GATA2 Regulates Constitutive PD-L1 and PD-L2 Expression in Brain Tumors

Encouraging clinical results using immune checkpoint therapies to target the PD-1 axis in a variety of cancer types have paved the way for new immune therapy trials in brain tumor patients. However, the molecular mechanisms that regulate expression of the PD-1 pathway ligands, PD-L1 and PD-L2, remain poorly understood. To address this, we explored the cell-intrinsic mechanisms of constitutive PD-L1 and PD-L2 expression in brain tumors. PD-L1 and PD-L2 expression was assessed by flow cytometry and qRT-PCR in brain tumor cell lines and patient tumor-derived brain tumor-initiating cells (BTICs). Immunologic effects of PD-L2 overexpression were evaluated by IFN-γ ELISPOT. CD274 and PDCD1LG2 cis-regulatory regions were cloned from genomic DNA and assessed in full or by mutating and/or deleting regulatory elements by luciferase assays. Correlations between clinical responses and PD-L1 and PD-L2 expression status were evaluated in TCGA datasets in LGG and GBM patients. We found that a subset of brain tumor cell lines and BTICs expressed high constitutive levels of PD-L1 and PD-L2 and that PD-L2 overexpression inhibited neoantigen specific T cell IFN-γ production. Characterization of novel cis-regulatory regions in CD274 and PDCD1LG2 lead us to identify that GATA2 is sufficient to drive PD-L1 and PD-L2 expression and is necessary for PD-L2 expression. Importantly, in TCGA datasets, PD-L2 correlated with worse clinical outcomes in glioma patients.. By perturbing GATA2 biology, targeted therapies may be useful to decrease inhibitory effects of PD-L2 in the microenvironment.

In vitro Luciferase assay. Bioluminescence imaging was performed in the Molecular Imaging Center. 4×10 3 cells/well were plated into 96 well plates. CD274 and PDCD1LG2 reporter luciferase vectors and pRLuc-N3 Renilla control were transfected into cells the next day. 48 hours later, D-luciferin and Coelenterazine were added and luminescence was analyzed by an IVIS 50 (PerkinElmer, Waltham, MA; Living Image 4.3.1).
Statistical analysis. BTIC experiments were repeated (n = 2) and all the other experiments were repeated (n ≥ 3). All statistical analyses were t-test or one-way ANOVA. GBM and LGG clinical and tumor mRNA (RNA-Seq V2) TCGA datasets were downloaded from www.cbioportal.org 20,21 . PD-L2/PD-L1 high and low expression status was defined as expression values ≥ or <median of PD-L2 and PD-L1, respectively (a commonly used, unbiased method of dichotomizing gene expression). Association between expression status of the two genes was examined by Mantel-Haenzel Chi-square test. Kaplan-Meier method was used to estimate empirical survival probabilities and generate KM curves. Log rank test was used to examine statistical significance of differences in OS and DFS between patient groups defined by PD-L1 or/and PD-L2. Hazard ratio with 95% confidence interval (CI) was reported from univariate Cox model. To determine whether PD-L2 expression status was independently associated with survival, multivariate Cox proportional hazards models were used, adjusting for age, gender and IDH1 mutation. All tests were 2-sided and P-value ≤0.05 was considered statistically significant. All statistical analyses were performed with SAS (version 9.4; SAS, Cary, NC).
Ethical approval. All procedures performed were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Animal studies were approved by the Animal Studies Committee at Washington University. BTIC lines as described 18 were generated from patients under informed consent in full adherence to tissue banking protocols approved by the Institutional Review Board at Washington University.
Novelty and impact. We identified high constitutive PD-L1 and PD-L2 expression in brain tumor cell lines and primary BTICs and characterized novel cis-regulatory regions for both genes. GATA2 upregulated both ligands and was necessary for PD-L2 expression. High PD-L1 and PD-L2 expression correlated with worse outcomes in GBM and low-grade glioma, while high PD-L2 independently correlated with shorter disease-free survival in low-grade glioma.

Results
High constitutive PD-L1 and PD-L2 expression in brain tumor cell lines. We identified cancer cell lines with high constitutive expression of PD-1 ligands to study cell intrinsic, rather than cytokine induced, regulation. We interrogated the Cancer Cell Line Encyclopedia (CCLE), an extensively-characterized human cancer cell line panel 19 . Analyzing 967 cell lines, we identified cancer lines that expressed PD-L1 and PD-L2; 350 cell lines expressed PD-L1 mRNA above the mean expression level, and 193 cell lines expressed PD-L1 mRNA more than 1 standard deviation above all CCLE lines (Fig. 1A). For PD-L2, 220 cell lines expressed PD-L2 mRNA above the mean, and 97 cell lines harbored PD-L2 expression greater than 1 standard deviation above the mean. Cell lines expressing high levels of PD-L1 and/or PD-L2 were derived from all cancers ( Supplementary Fig. 1A). Of 52 glioma cell lines, 34 harbored PD-L1 mRNA expression above the CCLE mean whereas 22 cell lines expressed PD-L2 mRNA above CCLE mean levels ( Fig. 1B). PD-L1 and PD-L2 expression levels were concordant in some cell lines but divergent in others. To confirm CCLE PD-L1 and PD-L2 expression, we assessed gene expression levels by qRT-PCR in select cell lines with either high or low gene expression. We confirmed high PD-L1 mRNA expression in 6 cell lines relative to 4 low-expressing cell lines (Fig. 1C, top). We also confirmed high constitutive PD-L2 mRNA expression in cell lines including GMS10, KALS1, and AM38 relative to low-expressing LN340 and LN464 (Fig. 1C, bottom). CH-157 and IOMM-Lee meningioma cell lines exhibited high expression of PD-L1 and PD-L2, respectively. Both PD-L1 and PD-L2 cell surface expression levels were evaluated by flow cytometry and were concordant with gene expression (Fig. 1D).
We next examined PD-L1 and PD-L2 gene expression by qRT-PCR in a panel of low passage (≤5 passages from initial culture) patient-derived brain tumor initiating cell (BTIC) lines established from patients with newly diagnosed GBM 18 . In 5 TERT mutant BTIC lines 24 , 4 exhibited constitutive PD-L1 and PD-L2 gene expression similar to high-expressing well-passaged cells (Fig. 1E). High PD-L1 and PD-L2 mRNA in the representative B36 BTIC line was concordant with cell surface expression compared to the lower-expressing B49 cell line (Fig. 1F). These data show that a subset of cancer cell lines, including primary BTICs, exhibit high constitutive levels of both PD-L1 and PD-L2. In addition, analysis of TCGA datasets revealed that PD-L2 was also expressed in human cancers including GBM and low-grade glioma (LGG)( Supplementary Fig. 1B), lung adenocarcinoma, melanoma, and renal cell carcinoma ( Supplementary Fig. 1C).

PD-L2 inhibits Neoantigen-Specific T Cell function.
Although the PD-L1 inhibitory functions are well-established, the effects of tumor cell PD-L2 overexpression on T cell function have not been well studied. We tested the effects of PD-L1 and/or PD-L2 overexpression in the GL261 brain tumor model in which the GL261-derived mutant Imp3-D81N 8-mer is an immunogenic neoantigen 22 . GL261 cells express low levels of PD-L1 that increase in response to IFN-γ stimulation, whereas PD-L2 is not expressed or induced ( Fig. 2A). To test the effects of both PD-1 ligands on GL261 immunogenicity, we overexpressed PD-L1 (GL261.PD-L1), PD-L2 (GL261.PD-L2), or both ligands (GL261.PD-L1/2) and confirmed their individual and combined overexpression www.nature.com/scientificreports www.nature.com/scientificreports/ by flow cytometry (Fig. 2B). Compared to parental GL261, each cell line inhibited IFN-γ production by Imp3 neoantigen-specific tumor infiltrating lymphocytes by over 50% (Fig. 2C). Thus, tumor cell overexpression of PD-L2 inhibits neoantigen-specific T cell function similar to PD-L1 overexpression and confirms the inhibitory function ascribed to PD-L2 in non-tumor settings 11 .
Identification of novel PD-L1 regulatory elements. After confirming that human PD-L1 (Supplementary 2A,B) and human PD-L2 were upregulated by IFN-γ (Fig. 3A), we examined mechanisms that www.nature.com/scientificreports www.nature.com/scientificreports/ controlled baseline, rather than inducible, PD-1 ligand overexpression. We tested the hypothesis that constitutive expression of PD-L1 and PD-L2 in brain tumor cells is caused, at least partially, by transcriptional activity of cis-regulatory elements near transcription start sites (TSS) of CD274 and PDCD1LG2. To identify candidate elements, we examined the University of California Santa Cruz Genome Browser (UCSCGB). The UCSCGB displays human genomic datasets including data from the ENCODE Project 25 . We assessed (a) areas of increased histone acetylation marks (H3K27Ac) suggesting active regulatory regions, (b) regions of increased DNAse www.nature.com/scientificreports www.nature.com/scientificreports/ hypersensitivity accessible to TF binding, and (c) annotated TF binding sites measured by chromatin immunoprecipitation (CHiP) sequencing. When we integrated these datasets for CD274, we identified 3 candidate regulatory elements relative to the TSS using the Genome Reference Consortium Human Build 37: PD-L1.Pr1 (−4167 to +538), PD-L1.Pr2 (+4564 to +5691), and PD-L1.Pr3 (+8572 to +10276) (Fig. 3B). To test their regulatory activity, we assessed their ability to drive luciferase expression. Each element drove luciferase activity in PD-L1 high-expressing lines (IOMM-Lee, KALS1 and AM38) between 3 and 100-fold relative to low-expressing lines (Fig. 3C). Because PD-L1.Pr1 and.Pr2 included regions previously shown to drive PD-L1 expression via ISRE/IRF-1 9 and AP-1 26 elements, respectively, we focused on the novel Pr3 region located between exons 2 and 3. To identify the Pr3 region driving activity, we generated a truncation retaining a predicted FOS binding site but lacking 979 bp (PD-L1 Pr3M) including predicted GATA2 TF binding sites (Fig. 3D). In contrast to the full PD-L1.Pr3 construct, luciferase activity in cell lines transduced with PD-L1.Pr3M was decreased by over 10-fold (Fig. 3E). These data show that several cis-regulatory regions in CD274 drive transcriptional activity and identify a novel regulatory region.
Identification of Novel PD-L2 regulatory elements. Compared to PD-L1, little is known regarding PD-L2 regulation. To determine the molecular basis of constitutive PD-L2 expression observed in our cell line panel, we took a similar approach to our study of CD274 regulation. Integrating ENCODE data in the UCSCGB, we identified 2 adjacent but distinct candidate regulatory regions defined by H3K27Ac mark density, DNase hypersensitivity, and TF binding sites. We cloned 3 elements from IOMM-Lee cell line genomic DNA relative to the transcription start site: PD-L2.Pr1 (−1145 to −495), PD-L2.Pr2 (−525 to +780), and the PD-L2.Pr1 + 2 region encompassing both sites (−1145 to +780) (Fig. 3F). To determine activity, each was cloned into the pGL3-Promoter luciferase vector and transduced into PD-L2 high-expressing (IOMM-Lee, KALS1, and AM38) and low-expressing (YKG1, LN340, and LN464) cell lines and assayed for luciferase activity. The PD-L2.Pr1 + 2 construct drove normalized luciferase activity 15-150x higher in high-expressing lines compared to low-expressing lines, demonstrating that luciferase activity driven by this element was consistent with endogenous PD-L2 expression levels observed (Fig. 3G, left panel). Moreover, the PD-L2.Pr1 construct drove identical levels of luciferase activity in the high PD-L2 IOMM-Lee and AM38 cell lines compared to the low PD-L2 LN340 and LN464 cell lines. However, when the same cell lines were transfected with the PD-L2.Pr2 constructs, luciferase activity was significantly diminished to about 18% of levels driven by the Pr1 + 2 region in IOMM-Lee and about 2.5% levels driven by the Pr1 + 2 region in AM38 cell lines (Fig. 3G, right panel).
To test the hypothesis that regulatory elements harbored within the PD-L2.Pr1 region were necessary for activity in PD-L2 high-expressing cell lines, we generated mutant constructs of the PD-L2.Pr1 element based on predictions from both the UCSCGB and the PROMO TF prediction resource. Based on TF binding sites that were unique to PD-L2.Pr1 and not found on PD-L2.Pr2, we focused on the GATA2/3 and STAT1 TF binding sites. To test their contribution to PD-L2 Pr1 driven luciferase activity, we generated  (Fig. 3H). The pGL3 constructs were introduced into high-expressing (IOMM-Lee, AM-38) and low-expressing (LN340, LN464) cell lines to determine the effect of each alteration on luciferase activity. The PD-L2.Pr1 construct drove robust luciferase activity in high-expressing cell lines that increased when the STAT1 TF binding site was truncated (Fig. 3I, top panel). In contrast, luciferase activity was nearly extinguished when the GATA2/3 binding site was truncated from PD-L2.Pr1. Luciferase activity was decreased significantly when the GATA2/3 TF binding site was deleted from the PD-L2.Pr1(ΔSTAT1) construct (Fig. 3I, bottom panel). These experiments show that the PD-L2.Pr1 region contains elements that drive constitutive transcriptional activity and suggests that GATA2/3 contributes to this functional role.

GATA2 Upregulates PD-L1 and PD-L2 and is necessary for constitutive PD-L2 expression.
Because regulatory elements predicted to bind the GATA2/3 TFs were required for high luciferase activity in both PD-L1 and PD-L2 regulatory regions, we tested the hypothesis that GATA2 overexpression was sufficient to drive endogenous PD-L1 and PD-L2 gene and cell surface expression. The LN340 and LN464 cell lines, which expressed low basal levels of these ligands, were transduced with the pBabe.GATA2 or pBabe.puro retroviruses ( Supplementary Fig. 3A,B) and assayed for PD-L1 and PD-L2 gene expression and surface protein levels. In both lines, PD-L1 and PD-L2 gene expression measured by qRT-PCR was significantly induced following GATA2 overexpression relative to negative controls (Fig. 4A). Moreover, LN340 and LN464 cells overexpressing GATA2 (Fig. 4B, dark line) upregulated cell surface PD-L1 and PD-L2 by flow cytometry compared to negative controls (Fig. 4B, light line). Because the GATA2 TF binding site shares some overlap with predicted GATA3 TF binding site, we tested the possibility that GATA3 overexpression also drives PD-L2 expression levels. However, when GATA3 was overexpressed, neither PD-L2 mRNA (data not shown) nor cell surface (Supplementary Fig. 3D) changed in LN340 cells compared to the negative control. Thus, overexpression of the GATA2 TF was sufficient to increase PD-L1 and PD-L2 gene expression and protein levels. We next examined whether GATA2 expression was necessary to regulate PD-L1 and PD-L2 levels. To determine the effects of decreased GATA2 expression, we targeted GATA2 transcripts with 2 shRNAs and a control shRNA in IOMM-Lee and AM38 cells (Fig. 4C,D). Although GATA2 overexpression was sufficient to upregulate PD-L1, GATA2 knockdown was not sufficient to decrease cell surface expression of PD-L1 (data not shown). However, GATA2 knockdown using 2 distinct shRNA constructs led to decreased gene expression (Supplementary Fig. 3E) and cell surface expression of PD-L2 in both IOMM-Lee (Fig. 4C) and AM38 (Fig. 4D), PD-L2 high-expressing cell lines concordant with the level of GATA2 protein knockdown observed by western blot. Thus, increased GATA2 expression is sufficient to increase the cell surface levels of both PD-L1 and PD-L2 and is necessary for constitutive high expression of PD-L2.

PD-1 Ligand expression is associated with worse clinical outcomes in glioma.
We evaluated the effects of PD-L1 and PD-L2 gene expression on clinical outcomes in both GBM and LGG, brain tumors for which there are genomic and clinical data from TCGA 27,28 . PD-L1 and PD-L2 "high" or "low" expression status was Scientific RepoRtS | (2020) 10:9027 | https://doi.org/10.1038/s41598-020-65915-z www.nature.com/scientificreports www.nature.com/scientificreports/ determined by dichotomizing gene expression defined as ≥ or < the median of each gene's median expression value, respectively. Because we found that expression levels of PD-L1 and PD-L2 were highly concordant in LGG and GBM (Suppl. Fig. 4A), Mantel-Haenszel Chi-square test P < 0.0001), we incorporated PD-L1 expression levels into our analysis. Although individual PD-L1 ligand expression status did not significantly influence disease-free survival (DFS) in GBM (data not shown), GBM patients with high PD-L2 expression showed a trend of shorter DFS than patients with low expression (Fig. 5A, left)(log rank test P = 0.0548). DFS of GBM patients www.nature.com/scientificreports www.nature.com/scientificreports/ were then stratified by PD-L1 and/or PD-L2 into 3 groups by expression-i.e., both high, both low, or only one highly expressed. GBM patients with high PD-L1/PD-L2 harbored a statistically significant decreased DFS compared to patients who harbored both low expression levels (both high vs. both low: HR, 95% CI = 1.95, 1.21~3.15, log rank test P = 0.0072). However, after adjusting for age, gender, and IDH1 status, PD-L1 and PD-L2 expression levels alone were not independently associated with disease-free survival in multivariate Cox proportional hazards models (data not shown). Multivariate analysis for DFS shows PD-L2 is an independent factor in LGG patients after adjusting by covariates of age, gender and IDH1 mutation (p = 0.0062) (left); Multivariate analysis shows PD-L1/2 both high is independently associated with shorter DFS in LGG patients compared to "both low" (p = 0.0055) or "one high" (p = 0.0452) after adjusting by covariates age, gender and IDH1 mutation (right).
LGG patients with both high PD-L1 and PD-L2 expression levels harbored statistically significant shorter OS and DFS compared to patients with either low expression of one or both ligands (Fig. 5C, left and right panels, respectively). In multivariate analsyis, high PD-L2 levels alone were independently associated with worse DFS even after adjusting for age, gender, and IDH1 status (HR, 95% CI = 2.98, 1.36~6.50, p = 0.0062, Fig. 5D, left), although PD-L2 status alone was not independently statistically significant for OS in LGG (Supplementary 4B). Finally, high dual expression of PD-L1/PD-L2 compared to dual low (p = 0.0055) or singly high PD-L1 or PD-L2 expression (p = 0.0452) was independently associated with shorter DFS when adjusted for age, gender, and IDH1 status in a multivariate Cox proportional hazards model (Fig. 5D, right). Thus, high PD-L2 expression was associated with worse clinical outcomes in LGG and was an independent prognostic indicator of worse DFS in LGG both alone and with PD-L1.

Discussion
In this study, we investigated the molecular basis of cell autonomous expression of immunoregulatory ligands PD-L1 and PD-L2. We demonstrated that a subset of cancer cell lines and patient-derived BTICs expresses constitutive high levels of both proteins. By analyzing the genomic regulatory datasets in the ENCODE compendium and interrogating candidate cis-regulatory elements in targeted luciferase assays, we identified novel transcriptionally active regulatory elements in both CD274 and PDCD1LG2 and showed that GATA2 directly regulates PD-L1 and PD-L2 expression. We also observed that both high PD-L1 and PD-L2 expression was associated with worse clinical outcomes in GBM and LGG; both high PD-L1/PD-L2 levels were independently associated with shorter DFS in LGG.
We analyzed the CCLE to focus on cell autonomous PD-L1 and PD-L2 expression. The CCLE and others have been leveraged typically to study non-immunologic cancer biology. However, the extensive characterization of the CCLE facilitated our studies of PD-L1 and PD-L2, this approach may be useful to study other immunoregulatory ligands. Our observation that a subset of patient-derived BTICs also expressed constitutive PD-L1/2 suggests that this finding was not an artifact of long-term cell passage. Although PD-L1 has been characterized in GBM in recent work 4,5 , PD-L2 expression in GBM has not been described. Ongoing work is directed at extending our observations to fixed tissue. Moreover, although it has been reported that meningiomas overexpress PD-L1, we demonstrate that 2 meningioma cell lines express constitutive levels of PD-L2. While we focused on brain tumors in this study, a subset of all cancer types harbor PD-L2 overexpression. This observation is consistent with a growing number of studies of PD-L2 in other cancers [12][13][14][15][16]29 . Further work is necessary to understand what PD-L2 expression signifies within the broader context of the tumor microenvironment.
CD274 and PDCD1LG2 are located adjacent to each other on chromosome 9p24.1, possibly the result of a gene duplication event. However, these genes appear to be differentially regulated. We confirmed previously identified ISRE/IRF-1 and AP-1 TF binding sites 5' to exon 1 and exon 2, respectively 9,26 as these regions were contained within our PD-L1 Pr1 and Pr2 constructs. Thus, we focused on a novel region within intron 1 (PD-L1 Pr3). Although we found the GATA2 TF to be sufficient to upregulate PD-L1, nearby genomic regions are also known to contribute to PD-L1 regulation. In addition, it has become clear that PD-L1 regulation is highly complex. Further work will be important to clarify how these distinct pathways intersect in the regulation of PD-L1.
Compared to PD-L1, the regulation of PD-L2 has been underexplored. Our data corrobrate recent work that interferons upregulate PD-L2 30 , and IL-4 can also induce the expression of PD-L2 in esophageal cancer 16 . We provide evidence that PD-L2 can also be expressed constitutively. We showed that GATA2 is both necessary and sufficient to modulate this expression. GATA2 is one of 6 members of the GATA pioneer TF family involved in development and differentiation 31 . GATA2 regulates stem cell progenitor development and hematopoietic maintenance 32 . GATA2 is also overexpressed in AML and drives prostate cancer pathogenesis, especially in castration-resistant settings 31 . One hypothesis is that GATA2 overexpression drives PD-L2 expression that could inhibit anti-tumor immunity. There are species-specific differences in PD-L2 regulation, and thus further work is necessary in antigenically defined human systems to further define the immunologic consequences of PD-L2 overexpression. Recent work demonstrated that the repulsive guidance molecule b (RGMb) represents another receptor for PD-L2 separate from PD-1 33 , and thus PD-L2 expression may have non-overlapping downstream immunobiological effects from PD-L1. Ongoing work is directed at understanding the molecular basis of GATA2 overexpression and/or upstream pathway activation as well as the other concomitant biological effects its dysregulation may impose in GBM and LGG.
Our data show that PD-L2 transcriptional overexpression correlates with worse clinical outcomes in GBM and LGG. In particular, these data are correlated with decreased DFS in LGG both independently and together with PD-L1 overexpression. Further work is necessary to validate these data on the protein level similar to PD-L1. Additional study is needed to determine the influence of PD-L2 expression on the natural history of brain tumors and to explore whether it may serve as a biomarker of response to immunotherapies. PD-L2 expression has been previously recognized as a biomarker of host immune responses in melanoma 34 , and PD-L2 expression was also predictor of positive clinical responses to anti-PD-1/Keytruda in the Keynote-12 trial for head and neck cancer patients with recurrent or metastatic disease 12 . Because PD-L2 interacts with other receptors such as RGMb, its inhibition may not be achieved exclusively by blocking antibodies to the PD-1 receptor, and thus, there may be merit to exploring efforts to inhibit PD-L2. Finally, it will be important to consider whether the mechanisms that drive constitutive PD-L1 and/or PD-L2 expression-such as GATA2-can be inhibited in a way that decreases the immunosuppressive microenvironment and creates a more permissive context for immune-based therapies.