Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP

Article

Blockade of the Phagocytic Receptor MerTK on TumorAssociated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP Graphical Abstract

Authors Yi Zhou, Mingjian Fei, Gu Zhang, ..., Søren Warming, Merone Roose-Girma, Minhong Yan

Correspondence [email protected]

In Brief Zhou et al. generate an antibody that selectively inhibits efferocytosis by the phagocytic receptor MerTK and show that MerTK blockade increases tumor immunogenicity and potentiates antitumor immunity via the transfer of tumorderived cGAMP into tumor-associated macrophages (TAMs) through the ATPgated channel P2X7R and subsequent STING activation.

Highlights d

Antibody blockade of MerTK prevents apoptotic cell clearance by macrophages

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MerTK blockade induces tumor-cGAS- and host-STINGdependent type I IFN response

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Extracellular ATP facilitates transfer of tumor-derived cGAMP to TAMs via P2X7R

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MerTK blockade increases tumor immunogenicity and enhances anti-PD-1/PD-L1 therapy

Zhou et al., 2020, Immunity 52, 1–17 February 18, 2020 ª 2020 Elsevier Inc. https://doi.org/10.1016/j.immuni.2020.01.014

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Immunity

Article Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP Yi Zhou,1,3 Mingjian Fei,1,3 Gu Zhang,1 Wei-Ching Liang,1 WeiYu Lin,1 Yan Wu,1 Robert Piskol,1 John Ridgway,1 Erin McNamara,1 Haochu Huang,1 Juan Zhang,1 Jaehak Oh,1 Jaina M. Patel,2 Diana Jakubiak,1 Jeff Lau,1 Beth Blackwood,1 Daniel D. Bravo,1 Yongchang Shi,1 Jianyong Wang,1 Hong-Ming Hu,2 Wyne P. Lee,1 Rajiv Jesudason,1 Dewakar Sangaraju,1 Zora Modrusan,1 Keith R. Anderson,1 Søren Warming,1 Merone Roose-Girma,1 and Minhong Yan1,4,* 1Genentech

Inc., South San Francisco, CA, USA Immunobiology Laboratory, Earle A. Chiles Research Institute, Portland, OR, USA 3These authors contributed equally 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2020.01.014 2Cancer

SUMMARY

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or antiPD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas
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mice. Abolishing cGAMP production in Cgas
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tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy. INTRODUCTION The most clinically developed cancer immunotherapies target the inhibitory pathways that negatively regulate T cell activation and effector functions (Buchbinder and Desai, 2016; Ribas and Wolchok, 2018). Whereas durable responses have been observed in some patients, the overall response rate is still low (Dempke et al., 2017). The immunological contexture of a tumor appears to be prognostic for therapeutic outcome. Tumors with pre-existing CD8+ T cell infiltrates tend to exhibit better clinical

response (Galon et al., 2016; Page`s et al., 2009; Tumeh et al., 2014). Multiple mechanisms might contribute to the poor T cell infiltration and activity in non-responding tumors. The suppression of innate immune sensing of tumor-derived factors could be one of them. In homeostasis, apoptosis is thought to be immunologically quiescent (Arandjelovic and Ravichandran, 2015; Nagata, 2018). Apoptotic cells are cleared before their plasma membrane integrity is compromised and cellular contents are released, thus preventing inappropriate inflammatory response. Clearance of apoptotic cells in different tissues is performed by phagocytic cells such as macrophages and involves various receptors including MER proto-oncogene tyrosine kinase (MerTK). MerTK belongs to a receptor tyrosine kinase family that includes Tyro3 and Axl and is uniquely important for maintaining tissue homeostasis and tissue remodeling (Lemke, 2013; Rothlin et al., 2015). Aided by bridging molecule growth arrest specific 6 (GAS6) or protein S, MerTK facilitates the removal of dying and/or damaged cells that display the ‘‘eat me’’ signal, phosphatidylserine (PtdSer), on the cell surface. MerTK-expressing macrophages engulf apoptotic cells via efferocytosis (Graham et al., 2014). In malignant solid tumors, uncontrolled proliferation can cause elevated cellular stress and increased apoptosis of cancer cells. MerTK-dependent clearance of dying cells by tumor-associated macrophages (TAMs) might inhibit immune activation in response to tumor cell death. Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling has been implicated in innate immune sensing of cancer cells (Deng et al., 2014; Wang et al., 2017; Woo et al., 2014). The cytosolic DNA sensor cGAS is activated by double stranded DNA (dsDNA) and catalyzes the synthesis of cyclic GMP-AMP (cGAMP) (Ablasser et al., 2013; Sun et al., 2013). As a second messenger, cGAMP activates the adaptor protein STING, which in turn triggers a TANK-binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3)-dependent signaling process leading to the production of pro-inflammatory cytokines, including type I interferons (IFNs) (Chen et al., 2016; Wu et al., 2013). Current data support an important role of this pathway in anti-tumor immunity. STING signaling in immune Immunity 52, 1–17, February 18, 2020 ª 2020 Elsevier Inc. 1

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

cells is needed for type-I-IFN-dependent spontaneous T cell priming, which is a key determinant for therapeutic efficacy of immune checkpoint inhibitors (Wang et al., 2017; Woo et al., 2014) and radiation therapy (Deng et al., 2014). The mechanisms that activate cGAS-STING signaling in the tumor context remain unclear, though some evidence suggests that DNA from dying tumor cells is the initial trigger of cGAS-STING signaling and other studies pointing to the transfer of cGAMP from tumor cells to immune cells in the tumor microenvironment (TME) (Ahn et al., 2018; Marcus et al., 2018; Schadt et al., 2019). Here, we examined the effect of blocking MerTK-mediated efferocytosis on innate immune sensing of cancer cell death and the associated anti-tumor immune response. Blockade of MerTK-mediated phagocytic clearance of dying tumor cells resulted in enhanced anti-tumor immune responses that were dependent on type I IFN. The induction of type I IFN response was driven by tumor-cell-expressed cGAS that transactivated STING in host cells. The entry of tumor-cell-produced cGAMP into host cells was facilitated by the ATP-gated P2X purinoceptor 7 (P2X7) receptor (P2X7R). Thus, blockade of MerTK presents a therapeutic avenue to increase tumor immunogenicity and improve cancer immunotherapy. RESULTS Developing a Functional Antibody to Block MerTKMediated Efferocytosis To explore the therapeutic potential of targeting MerTK, we developed a function-blocking antibody (Figure 1A). The selected antibody specifically interacted with MerTK but not Tyro3 and Axl or other surface proteins on macrophages (Figures 1B and S1A) and inhibited ligand-induced MerTK signaling in macrophages as measured by MerTK auto-phosphorylation and downstream pAKT (Figures S1B and S1C). We further characterized the anti-MerTK antibody in a biologically relevant cellbased assay assessing apoptotic cell uptake by macrophages. We used mouse peritoneal macrophages as phagocytes and pHrodo-labeled apoptotic thymocytes as target cells. pHrodo is a pH-sensitive dye that increases fluorescence intensity in acidic environments and can serve as an indicator of phagocytic events associated with acidification (Figure 1C). Anti-MerTK treatment substantially reduced the engulfment of apoptotic thymocytes by peritoneal macrophages (Figure 1D). To further validate the antibody in vivo, we treated mice with dexamethasone to induce apoptosis in the thymus. Apoptotic and dead thymocytes were readily detected 8 h after dexamethasone administration. By 24 h, most of the dying or dead cells were cleared by thymic resident macrophages (Figure S1D). In comparison, the clearance was mostly compromised in mice treated with antiMerTK antibody (Figure 1E). This in vivo result was consistent with the defective efferocytosis observed in MerTK-deficient mice (Scott et al., 2001), demonstrating the functional effectiveness of the anti-MerTK antibody. MerTK Blockade Causes Accumulation of Apoptotic Cells in Tumors TAMs are among the most abundant tumor-infiltrating immune cells across multiple indications and tumor types (Thorsson et al., 2018). We analyzed MC38 syngeneic murine colon adeno2 Immunity 52, 1–17, February 18, 2020

carcinoma tumors growing in wild-type (WT) or Mertk
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mice and detected specific expression of MerTK in TAMs but not tumor cells (Figure 2A). Flow-cytometric analysis further confirmed that MerTK was predominantly expressed by TAMs, but not other cell types, including tumor-associated dendritic cells (DCs) or monocytes (Figures 2B and S2A). In addition, analysis of data compiled by The Cancer Genome Atlas (TCGA) showed that MerTK expression exhibited greater correlation with the abundance of TAMs compared with other immune cell types in human cancers (Figure S2B), consistent with MerTK being mainly expressed by TAMs. In in vitro efferocytosis assay, TAMs from MC38 tumors engulfed apoptotic cells and antiMerTK antibody inhibited this uptake (Figure S2C). To explore the role of MerTK in clearing dying cells in tumors, we treated MC38-tumor-bearing mice with anti-MerTK antibody. Tumors were collected 24 h after antibody treatment. We quantified apoptotic cells by immunofluorescence staining of tumor sections by using an antibody recognizing the cleaved-Caspase 3 (c-Casp3), a hallmark of apoptosis. MerTK inhibition resulted in an increased number of apoptotic cells in tumors, consistent with impaired dying cell clearance (Figures 2C and 2D). Compromised removal of dying cells could lead to the progression to secondary necrosis and subsequent release of intracellular contents, including DNA. Cell-free DNA (cfDNA) in blood circulation is released by damaged or dead cells (Wan et al., 2017). In cancer patients or tumor-bearing mice, a subpopulation of cfDNA is tumor derived, called circulating tumor DNA (ctDNA). In the MC38 tumor model, we were able to utilize a SNP to distinguish host-derived cfDNA from tumor-derived ctDNA. Three days after anti-MerTK treatment, we detected increased ctDNA in the plasma of tumor-bearing mice (Figure 2E). Anti-MerTK treatment also increased host-derived cfDNA in blood circulation (Figure 2E). Thus, MerTK promotes the rapid clearance of apoptotic cells by TAMs in the TME. MerTK Blockade Enhances Anti-tumor Response Uncleared dying cells release intracellular contents as a result of progressive loss of plasma membrane integrity. Some of these released molecules can serve as damage-associated molecular patterns (DAMPs) and be recognized by the immune system, triggering an inflammatory response. Given the observed effect of MerTK blockade on dying cell clearance in tumors, we investigated the effect of anti-MerTK antibody on tumor growth. AntiMerTK treatment as a single agent modality at an early stage of tumor progression reduced the growth of MC38 tumors (Figure 3A). In comparison, anti-MerTK treatment did not affect tumor growth in Mertk
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mice (Figure S3A), validating targetspecific anti-tumor response. Mice that had previously rejected tumors after anti-MerTK treatment were protected upon re-challenge, whereas in the control group of naive mice, only 3 out of 15 mice were tumor-free (Figure S3B), indicating that the mice with prior anti-MerTK treatment developed a long-term anti-tumor memory. Given that the anti-tumor effect of MerTK blockade is likely linked to the impaired dying cell clearance, the prediction would be that tumors that are intrinsically less prone to apoptosis should be less sensitive to anti-MerTK treatment. To test this hypothesis, we generated Bax
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Bak
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MC38 cells (Figure S3C). In cell culture, Bax
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Bak
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MC38 cells had similar growth rate as the parental cells, but were resistant to apoptosis induced by

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 1. Developing a Functional Antibody to Block MerTK-Mediated Efferocytosis (A) Schematic depiction of MerTK-dependent efferocytosis. Anti-MerTK antibody blocks the interaction between MerTK and Gas6 or ProS. (B) Flow-cytometric analysis of the expression of MerTK on WT and Mertk
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peritoneal macrophages (MF) by using AF488-conjugated anti-MerTK antibody. Data are representative of two independent experiments. (C) Schematic depiction of in vitro efferocytosis assay. Apoptotic thymocytes (ACs) labeled with pH-sensitive pHrodo dye were co-cultured with MF. Unengulfed ACs were washed away after co-culture. Engulfed ACs exhibited increased fluorescence intensity (red) in acidic environment. (D) Anti-MerTK antibody blocked the uptake of ACs (red) by MF (green). Data are representative of three independent experiments. Scale bar, 50 mm. (E) Evaluating the in vivo clearance of apoptotic cells in the thymus. Mice were dosed with a control antibody (control Ab) or anti-MerTK antibody (anti-MerTK) 1 h prior to dexamethasone treatment. Thymic tissues were harvested 24 h after dexamethasone administration and analyzed by flow cytometry (n = 4 mice). FITCVAD-FMK and PI were used to detect active caspases-positive apoptotic cells and dead cells, respectively. Data are representative of two independent experiments. All mice were in C57BL/6N background. Abbreviation is as follows: Rx, antibody treatment. In (D) and (E), error bar represents mean ± SD. ***p < 0.001, unpaired two-tailed Student’s t test. See also Figure S1.

Immunity 52, 1–17, February 18, 2020 3

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 2. MerTK Blockade Causes Accumulation of Apoptotic Cells in Tumors (A) Tumor sections of MC38 tumors growing in WT and Mertk
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mice were stained with anti-CD68 (green) and anti-MerTK (red) antibodies. MerTK staining signal was not detected in MC38 tumors growing in Mertk
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mice, indicating that MC38 tumor cells do not express MerTK. (B) Flow-cytometric analysis of MerTK expression in different immune cell subsets from MC38 tumors. The anti-MerTK antibody is in blue, and the isotype control antibody is in gray. (C) Representative fluorescence microscopy images of MC38 tumor sections stained with anti-cleaved Caspase 3 (c-Casp3) antibody. MC38 tumors were collected 24 h after antibody treatment. (D) Quantification of c-Casp3+ cells in non-necrotic regions of MC38 tumors (control Ab, n = 5 mice; anti-MerTK, n = 6 mice). (E) Quantification of ctDNA and host-derived cfDNA in the plasma collected from MC38-tumor-bearing mice treated with a control Ab or anti-MerTK (control Ab, n = 9 mice; anti-MerTK, n = 10 mice). Data are representative of two independent experiments. Mice used in (A)–(D) were in C57BL/6N background. Mice in (E) were in C57BL/6J background to differentiate host- versus tumor-derived cfDNA. Abbreviation is as follows: Rx, antibody treatment. In (D) and (E), error bar represents mean ± SD. *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test. Scale bars are as follows: 50 mm in (A), 1 mm in (C), and 100 mm in (C) insert. See also Figure S2.

apoptotic insults, e.g., UV and Bcl2 and Mcl1 inhibitors (Figure S3D). Treatment with anti-MerTK antibody had no effect on the growth of Bax
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Bak
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MC38 tumors (Figure S3E), consistent with the notion that prolonged accumulation of dying tumor cells drives the anti-tumor response of MerTK blockade. Next, we tested the potential of anti-MerTK antibody in treating fully established tumors (typically two weeks after tumor inoculation). In this intervention setting, anti-MerTK antibody alone had a marginal effect (Figure 3B). Both clinical and preclinical studies have shown checkpoint inhibitors such as antiprogrammed death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) antibodies enhance anti-tumor immune responses by removing a key inhibitory signal that regulates T cell activation. We therefore combined anti-MerTK with 4 Immunity 52, 1–17, February 18, 2020

anti-PD-L1 antibodies to treat established tumors. Single-agent anti-PD-L1 treatment exhibited modest anti-tumor activity. In contrast, combination of anti-MerTK and anti-PD-L1 treatment (Figure 3B) or anti-MerTK and anti-PD-1 treatment (Figure 3C) resulted in robust anti-tumor responses. Given that the effect of MerTK blockade is likely linked to the failed clearance of dying cells in tumors (Figures 2C and 2D), we investigated the anti-tumor response mediated by anti-MerTK treatment in combination with a cytotoxic agent that increases tumor cell death, gemcitabine. In the MC38 tumor model, a single dose of gemcitabine had little effect on tumor growth and moderately improved anti-PD-1 therapy (Figure 3C). In contrast, addition of anti-MerTK antibody to the combination therapy of gemcitabine plus antiPD-1 antibody resulted in complete regression of all treated

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 3. MerTK Blockade Enhances Anti-tumor Response (A) Assessing the growth of MC38 tumors in mice treated with a control Ab or anti-MerTK. Treatment started 4 days after tumor cell inoculation (n = 10 mice). Data are representative of two independent experiments. (B) Assessing the growth of MC38 tumors in mice treated with the indicated antibodies (n = 10 mice). Treatment started 14 days after tumor cell inoculation. Data are representative of two independent experiments. (C) Assessing the growth of MC38 tumors in mice treated with the indicated therapies (control Ab group, n = 15 mice; anti-PD-1 + anti-MerTK group, n = 8 mice; other groups, n = 10 mice). Abbreviation is as follows: Gem, gemcitabine. Data are representative of two independent experiments. (D) Assessing the growth of E0771 tumors in mice treated with the indicated antibodies (n = 10 mice). Treatment started 15 days after tumor cell inoculation. All mice were in C57BL/6N background. Abbreviation is as follows: Rx, antibody treatment. Both individual tumor growth curves and linear mixed-effects (LME)fitted tumor growth curves of each group are presented. See also Figure S3.

tumors. Anti-MerTK treatment similarly improved the efficacy of anti-PD-L1 antibody in another syngeneic tumor model, the E0771 murine model of triple negative breast cancer (Figure 3D).

Together, these results demonstrate the potential of targeting MerTK-mediated dying cell clearance to improve cancer immunotherapies. Immunity 52, 1–17, February 18, 2020 5

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 4. MerTK Blockade Induces Rapid Local Type I IFN Response in Tumors (A) Transcriptome analysis by RNA-seq of TAMs isolated from mice treated with a control Ab or anti-MerTK (n = 5 mice). Heatmap shows the changes in gene expression. (B) Gene set enrichment analysis of IFN-a response genes using data from (A). (C) RT-qPCR analysis of mRNA expression of Ifnb1 and representative ISGs in TAMs (control Ab, n = 9 mice; anti-MerTK, n = 7 mice,). Data are representative of two independent experiments. (D) Measurement of IFN-b protein in tumor homogenates (control Ab, n = 8 mice; anti-MerTK, n = 9 mice,). Tumors were collected three days after antibody treatment. Data are representative of two independent experiments. (legend continued on next page)

6 Immunity 52, 1–17, February 18, 2020

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

MerTK Blockade Induces Rapid Local Type I IFN Response in Tumors We next investigated the mechanism underlying the anti-tumor effect of MerTK blockade. Because, in tumors, MerTK is predominantly expressed on TAMs, we first examined the effect of MerTK inhibition on TAMs. We isolated TAMs from established MC38 tumors (Figures S4A and S4B) and performed transcriptome analysis. Treatment of TAMs with anti-MerTK antibody for 24 h resulted in changes in gene expression (Figures 4A and S4C), and gene set enrichment analysis (GSEA) revealed a prominent type I IFN gene signature in these cells (Figures 4B and S4D), which was confirmed by qPCR analyses of Ifnb1 and multiple interferon-stimulated genes (ISGs) (Figures 4C and S4E). Consistent with increased Ifnb1 expression, treatment of TAMs with anti-MerTK antibody increased IFN-b protein in tissue homogenates prepared from freshly dissected tumors (Figure 4D). Type I IFNs activate the autocrine and/or paracrine production of cytokines and chemokines that modulate innate and adaptive immune responses (Ivashkiv and Donlin, 2014; Zitvogel et al., 2015). After anti-MerTK treatment, besides the increased mRNA expression of ISGs in tumor samples (Figures S4F and S4G), we detected elevated protein expression of CCL3, CCL4, CCL5, CCL7, and CCL12 in tumor tissue homogenates (Figure 4E). The type I IFN response appeared to be restricted to the tumor site because no significant changes in ISG expression were detected in peripheral blood mononuclear cells (PBMCs) collected from tumor-bearing mice treated with anti-MerTK antibody (Figure 4F). Therefore, MerTK blockade locally transforms the TME toward a type-I-IFN-biased milieu. Type I IFN signaling positively regulates antigen presentation and T cell activation. Previous studies have also established its essential role in spontaneous and therapy-induced anti-tumor immune response (Diamond et al., 2011; Fuertes et al., 2011; Minn and Wherry, 2016; Zitvogel et al., 2015). To determine the functional importance of type I IFN signaling for the anti-tumor effect triggered by anti-MerTK treatment, we utilized a function-blocking antibody against interferon-alpha/beta receptor 1 (IFNAR1). Anti-IFNAR1 treatment completely abolished the anti-tumor activity of anti-MerTK antibody (Figure 4G). Therefore, the anti-tumor effect of MerTK blockade depends on intact type I IFN signaling. Besides the well-established role in phagocytic clearance of dying cells, MerTK signaling is linked to changes in cytokine production, including interleukin-10 (IL-10), transforming growth factor-b (TGF-b), IL-6, and IL-12 (Akalu et al., 2017; Cook et al., 2013; Crittenden et al., 2016; Stanford et al., 2014). In our tumor studies, we did not observe any significant changes in these cytokines after anti-MerTK treatment (Figure S4H). A previous study

has suggested that Tyro3-Axl-MerTK activation might suppress type I IFN signaling through increasing the expression of negative regulators such as suppressor of cytokine signaling 1 (SOCS1) and SOCS3 (Rothlin et al., 2007). Again, in our tumor studies, we did not detect any significant changes in the expression of SOCS1, SOCS3, or type I IFN receptors (Figures S4I and S4J). MerTK Blockade Enhances Anti-tumor T Cell Response Having demonstrated that MerTK blockade induced the production of IFN-b, we next investigated the source of IFN-b. In MC38 tumors, we found that the increase of Ifnb1 mRNA expression was restricted to CD45+ immune cells. In addition, the basal mRNA expression of IFN-b was much higher in CD45+ immune cells than in CD45
cells (Figure 5A). Within the tumor-infiltrating immune cells, we focused on TAMs and tumor-infiltrating DCs. In MC38 tumors, TAMs were considerably more abundant than DCs (Figure S5A). In response to MerTK blockade, Ifnb1 mRNA expression was increased in TAMs, but not DCs (Figure 5A). To further confirm that TAMs were the major source of IFN-b, we depleted TAMs by using an antibody against colony-stimulating factor 1 receptor (CSF1R), which is key to the survival of macrophages. Anti-CSF1R treatment effectively depleted TAMs but had little effect on tumor-infiltrating DCs (Figure S5B). In addition, in anti-CSF1R-antibody-treated tumors, the type I IFN response induced by anti-MerTK treatment was lost (Figure 5B). Batf3-dependent DCs are needed for priming naive CD8+ T cell against tumor-derived antigens. Batf3
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mice are defective in rejecting highly immunogenic tumors or responding to checkpoint inhibitors and immunostimulatory antibodies (Fuertes et al., 2011; Hildner et al., 2008; Salmon et al., 2016; Sa´nchez-Paulete et al., 2016). We found that the tumor inhibiting activity of anti-MerTK antibody was also lost in Batf3
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mice (Figure S5C), indicating that the anti-tumor CD8+ T cell response initiated by Batf3-dependent DCs is a prerequisite for the anti-tumor effect of MerTK blockade. On the other hand, anti-MerTK treatment was still able to induce the type I IFN response in tumors growing in Batf3
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mice (Figure S5D). This observation demonstrated that Batf3-dependent DCs were dispensable for the type I IFN production after MerTK blockade, consistent with our finding that TAMs were the major source of IFN-b. Type I IFNs positively regulate various aspects of antigen-presenting cells (APCs), including maturation, antigen presentation, and cross-priming of cytotoxic T cells (Zitvogel et al., 2015). Given that MerTK blockade induced a type I IFN response within the tumor, we investigated whether antigen presentation by TAMs and tumor-associated DCs was enhanced. To study antigen presentation, we used a tumor model of MC38 tumors expressing ovalbumin (MC38.OVA). In the MC38.OVA tumor

(E) Assessing protein expression of chemokines in tumor homogenate (control Ab, n = 8 mice; anti-MerTK, n = 9 mice). Tumors were collected three days after antibody treatment. Data are representative of two independent experiments. (F) RT-qPCR analysis of mRNA expression of representative ISGs in PBMCs (control Ab, n = 7 mice; anti-MerTK, n = 9 mice,). Data are representative of two independent experiments. (G) Assessing the growth of MC38 tumors in mice treated with the indicated antibodies. Anti-IFNAR1 antibody abolished the anti-tumor activity of anti-MerTK treatment (n = 10 mice). Both individual tumor growth curves and LME-fitted tumor growth curves of each group are presented. Data are representative of two independent experiments. All mice were in C57BL/6N background. Abbreviations is as follows: Rx, antibody treatment. In (C)–(F), error bar represents mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; unpaired two-tailed Student’s t test. See also Figure S4.

Immunity 52, 1–17, February 18, 2020 7

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 5. MerTK Blockade Enhances Anti-tumor T Cell Response (A) RT-qPCR analysis of mRNA expression of Ifnb1 in different cell populations in tumors (CD45+ and CD45
, n = 4 mice; TAM and DC, n = 5 mice). (B) RT-qPCR analysis of mRNA expression of representative ISGs in MC38 tumors treated with indicated antibodies. (C) Flow-cytometric analysis of the expression of H-2Kb-SIINFEKL on TAMs and CD103+ DCs and CD11b+ DCs in MC38.OVA tumors (n = 6). Non-OVA MC38 tumors were used as negative controls for detection specificity. Data are presented as median fluorescence intensity (MFI) subtracted by baseline signal (MFI of isotype control). ND, not detectable. Data are representative of two independent experiments. (legend continued on next page)

8 Immunity 52, 1–17, February 18, 2020

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

model, the presentation of an ovalbumin-derived SIINFEKL peptide in complex with mouse major histocompatibility complex (MHC) class I H-2Kb molecule can be readily monitored. AntiMerTK treatment increased the H2Kb-SIINFEKL presentation and CD86 (a co-stimulatory molecule for T cell activation) expression on TAMs, but not CD103+ DCs. (Figures 5C, 5D, and S5E). Furthermore, TAMs purified from MC38.OVA tumors treated with anti-MerTK antibody showed increased capability to cross-prime OVA-specific CD8+ T cells (OT-I cells) ex vivo (Figure S5F). TAMs often display the alternatively activated or ‘‘M2-like’’ phenotypes and have properties of suppressing inflammatory response and promoting tumor growth (Mantovani et al., 2002). Anti-MerTK treatment decreased the expression of CD206, an ‘‘M2-like’’ macrophage marker, on TAMs (Figure S5G). These findings suggest that MerTK blockade induces an immunogenic reprogramming of the TME, which in turn enhances the adaptive T cell response. Tumor-infiltrating lymphocyte (TIL) clonality reflects the frequency of T cells with a specific T cell receptor (TCR) chain usage at the tumor site. Anti-MerTK treatment led to an increase in TIL clonality and the frequency of top 10 TCR clones, indicating clonal expansion of antigen-specific TILs (Figure 5E). In addition, anti-MerTK treatment increased the intratumoral frequency of total CD8+ T cells, as well as tumor-antigen-specific CD8+ T cells, e.g., T cells that recognize p15e, an endogenously expressed retroviral tumor antigen in MC38 tumor cells (Yang and Perry-Lalley, 2000) (Figure 5F). Furthermore, anti-MerTK treatment increased the frequency of intratumoral Ki67+ proliferating CD8+ T cells (Figure S5H). In comparison, no significant changes in the frequency of Ki67+ or tetramer+CD8+ T cells were found in the tumor draining lymph nodes (dLNs) (Figure S5H), indicating that MerTK blockade primarily enhances CD8+ T cell responses at the tumor site. This result is consistent with the aforementioned type I IFN response observed in tumors growing in Batf3
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mice after anti-MerTK treatment (Figure S5D) and TAMs being the major source of IFN-b (Figure 5A). To demonstrate that the enhanced CD8+ T cell response was responsible for the anti-tumor effect, we depleted CD8+ T cells with an anti-CD8a antibody. Depletion of CD8+ T cells abolished the anti-tumor effect of anti-MerTK treatment (Figure 5G). Therefore, MerTK blockade triggers a CD8+ T-cell-dependent anti-tumor response. Tumor cGAS Dictates Anti-tumor Effect of MerTK Blockade The STING pathway has emerged as a key signaling mechanism that drives the anti-tumor type I IFN response (Deng et al., 2014; Woo et al., 2014). To determine the role of STING signaling for the anti-tumor effect of MerTK blockade, we carried out tumor

studies in STING-defective (Stinggt/gt) mice (Sauer et al., 2011). In contrast to WT mice, anti-MerTK treatment failed to increase the expression of ISGs in tumors growing in Stinggt/gt mice (Figure 6A). Furthermore, in Stinggt/gt mice the anti-tumor effect of MerTK or PD-L1 inhibition was lost (Figures 6B and S6A), demonstrating the requirement for STING in host cells. Although STING activation is important for innate immune sensing of tumor DNA (Woo et al., 2014; Xu et al., 2017), it remains an open question how tumor DNA triggers STING activation in host cells. We confirmed in vitro that loss of cGAS or STING in macrophages abolished the production of IFN-b in response to transfected DNA (Figure S6B). To examine the role of cGAS in host cells in vivo, we performed tumor studies in Cgas
/
mice. Surprisingly, anti-MerTK treatment still induced a type I IFN response and inhibited MC38 tumor growth (Figures 6C and 6D), indicating cGAS in host cells is dispensable for the MerTK blockade effect. This apparent discrepancy between loss of STING and loss of cGAS in host cells prompted us to investigate the possible involvement of cGAS expressed by tumor cells. We generated Cgas
/
MC38 cells by CRISPR/Cas9 technology and confirmed the cells lost the ability to produce cGAMP (Figures S6C and S6D). To investigate the significance of tumor-derived cGAS in vivo, we carried out tumor studies with Cgas
/
MC38 cells. After anti-MerTK treatment, the type I IFN response observed in WT MC38 tumors was completely lost in Cgas
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MC38 tumors (Figure 6E). In addition, cGAS deficiency rendered tumors resistant to anti-MerTK treatment (Figure 6F). The anti-tumor effect of anti-PD-L1 antibody was also lost in Cgas
/
MC38 tumors (Figure S6E). To rule out the potential off-target effect of gene-editing, we reconstituted Cgas
/
MC38 tumor cells with WT cGAS or catalytically inactive cGAS (GS > AA mutant) (Figure S6F). Reconstitution with WT cGAS substantially elevated the baseline ISG expression in tumors, to an amount even higher than the WT MC38 tumors treated with anti-MerTK antibody (Figure S6G). This increase was likely due to the much higher expression of exogenous cGAS compared with the endogenous cGAS in WT MC38 (Figures S6F and S6G). In fact, WT cGAS-reconstituted tumor cells failed to form sustained tumors even in the absence of anti-MerTK treatment. In contrast, reconstitution with the GS > AA mutant cGAS failed to restore the type I IFN response and anti-tumor effect after MerTK inhibition (Figures S6G and S6H), indicating a critical role of the enzymatic activity of tumor cGAS. Therefore, cGAS expressed in tumor cells dictates the anti-tumor effect of MerTK blockade. Type I IFN Response Induced by MerTK Blockade Requires ATP-gated P2X7R Channel In stressed or dying tumor cells, self-DNA released from damaged nuclei or mitochondria can get access to and activate

(D) Flow-cytometric analysis of the expression of CD86 on TAMs and CD103+ DCs and CD11b+ DCs in MC38.OVA tumors (n = 8). Data are representative of two independent experiments. (E) TCR clonality analysis of tumor-infiltrating T cells in MC38 tumors (control Ab, n = 9 mice; anti-MerTK, n = 6 mice). (F) Flow-cytometric analysis of the frequency of CD8+ and CD4+ T cells, and p15e tetramer-reactive T cells in MC38 tumors (n = 10 mice). (G) Assessing the growth of MC38 tumors in mice treated with the indicated antibodies. CD8+ T cell depletion by using an anti-CD8 antibody abolished the antitumor activity of anti-MerTK treatment (n = 10 mice). Both individual tumor growth curves and LME-fitted tumor growth curves of each group are presented. All mice were in C57BL/6N background. Abbreviations are as follows: Rx, antibody treatment; MFI, median fluorescence intensity. In (A)–(F), error bar represents mean ± SD. *p < 0.05, **p < 0.01; n.s., not significant; unpaired two-tailed Student’s t test. See also Figure S5.

Immunity 52, 1–17, February 18, 2020 9

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 6. Tumor CGAS Dictates Anti-tumor Effect of Anti-MerTK (A) RT-qPCR analysis of mRNA expression of representative ISGs in MC38 tumors growing in Stinggt/gt mice (n = 10 mice). (B) Assessing MC38 tumor growth in WT or Stinggt/gt mice treated with a control Ab or anti-MerTK (n = 10 mice). Data are representative of two independent experiments. (C) RT-qPCR analysis of mRNA expression of representative ISGs in MC38 tumors growing in Cgas
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mice (n = 9 mice). (D) Assessing MC38 tumor growth in WT or Cgas
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mice treated with a control antibody or anti-MerTK antibody (control Ab, n = 8 mice; anti-MerTK antibody, n = 10 mice). Data are representative of two independent experiments. (E) RT-qPCR analysis of mRNA expression of representative ISGs in Cgas
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MC38 tumors growing in WT mice (n = 10 mice). (F) Assessing the growth of WT or Cgas
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MC38 tumors in WT mice treated with a control Ab or anti-MerTK (n = 10 mice). Data are representative of two independent experiments. Mice in (A) and (B) were bred in C57BL/6J background. Mice in (C)–(F) were in C57BL/6N background. Abbreviation is as follows: Rx, antibody treatment. In (A), (C), and (E) error bars represents mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; unpaired two-tailed Student’s t test. In (B), (D), and (F), both individual tumor growth curves and LME-fitted tumor growth curves of each group are presented. See also Figure S6.

€ck et al., the cytosolic DNA sensor cGAS (Ahn et al., 2018; Glu 2017; Harding et al., 2017; Mackenzie et al., 2017; McArthur et al., 2018; Riley et al., 2018; Rongvaux et al., 2014; White et al., 2014; Yang et al., 2017), leading to the production of 10 Immunity 52, 1–17, February 18, 2020

cGAMP. We confirmed that unlike the parental MC38 cells, Cgas
/
MC38 cells were unable to produce cGAMP in response to DNA stimulation (Figure S6D). The finding that anti-MerTK treatment failed to induce STING-dependent type I IFN response

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

in Cgas
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MC38 tumors suggests that tumor-cell-derived cGAMP is responsible for the activation of STING in host cells. However, it remains an open question how tumor-derived cGAMP reaches the cytosol of immune cells. Extracellular ATP (eATP) is broadly used for intercellular communication via different purinoreceptors that detect the eATP. Different from other purinoreceptors, P2X7R uniquely requires a very high concentration of ATP for its activation (half maximal effective concentration [EC50] R 100 mM) (Bianchi et al., 1999; Surprenant et al., 1996). In homeostasis, the concentration of eATP is low. A large increase in eATP can occur when cells are damaged or become necrotic. Indeed, it has been shown that eATP concentration can be in the range of hundreds of micromolar in the interstitium of inflamed tissues and tumors (Di Virgilio et al., 2018b; Pellegatti et al., 2008), which would potentially open the ATP-gated P2X7R channel and allow the direct passage of nanometer-sized molecules (Savio et al., 2018). In the current work, MerTK blockade resulted in the accumulation of apoptotic cells in the tumors (Figures 2C and 2D). Left uncleared, apoptotic cells can progress to secondary necrotic cells and leak intracellular constituents, including ATP and cGAMP. This raises an interesting possibility that the extracellular cGAMP released from damaged and/or dead tumor cells can enter host cells through the ATP-gated P2X7R channel to activate STING. To test this hypothesis, first we used an in vitro system of cultured bone-marrow-derived macrophages (BMDMs). The production of IFN-b by BMDMs was measured after incubation with cGAMP of different concentrations (Figure 7A). A high concentration of cGAMP was needed to induce IFN-b. Although ATP by itself had little effect, it greatly enhanced the IFN-b production even at a much lower concentration of cGAMP (Figure 7A). Then we performed similar assays with Cgas
/
and Stinggt/gt BMDMs. The IFN-b production was not affected by the loss of cGAS but was completely abolished in Stinggt/gt BMDMs (Figure S7A), confirming that the IFN-b production was STING-dependent in the experimental setting. Besides measuring IFN-b protein, we examined the messenger RNA (mRNA) expression of Ifnb1 and ISGs. Indeed, exogenous ATP enhanced the activity of cGAMP to induce the expression of Ifnb1 and ISGs (Figure S7B). As a control experiment, instead of adding cGAMP to the culture medium, we delivered cGAMP at a low concentration to macrophages by electroporation. In this setting, ATP had no effect on IFN-b production (Figure S7C), ruling out the possibility that ATP triggers a signaling cascade that modulates IFN-b production in response to intracellular cGAMP. Recently, CRISPR screens in cultured cells identified solute carrier family 19 member 1 (SLC19A1) as a transporter for cGAMP in human cells (Luteijn et al., 2019; Ritchie et al., 2019). ATP treatment did not affect the expression of SLC19A1 on BMDMs (Figure S7D). In addition, folinic acid (FA), a competitive inhibitor of SLC19A, did not affect IFN-b production (Figure S7E), indicating that SLC19A1 is unlikely involved in the ATP effect in this context. To address whether the observed effect of ATP was mediated by P2X7R, we used A740003, a highly specific P2X7R antagonist (Honore et al., 2006). A740003 effectively abrogated the ability of ATP to enhance IFN-b production (Figure 7B). To further establish the role of P2X7R, we used BMDMs derived from P2x7r
/
mice (Figure S7F). We used fluorescein-conjugated cGAMP to

monitor its entry into macrophages by fluorescence-activated cell sorting (FACS) analysis. ATP enhanced cGAMP uptake by WT BMDMs but not P2x7r
/
BMDMs (Figure 7C). Upon activation by intracellular cGAMP, STING recruits and activates TBK1, which in turn phosphorylates the transcription factor IRF3. Indeed, in WT BMDMs, ATP and cGAMP synergistically increased the phosphorylation of TBK1 (p-TBK1) and IRF3 (p-IRF3), which was completely absent in P2x7r
/
BMDMs (Figure 7D). Finally, we measured IFN-b production as a functional readout of STING activation. At a low cGAMP concentration (1 mM), ATP enhanced in a dose-dependent manner the IFN-b production by WT BMDMs but not P2x7r
/
BMDMs (Figure 7E). In comparison, when BMDMs were treated with a high concentration of cGAMP (10 mM), a similar amount of IFN-b was produced regardless of the genotype of BMDMs, ruling out a general defect in P2x7r
/
BMDMs (Figure S7G). P2X7R activation by ATP can also lead to inflammasome activation and subsequent pore formation by members of Gasdermin family (Swanson et al., 2019). To test whether Gasdermin pores are involved, we used Gsdmd
/
Dfna5
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macrophages. Gsdmd
/
Dfna5
/
macrophages exhibited a similar response to ATP and cGAMP treatment in comparison to WT macrophages (Figure S7H), suggesting that under the experimental setting Gasdermin pores do not contribute to ATP-mediated enhancement of IFN-b production by extracellular cGAMP. Together, our findings support the notion that ATP-gated P2X7R channel allows the entry of extracellular cGAMP into the cytosol of macrophages to stimulate STING-dependent production of IFN-b. To demonstrate the in vivo significance of our in vitro findings, we first examined the expression of P2X7R on TAMs and tumorinfiltrating DCs. Flow-cytometric analysis showed that much more P2X7R was expressed on TAMs than on DCs (Figure S7I). This result was consistent with our finding that TAMs but not DCs were the major source of increased IFN-b production after antiMerTK treatment. Next, we investigated the effect of antagonizing P2X7R in the context of MerTK blockade in tumors. In the MC38 tumor model, administration of the P2X7R antagonist A740003 abolished the type I IFN response induced by antiMerTK treatment (Figure S7J). Given that A740003 can have a global effect on P2X7R in both tumor cells and host cells, we performed additional tumor studies in P2x7r
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mice. Anti-MerTK treatment failed to induce the type I IFN response in tumors growing in P2x7r
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mice (Figure 7F). Furthermore, the anti-tumor effect of anti-MerTK treatment was diminished in P2x7r
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mice (Figure 7G). To further demonstrate the importance of extracellular ATP in the tumor milieu, we decided to deplete extracellular ATP in vivo. To this end, we generated MC38 tumor cells overexpressing CD39, an ectonucleotidase that hydrolyzes extracellular ATP (Figure S7K) (Di Virgilio et al., 2018a). Cultured CD39-overexpressing MC38 (MC38.CD39) cells effectively degraded ATP added to the culture medium (Figure S7L). In addition, after anti-MerTK treatment, the type I IFN response (Figure S7M) and tumor growth inhibition effect (Figure S7N) were lost in MC38.CD39 tumors. These tumor study results, together with our in vitro findings, support a role of extracellular ATP and P2X7R in facilitating tumor-derived extracellular cGAMP to stimulate STING-dependent type I IFN response. When MerTKmediated dying cell clearance is blocked, more cGAMP and Immunity 52, 1–17, February 18, 2020 11

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Figure 7. Type I IFN Response Induced by MerTK Blockade Requires ATP-gated P2X7R Channel (A and B) Measuring IFN-b protein production by BMDMs. (A) WT BMDMs were treated with increasing concentrations of cGAMP in the presence or absence of 0.5 mM ATP. Data are representative of two independent experiments. (B) WT BMDMs were treated as indicated. We used1 mM cGAMP, 0.5 mM ATP, or 100 mM A740003 (P2X7R antagonist). (C) WT or P2x7r
/
BMDMs were incubated with 1 mM fluorescein-conjugated cGAMP in the presence or absence of 0.5 mM ATP. Flow cytometric measurements of fluorescent intensity in BMDMs are shown. Data are representative of two independent experiments. (D) Western blot analysis of p-TBK1, p-IRF3, and total TBK1 and IRF3 in WT or P2x7r
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BMDMs stimulated as indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. We used 5 mM cGAMP or 0.5 mM ATP. Data are representative of two independent experiments. (E) WT or P2x7r
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BMDMs stimulated with 1 mM cGAMP with increasing concentrations of ATP. IFN-b protein production by BMDMs was measured. Data are representative of two independent experiments. (F) RT-qPCR analysis of mRNA expression of representative ISGs in MC38 tumors growing in WT or P2x7r
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mice treated with a control Ab or anti-MerTK (WT mice treated with control Ab, n = 9; P2x7r
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mice treated with anti-MerTK, n = 9; other groups, n = 10). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; unpaired two-tailed Student’s t test. (G) Assessing MC38 tumor growth in P2x7r
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mice treated with a control Ab or anti-MerTK (WT treated with control Ab, n = 9; P2x7r
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mice treated with antiMerTK, n = 9; other groups, n = 10). Both individual tumor growth curves and LME-fitted tumor growth curves of each group are presented. All mice were in C57BL/6N background. Data are representative of two independent experiments. See also Figure S7.

ATP are released from uncleared dead cells, ultimately leading to an increased type I IFN response. DISCUSSION Currently most cancer immunotherapy strategies focus on T cells. However, given that innate immunity plays a critical role in the induction and maintenance of adaptive immunity, a 12 Immunity 52, 1–17, February 18, 2020

more effective therapeutic strategy should incorporate both arms of the immune system. In this study, we identified an immune evasion mechanism by which cancers employ TAMs to suppress innate immune sensing of tumors via engaging MerTK-dependent phagocytic clearance of dying tumor cells. In this context, MerTK signaling acts as an innate immune checkpoint. Selective inhibition of MerTK with a function-blocking antibody led to rapid induction of local type I IFN response,

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

enhanced anti-tumor T cell immunity, and improved the efficacy of anti-PD-1 and anti-PD-L1 therapies. Defective STING signaling is found in cancer cells because of suppressed expression of cGAS or STING (Xia et al., 2016a; Xia et al., 2016b). This tumor-intrinsic defect might limit self-DNAinduced type I IFN response. Acquisition of tumor DNA by immune cells has been proposed as the mechanism for extrinsic STING activation (Deng et al., 2014; Wang et al., 2017; Woo et al., 2014). The assumption has been that somehow tumor DNA gets access to and activates cGAS in immune cells. A recent study showed that cGAMP produced through intrinsic cGAS activation within stressed or dying cells can activate extrinsic STING signaling in phagocytes (Ahn et al., 2018). From another study, it is suggested that tumor-derived cGAMP can trigger STING activation in non-cancer cells, induce type I IFN production, and activate natural killer (NK)-mediated tumor lysis (Marcus et al., 2018). However, the exact mechanism of how cGAMP is transferred from tumor cells to immune cells remains elusive. In principle, immune cells can acquire cGAMP via phagocytosis or pinocytosis (Ahn et al., 2018; Wang et al., 2017). In this scenario, however, cGAMP has to escape the endolysosomal compartments. Gap junctions have been proposed as an alternative mechanism for cGAMP horizontal transfer from cancer cells to immune cells in an in vitro setting (Schadt et al., 2019). Cell-culture-based assays identify SLC19A1 as a cGAMP transporter in certain cell types (Luteijn et al., 2019; Ritchie et al., 2019). However, the in vivo roles of gap junctions and SLC19A1 have yet to be established. Our study reveals a new mechanism of extrinsic STING activation by which extracellular cGAMP released from tumor cell enters the immune cells via the ATPgated P2X7R channel. In MC38 tumors, TAMs express more P2X7R than DCs, potentially more readily allowing cGAMP entry in TAMs. This finding might explain why TAMs were the major producers of IFN-b upon MerTK blockade. In addition, due to their abundant presence in the TME, TAMs are likely in close proximity to dying or dead tumor cells that produce and release cGAMP and ATP. Although DCs, in particular CD103+ DCs, are in general better APCs, tissue resident macrophages and TAMs can also function to effectively cross-present antigens and activate CD8+ T cells under certain biological settings (Be´ne´chet et al., 2019; Bernhard et al., 2015; Kerkar et al., 2011; Muraoka et al., 2019). In the context of MerTK blockade in tumors, we observed increased tumor-derived antigen presentation and enhanced cross-priming capabilities in TAMs, but not CD103+ DCs. Type I IFNs are key regulators of antigen presentation. In our study, the increase of Ifnb1 mRNA in TAMs was statistically significant, but the degree of change was modest. In addition, the expression of IFN-b protein in the tumor milieu was low. Therefore, it is likely that antigen presentation is predominantly modulated in an autocrine fashion by IFN-b produced by TAMs. As a result, tumor-infiltrating DCs, which did not show an increase in IFN-b production, would not benefit from MerTK blockade to further enhance the anti-tumor response. However, we cannot rule out the possibility that tumor-infiltrating DCs might still play a constitutive role to support CD8+ T cells (Broz et al., 2014; Chow et al., 2019; Hildner et al., 2008; Roberts et al., 2016; Salmon et al., 2016; Spranger et al., 2017).

During tumor progression, to avoid alarming the immune system, dying tumor cells are rapidly disposed of by TAMs, eliminating the source of extracellular cGAMP and ATP. Blocking MerTK-mediated phagocytic clearance of apoptotic cells potentially prolongs the cGAMP production in dying tumor cells and increases the release of cGAMP and ATP from the uncleared dead tumor cells. Immunogenic cell death (ICD) has been explored to engage the adaptive arm of the immune system and improve cancer therapy. ICD is associated with the emission of endogenous danger signals or DAMPs from dying cells, including ATP. Mechanistically, it has been shown that ATP induces an IL-1b-dependent adaptive immunity against tumors (Bezu et al., 2015; Ghiringhelli et al., 2009). Here, we showed that ATP facilitated cGAMP entry via the P2X7R channel into host immune cells to boost the innate immune sensing of tumor cells and the production of type I IFNs. In addition, our study defines cGAMP as an extracellular ‘‘danger signal’’ that spatiotemporally coordinates with extracellular ATP to activate STING-dependent type I IFN response. Our work provides mechanistic insight into developing new therapeutic strategy that employs a highly effective cGAMPinducing agent. In the current study, we showed that whereas the host STING was critical for the anti-tumor immune response upon MerTK blockade, the tumor-derived cGAS drove the transactivation of STING. These findings provide mechanism-based guidance for patient stratification and selection for anti-MerTK therapy. We found that CD8 depletion abolished the anti-tumor effect of MerTK blockade. Therefore, besides the contribution by NK cells (Marcus et al., 2018), CD8+ T cells are critically important for the anti-tumor effect triggered by tumor-derived cGAMP. Targeting PD-1 or PD-L1 increases the density and functionality of TILs. However, TIL clonality usually is not affected by inhibiting PD-1 or PD-L1 (Juneja et al., 2017; Roh et al., 2017; Tumeh et al., 2014; Weir et al., 2016). Interestingly, recent studies suggest that a robust T cell response against only a few antigens might be therapeutically more desirable than a diverse response against many different antigens (Roh et al., 2017; Thorsson et al., 2018; Tumeh et al., 2014). Therefore, our finding of increased TIL clonality upon MerTK blockade offers a strong rationale for combination therapy targeting both MerTK and PD-1 or PD-L1. The clinical success of systemic type I IFN therapy for cancer has been limited due to the complex activities and side effects influenced by dosing and duration (Hervas-Stubbs et al., 2011; Trinchieri, 2010). STING agonists are being explored for cancer immunotherapy in pre-clinical and clinical settings (Corrales et al., 2015; Demaria et al., 2015; Fu et al., 2015). Given the broad tissue expression of STING, STING agonists are only being investigated by intratumoral injection to avoid the toxicity associated with systemic type I IFN response. In contrast, the type I IFN response after systemic MerTK blockade was mainly restricted to the tumor site, which was likely attributed to the high rate of tumor cell apoptosis and active MerTK engagement in the TME. The tumor-selective effect of anti-MerTK therapy might offer more flexibility in clinical development. We are aware of the limitations of our current study. The mechanistic studies were mainly carried out in the MC38 tumor model. Immunity 52, 1–17, February 18, 2020 13

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

The importance and contribution of the mechanism delineated in MC38 tumors to other tumor models remain to be determined. It is also possible that in a different context blockade of MerTKmediated efferocytosis might rely on endogenous danger signals or DAMPs other than ATP and cGAMP. Another key question is the translatability of findings from mouse tumor studies to human diseases. In summary, the effects of MerTK blockade on anti-tumor immunity highlight the potential of exploiting dying cell clearance for cancer immunotherapy. Targeting this mechanism offers new opportunities for harnessing the immune system to improve cancer therapy.

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. immuni.2020.01.014. ACKNOWLEDGMENTS We thank M. Wang, R. Corpuz, and I. Lehoux for protein production and purification; E. Wonder, M. Long, and W. Ortiz for animal husbandry; K. Hotzel and F. Peale for pathology support; K. Totpal for cell bank assistance; B. Alicke and B. Forrest for statistical analysis support; and members of the Microinjection, Genetic Analysis, Microscopy, and FACS laboratories for technical assistance. Funding source: Genentech Inc., United States. AUTHOR CONTRIBUTIONS

STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B Cell Culture B Isolation of Mouse Primary Macrophages METHOD DETAILS B Antibodies B Anti-MerTK Antibody Specificity Determination by BIAcore B pAKT Assay B MerTK Autophosphorylation Assay and Immunoblot Analysis B In vitro Efferocytosis Assay B In vivo Efferocytosis Assay B Tumor Studies B Histology, Immunofluorescence and Confocal Imaging B Image Analysis for c-Casp3 Quantification in Whole Tumor Sections B Tumor Digestion and Flow Cytometry B Isolation of tumor-associated macrophages (TAMs) B RNA Sequencing B Gene Set Analysis B TCGA Data Processing and Estimation of Immune Cell Subsets B Quantification of Cell-free DNA (cf.DNA) and Circulating Tumor DNA (ctDNA) B Quantification of Gene Expression in Whole Tumor B Quantification of Cytokines/chemokines in Tumor Homogenate B TCR Sequencing + B Measurement of Cross-priming of CD8 T Cells by Enzyme-linked Immunospot (ELISPOT) Assay B Generation of Knockout Cell Lines by CRISPR/Cas9 Technology B Lentivirus Production and Transduction B 2’30 cGAMP Measurement by LC-MS/MS B BMDMs in vitro Stimulation B Quantification of Extracellular ATP in Cell Culture QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

14 Immunity 52, 1–17, February 18, 2020

Y.Z. and M.F. contributed to study design, experiment execution, data collection, and analysis. M.F., W-C.L., W.Y.L., and Y.W. directed and performed antibody generation and characterization. R.P. performed bioinformatics analyses. G.Z. designed and performed efferocytosis assays. J. R. contributed to generating mutant cell lines. E.M., H.H., J.Z., W.P.L., D.J., J.L., and B.B. contributed to the design and execution of tumor efficacy and pharmacodynamics experiments. J.O. contributed to the bone marrow chimera experiments. D.D.B., Y.S., and J.W. carried in vitro antibody characterization. J.M.P. and H-M.H. provided cell lines for in vitro studies. R.J. performed image analysis. D.S. performed measurement of cGAMP. Z.M. contributed to RNAseq experiment. K.R.A., S.W. and M.R.-G. contributed to generating Mertk
/
and cGAS
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mice. M.Y. directed the study and designed experiments. Y.Z. and M.Y. wrote the paper with inputs from all authors. DECLARATION OF INTERESTS All authors, except J.P. and H-M.H., were employees of Genentech. Received: June 25, 2019 Revised: December 3, 2019 Accepted: January 22, 2020 Published: February 11, 2020 REFERENCE Ablasser, A., Goldeck, M., Cavlar, T., Deimling, T., Witte, G., Ro¨hl, I., Hopfner, K.P., Ludwig, J., and Hornung, V. (2013). cGAS produces a 20 -50 -linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384. Ahn, J., Xia, T., Rabasa Capote, A., Betancourt, D., and Barber, G.N. (2018). Extrinsic phagocyte-dependent STING signaling dictates the immunogenicity of dying cells. Cancer Cell 33, 862–873. Akalu, Y.T., Rothlin, C.V., and Ghosh, S. (2017). TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy. Immunol. Rev. 276, 165–177. Anderson, K.R., Haeussler, M., Watanabe, C., Janakiraman, V., Lund, J., Modrusan, Z., Stinson, J., Bei, Q., Buechler, A., Yu, C., et al. (2018). CRISPR off-target analysis in genetically engineered rats and mice. Nat. Methods 15, 512–514. Arandjelovic, S., and Ravichandran, K.S. (2015). Phagocytosis of apoptotic cells in homeostasis. Nat. Immunol. 16, 907–917. Be´ne´chet, A.P., De Simone, G., Di Lucia, P., Cilenti, F., Barbiera, G., Le Bert, N., Fumagalli, V., Lusito, E., Moalli, F., Bianchessi, V., et al. (2019). Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming. Nature 574, 200–205. Bernhard, C.A., Ried, C., Kochanek, S., and Brocker, T. (2015). CD169+ macrophages are sufficient for priming of CTLs with specificities left out by crosspriming dendritic cells. Proc. Natl. Acad. Sci. USA 112, 5461–5466. Bezu, L., Gomes-de-Silva, L.C., Dewitte, H., Breckpot, K., Fucikova, J., Spisek, R., Galluzzi, L., Kepp, O., and Kroemer, G. (2015). Combinatorial strategies for the induction of immunogenic cell death. Front. Immunol. 6, 187.

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies anti-MerTK, mIgG2a LALAPG

This paper

N/A

anti-gp120, mIgG2a LALAPG

Genentech

N/A

anti-PD-L1, mIgG2a LALAPG

Genentech

N/A

anti-CD8a (clone clone 53-6.7)

BioXCell

Cat#BP0004-1; RRID: AB_1107671

anti-IFNAR1 (clone MAR1-5A3)

BioXCell

Cat#BP0241; RRID: AB_2687723

anti-PD-1 (clone RMP1-14)

BioXCell

Cat#BP0146; RRID: AB_10949053

anti-CSF1R (clone AFS98)

BioXCell

Cat#BE0213; RRID: AB_2687699

Rat anti-mouse F4/80 (clone BM8)

BMA Biomedicals

Cat#T-2028; RRID: AB_1227366

Rabbit anti-mouse cleaved caspase 3

Cell signaling

Cat#9664; RRID: AB_2070042

Rat anti-mouse CD68 (clone FA-11)

BioRad

Cat#MCA1957; RRID: AB_322219

Rabbit anti-mouse MerTK

This paper

N/A

Anti-Phosphotyrosine Antibody, clone 4G10, Biotin Conjugate

Millipore

Cat#16-103; RRID: AB_310777

Rabbit anti-mouse cGAS (clone D3080)

Cell signaling

Cat#31659S; RRID: AB_2799008

Rabbit anti-mouse STING (clone D2P2F)

Cell signaling

Cat#13647S; RRID: AB_2732796

Rabbit anti-mouse GAPDH (clone D16H11)

Cell signaling

Cat#5174; RRID: AB_10622025

Rabbit anti-mouse Bax (clone D3R2M)

Cell signaling

Cat#14796; RRID: AB_2716251

Rabbit anti-mouse Bak (clone D4E4)

Cell signaling

Cat#12105; RRID: AB_2716685

Rabbit anti-mouse phospho-IRF3 (clone D6O1M)

Cell signaling

Cat#29047; RRID: AB_2773013

Mouse anti-mouse IRF-3 (clone 12A4A35)

BioLegend

Cat#655702; RRID: AB_2562202

Mouse anti-mouse TBK1 (clone E9H5S)

Cell signaling

Cat#51872; RRID: AB_2799403

Rabbit anti-mouse phosphor-TBK1 (clone D52C2)

Cell signaling

Cat#5483; RRID: AB_10693472

Goat anti-mouse MerTK

R&D

Cat#AF591; RRID: AB_2098565

FITC anti-CD11b (clone M1/70)

eBioScience

Cat#11-0112-82; RRID: AB_464935

FITC-VAD-FMK

Promega

Cat#G7461

BUV395 anti-CD45 (clone 30-F11)

BD Biosciences

Cat#564279; RRID: AB_2651134

PE-Cy7 anti-CD11c (clone HL3)

BD Biosciences

Cat#558079; RRID: AB_647251

BUV563 anti-Ly6G (clone 1A8)

BD Biosciences

Cat# 65707; RRID: AB_2739334

V500 anti-MHC Class II (clone M5/114.15.1)

BD Biosciences

Cat#562366; RRID: AB_11153488

BUV737 anti-CD24 (clone M1/69)

BD Biosciences

Cat#565308; RRID: AB_2739174

BUV737 anti-CD4 (clone GK1.5)

BD Biosciences

Cat#564298; RRID: AB_2738734

BV786 anti-CD86 (clone GL1)

BD Biosciences

Cat#740877; RRID: AB_2740528

Pacific Blue anti-CD8a (clone 53-6.7)

BD Biosciences

Cat#558106; RRID: AB_397029

BV711 anti-CD103 (M290)

BD Biosciences

Cat#564320; RRID: AB_2738743

BV605 anti-Ly6C (clone HK1.4)

Biolegend

Cat#128036; RRID: AB_2562353

BV421 anti-CD64 (clone X54-5/7.1)

Biolegend

Cat#139309; RRID: AB_2562694

BV785 anti-CD90.2 (clone 30-H12)

Biolegend

Cat#105331; RRID: AB_2562900

PE-Cy7 anti-CD8b (clone 53-5.8)

Biolegend

Cat#140416; RRID: AB_2564385

FITC anti-CD335 (clone 29A1.4)

Biolegend

Cat#137606; RRID: AB_2298210

BV650 anti-CD206 (clone C068C2)

Biolegend

Cat#141723; RRID: AB_2562445

BV650 anti-F4/80 (clone BM8)

Biolegend

Cat#123149; RRID: AB_2564589

APC anti-H2Kb bound SIINFEKL

Biolegend

Cat#141606; RRID: AB_11219595

APC H2Kb p15e tetramer

MBL International

Cat#TB-M507-2

PerCP-eFluor 710 anti-Ki67 (clone SolA15)

ThermoFisher Scientific

Cat#46-5698-82; RRID: AB_11040981

Biotin anti-IFNAR1 (clone MAR1-5A3)

Biolegend

Cat#127306; RRID: AB_1134249

APC anti-IFNAR2

R&D

Cat#FAB1083A; RRID: AB_10730826 (Continued on next page)

e1 Immunity 52, 1–17.e1–e9, February 18, 2020

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Continued REAGENT or RESOURCE

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IDENTIFIER

PE H2Kb-OVA tetramer

MBL International

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AF488 conjugated anti-MerTK

This paper

N/A

AF488 conjugated anti-gp120

This paper

N/A

HRP–conjugated Donkey anti-Rabbit IgG

GE Healthcare

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HRP-conjugated Sheep anti-Mouse IgG

GE Healthcare

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HRP–conjugated Streptavidin

Jackson ImmunoResearch

Cat#016-030-084; RRID: AB_2337238

Rat anti-mouse CD16/CD32 (Mouse BD Fc Block)

BD Biosciences

Cat#553141; RRID: AB_394656

APC anti-P2X7R (clone 1F11)

Biolegend

Cat#148706; RRID: AB_2650954

PE anti-CD39 (clone Duha59)

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BUV395 anti-CD45.2 (clone 104)

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Cat#564616; RRID: AB_2738867

PE anti-CD45.1 (clone A20)

Biolegend

Cat#110708; RRID: AB_313497

Genentech

N/A

Recombinant mouse M-CSF

GIBCO

Cat#PMC2044

Recombinant mouse Tyro3 Fc tagged

R&D

Cat#759-DT-100

Recombinant mouse Axl Fc tagged

R&D

Cat#7477-AX-050

Recombinant mouse MerTK Fc tagged

R&D

Cat#591-MR-100

human GAS6-Fc fusion protein

This paper

N/A

Thioglycolate

Sigma

Cat#B2551

Dexamethasone

Sigma

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pHrodo Red succinimidyl ester

ThermoFisher Scientific

Cat#P36600

Propidium Iodide (PI)

Biochemika

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Matrigel

Corning

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Gemcitabine

Selleckchem

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40 ,6-diamidino-2-phenylindole (DAPI)

Invitrogen

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VECTASHIELD antifade mounting medium

Vector Laboratories

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ACK lysing buffer

Lonza

Cat#10-548E

Lymphocyte M media

Cedarlane Labs

Cat#CL5031

HaltTM Protease and Phosphatase Inhibitor Cocktail

ThermoFisher Scientific

Cat#78446

Alt-R S.p. Cas9 Nuclease V3

IDT

Cat#1081058

Bacterial and Virus Strains pLenti6.3 lentiviral vector Chemicals, Peptides, and Recombinant Proteins

Lipofectamine 3000

Life Technologies

Cat#L3000001

Polybrene

Sigma

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50 ppp-dsRNA

Invivogen

Cat#tlrl-3prna

DMXAA

Invivogen

Cat#tlrl-dmx

ATP

ThermoFisher

Cat#18330019

cGAMP

Invivogen

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Fluorescein-conjugated cGAMP

BIOLOG

Cat#C178

A740003

TOCRIS

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Phospho-AKT-1 (Ser473) HTRF Kit

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mouse Tumor Dissociation Kit

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Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

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RNeasy Plus Mini Kit

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BCA Protein Assay Kit

Pierce

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Mouse MCP-3 Instant ELISA Kit

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mouse IFNg ELISPOT Kit

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This paper

NCBI GEO: GSE119952

MC38

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E0771

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293T

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N/A

J774A.1

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N/A

Deposited Data RNA-sequencing raw data and gene expression quantifications Experimental Models: Cell Lines

Experimental Models: Organisms/Strains C57BL/6N

Charles River Laboratories

Cat#C57BL/6NCrl

C56BL/6J

The Jackson Laboratory

Cat#000664; RRID: IMSR_JAX:000664

cGAS
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This paper

N/A

B6.129S(C)-Batf3tm1Kmm/J

The Jackson Laboratory

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C57BL/6J-Tmem173gt/J

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Cat#017537; RRID: IMSR_JAX:017537

MerTK
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This paper

N/A

Genentech

N/A

Genentech

N/A

P2x7r


/


Gsdmd/Dfna5 dKO Oligonucleotides qRT-PCR taqman probes, see Table S1

ThermoFisher Scientific

see Table S1

cGAS CRIPSR/Cas9 targeting sequence: 50 - AGATCCGCGT AGAAGGACGA-30

This paper

N/A

Bax CRISPR/Cas9 targeting sequence: 50 -TAGGTAGGC TCATAACCCTG-30 , 50 - GTGCGATGCTACTAGTGTGG-30

This paper

N/A

Bak1 CRISPR/Cas9 targeting sequence: 50 -ACAAGGACCA GGTCCCCCGA-30 , 50 - GTACTTAATGAGGTTCTGAG-30

This paper

N/A

Lonza

Cat#VACA-1003

FlowJo

Flowjo

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Graph Prism 7

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R

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Microsoft

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Fiji

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https://fiji.sc/

Developer XD image analysis software

Definiens

https://www.definiens.com/

Recombinant DNA pmaxGFP Software and Algorithms

e3 Immunity 52, 1–17.e1–e9, February 18, 2020

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Minhong Yan ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice All animal studies conforming to ethical compliance were approved by the Genentech and Earle A. Chiles Research Institute Animal Care and Use Committee. For all tumor studies, female mice between 8 and 12 weeks of age were used. Animals were housed under specific-pathogen free conditions at the Genentech lab animal facility. C57BL/6N mice were purchased from the Charles River Laboratories. C57BL/6J, Batf3
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and Stinggt/gt mice (Sauer et al., 2011) were purchased from the Jackson Laboratory. P2x7r
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(Qu et al., 2011) and Gsdmd
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Dfna5
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(Kayagaki et al., 2015) mice were described previously. Mice harboring a sequence with stop codons in all reading frames in Mertk exon 2 were obtained by cytoplasmic co-injection of Cas9 mRNA, sgRNA, and an ssDNA oligo donor into C57BL/6N zygotes. The resulting mosaic founders were analyzed for editing at the Mertk locus and at the top 11 algorithm-predicted off-targets, as previously described (Anderson et al., 2018). Mosaic founders without off-targets were bred to C57BL/6N to generate F1 heterozygous progeny for subsequent intercrossing. The sgRNA sequence used to target Mertk exon 2 is 50 -gGAATGGCCTGTGGTTGACT-30 (50 mismatch indicated by lowercase g) and the stop cassette inserted at genomic position GRCm38/mm10 chr2:128,729,258 has the following sequence: 50 -cgcTAAgTGAcTGAccaccgc-30 (Stop codons in upper case, flanking homology sequences are not included). cGAS
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mice were generated by CRIPSR-mediated deletion of exon 2. The sgRNA sequences used to target cGAS exon 2 are 50 - GTATACTGTTCCAACACAGG-30 and 50 -TGACCGCACGACTTACCCTG-30 . Cell Culture MC38, MC38.OVA, E0771, 293T and J774A.1 macrophages (all from Genentech cell bank) were cultured in RPMI plus 10% heatinactivated fetal bovine serum (HI-FBS). All cells were tested for mycoplasma using two methods to avoid false positive/negative results: Lonza Mycoalert and Stratagene Mycosensor. All cell lines used were not found in the International Cell Line Authentication Committee (ICLAC) misidentified cell lines database. In some cases, an IncuCyte ZOOM System was used to capture time-lapse images every 30 min and calculate the confluency over time. Isolation of Mouse Primary Macrophages Female mice age between 8 and 12 weeks were used for primary macrophage isolation. Cells were harvested from mouse peritoneal cavity in 10 mL PBS with 4% HI-FBS, and plated on culture dishes in RP10 medium (RPMI supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 2 mM L-Glutamine, 10 mM MEM non-essential amino acids solution, 55 mM b-mercaptoethanol, 100 units/mL penicillin and 100 mg/mL streptomycin). After incubation at 37 C for 2 h, non-adherent cells were removed, and the adherent macrophages were cultured overnight prior to in vitro assays. For MerTK autophosphorylation assay, 1 mL aged 4% Brewer’s thioglycolate medium (Sigma) was injected into mice four days before peritoneal lavage. To generate BMDM cells, bone marrow cells were flushed out of dissected femurs, and differentiated in RP10 medium with 40 ng/mL M-CSF (GIBCO) for 5–7 days. METHOD DETAILS Antibodies Anti-MerTK antibodies were generated from New Zealand White rabbits immunized with recombinant murine MerTK protein using a protocol based on a previous report (Seeber et al., 2014). Briefly, B cell clones were selected based on binding to recombinant MerTK proteins by ELISA, and MerTK-expressing cells by FACS. Variable regions of light chain and heavy chain of positive clones were amplified by PCR and cloned into expression vectors. Recombinant antibodies were expressed in Expi293 cells, and purified with protein A. An antibody that selectively blocked murine MerTK was reformatted into mIgG2a, LALAPG (effectorless variant [Lo et al., 2017]) for all studies described in the current work. Anti-PD-L1 antibody (mIgG2a, LALAPG), anti-gp120 antibody (as a control) (mIgG2a, LALAPG) and Alexa Fluor 488 (AF488)-conjugated anti-MerTK antibody and control antibody were also prepared at Genentech. Anti-CD8a antibody (clone 53-6.7), anti-IFNAR1 antibody (clone MAR1-5A3), anti-CSF1R antibody (clone AFS98) and anti-PD-1 antibody (clone RMP1-14) were purchased from BioXCell. Anti-MerTK Antibody Specificity Determination by BIAcore To determine the binding specificity of anti-MerTK antibody, surface plasmon resonance (SPR) BIAcore-T200 instrument was used. Series S sensor chip Protein A (GE Healthcare) was first applied to capture Fc-tagged mouse Tyro3/Dtk (759-DT-100, R&D), Axl (7477-AX-050, R&D), and MerTK (591-MR-100, R&D) on different flow cell (FC) to achieve approximately 100 response unit (RU), followed by injection of 100 nM anti-MerTK Fab fragments (Genentech) in HBS-EP buffer (100 mM HEPES pH7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Surfactant P20) with a flow rate of 100 ml/min at 37 C. The sensorgrams were recorded and adjusted to determine the binding response (BIAcore T200 evaluation software version 2.0).

Immunity 52, 1–17.e1–e9, February 18, 2020 e4

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

pAKT Assay J774A.1 macrophages from an exponentially growing culture were seeded at a density of 2.0 3 105 cells/well on a 96-well plate in RPMI medium + 10% FBS. The following day, cells were washed with 200 mL of serum free RPMI twice, and incubated in 200 mL of serum free RPMI for 4 h. After serum starvation, 10 mg/mL recombinant human GAS6-Fc protein (Genentech) were added and incubated for 20 min. Phospho-AKT (pAKT) measurements were taken from treated cell lysates using the Phospho-AKT-1 (Ser473) HTRF Kit (Cisbio, #63ADK078PEG) following the manufacturer’s instructions (standard protocol for two-plate assay protocol in 20 mL final volume). MerTK Autophosphorylation Assay and Immunoblot Analysis Thioglycolate-elicited peritoneal macrophages were serum-starved for 6 h. Cells were then treated with 10 mg/mL control antibody or anti-MerTK antibody for 15 min prior to stimulation with 10 mg/mL hGAS6-Fc for 15 min and processed for protein analysis. AntiMerTK immunoprecipitation was performed as previously described (Zago´rska et al., 2014). Briefly, cells were lysed in lysis buffer containing 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.27 M sucrose, 0.1% b-mercaptoethanol, with 1x protease and phosphatase inhibitors (ThermoFisher Scientific), and cell lysates were immunoprecipitated with anti-MerTK antibody (R&D, AF591) for overnight and incubated with protein A/G-Sepharose (1:1) (Sigma) for 1 h. Immunoprecipitates were washed and eluted in LDS buffer and separated by electrophoresis. Primary antibodies used for immunoblot analysis included: antibody to phosphorylated tyrosine (Millipore, 4G10-Biotin 16-103), anti-MerTK antibody (Genentech), anti-cGAS antibody (Cell signaling, 31659), anti-STING antibody (Cell signaling, 13647), anti-Bax antibody (Cell signaling, 14796), anti-Bak antibody (Cell signaling, 12105), anti-phospho-IRF3 antibody (Cell signaling, 29047), anti-IRF3 antibody (Biolegend, 655702), anti-phospho-TBK1 antibody (Cell signaling, 5483), anti-TBK1 antibody (Cell signaling, 51872), and anti-GAPDH antibody (Cell signaling, 5174). The secondary antibodies used were horseradish peroxidase (HRP)–conjugated Donkey anti-Rabbit IgG (GE Healthcare, NA934), HRP-conjugated Sheep anti-Mouse IgG (GE Healthcare, NA931), and HRP–conjugated Streptavidin (Jackson ImmunoResearch, 016-030-084). Chemiluminescent signal was developed with ECL Prime Western Blotting Detection Reagents (GE Healthcare, RPN2232) and detected by an Azure c500 Infrared Western Blot Imaging System (Azure Biosystems). In vitro Efferocytosis Assay Thymus was harvested from 4–6 weeks old C57BL/6N mice and minced to yield a single-cell suspension. Apoptosis of thymocytes was induced by 2 mM dexamethasone at 37 C for 5 h. Membrane integrity and exposure of phosphatidylserine on cell surface were assessed using APC Annexin V Apoptosis Detection Kit with PI (Biolegend). Apoptotic thymocytes were labeled with 1 mg/mL pHrodo Red succinimidyl ester (ThermoFisher Scientific). pHrodo Red dye is pH-sensitive and increases its fluorescent intensity dramatically as pH decreases from neutral to acidic, and therefore labels engulfed apoptotic cells. Macrophages were pre-incubated with 20 mg/mL control antibody or anti-MerTK antibody one h prior to adding pHrodo Red-labeled apoptotic cells. After 45 min incubation, unengulfed apoptotic cells were washed away, and macrophages were labeled with FITC-conjugated anti-CD11b antibody (eBioscience, clone M1/70). After fluorescence images were taken, the cells were detached from the cell culture plate for quantification by FACS analysis. In vivo Efferocytosis Assay To induce thymocyte apoptosis, dexamethasone was dosed by intraperitoneal injection as previously described (Seitz et al., 2007). To assess the accumulation of apoptotic cells, thymic tissue was collected and dissociated into single-cell suspension. Cells were stained with FITC-VAD-FMK (used as affinity labels of the enzymatic active center of caspases, 1:500 in PBS, Promega, Cat#G7461) and propidium iodide (PI, 1:1,000, Biochemika, Cat # 70335) to detect active caspases-positive apoptotic cells and dead cells, respectively. The stained cells were analyzed by FACS analysis. To evaluate the in vivo activity of anti-MerTK antibody, C57BL/ 6N mice were dosed with 20 mg/kg control or anti-MerTK antibody one h prior to dexamethasone injection. Tumor Studies Female C57BL/6N mice were inoculated subcutaneously on the lower right flank with 0.1 million MC38 cells or 5 million MC38.OVA cells in HBSS/Matrigel (1:1, v/v). For E0771 tumor model, 0.1 million E0771 cells were inoculated to the 5th mammary fat pad of female C57BL/6N mice. Animals were grouped based on weight and tumor volume to ensure similar weight and starting tumor volume distribution before treatment. Anti-MerTK antibody was administered via intraperitoneal (IP) injection at a dose of 20 mg/kg. Isotype-matched anti-gp120 antibody was used as control antibody. In some studies, anti-PD-L1 antibody was administered at 10 mg/kg, anti-PD-1 antibody at 8 mg/kg, and gemcitabine at 120 mg/kg. Tumors were monitored twice a week. Mice were humanely euthanized if ulceration occurred or tumor volume reached 2,000 mm3. Tumor volume was calculated as (length x width2)/2. A linear mixed effects (LME) modeling was performed to analyze repeated-measurements of tumor volume over time from the same animals to properly account for both latitudinal variability (differences in tumor volumes at each study day) and longitudinal variability (differences in how individual tumors change over the course of the study) (Wood, 2017). LME modeling also takes accounts for missing values from study dropouts due to ulceration or tumor volume exceeds 2,000 mm3. Tumors tend to exhibit exponential growth for the most part, and therefore, tumor volumes were subjected to natural log transformation before being analyzed. Changes in ln(tumor volumes) over time for all groups are described by fits (regression splines with auto-generated spline bases). Both individual tumor growth curves and LME-fitted tumor growth curves of each group are presented for each tumor growth efficacy studies. e5 Immunity 52, 1–17.e1–e9, February 18, 2020

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Histology, Immunofluorescence and Confocal Imaging Tumors were fixed in 4% paraformaldehyde/PBS overnight at 4 C, cryoprotected in 30% sucrose/PBS solution, and frozen in the Optimal Cutting Temperature Compound (Tissue-Tek). Alternatively, tumors were fixed in 10% neutral buffered formalin, dehydrated in 70% ethanol and embedded in paraffin with Leica EG1160 station. Immunodetection studies were performed on 12 mm frozen sections with the following primary antibodies: rat anti-mouse CD68 antibody (Bio-Rad, clone FA-11, MCA1957) and rabbit antimouse MerTK antibody (Genentech). To detect apoptosis in situ, an antibody against cleaved-Caspase 3 (Cell signaling, 9664) was used to stain 5 mm paraffin sections. Alexa fluorophores-conjugated secondary antibodies were from Invitrogen. Tissue sections was counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, Invitrogen), and mounted in VECTASHIELD antifade mounting medium (Vector Laboratories). Images were taken with a Leica SP5 confocal microscope or scanned with a slide scanner (3DHISTECH). Image Analysis for c-Casp3 Quantification in Whole Tumor Sections Images of entire tumor sections were acquired at 20X magnification using the Pannoramic SCAN 150 digital slide scanner (3D Histech, Budapest, Hungary). Image analysis was performed using custom algorithms developed in Developer XD image analysis software (Definiens, Munich, Germany). Automated quantification was performed only in tissue regions with normal cell density and positive DAPI expression. Necrotic regions were automatically excluded based on nuclear morphology and defined as large areas of low cell density (Dull et al., 2018). For quantification of c-Caspase 3 expression within non-necrotic regions, Texas Red background fluorescent signal was equalized using a top-hat filter. The total area of c-Caspase 3 positive pixels was segmented and normalized to DAPI+ pixel area. Tumor Digestion and Flow Cytometry Tumors were dissected and dissociated using mouse Tumor Dissociation Kit (Miltenyi Biotec) to obtain single cell suspension following the manufacturer’s protocol. After lysis of red blood cells using ACK lysing buffer (Lonza), cells were filtered through a 70 mm cell strainer. Cells were blocked with mouse BD Fc BlockTM (BD Biosciences), and then stained with following fluorochrome-conjugated antibodies: anti-MerTK antibody (clone DS5MMER), anti-Ki67 antibody (clone SolA15) from ThermoFisher Scientific, anti-CD11b antibody (clone M1/70) from eBioscience; anti-CD45 antibody (clone 30-F11), anti-CD11c antibody (clone HL3), anti-Ly6G antibody (clone 1A8), anti-MHC Class II antibody (M5/114.15.1), anti-CD24 antibody (clone M1/69), anti-CD4 antibody (clone GK1.5), anti-CD86 antibody (clone GL1), anti-CD8a antibody (clone 53-6.7) from BD Biosciences; anti-Ly6C antibody (clone HK1.4), anti-CD64 antibody (clone X54-5/7.1), anti-CD90.2 antibody (clone 30-H12), anti-CD8b antibody (clone 53-5.8), anti-CD335 antibody (clone 29A1.4), anti-CD206 antibody (clone C068C2), anti-F4/80 antibody (clone BM8), anti-IFNAR1-biotin (clonal MAR15A3), anti-P2X7R antibody (clone 1F11), anti-CD39 antibody (clone Duha59) from Biolegend; anti-IFNAR2 polyclonal antibody from R&D Systems. The anti-H-2Kb-SIINFEKL antibody (Biolegend, clone 25-D1.16) was used to specifically detect OVA-derived peptide SIINFEKL bound to MHC class I H-2Kb, but not H-2Kb bound to other peptides by incubating titrated 25-D1.16 antibody with cells on ice for 30 min. Antigen-specific T cells in tumors and dLNs were detected using p15 tetramer for MC38 tumor models or iTAg H-2Kb OVA (SIINFEKL) tetramer for MC38.OVA tumor models (MBL International) according to manufacturer’s instructions. Multicolor analysis was performed on a BD FACSymphony analyzer, and data were analyzed with Flowjo software (FlowJo LLC). Gating strategies for analysis of immune cell populations have been described previously (Broz et al., 2014; Salmon et al., 2016). Isolation of tumor-associated macrophages (TAMs) Tumors were harvested and dissociated into single cell suspensions. Live cells were enriched using Lymphocyte M media (Cedarlane Labs). CD335+, Siglec F+, Ly6G+ and Ly6C+ cells were labeled with biotin-conjugated antibodies and depleted with anti-biotin MACSiBeadTM Particles (Miltenyi Biotec). TAMs were then purified with anti-F4/80 Microbeads (Miltenyi Biotec). The purity of isolated TAMs was confirmed to be > 90% as assessed by FACS. RNA Sequencing Total RNA from purified TAMs was extracted using RNeasy Plus Micro Kit (QIAGEN). Five replicate samples were collected for each treatment condition. QC of total RNA was done to determine sample quantity and quality. The concentration of RNA was determined using NanoDrop 8000 (Thermo Scientific) and the integrity of RNA was determined by Fragment Analyzer (Advanced Analytical Technologies). 100 ng of total RNA was used as input material for library preparation using TruSeq Stranded Total RNA Library Prep Kit (Illumina). Size of the libraries was confirmed using High Sensitivity D1K screen tape (Agilent Technologies) and their concentration was determined by qPCR-based method using Library quantification kit (KAPA). The libraries were multiplexed and sequenced on Illumina HiSeq4000 (Illumina). We obtained on average 50 million single-end RNA-seq reads (50bp) per sample. Reads were first aligned to ribosomal RNA sequences to remove ribosomal reads. The remaining reads were aligned to the mouse reference genome (NCBI Build 38) using GSNAP (Wu and Nacu, 2010) version ‘2013-10-10’, allowing a maximum of two mismatches per 50 base pair sequence (parameters: ‘-M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1–pairmax-rna = 200000–clip-overlap’). Transcript annotation was based on the RefSeq database (NCBI Annotation Release 104). To quantify gene expression levels, the number of reads mapped to the exons of each RefSeq gene was calculated using the HTSeqGenie R package. Read counts were scaled by library size, quantile normalized, and precision weights calculated using the ‘‘voom’’ R package (Law et al., 2014). Subsequently, differential expression analysis on the normalized count data was performed using the ‘‘limma’’ R package (Ritchie et al., 2015) by contrasting anti-MerTK antibody treated samples with control antibody (anti-gp120) treated samples, respectively. Gene expression levels were Immunity 52, 1–17.e1–e9, February 18, 2020 e6

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

considered significantly different across groups if we observed an |log2-fold change| R 1 (estimated from the model coefficients) associated with an FDR adjusted P value % 0.05. In addition, gene expression was obtained in the form of normalized Reads Per Kilobase gene model per Million total reads (nRPKM) as described previously (Srinivasan et al., 2016). Gene Set Analysis We performed Quantitative Set Analysis for Gene Expression (QuSAGE) (Yaari et al., 2013) to identify relevant biological processes associated with MerTK inhibition. For that purpose, we contrasted anti-MerTK antibody treated samples with control antibody treated samples. We then calculated the gene set activity (i.e. the mean difference in log2 expression of the individual genes that compose the set) for all sets present in MSigDB (Liberzon et al., 2011). To determine statistical significance of gene set activity, false discovery rate (FDR)-adjusted p values were obtained by comparing the probability distribution function of log2-fold changes in a given gene set to a baseline value of zero using a one-sided test. In addition, we assessed the upregulation of the interferon alpha response (Moserle et al., 2008) using Gene Set Enrichment Analysis (GSEA) v3.0 (Subramanian et al., 2005). Significance of the enrichment (shown as false discovery rate - FDR) was determined through 1,000 permutations of random gene sets. TCGA Data Processing and Estimation of Immune Cell Subsets Expression data in TCGA samples were obtained as described by Daemen et al. (Daemen et al., 2018). Gene expression in form of RPKMs served as input for the TIMER software to calculate relative levels of six tumor-infiltrating immune subsets (Li et al., 2017). We ensured that MerTK was not part of the signatures used to estimate immune set abundance. Subsequently, Pearson correlation coefficients between gene expression level and immune cell type estimates were computed for each cell type and indication. Quantification of Cell-free DNA (cf.DNA) and Circulating Tumor DNA (ctDNA) MC38 tumor cells were inoculated into C57BL/6J mice and allowed tumors to establish. Three days post treatment, whole blood was collected by cardiac puncture into Cell-free DNA BCT tubes (Streck). Plasma was obtained by a double spin procedure (1,600 g for 10 min, separation, followed by 16,000 g for 10 min). cf.DNA (12.5 mL for 200 mL of plasma) was obtained using MagMAXTM Cell-Free DNA Isolation Kit (ThermoFisher Scientific) following the manufacturer’s protocol. To assay the levels of host-derived cf.DNA and MC38-derived ctDNA, multiplexing droplet digital PCR (Bio-Rad Laboratories) was performed using an assay containing primers and probes targeting SNPs of gene Jmjd1c (rs13480628, ThermoFisher Scientific). C57BL/6J mice and MC38 cells express a ‘‘T’’ and a ‘‘C’’ allele at this locus, respectively. For droplet digital PCR, 4 mL of isolated cf.DNA was used in each 20 mL-reaction, and each sample was analyzed in duplicates. Sample analysis was performed using QuantaSoft software (Bio-Rad Laboratories), and target DNA (copies/mL of plasma) was calculated as the quantitative outcome. Size of isolated cf.DNA was also confirmed to be predominantly 170bp using Agilent Bioanalyzer 2100. Quantification of Gene Expression in Whole Tumor Three days after antibody treatment, tumors were homogenized in RLT Plus Lysis buffer (QIAGEN) in gentleMACS M tubes using gentleMACS Dissociator (Miltenyi Biotec) following the manufacturer’s protocol. RNA was extracted using RNeasy Plus Mini Kit (QIAGEN). Gene expression was examined using Taqman Gene Expression Assays and Taqman RNA-to-Ct 1-Step Kit (ThermoFisher Scientific). The results were normalized to housekeeping genes, and calculated as fold changes using the 2
DDCt method. The Taqman assays used in this study are shown in Table S1. Quantification of Cytokines/chemokines in Tumor Homogenate Three days after treatment, tumors were homogenized in PBS supplemented with HaltTM Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific) in gentleMACS M Tubes (Miltenyi Biotec) using gentleMACS Dissociator (Miltenyi Biotec) following the manufacturer’s protocol. For every 100 mg of tumor tissue, 500 mL buffer was used. Tumor homogenates were clarified by centrifugation at 12,000 g for 20 min at 4 C. Homogenates were normalized based on total protein concentrations determined by BCA Protein Assay Kit (Pierce). IFNb and CCL7 (MCP-3) were assayed using High Sensitivity Mouse IFN Beta ELISA Kit (PBL Assay Science) and Mouse MCP-3 Instant ELISA Kit (Invitrogen), respectively. Other cytokines/chemokines were assayed using MILLIPLEX MAP Mouse Cytokine/chemokine Magnetic Beads Penal-Premixed 15-Plex and 32-Plex (Millipore). Cytokine/chemokine results were expressed as pg/mg of total protein in tumor homogenate. TCR Sequencing Tumor-infiltrating T cells were enriched using Dynabeads Mouse Pan T Kit (ThermoFisher Scientific). Genomic DNA from enriched T cells was extracted using AllPrep DNA/RNA/Protein Mini Kit (QIAGEN) and subjected to TCRb CDR3 sequencing using Immunoseq platform at survey level (Adaptive Biotechnologies). Sequencing results were analyzed using ImmunoSEQ Analyzer (Adaptive Biotechnologies). Clonality scores were calculated as 1-(entropy)/log2(number of productive unique sequences), where the entropy takes into account the varying clone frequency. For each sample, all TCR clones were ranked based on the productive frequency. The productive frequency of top 10 TCR clones represents the frequency of 10 most dominantly expanded clones.

e7 Immunity 52, 1–17.e1–e9, February 18, 2020

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

Measurement of Cross-priming of CD8+ T Cells by Enzyme-linked Immunospot (ELISPOT) Assay Established MC38.OVA tumors were treated with 20 mg/kg control antibody or anti-MerTK antibody. Tumors were harvest 4 days after treatment and dissociated into single cell suspension for cell surface marker staining. TAMs and DCs were sorted out by FACS. CD8+ OT-I T cells were purified from spleens and lymph nodes of OT-I mice with Naive CD8a+ T Cell Isolation Kit (Miltenyi) following manufacturer’s instructions. To measure cross-priming capacities of TAMs and DCs, 20,000 purified TAMs were cocultured with 100,000 OT-I CD8+ T cells. Due to scarcity of DCs sorted from tumors, 4,000 DCs were used to co-culture with 20,000 OT-I CD8+ T cells. After 60 h of co-culture, ELISPOT assay was performed with mouse IFNg ELISPOT Kit (BD Biosciences) following manufacturer’s protocol. IFNg+ spots were enumerated with an AID ELISPOT reader (AID). Generation of Knockout Cell Lines by CRISPR/Cas9 Technology MC38 cells were transfected with Cas9/gRNA expressing plasmid (kindly provided by Dr. Ben Haley at Genentech) or Cas9/gRNA ribonucleoprotein complex (IDT) by electroporation with Nucleofector II (Lonza) according to manufacturer’s recommendation. Single cell clones were screened for the loss of target protein by western blotting, and genomic alternations were verified by Sanger sequencing. CRISPR/Cas9 targeting sequences are: mouse cGAS, 50 - AGATCCGCGTAGAAGGACGA-30 ; mouse Bax, 50 -TAGG TAGGCTCATAACCCTG-30 , 50 - GTGCGATGCTACTAGTGTGG-30 ; mouse Bak1, 50 -ACAAGGACCAGGTCCCCCGA-30 , 50 - GTACT TAATGAGGTTCTGAG-30 . Lentivirus Production and Transduction DNA sequences encoding WT cGAS, GS > AA mutant cGAS and mouse CD39 were synthesized by GenScript and cloned into pLenti6.3 vector (kindly provided by Dr. Ben Haley at Genentech) and co-transfected into 293T cells with packaging plasmids delta8.9 and VSVG (Genentech) using lipofectamine 3000 (Life Technologies). Target cells were spin infected with lentiviral supernatant in the presence of 8 mg/mL polybrene (Sigma). Infected cells were passaged to sufficiently dilute live virus, and p21 ELISA (Clontech Lenti-X p24 Rapid Titer Kit) was performed to ensure viral clearance before cells were used for any in vitro and in vivo studies. Transduced cells were purified by sorting. 2’30 cGAMP Measurement by LC-MS/MS Cell pellets were processed and analyzed for 2’30 cGAMP as described previously (Jønsson et al., 2017). Briefly, cell pellets (around 10 million cells) were added with 300 mL of 2% acetic acid in 80:20 (v/v) methanol:water and lysed by sonication in water bath at room temperature for 15 min. Samples were then centrifuged at 10,000 3 g for 5 min and supernatant was aliquoted into a new sample tube. Pelleted precipitate was again lysed as described above with 200 mL of 2% (v/v) acetic acid in water. Samples were centrifuged again and supernatant was aliquoted and both supernatants were mixed and processed using OASIS WAX SPE (60 mg, 30 mm, 96 well plate, Waters Corporation, Milford, MA, USA) as described below. SPE columns were conditioned with 1 mL of methanol followed by 1 mL of water. 200 mL of the above mixed supernatant was loaded onto SPE and washed with 0.75 mL of water and 0.75 mL methanol. 2’30 cGAMP was then eluted with 0.75 mL of 20% (v/v) NH3 solution (25% NH4OH) in methanol. SPE elutes were then dried under nitrogen flow and reconstituted with 60 mL of 0.1% (v/v) formic acid in water. 10 mL was injected for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Liquid chromatography (LC) system included Shimadzu Nexera series UHPLC system (Shimadzu, Kyoto, Japan) consisting of LC pumps (Model LC-30AD) with online degasser was used to deliver the mobile phases with 20 mM ammonium acetate and 20 mM ammonium hydroxide in water (A) and Acetonitrile (B) at a flow rate of 0.4 mL/min. Luna NH2 (50 3 2 mm, 5mm, 100 A˚, Phenomenex, Torrance, CA, USA) was used as LC column and 2’30 cGAMP was eluted using gradient LC conditions. Gradient LC flow started with 50% B to 45% B in 1.5 min; followed by a linear decrease to 35% B in 0.2 min and held at 35% B for 0.3 min, and to 10% B in 1.5 min and held at 10% B for 1.5 min followed by decrease to 5% B in 1 min. Gradient was returned to 50% B in 0.5 min and held at 50% B for 0.9 min for column equilibration. Total run time was 7.4 min. The column oven (Model CTO20AC) temperature was maintained at 40 C. 2’30 cGAMP was eluted at 3.7 min. MS analysis was performed on a linear quadrupole ion trap mass spectrometer (QTRAP 6500, AB Sciex Instruments, Redwood City, CA, USA) equipped with a Turbo V ion source operated under standard electrospray ionization (ESI) conditions. Multiple reaction monitoring (MRM) scan mode was used for the mass spectrometric detection and quantification of the 2’30 cGAMP in positive mode. The protonated parent to the MS2 fragment ions MRM transitions were used for quantitation and qualification of 2’30 cGAMP analyte. These MRMs included quantifier transition m/z 674.9 / 524.0 (Collision energy (CE): 31V and Collision cell exit potential (CXP): 14V) and other qualifier transitions included m/z 674.9 / 312.0 (CE: 53, CXP: 28), 674.9 / 136 (CE: 41, CXP: 12), 674.9 / 524.0 (CE: 39, CXP: 14). DP: Declustering potential (DP) and Entrance potential (EP) were 110 and 10 V respectively. ESI source parameters included ion spray voltage of 5.0ckV, desolvation temperature of 600 C, pressures of curtain gas pressure at 15 psi, Nebulizer gas (GS1) at 60 psi and Heater gas (GS2) of 50 psi. Semiquantitative 2’30 cGAMP results were obtained using analyte calibration curve (MS pear area versus concentration) samples in 2% (v/v) acetic acid in 2:3 methanol:water and processed in the same manner as described above for cell pellet samples. Calibration curve included blanks, 1, 5, 10, 50, 100 and 500 ng/mL. SPE recovery was also evaluated at two concentration levels (50, 500 ng/mL in 2% (v/v) acetic acid in 2:3 methanol:water) by spiking before and after SPE and was calculated consistently at an average of 90%. Analyst software (Version 1.6.3; AB Sciex Instruments, Redwood City, CA, USA) was used for data acquisition, and MultiQuant software (Version 3.0.2; AB Sciex Instruments, Redwood City, CA, USA) was used for data analysis.

Immunity 52, 1–17.e1–e9, February 18, 2020 e8

Please cite this article in press as: Zhou et al., Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7RDependent STING Activation by Tumor-Derived cGAMP, Immunity (2020), https://doi.org/10.1016/j.immuni.2020.01.014

BMDMs in vitro Stimulation BMDMs from WT, cGAS
/
or Stinggt/gt mice were transfected with plasmid DNA (pmaxGFP, Lonza) or 50 ppp-dsRNA (Invivogen), or treated with 10 mg/mL DMXAA (Invivogen) for overnight, respectively. To examine the effect of extracellular ATP on IFNb production in response to cGAMP stimulation, BMDMs were treated with different concentrations of ATP and cGAMP for 24 h. Concentrations of ATP and cGAMP for particular experiments are indicated in figure legends. Fluorescein-conjugated cGAMP (BIOLOG C178) was used to directly monitor cGMAP uptake. In some cases, 100 mM A740003 (TOCRIS) was added to assess the effect of blocking P2X7R. 125 mM folinic acid (Sigma) was used as competitive inhibitor for SLC19A1. Electroporation was performed with the Nucleofector Kit V (Lonza) according to manufacturer’s recommendations. IFNb protein in culture supernatant was measured with the High Sensitivity Mouse IFN Beta ELISA Kit (PBL Assay Science). Quantification of Extracellular ATP in Cell Culture MC38.CD39 cells or parental MC38 cells were incubated with an increasing concentration of extracellular ATP (ThermoFisher Scientific) for 1 h. ATP Determination Kit (ThermoFisher Scientific) was used to detect remaining ATP concentration in the cell culture supernatant. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical details of experiments are indicated in the figure legends, text or methods. All data analyses were performed with Prism software (GraphPad). All data are presented as mean ± standard deviation (SD), and error bars represent the SD of at least three biological replicates, unless otherwise indicated in the figure legends. No statistical analysis was used to predetermine sample size. For in vivo studies, animals were randomized to ensure similar tumor volume distribution in different treatment groups. The majority of the datasets passed the D’Agnostino & Pearson omnibus normality test and were analyzed by unpaired two-tailed Student’s t test. Welch’s correction was performed when the variances were significantly different. For datasets where the N was too small for the normality test, normal distribution was assumed based on data distribution. p % 0.05 was considered significant. DATA AND CODE AVAILABILITY The RNA-sequencing data generated during this study are available at the NCBI GEO: GSE119952: https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE119952.

e9 Immunity 52, 1–17.e1–e9, February 18, 2020