Hedgehog-induced PD-L1 on tumor-associated macrophages is critical for suppression of tumor-infiltrating CD8+ T cell function

The programmed death-1 (PD-1) and the PD ligand 1 (PD-L1) interaction represents a key immune checkpoint within the tumor microenvironment (TME), and PD-1 blockade has led to exciting therapeutic advances in clinical oncology. Although IFN-γ–dependent PD-L1 induction on tumor cells was initially thought to mediate the suppression on effector cells, recent studies have shown that PD-L1 is also expressed at high level on tumor-associated macrophages (TAMs) in certain types of tumors. However, the precise role of PD-L1 expression on TAMs in suppressing antitumor immunity within the TME remains to be defined. Using a myeloid-specific Pdl1-knockout mouse model, here we showed definitive evidence that PD-L1 expression on TAMs is critical for suppression of intratumor CD8+ T cell function. We further demonstrated that tumor-derived Sonic hedgehog (Shh) drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8+ T cell function, leading to tumor progression. Mechanistically, Shh-dependent upregulation of PD-L1 in TAMs is mediated by signal transducer and activator of transcription 3, a cascade that has not been previously reported to our knowledge. Last, single-cell RNA sequencing analysis of human hepatocellular carcinoma revealed that PD-L1 is mainly expressed on M2 TAMs, supporting the clinical relevance of our findings. Collectively, our data revealed an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing antitumor immunity within the TME, which could lead to the development of new immunotherapeutic strategies for treating cancer.


Introduction
The programmed death-1/programmed death ligand 1 (PD-1/PD-L1) axis of interaction is one of the most important immunosuppressive mechanisms within the tumor microenvironment (TME), and targeting this mechanism has led to exciting therapeutic advances in clinical oncology (1)(2)(3)(4)(5)(6). While PD-1 was mainly found on the intratumoral lymphocyte population, PD-L1 expression has been observed on a diverse group of cells, including tumor cells, myeloid cells, lymphocytes, and stromal cells (7). Although IFN-γ-dependent PD-L1 induction on tumor cells was initially found and commonly thought to mediate the suppression on effector cells (8), recent reports have shown that PD-L1 is also expressed on stromal cells, especially tumor-associated macrophages (TAMs,. Additionally, PD-L1 on TAMs is more stable and less dependent on IFN-γ (12). Furthermore, PD-L1 expression was more frequently detected on immune cells than on malignant cells in hepatocellular carcinoma (HCC), non-small cell lung cancer, urothelial carcinoma, and esophageal squamous cell carcinoma (13)(14)(15). However, it has been difficult to query the precise role of PD-L1 on each of the cell populations within the TME due to the lack of a conditional ready Pdl1 fl/fl mouse model that allows for lineage-specific deletion of Pdl1. Here we showed that we have successfully generated, to the best of our knowledge, the first Pdl1 fl/fl model in pure C57BL/6 background. After breeding to LysM-cre mice expressing Cre recombinase in myeloid cells (16), these mice showed a complete deletion of Pdl1 in myeloid lineage cells. This allowed us to directly study the role of TAM-derived PD-L1 in subverting adaptive immune responses within the TME.
The programmed death-1 (PD-1) and the PD ligand 1 (PD-L1) interaction represents a key immune checkpoint within the tumor microenvironment (TME), and PD-1 blockade has led to exciting therapeutic advances in clinical oncology. Although IFN-γ-dependent PD-L1 induction on tumor cells was initially thought to mediate the suppression on effector cells, recent studies have shown that PD-L1 is also expressed at high level on tumor-associated macrophages (TAMs) in certain types of tumors. However, the precise role of PD-L1 expression on TAMs in suppressing antitumor immunity within the TME remains to be defined. Using a myeloid-specific Pdl1-knockout mouse model, here we showed definitive evidence that PD-L1 expression on TAMs is critical for suppression of intratumor CD8 + T cell function. We further demonstrated that tumor-derived Sonic hedgehog (Shh) drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8 + T cell function, leading to tumor progression. Mechanistically, Shh-dependent upregulation of PD-L1 in TAMs is mediated by signal transducer and activator of transcription 3, a cascade that has not been previously reported to our knowledge. Last, single-cell RNA sequencing analysis of human hepatocellular carcinoma revealed that PD-L1 is mainly expressed on M2 TAMs, supporting the clinical relevance of our findings. Collectively, our data revealed an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing antitumor immunity within the TME, which could lead to the development of new immunotherapeutic strategies for treating cancer.
JCI Insight 2021;6(6):e146707 https://doi.org/10.1172/jci.insight.146707 The Hedgehog (Hh) signaling pathway plays an important role in tumorigenesis in many types of human cancer (17). Binding of Sonic hedgehog (Shh), Desert hedgehog (Dhh), or Indian hedgehog (Ihh) to the transmembrane protein Patched-1 on target cells leads to the release of Smoothened (Smo) and activation of downstream signaling events mediated by the Gli family of transcription factors (18). We have recently demonstrated an important role for Hh signaling pathway in promoting M2 polarization of TAMs, leading to a reduction in CD8 + T cell recruitment to the TME (19). The immunosuppressive M2 phenotype of TAMs is also closely correlated with PD-L1 expression in several cancer types (20)(21)(22)(23). However, what regulates the PD-L1 upregulation on M2 TAMs remains to be determined.
In this study, we first showed that tumor stroma-derived PD-L1 is important for suppression of intratumor CD8 + T cells and that the majority of PD-L1-expressing cells in the hepatoma stroma were TAMs. Using a newly generated myeloid-specific Pdl1-knockout model, we demonstrated that deletion of Pdl1 in TAMs rescued intratumor CD8 + T cell function and suppressed tumor growth, providing proof for the critical role TAM-derived PD-L1 plays in suppressing intratumor CD8 + T cell function. We further found that Hh signaling regulates PD-L1 expression in TAMs and that tumor-derived Shh drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8 + T cell effector function, resulting in accelerated tumor progression. Last, we identified that signal transducer and activator of transcription 3 (Stat3) mediates the downstream effects of Hh in TAMs to regulate PD-L1 expression. Single-cell RNA (scRNA) sequencing analysis of human HCC revealed that PD-L1 is mainly on M2 TAMs, supporting the clinical relevance of our findings. Collectively, our data revealed an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing antitumor immunity within the TME, which could lead to the development of new immunotherapeutic strategies for treating cancer.

Results
Tumor stroma-derived PD-L1 is critical for suppression of intratumor CD8 + T cells. To first investigate whether non-tumor-derived PD-L1 plays a role in suppressing intratumor CD8 + T cell function and tumor growth in HCC, we generated mouse hepatoma Hepa1-6 cells with Pdl1 deletion (referred to as Pdl1-KO) using CRISPR/Cas9-mediated gene knockout (24). Pdl1-WT Hepa1-6 cells were created using a lentiviral CRIS-PR/Cas9 vector containing a nontargeting guide RNA (gRNA) sequence. Pdl1-WT and Pdl1-KO Hepa1-6 cells were subcutaneously inoculated in C57BL/6 mice, and a cohort of mice bearing the Pdl1-WT tumor were further treated with 10 mg/kg anti-PD-L1 antibodies 3 times per week starting at day 14 postinoculation. On day 28 at sacrifice, we observed no significant tumor growth reduction (P = 0.08) in mice bearing the Pdl1-KO tumor compared to Pdl1-WT bearing mice. However, treatment of Pdl1-WT tumor-bearing mice with anti-PD-L1 antibodies resulted in significant (P < 0.005) reduction in tumor growth compared with untreated mice ( Figure 1A). Assessment of the tumor-infiltrating CD8 + T cells revealed no significant change in CD8 + T cell numbers within the tumor stroma regardless of Pdl1 deletion in tumor cells or with PD-L1-blocking antibodies ( Figure 1B). However, CD8 + T cells in the tumors treated with anti-PD-L1 antibodies demonstrated a marked increase in effector function measured by IFN-γ and granzyme B (GzmB) production. When compared with the Pdl1-KO samples, intratumor CD8 + T cells in mice treated with anti-PD-L1 antibodies produced significantly (P < 0.05) higher levels of IFN-γ and GzmB measured by fluorescence-activated cell sorting (FACS, Figure 1C). These data suggested that non-tumor-derived PD-L1 plays an important role in subverting intratumor CD8 + T cell function.
TAMs are the main PD-L1-expressing cells within the TME. We then proceeded to identify the PD-L1 + populations within the TME of HCC. We found that 72.3% of PD-L1 + cells were CD11b + F4/80 + TAMs, with CD11b + F4/80other myeloid cells and CD11bnonmyeloid cells making up 15.3% and 12.4%, respectively ( Figure 2A). Immunofluorescence staining of the subcutaneously inoculated Hepa1-6 tumor samples further revealed substantial overlap between PD-L1 + cells (shown in green) and F4/80 + TAMs (shown in red, Figure 2B). These results indicated that TAMs are the main PD-L1-expressing cells with the TME.
Generation and characterization of myeloid-specific Pdl1 conditional knockout mice.
To study the precise role of TAM-derived PD-L1 in suppressing antitumor immunity in vivo, we generated Pdl1 conditional ready (referred to as Pdl1 fl/fl ) mice in a pure C57BL/6 background. Briefly, Pdl1 fl/fl mice were created by replacing the first 2 coding exons with a new gene segment with exons 2 and 3 plus a neomycin resistance cassette (Neo r ) flanked by LoxP sites via homologous recombination ( Figure 3A). Successful integration was confirmed by digesting genomic DNA of embryonic stem cells with EcoRI and performing Southern blotting ( Figure 3B). The genotype of the mice was further confirmed by PCR of tail samples (data not shown). We JCI Insight 2021;6(6):e146707 https://doi.org/10.1172/jci.insight.146707 then generated the Pdl1 conditional knockout mice allowing for myeloid lineage deletion of Pdl1 by crossing Pdl1 fl/fl mice with LysM-cre mice, producing LysM-cre + Pdl1 fl/fl (referred to as Pdl1 ΔM ). Deletion of Pdl1 in myeloid cells in these mice was first assessed by quantitative real-time PCT (qRT-PCR) using bone marrow-derived macrophages (BMDMs) from Pdl1 fl/fl and Pdl1 ΔM and primers specific for mouse Pdl1 mRNA ( Figure 3C). Knockout status was confirmed with FACS staining of CD11b and PD-L1 on peripheral blood cells from Pdl1 fl/fl and Pdl1 ΔM mice ( Figure 3D).
TAM-derived PD-L1 is critical for suppressing intratumor CD8 + T cell function. We then proceeded to investigate whether TAM-derived PD-L1 expression affected tumor growth and intratumor CD8 + T cell function in vivo. Hepa1-6 tumor cells were inoculated in Pdl1 fl/fl and Pdl1 ΔM , and tumor growths were monitored over time. We found that Pdl1 ΔM mice had a significant (P < 0.005) reduction in tumor growth, compared with Pdl1 fl/fl tumor-bearing mice ( Figure 4A). This was accompanied by a significant (P < 0.05) increase in the levels of IFN-γ and GzmB production by intratumor CD8 + T cells compared with the control ( Figure 4B). However, no significant difference was detected in the percentages of the intratumor CD8 + T cells compared to control ( Figure 4C). Collectively, these results suggest that PD-L1 expression on TAMs is critical for suppressing intratumor CD8 + T cell function.  physiological environment in which the HCC normally arise, we utilized mice deficient for the multidrug resistance gene 2 (Mdr2 -/-) as an autochthonous model of HCC previously described (25,26). After generating LysM-cre + Smo F/F Mdr2 -/mice (referred to as Smo ΔM Mdr2 -/-), we also found a consistently increased production of IFN-γ and GzmB in intratumor CD8 + T cells isolated from Smo ΔM Mdr2 -/tumors ( Figure  5C), which was correlated with a reduction of PD-L1 expression on Smo ΔM Mdr2 -/-TAMs ( Figure 5D). Collectively, these results revealed an important role for Hh signaling in promoting PD-L1 expression on TAMs and suppressing intratumor CD8 + T cell effector function.
Tumor-derived Shh ligand is critical for PD-L1 upregulation on TAMs. To further confirm that Hh signaling can directly promote PD-L1 expression on macrophages, we treated BMDMs from C57BL/6 mice with 5 ng/mL of Shh ligands for 24 hours and measured expression of PD-L1 via flow cytometry. We observed that treatment of BMDMs with Shh significantly upregulated PD-L1 expression (P < 0.0005; Figure 6A). Additionally, in vitro coculturing of CD3/CD28-activated CD8 + T cells with Pdl1 fl/fl BMDMs in the presence of Shh showed significantly suppressed IFN-γ and GzmB productions (P < 0.005) when compared with untreated samples. However, when Pdl1 was deleted in macrophages, no reduction was observed in the presence or absence of Shh ( Figure 6B), indicating the suppressive effects of Shh-induced M2 macrophages on CD8 + T cells are mediated through PD-L1.
Furthermore, we have previously generated Shh-KO Hepa1-6 hepatoma and LLC1 Lewis lung carcinoma cells using the CRISPR/Cas9 technology to study whether tumor-derived Shh ligands are the major source of Hh signaling within the TME (19). Upon assessing the expression of PD-L1 on TAMs in Shh-KO tumor samples, we found that deletion of Shh in tumor cells also resulted in reductions of PD-L1 expression on TAMs, both in the Hepa1-6 ( Figure 6C) and in the LLC1 (P < 0.005; Figure 6E) tumor models. Last, this was correlated with improved intratumor CD8 + T cell effector cell functions demonstrated by increased IFN-γ and GzmB productions in the Shh-KO samples measured by FACS (Figure 6, D and F). This further supported a critical role for tumor-derived Shh in TAM PD-L1 expression and suppression of intratumor CD8 + effector cell functions.
Hh-induced PD-L1 upregulation in TAMs is mediated by Stat3. We next sought to understand the mechanisms by which Shh induces PD-L1 expression in TAMs. Previous studies have demonstrated that several transcription factors are associated with PD-L1 upregulation in various cell types, including c-Jun (gene symbol: Jun), c-Myc (gene symbol: Myc), Stat1, Stat3, and NF-κB (gene symbol: Nfkb1; ref. 27). Thus, we surveyed the expression levels of these transcription factors in BMDMs treated with Shh and found that Stat3 mRNA expression was significantly elevated when compared with untreated control (P < 0.05; Figure 7A). We also found that Stat3 mRNA levels were significantly reduced in Smo ΔM TAMs, compared with Smo fl/fl TAMs ( Figure 7B), suggesting that Stat3 could be mediating the downstream effects of Shh in TAMs. Through in silico promoter analysis, we found a consensus Gli-binding sequence (GCCCCG-CCCC) at the -1076 to -1067 position upstream of the transcription start site of Stat3 (28). Using the chromatin immunoprecipitation (ChIP) method, we found increased Gli1 occupancy at that site when BMDMs were treated with Shh. Such binding was reduced to baseline when BMDMs were treated with 5   (32). The pipeline of scRNA sequencing analysis has been described previously (33,34). Briefly, we obtained scRNA transcriptomes of 9493 cells after quality control steps and conducted normalization and principal component analysis (PCA) on the 3000 most variable genes. We then performed uniform manifold approximation and projection (UMAP) dimension reduction analysis, which revealed 24 unique clusters of cells annotated based on known cell lineagespecific markers -including 6 clusters of hepatocytes (HC), 2 clusters of bile ductal cells (BDC), 2 clusters of TAMs, 1 cluster of plasmacytoid dendritic cells (pDC), 3 clusters of B cells, 1 cluster of CD4 + cells, 1 cluster of Foxp3 + T regulatory (Treg) cells, 2 clusters of CD8 + T cells, 1 cluster of natural killer (NK) cells, 2 clusters of fibroblasts (FB), and 3 clusters of endothelial cells (EC; Figure 8A). Analyzing expressions of SHH, IHH, and DHH across all clusters revealed that HCs are the main source of SHH ( Figure 8B). More importantly, PD-L1 (gene symbol: CD274) was mainly expressed in TAMs ( Figure 8C), similar to what we have observed in our mouse models. Further analysis of the TAM population revealed 2 distinctive groups -M2-high and M2-low ( Figure 8D). The M2-high group was characterized by higher expressions of CD163 (scavenger receptor), MRC1 (CD206, mannose receptor), and MMP9, which are 3 genes associated with the TAM M2 phenotype (35). The M2-low group was defined by higher expressions of TIMP1 and LGALS2, which are associated with antiangiogenic and proinflammatory functions of macrophages in humans (36,37). PD-L1 expression was mainly found in the M2-high group, further suggesting that M2-polarized TAMs can contribute to intratumor immunosuppression through PD-L1 in human HCC and IHCC.

Discussion
In this study, we provided proof that TAM-derived PD-L1 expression is critical for suppressing intratumor CD8 + T cell function in vivo. We further demonstrated that Hh signaling regulates PD-L1 expression in TAMs and that tumor-derived Shh drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8 + T cell effector function. Mechanistically, intracellular Hh signaling activated Stat3 to regulate PD-L1 expression in TAMs. Last, analysis of scRNA sequencing results of human HCC samples supported that PD-L1 is mainly expressed on TAMs within the TME and that PD-L1 expression on TAMs is strongly correlated with the M2-TAM phenotype. Thus, our findings identified an important and potentially novel role for paracrine Hh signaling in promoting TAM PD-L1 expression mediated by Stat3, resulting in intratumor CD8 + T cell dysfunction and increased immunosuppression within the TME.
Although IFN-γ-dependent PD-L1 upregulation on tumor cells was thought to mediate the suppression on intratumor CD8 + T cells in certain tumors (8), recent studies have suggested PD-L1 expression was more frequently detected on immune cells than on malignant cells in HCC, non-small cell lung cancer, urothelial carcinoma, and esophageal squamous cell carcinoma (13)(14)(15). However, the precise role of PD-L1 expression on TAMs in suppressing antitumor immunity required further investigation. Here using conditional Pdl1-knockout mice, we found that TAM-derived PD-L1 upregulation is critical for suppressing intratumor CD8 + T cell function, leading to tumor progression in vivo. In addition, tumor-derived PD-L1 expression was found to be low and noncontributory to intratumor CD8 + T cell suppression. This is consistent with a recent report in HCC that tumor environmental factors induce PD-L1 expression on monocytes/ macrophages in the peritumor stroma, and high percentages of these PD-L1 + monocytes/macrophages are correlated with disease progression and poor survival in patients (9). These previous studies prompted us to create a conditional ready Pdl1 mouse model that allows us to knock out Pdl1 in myeloid cells. Using this model, we were able to provide the first definitive proof to our knowledge that PD-L1 expression on TAMs plays a more critical role in suppressing CD8 + T cell effector function than tumor-derived PD-L1. This model will also be useful in elucidating the functions of PD-L1 in different cell populations and in various disease processes. It also has important mechanistic and clinical implications as PD-L1 expression pattern may be used to stratify patients for response to PD-1/PD-L1 treatments.
What then regulates the expression of PD-L1 in TAMs? A recent report highlighted that TAMs accumulate in PD-L1 hi human HCC tumors and only few of these PD-L1 hi samples displayed IFN-γ hi signatures (38), suggesting that there are IFN-γ-independent mechanisms regulating PD-L1 expression in TAMs. Here we revealed that the Hh signaling pathway in TAMs is active and important for upregulation of PD-L1 expression, which has not been reported in previous literature to our knowledge. We also demonstrated that the Shh, produced by tumor cells, is responsible for driving the immunosuppressive phenotype of TAMs characterized by high PD-L1 expression to facilitate its own immune evasion, suggesting the importance of communication between tumor cells and TAMs to promote tumor growth. Together, our study not only revealed the importance of paracrine Hh signaling in modulating the TME to facilitate cancer progression but also identified Shh as a potentially new upstream signaling cascade that regulates PD-L1 expression in TAMs.

Figure 5. Loss of Smo in myeloid cells interferes with PD-L1 expression in TAMs and promotes intratumor CD8 + T cell effector functions in vivo. (A) Smo ΔM intratumor CD8 + T cells produced higher levels of IFN-γ and GzmB than
How does Shh signaling pathway regulate the expression of PD-L1? Here we provide what we believe is the first evidence to support that Shh pathway transcription factor Gli1 regulates Stat3 expression transcriptionally. Consequently, Stat3 drives the downstream effects of Hh signaling in TAM PD-L1 upregulation, eventually resulting in the functional suppression of CD8 + TILs. Stat3 is a key transcription factor that mediates macrophage M2 polarization (39). Murine studies using a myeloid-specific Stat3-knockout model demonstrated the antiinflammatory function of Stat3, characterized by impaired bactericidal activity and increased IL-10 production (40). In tumor studies with HCC, lung carcinoma, and melanoma, it was also shown that inhibition of Stat3 in macrophages abrogated their M2 polarization and protumorigenic effects (41,42). Furthermore, there is additional evidence indicating Stat3 can regulate the expression of PD-L1 in various cell types (43,44). Our finding is also consistent with a previous report that PD-L1/2 overexpression was dependent on activation of Stat3 in TAMs in human lymphoma (45). Collectively, our data revealed a potentially novel role for the Shh-Gli1-Stat3 signaling cascade in promoting TAM-derived PD-L1 upregulation and intratumor immunosuppression. However, it remains to be explored whether there is crosstalk between the IFN-γ and Shh pathways through Stat3 or other intracellular mediators in driving PD-L1 expression in TAMs.
Our findings are clinically relevant because we have also shown in our analysis of scRNA sequencing results that human HCC cells produce SHH and that PDL1 (CD274) expression is mainly found in TAMs. A query of other human cancers using The Cancer Genome Atlas PanCancer studies revealed that, in addition to HCC, colorectal carcinoma, renal cell carcinoma, pancreatic adenocarcinoma, gastric cancer, and cholangiocarcinoma were the highest SHH-expressing cancer types (46). This suggests an exciting therapeutic potential of Hh inhibitors in treating a broad range of human cancers. Indeed, pharmacologic inhibition of Hh signaling with small molecule inhibitors also showed effectiveness in reducing M2 polarization and impressive synergism with immune checkpoint inhibitors in models of HCC and lung cancer in our previous investigation (19). Further investigations are needed to translate this combinational strategy in clinical trials in treating patients with Shh-expressing cancers.
In conclusion, we have identified a critical role for Shh in promoting PD-L1 upregulation on TAMs and that TAM-derived PD-L1 in the TME of HCC is a major and important source for PD-L1/PD-1 axis-mediated suppression on intratumor CD8 + T cells. We further demonstrated that Shh-activated TAMs signal through intracellular Stat3, which results in PD-L1 upregulation. Our findings are novel and could potentially provide therapeutic insights in the development of novel chemotherapeutic and/or immunotherapeutic strategies for the treatment of HCC and other Shh-expressing human cancers.

Methods
Animals. Animals used in our studies were described previously (19). In addition, B6.129S1-Stat3 tm1Xyfu /J (Stat3 fl/fl ) was purchased from The Jackson Laboratory. Prior to arriving, Stat3 fl/fl mice were backcrossed to C57BL/6 mice in-house for at least 9 generations at The Jackson Laboratory. Backcrossed  The neomycin-resistant ES clones were screened for homologous recombinants using PCR primers flanking the 5′ and 3′ recombination sites. Positive clones were subsequently confirmed by Southern blot analysis after restriction digest of genomic DNA with EcoRI and hybridization with a 32 P-radioisotope-labeled probe against a 772 bp sequence in the intron 3 region of the Pdl1 gene. ES cells from a confirmed clone were eventually injected into blastocysts derived from C57BL/6 mice, and these blastocysts were transferred to pseudopregnant C57BL/6 females. Chimeric offspring were identified by genotyping using PCR primers flanking the 5′ and 3′ recombination sites. Mice heterozygous for the floxed Pdl1 allele were mated with C57BL/6 mice for 1 additional generation to ensure germline transmission. Offspring from the additional backcrossing were mated with mice expressing Cre under the control of the LysM promoter (LysM-cre) to generate LysM-cre + Pdl1 fl/fl (referred to as Pdl1 ΔM ).
Tumor models. Hepa1-6 hepatoma or LLC1 lung carcinoma cells were injected subcutaneously into each mouse in the right hind limb region in 100 μL HBSS with a 27-gauge needle syringe. For treatments, beginning 14 days after injection of tumor cells, mice were injected intraperitoneally with 200 μL (200 μg/ mouse) anti-PD-L1 antibody (Bio X Cell) thrice weekly until humane endpoints were reached. Both male and female mice were used. Mice were 6 to 8 weeks of age. There was no systematic means of randomization of mice. Three-digit codes identified the mice, and the experiment was carried out blindly throughout. To estimate the volume of the growing tumor mass, diameters of both the length (a) and the width (b) of the mass were measured every 3-4 days, after which the tumor volume (V) was calculated according to the formula V = ab 2 /2, as described previously (50). When experimental endpoints were met or when the longer axis of each tumor was more than 20 mm in diameter, all the mice were euthanized according to the NIH guidelines. Tumors were resected and transferred to 2 mL RPMI 1640 medium on ice. Tumor size (mm) was measured with a ruler. The tumors from all experiments were then processed for FACS analysis or sorting on the same day or frozen in O.C.T. Compound (VWR) for cryosectioning.
Isolation of BMDMs. To prepare macrophages, mice were sacrificed and disinfected with 70% ethanol. Both lower extremities were excised, and the long bones -femur and tibia -were separated from muscular layers and placed in RPMI medium. To extract BMDMs, 10 mL RPMI medium was used to flush out each bone using a 25-gauge needle, and cells were gently dissociated by pipetting. A 70 μm nylon BD Falcon cell strainer was placed atop a 50 mL BD Falcon tube, and the suspension was filtered into the 50 mL tube. The resultant suspension was centrifuged at 300g for 5 minutes. The supernatant was then aspirated. ACK lysis buffer (5 mL) was then added, and the contents were incubated for 2 minutes at room temperature. To quench the lysis reaction, 10 mL RPMI was added. The content was then centrifuged at 300g, and the cells were washed 2 additional times with 1× HBSS. The cells were plated at 1 × 10 6 cells per well in a sterile 6-well tissue culture plate in 2 mL BMDM culture medium plus 10 ng/mL M-CSF to obtain mature BMDMs. On day 2, supernatants were aspirated and replenished with fresh culture medium with M-CSF. On day 5, 2 mL fresh culture medium with M-CSF was added. Purity of the BMDMs (>95% F4/80 + cells) was confirmed by flow cytometry.
In vitro coculturing of BMDMs and CD8 + T cells. CD8 + T cells were purified from C57BL/6 mouse spleens and cocultured with mature Pdl1 fl/fl and Pdl1 ΔM BMDMs with or without 5 ng/mL Shh ligands in the presence of 2 μg/mL CD3e and CD28 antibodies (BD Pharmingen clones 145-2C11 and 37.51, respectively) at different ratios and different time points (6,12,24, and 48 hours). Data shown here were obtained from the optimal conditions of 10:1 T cell/BMDM ratio and 24-hour coculture period.
Antibodies Statistics. Results are expressed as mean ± SEM. Comparison between groups was performed by Kruskal-Wallis, Wilcoxon-Mann-Whitney, 1-and 2-way ANOVA, and 2-tailed Student's t test. All statistical analyses were performed with JMP Version 12 software (SAS Software). P values of less than 0.05 were considered significant.
Study approval. All animal experiments were performed in agreement with the protocols approved by the Institutional Animal Care and Use Committee of Duke University, Durham, North Carolina, USA, and The Ohio State University, Columbus, Ohio, USA.

Author contributions
AJP designed and performed the experiments, analyzed the data, and wrote the manuscript. RD assisted with performing the experiments and contributed essential ideas and discussion. RL, DMB, RAB, ZL, and XH participated in designing various parts of the study and in discussion and interpretation of the results. YY assisted with designing the experiments, supervised the work, and wrote the manuscript.