from rationally integrating known immune or signaling circuits into novel, synergistic therapeutic strategies. Similarly, our study identifies CD24 as a new, functionally relevant phagocytosis checkpoint and establishes its dual blockade with CD47 as a mechanistically validated and translationally relevant approach for glioblastoma. This represents a conceptual advance in understanding and therapeutically exploiting innate-adaptive immune synergy in brain tumors.\n• No demonstration of direct causality: The study infers a mechanism from correlations and phenotypic observations, but does not directly link the pathway activation to the functional outcome (e.g., chromatin accessibility, transcription factor binding, or downstream effector knockout).\nResponse: We thank the reviewer for this important comment regarding mechanistic causality. We have established the functional requirement of the cGAS-STING pathway for dual blockade–mediated antitumor activity through both in vitro and in vivo experiments using STING-knockout (STING / ) mice. To further strengthen this causal link, we additionally performed a survival study using Ifnar1-knockout (Ifnar1 / ) mice in the GL261 model (Fig. 4g).\nActivation of the cGAS–STING pathway in macrophages⁻ i⁻nduces type I interferon release, which acts through IFNAR1 signaling on T cells to promote their priming and effector function. In Ifnar1 / mice, despite receiving the same dual-blockade treatment as wild-type controls, survival benefit was completely abrogated, demonstrating that type I interferon signaling is ⁻ ⁻essential for the therapeutic efficacy of CD24/CD47 dual blockade.\nWhile additional studies such as chromatin accessibility or transcription factor-binding analyses could provide deeper mechanistic insight into how interferon responses are transcriptionally enhanced, such detailed molecular characterization is beyond the scope of the current translational study, which focuses on validating the therapeutic potential of this combination strategy. We are actively pursuing these mechanistic investigations in a separate follow-up study aimed at dissecting the transcriptional and epigenetic regulation of the IFN response under dual phagocytosis-checkpoint blockade.\nFig. 4g\n• No temporal analysis: The authors do not assess whether changes in gene expression precede phenotypic outcomes (e.g., phagocytosis, tumor shrinkage). Time-course analysis would help establish causality.\nResponse: We thank the reviewer for this valuable suggestion. To address this point, we performed a temporal analysis of cGAS-STING pathway activation by western blotting in GL261 and bone marrow-derived macrophage (BMDM) co-cultures (Supplementary Fig. S16). Under dual-blockade conditions, phosphorylated STING (p-STING) became distinctly detectable at 6 hours post-treatment and remained elevated up to 24 hours, whereas phosphorylated IRF3 (p-IRF3) was modestly induced at 6 hours and peaked at 24 hours before declining by 48 hours. These kinetics indicate that cGAS-STING pathway activation precedes the onset of downstream functional responses, including enhanced phagocytosis and immune activation, which occur after 24 hours. Together, these data establish a temporal sequence linking early innate-sensing events to later phenotypic outcomes, thereby supporting the mechanistic causality of dual checkpoint blockade.\nSuppl. Fig. S16\nAdditional Critiques\n1. Line 68… The assumption that SB28 may be a progenitor like cell lines and the references cited are incorrect\nResponse: We thank the reviewer for pointing this out and for the opportunity to clarify our description of the SB28 model. We had cited Letchuman et al., Neurosurgical Focus (2022; 52: E5; doi:10.3171/2021.11.FOCUS21556), which described SB28 as follows:\n“Developed through transfection of the Sleeping Beauty (SB) transposon-flanked proto-oncogene, the SB28 cell line was initially induced in a neonatal C57BL/6 mouse. Since the model is driven by transfection of the human oncogene short hairpin targeting P53, PDGFB, and NRAS, it bears genetic resemblance to the group of GBMs once defined as proneural.”\nOur original use of the term “progenitor-like” was based on this description. However, we agree that this characterization may be ambiguous, as recent transcriptomic and immunogenomic studies have demonstrated that SB28 tumors exhibit mesenchymal-like molecular and immune features, including high CD44 and STAT3 expression, low MHC-I/II, and limited T-cell infiltration. To address this concern and improve accuracy, we have revised the text to state that:\n“The SB28 model was generated using PDGFB, NRAS, and p53-silencing constructs, drivers commonly associated with the proneural subtype but displays mesenchymal-like molecular and immune characteristics.”\nThis revised phrasing more accurately reflects both the genetic lineage and functional phenotype of SB28 and aligns with the current consensus in the field.\n2. Line 84: Misleading sentence, how is it optimal if there's no significant advantage\nResponse: We agree with the reviewer that the term “optimal” was not appropriate in the absence of a statistically significant difference. We have revised the wording to “highest numerical value” to address this comment (Line 85).\n5:3 and 1:3 are vastly different ratios, why is there such a difference to achieve an optimal phagocytosis\nResponse: CD24 expression levels vary across different cell lines, which may influence their sensitivity to CD24 blockade. As shown in Fig. 2a, SB28 reached a plateau at a lower anti-CD24 dose, whereas GL261 required a higher antibody concentration to achieve comparable effects. This difference explains why distinct antibody combination ratios were required to maximize phagocytosis in each model.\nFig. 2a\n3. How is their \"relative\" phagocytic index calculated.\nResponse: The relative phagocytic index was calculated by dividing the phagocytosis ratio of each treatment condition by the phagocytosis ratio of the IgG control within the same cell line. We also updated the methods section to enhance clarity: “For concentration-response experiments, relative phagocytic index was determined by normalizing the phagocytosis ratio of each treatment condition to that of the IgG control within the same cell line.”\n4. Line 98: This is an insufficient experiment\n1. Increase of ova presentation on macrophages makes sense and is a logical assay\n2. Using ova specific CD4 and CD8 cells does nothing to prove the crosstalk between innate and adaptive immunity. We need to see an in vivo assay demonstrating increased ova-specific CD4 and CD8 in response to ova expressing tumor cells.\nResponse: We thank the reviewer for this valuable comment and fully agree that additional in vivo evidence strengthens the link between macrophage-mediated innate activation and adaptive T cell responses. To directly address this point, we analyzed OVA-specific CD8 T cell responses in vivo using tetramer staining of splenocytes from GL261-cOVA tumor-bearing mice ⁺following antibody treatment (Fig. 3i).\nOur results show that neither anti-CD24 nor anti-CD47 monotherapy increased the proportion of OVA-tetramer CD8 T cells compared with the IgG control. In contrast, the dual-blockade treatment significantly expanded OVA-specific CD8 T cells in the spleen, indicating ⁺ ⁺enhanced antigen-specific T cell priming. These findings demonstrate that dual CD24/CD47 ⁺blockade promotes macrophage-mediated phagocytosis and antigen presentation, which in turn drive systemic expansion of tumor antigen-specific CD8 T cells, thereby providing in vivo evidence of innate-adaptive immune crosstalk.\n⁺\nFig. 3i\n5. In Fig 2, while the combination treatment leads to a modest increase in phagocytosis it seems to increase antigen presentation in a significant way. Please explain\nResponse: We appreciate the reviewer’s careful observation. According to other reports (Liu et al., ACS nano, 2025. doi: 10.1021/acsnano.5c09460) and our previous study (Lee et al., Nature biomedical engineering, 2024. doi: 10.1038/s41551-024-01194-7), phagocytosis and antigen presentation do not necessarily increase in a strict one-to-one fashion. While dual blockade induces a modest but clear increase in phagocytosis compared with other treatments, it synergistically activates the cGAS-STING and NF-κB pathways (Fig. 2i). Activation of these pathways promotes antigen processing, resulting in higher levels of antigen presentation.\n6. The cell line QPP8 appears to be most susceptible to phagocytosis however its has not been used in any of the subsequent studies.\nResponse: We have conducted the survival study using the QPP8 cell line (Suppl. Fig. S31). In this experiment, dual blockade significantly prolonged the survival of QPP8 tumor-bearing mice compared with the IgG controls.\nSuppl. Fig. S31\n7. Line 101: This experiment is insufficient to show cross-presentation as it's simply showing that activation of macrophages can cause secretion of cytokines that promote CD4, CD8 activity. The real issue that needs is addressing is whether these macrophages can prime an adaptive immune response by presenting neo-antigens from the ingested tumor cells\nResponse: We thank the reviewer for this important comment. We agree that our initial data were insufficient to conclusively demonstrate cross-presentation and adaptive T cell priming. To address this, we performed additional in vivo analyses of antigen-specific CD8 T cells using the GL261-cOVA tumor model, as detailed in our response to Comment #4 (Fig. 3i).\n⁺\nIn this experiment, dual CD24/CD47 blockade significantly increased the frequency of OVA-tetramer CD8 T cells in the spleen compared with the IgG and monotherapy groups, which showed no change. These results indicate that macrophage-mediated phagocytosis and ⁺ ⁺antigen processing under dual blockade led to effective cross-presentation and priming of antigen-specific CD8 T cells, thereby providing direct in vivo evidence that this combination therapy enhances adaptive immune activation downstream of innate phagocytic signaling.\n⁺\n8. Results from Fig 2i are unclear whether they are looking at signal from macrophages and tumor cells. It might be better to carry out phospho-flow to characterize what is happening to the macrophages post phagocytosis.\nResponse: We thank the reviewer for this helpful comment and the opportunity to clarify our methodology. The western blot analysis in Fig. 2i was performed after enrichment of macrophages from the co-culture system to exclude potential signal contamination from tumor cells. Thus, the detected phosphorylation and pathway activation signals originate specifically from macrophages following co-culture with glioma cells.\nTo make this distinction clearer for readers, we have revised the figure legend to specify “enriched macrophages” as the sample source. While phospho-flow cytometry could indeed provide complementary single-cell resolution, our current approach effectively isolates the macrophage population and ensures that the observed cGAS-STING activation is macrophage-derived.\n9. Line 153: A rescue experiment of using wt-macrophages in Sting-KO mice is required to nail down their hypothesis.\nResponse: We thank the reviewer for this thoughtful suggestion. We agree that a rescue experiment involving the introduction of wild-type macrophages into STING-knockout (STING / ) mice would be conceptually informative. However, there are significant technical and biological limitations that preclude a clear interpretation of such an approach. Effective ⁻⁻engraftment would first require selective depletion of endogenous macrophages in STING / mice, but currently available depletion strategies (e.g., CSF1R blockade, clodronate liposomes, ⁻ ⁻or conditional knockouts) cannot selectively eliminate brain-resident macrophages without broadly affecting other myeloid populations and disrupting the tumor microenvironment.\nMoreover, bone-marrow-derived macrophages (BMDMs), while technically feasible for transfer, differ markedly from brain-resident macrophages in transcriptional and functional identity, making it difficult to attribute any observed rescue specifically to STING-dependent effects. For these reasons, although we appreciate the conceptual value of the experiment, it is not technically feasible or mechanistically definitive in the context of the current study. Instead, our existing STING / and Ifnar1 / mouse models already provide robust genetic evidence supporting the requirement of the STING-IFN axis for the therapeutic efficacy of dual blockade.\n⁻ ⁻ ⁻ ⁻\nReviewer #3 (Remarks to the Author):\nThis manuscript by Ha and colleagues addresses a critical roadblock in glioblastoma immunotherapy. To date immunotherapy approaches have failed in GBM, and activation of phagocytosis in immunosuppressed macrophage environment has not been possible. The authors have noted the highly GBM-associated checkpoint CD24 on GBM cells, and found that Ab inhibition of CD24, in conjunction with CD47 inhibition can synergistically induce innate and adaptive anti tumor responses in mouse and human GBM cells. Triple inhibition with addition of anti-PD-L1 (which is induced by anti-CD24) resulted in even further anti-GBM phagocytic activity by tumor associated macrophages and T-cells, and even anti-tumor memory. These results open a novel potential avenue for combination checkpoint inhibitor immunotherapy for human GBM.\nOverall the work is substantiated by carefully planned experiments. I see no significant flaws in experimental design.\nMinor comments:\nFig. 2D: very difficult to see arrows pointing out phagocytosis. Please enlarge.\nResponse: We thank the reviewer for this attentive comment. As suggested, we have revised Fig. 2d with enlarged arrows to improve readability.\nFig. 2d\nFig. 2F: Please provide arrows to identify cells undergoing phagocytosis.\nResponse: We appreciate the reviewer for pointing this out. As suggested, we have added arrows in Fig. 2f to highlight the regions of antigen presentation.\nFig. 2f\nGeneral comment: Although beyond the scope of this project, could the authors briefly discuss current clinical trials with anti-CD24 and potential utility for GBM...off target risks, etc?\nResponse: We are grateful to the reviewer for the insightful comment that helped us to improve the discussion. As suggested, we added an additional paragraph at the end of the discussion section.\nLine 248\n“Currently, two phase I trials are ongoing in patients with solid tumors and B cell non-Hodgkin’s lymphoma (NCT06028373, NCT05985083). In the clinical study of ATN-031 developed by Antennova, no dose-limiting toxicities have been reported in the initial five patients, and one heavily pretreated case showed tumor shrinkage on CT imaging after completion of initial dosing cohort. These preliminary results point to the clinical promise of CD24 blockade and further suggest that dual blockade with anti-CD47 may also have therapeutic applicability in the clinical setting. Given the poor outcomes of PD-1 monotherapy in many clinical trials, a triple combination incorporating dual phagocytosis checkpoint blockade and PD-1 inhibition may provide a strategy to overcome resistance. Since CD24 expression is largely absent from most normal tissues, significant off-target toxicity is not expected. However, it is expressed on certain immune subsets, including B cells and cDC1. Given the essential role of cDC1 in antitumor immunity, engineering antibodies for tumor-targeted delivery of CD24 blockade will be beneficial to improve clinical translation.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors did a very good and thorough job responding to my comments.", "reviewer_2": "While the authors convincingly demonstrate that dual blockade of CD24 and CD47 enhances macrophage phagocytosis and elicits a measurable anti-tumor immune response, the mechanistic underpinnings of this effect remain insufficiently defined. The manuscript reports activation of the cGAS–STING pathway following dual blockade, suggesting induction of a type I interferon–driven inflammatory milieu. However, the data stop short of establishing a causal link between this pathway and the observed adaptive immune changes—particularly the upregulation of PD-1 and PD-L1. The authors neither elucidate whether this checkpoint upregulation arises as a feedback response to STING-mediated IFN signaling nor explore whether it reflects activation-induced exhaustion of effector T cells or adaptive resistance of tumor cells. Without experiments dissecting how dual blockade intersects with canonical IFN–STAT1 or NF-κB signaling cascades to drive PD-(L)1 expression, the mechanistic narrative remains incomplete. Additional molecular analyses—such as transcriptional profiling of myeloid and tumor compartments, pathway inhibition studies, or cytokine neutralization assays—would substantially strengthen the causal framework connecting innate sensing to adaptive checkpoint regulation.\nPrimary Concern: Antigen-presentation claims rely almost entirely on an exogenous model antigen (cOVA/SIINFEKL), not endogenous GBM antigens\nThe paper’s central bridge from enhanced phagocytosis to adaptive immunity hinges on cOVA-engineered tumor cells and readouts specific to SIINFEKL–H-2Kᵇ (25-D1.16 and tetramers). This is a powerful assay system, but it is non-physiologic and risks over-inferring generalizable cross-presentation. The authors do not validate that dual CD24/CD47 blockade increases presentation of native GBM antigens (or even non-OVA model antigens) by macrophages or cDC1s, nor do they demonstrate peptide–MHC I/II loading from endogenous sources (e.g., immunopeptidomics, antigen-agnostic T cell assays). They explicitly co-culture macrophages with cOVA-expressing GBM cells and then quantify SIINFEKL–H-2Kᵇ on BMDMs, followed by OT-I/OT-II proliferation—excellent controls, but still OVA-dependent. Even the figure legend emphasizes H-2Kᵇ–SIINFEKL imaging as the presentation readout.\nThis is critical as cOVA short-circuits key biological chokepoints (processing, proteasomal trimming, transporter bias, source-protein abundance, PTMs). Without endogenous antigen validation, the work cannot firmly claim that dual blockade generally improves tumor-antigen cross-presentation or that the adaptive effects they observe will persist outside SIINFEKL systems.\nMinor Concerns\nLine 88: \"CD24 expression may be related to the origin of the cells\" is a unfounded statement with no evidence to back it up including the 2 references which don't talk about CD24 expression.\nThe authors are using the anti-msCD47 Mab (MIAP-301) where as previously it has been shown by willingham et al. that Clone MIAP410 has significantly higher activity. Hence it suggests that they are not fully harnessing the effects of CD47-SIRPa blockade. This confounds the interpretation of the results. Phagocytosis of CT2A cells has been shown in numerous publications upon CD47 blockade (PMID 40912739, 30602457, 34625564) If the authors could speculate why they see limited response in their hands would be very useful.\nSimilarly if they express CD24 in their CT2A cells do they restore sensitivity to anti-CD24 mAb treatment.\nIn Line 132.. The authors claim nuclear translocation of IRF3 where as they are showing whole cell lysate blots. How did they confirm nuclear translocation without sub-cellular fractionation.\nThe reproducibility of the western blot data is also unclear as there is no mention of the no of times the experiment was repeated and no quantification of the blots shown hence its unclear how the significance was evaluated.\nCompounding this, the authors themselves note less pronounced synergy in human GBM cell lines in vitro, yet they do not reconcile this with the strong murine GL261 findings; this raises concern that the platform’s efficacy may be murine-model–biased (GL261 is comparatively immunogenic; SB28/QPP8 are more suppressive and human-like)."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #2 (Remarks to the Author):\nWhile the authors convincingly demonstrate that dual blockade of CD24 and CD47 enhances macrophage phagocytosis and elicits a measurable anti-tumor immune response, the mechanistic underpinnings of this effect remain insufficiently defined. The manuscript reports activation of the cGAS–STING pathway following dual blockade, suggesting induction of a type I interferon–driven inflammatory milieu. However, the data stop short of establishing a causal link between this pathway and the observed adaptive immune changes—particularly the upregulation of PD-1 and PD-L1. The authors neither elucidate whether this checkpoint upregulation arises as a feedback response to STING-mediated IFN signaling nor explore whether it reflects activation-induced exhaustion of effector T cells or adaptive resistance of tumor cells. Without experiments dissecting how dual blockade intersects with canonical IFN–STAT1 or NF-κB signaling cascades to drive PD-(L)1 expression, the mechanistic narrative remains incomplete. Additional molecular analyses—such as transcriptional profiling of myeloid and tumor compartments, pathway inhibition studies, or cytokine neutralization assays—would substantially strengthen the causal framework connecting innate sensing to adaptive checkpoint regulation.\nWe thank the reviewer for this important comment regarding the mechanistic connection between cGAS-STING-type I interferon axis and the upregulation of adaptive immune checkpoints. To strengthen this causal link between innate and adaptive immunity, we performed additional in vivo experiments.\nFirst, we measured IFN-α levels in plasma collected from tumor-bearing mice following IgG and dual blockade treatment to determine dual blockade-mediated type I interferon responses. Consistent with activation of the cGAS-STING pathway, dual blockade treatment significantly elevated levels of systemic IFN-α compared with IgG-treated controls (Suppl. Fig. S28).\nSuppl. Fig. S28\nNext, we blocked type I interferon receptor signaling using an anti-IFNAR1 antibody in combination with IgG or dual blockade treatment and determined PD-1 and PD-L1 expression with IHC analysis. Notably, dual blockade-mediated upregulation of PD-1 and PD-L1 expressions was significantly abrogated in mice receiving IFNAR1 blockade, suggesting that type I interferon signaling is important in stimulating PD-1 and PD-L1 expressions following dual blockade (Suppl. Fig. S39).\nSuppl. Fig. S39\nPrimary Concern: Antigen-presentation claims rely almost entirely on an exogenous model antigen (cOVA/SIINFEKL), not endogenous GBM antigens\nThe paper’s central bridge from enhanced phagocytosis to adaptive immunity hinges on cOVA-engineered tumor cells and readouts specific to SIINFEKL–H-2Kᵇ (25-D1.16 and tetramers). This is a powerful assay system, but it is non-physiologic and risks over-inferring generalizable cross-presentation. The authors do not validate that dual CD24/CD47 blockade increases presentation of native GBM antigens (or even non-OVA model antigens) by macrophages or cDC1s, nor do they demonstrate peptide–MHC I/II loading from endogenous sources (e.g., immunopeptidomics, antigen-agnostic T cell assays). They explicitly co-culture macrophages with cOVA-expressing GBM cells and then quantify SIINFEKL–H-2Kᵇ on BMDMs, followed by OT-I/OT-II proliferation—excellent controls, but still OVA-dependent. Even the figure legend emphasizes H-2Kᵇ–SIINFEKL imaging as the presentation readout.\nThis is critical as cOVA short-circuits key biological chokepoints (processing, proteasomal trimming, transporter bias, source-protein abundance, PTMs). Without endogenous antigen validation, the work cannot firmly claim that dual blockade generally improves tumor-antigen cross-presentation or that the adaptive effects they observe will persist outside SIINFEKL systems.\nWe are grateful for this insightful comment. To address this concern, we conducted an additional experiment using an endogenous GL261 antigen model. Imp3 has been identified as a GL261-derived immunodominant neoantigen, with AALLNKLYA as its H-2Db-restricted epitope. Using an AALLNKLYA-H-2Db tetramer, we quantified antigen-specific T cells within splenic CD8+ T cell populations. Consistent with our observations in the OVA system, dual blockade markedly increased endogenous antigen-specific CD8+ T cell responses compared with IgG-treated controls (Suppl. Fig. S30).\nSuppl. Fig. S30\nMinor Concerns\nLine 88: \"CD24 expression may be related to the origin of the cells\" is a unfounded statement with no evidence to back it up including the 2 references which don't talk about CD24 expression.\nWe agree with the reviewer and have removed this statement.\nThe authors are using the anti-msCD47 Mab (MIAP-301) where as previously it has been shown by willingham et al. that Clone MIAP410 has significantly higher activity. Hence it suggests that they are not fully harnessing the effects of CD47-SIRPa blockade. This confounds the interpretation of the results. Phagocytosis of CT2A cells has been shown in numerous publications upon CD47 blockade (PMID 40912739, 30602457, 34625564) If the authors could speculate why they see limited response in their hands would be very useful.\nWe appreciate the reviewer’s comment regarding the choice of anti-CD47 antibody. We agree that the differences between clones could influence the outcome of blockade depending on the context of models and treatments. Regarding the efficacy of these clones, conflicting results have been reported by different groups over the years (PMID: 28100392 and PMID: 33244513). In our hands, we performed a parallel comparison of the two anti-CD47 clones in combination with anti-CD24 and found no significant difference between the two clones (Suppl. Fig. S3).\nSuppl. Fig. S3\nWe also would like to thank the reviewer for suggesting additional references to get more information about published studies in the field. Despite they provide informative background on CD47 blockade in various cancer cells, those studies are not directly comparable to our experimental system. Specifically, PMID 40912739 reported that “Anti-CD47 treatment significantly increased the phagocytosis of SB28 cells, but not CT-2A cells compared with control (figure 2G–J)”, highlighting the limited efficacy of CD47 monotherapy in the CT-2A model. In PMID 30602457, phagocytosis of CT-2A cells under CD47 was not assessed but the study examined changes in microglial composition. Finally, PMID 34625564 evaluated an oncolytic herpesvirus expressing a full-length anti-human CD47 antibody in hCD47-expressing tumor models, which is not directly comparable to murine CD47 blockade in syngeneic CT-2A systems.\nSimilarly if they express CD24 in their CT2A cells do they restore sensitivity to anti-CD24 mAb treatment.\nWe thank the reviewer for this insightful suggestion. To address this point, CT-2A cells were transfected to express CD24 and their response to the dual blockade was evaluated. CD24 transfection restored sensitivity of CT-2A cells to the dual blockade, with significantly increased phagocytosis compared to the parental cell line (Suppl. Fig. S7).\nSuppl. Fig. S7\nIn Line 132.. The authors claim nuclear translocation of IRF3 where as they are showing whole cell lysate blots. How did they confirm nuclear translocation without sub-cellular fractionation.\nWe would like to clarify that phosphorylation of STING and IRF3 was assessed by immunoblots (Fig. 2i, Suppl. Fig. S17), and nuclear translocation of IRF3 was confirmed by confocal microscopy (Fig. 2j). We have revised the text to enhance clarity: “In line with this mechanism, dual blockade induced significant STING and interferon regulatory factor 3 (IRF3) phosphorylation in macrophages co-cultured with GL261 cells, as assessed by immunoblotting (Fig. 2i, Suppl. Fig. S15), whereas this effect was absent in CT-2A co-cultures (Suppl. Figs. S17, S18). Confocal microscopy further demonstrated nuclear translocation of phosphorylated IRF3 (pIRF3) in macrophages co-cultured with GL261 cells but not CT-2A cells (Fig. 2j).”\nThe reproducibility of the western blot data is also unclear as there is no mention of the no of times the experiment was repeated and no quantification of the blots shown hence its unclear how the significance was evaluated.\nWe would like to clarify that all western blot experiments were performed using three independent biological replicates. To improve clarity and transparency, we have now included the all relevant blots in the source data file. In addition, as suggested by the reviewer, we have added quantification of the immunoblot signals which indicate that dual blockade significantly activates the pathways compared with other treatments (Suppl. Figs. S15, S18, S21, and S34). The immunoblots with significant uneven background signals (e.g. spill over from ladder) were excluded from quantitative analysis but still provided to show trend change. These blots that were not used for quantification have been clearly indicated in the source data files.\nSuppl. Fig. S15\nSuppl. Fig. S18\nSuppl. Fig. S21\nSuppl. Fig. S34\nCompounding this, the authors themselves note less pronounced synergy in human GBM cell lines in vitro, yet they do not reconcile this with the strong murine GL261 findings; this raises concern that the platform’s efficacy may be murine-model–biased (GL261 is comparatively immunogenic; SB28/QPP8 are more suppressive and human-like).\nWe appreciate the reviewer for the valuable comment. Dual blockade also demonstrated tumor inhibitory efficacy in the immunosuppressive and human-like SB28 and QPP8 models (Fig. 5j-l and Suppl. Fig. S40), suggesting that this therapeutic strategy may be extendable toward clinical settings.\nFig. 5j-l\nSuppl. Fig. S40\nIn our in vitro studies using human GBM cell lines, the antibodies were not humanized, which may limit optimal engagement of human Fcγ receptors and reduce the phagocytic activity. The development and evaluation of humanized antibodies would represent informative direction for future investigation. To acknowledge this limitation and outline future directions, we have revised the Discussion to include the following statements: “While this study establishes robust preclinical efficacy in murine models, future investigation using humanized antibodies and humanized mouse model will be required to extend these findings toward clinical translation.”"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have satisfactorily answered my queries. I commend them on their study"}, "author_response": "We are pleased that our responses have addressed all the queries satisfactorily. We sincerely appreciate the reviewer’s thoughtful comments throughout the review process, which have significantly helped improve the quality of our manuscript."}]

Decision letter