Purification of the scFvPD-1 from HEK293 cells

Abstract The GL261-luc2 and CT2A-luc syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM), but demonstrate distinct differences in response to immune checkpoint blockade. Whereas GL261-luc2 is readily responsive to several immunotherapies, CT2A-luc is broadly resistant to diverse immunotherapeutic modalities. To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their genomic and phenotypic profiles. The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of huGBM, despite neither line sharing the canonical genetic or histologic features of huGBM. Both models exhibited striking hypermutation and contained clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc), which have only been identified in <1% of huGBM tumors. CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery and interferon response pathways – confirmed at the genomic, transcriptomic, and proteomic levels. CT2A-luc uniquely contained multiple mutations in antigen presentation machinery genes that were computationally predicted to have deleterious biologic effects, including a clonal p.A275P missense mutation in Psmb8 (a subunit of the immunoproteasome, which degrades proteins into peptides for loading onto MHC class I) and a clonal p.Y488C missense mutation in Tap1 (which transports peptides into the endoplasmic reticulum for loading onto MHC class I). CT2A-luc also distinctly exhibited a single-copy loss of a chromosomal segment involving 4qC4 (FDR-adjusted p=0.04), which encompassed multiple type I IFN genes, as well as a single-copy loss of 10qD2-10qD3 (FDR-adjusted p=0.04), which contained Stat2, Stat6, and Ifng. Consistent with our observation of down-regulated IFN response pathways in CT2A-luc, phosphoproteomic analysis revealed decreased phosphorylation of several members of the JAK/STAT pathway in ex vivo CT2A-luc tumors, including Ptpn11 (i.e., Shp2), Il13ra1, and Stat3 - together suggesting reduced JAK/STAT signaling. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, all of which are known to play important roles as myeloid chemoattractants, in marked contrast to GL261-luc2. The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations for CT2A-luc. Thus, CT2A-luc may be an informative preclinical model of immunotherapy resistance due to its mesenchymal differentiation and antigen presentation machinery deficits. Citation Format: Bryan Iorgulescu, Neil Ruthen, Ryuhjin Ahn, Eleni Panagioti, Prafulla Gokhale, Martha Neagu, Maria Speranza, Benjamin Eschle, Kara Soroko, Raziye Piranlioglu, Meenal Datta, Shanmugarajan Krishnan, Kathleen Yates, Gregory Baker, Rakesh Jain, Mario Suva, Donna Neuberg, Forest White, E. Chiocca, Gordon Freeman, Arlene Sharpe, Catherine Wu, David Reardon. Antigen presentation deficiency and mesenchymal differentiation underlie resistance to immunotherapy in the murine syngeneic CT2A tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2990.

BackgroundThe GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy.MethodsTo decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics.ResultsThe transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants.ConclusionAlthough the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

Supplementary Methods and Legends

Engineering of NG34 to insert the sequence of the scFv against PD-1

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glio-blastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

In vivo comparison of scFvPD-1 production and viral gene expression in mouse and human GBM cells

Immune checkpoint blockers (ICBs) have revolutionized the treatment of some solid cancers but have failed to benefit the majority of glioblastoma (GBM) patients. Two reasons underlying limited ICB benefit are: 1) immune-related adverse events, and 2) resistance conferred by the tumor microenvironment. Here, we show that ICBs induce cerebral edema in patients and GBM mouse models. This edema results from an inflammatory response to ICB treatment that disrupts the blood-tumor-barrier, as confirmed by intravital imaging, mechanistic blocking studies, and single-cell RNA sequencing. Losartan – a commonly prescribed antihypertensive agent – controls ICB-induced edema, reprograms the immunosuppressive tumor microenvironment, and improves survival under ICB therapy. In combination with a standard of care regimen in mice mimicking clinical treatment (surgical resection, chemoradiation), losartan increases the percent of long-term surviving (cured) mice under ICB therapy from 16% to 43%. Finally, a bihemispheric “resect-and-response” model to establish predictive biomarkers from the tumor microenvironment reveals that cured mice have an immunostimulatory (“hot”) immune tumor compartment prior to therapy. These results provide the basis for clinical testing of adding to losartan to ICB treatment for GBM patients.

Abstract Purpose: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy. Experimental Design: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors. Results: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti–PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti–PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30–3.68; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23–3.16; P = 0.005]. Conclusions: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.

Background Increasing data indicate that corticosteroids can exert a detrimental effect on immunotherapy for oncology patients. Dexamethasone, a uniquely potent corticosteroid, is frequently administered to brain tumor patients to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in glioblastoma patients – particularly in the context of immunotherapy. Methods We evaluated the dose-dependent effects of dexamethasone when administered with PD-1 blockade and/or radiotherapy on survival and tumor response in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A glioblastoma tumors. The immune microenvironment was comprehensively profiled using flow cytometry analysis. Clinically, the effect of dexamethasone on survival was evaluated in 181 IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors using multivariable Cox regression. Results Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner (figure 1). Concurrent dexamethasone also abrogated survival following anti-PD-1 with or without radiotherapy in immunoresistant CT-2A models (figure 2). Dexamethasone decreased T lymphocyte numbers (figure 3) by increasing apoptosis (figure 4), in addition to decreasing lymphocyte functional capacity (figure 3C/D). Myeloid and NK cell populations were also generally reduced by dexamethasone (figure 3). Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment by relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival, regardless of dose (referent no dexamethasone; <2 mg HR 2.16, 95%CI: 1.30–3.68, p=0.003; ≥2 mg HR 1.97, 95%CI: 1.23–3.16, p=0.005; table 1 and figure 5). Conclusions We demonstrate that concurrent dexamethasone administration, even at a low dose, limits the therapeutic benefit of anti-PD-1 therapy both in mouse glioblastoma models and in a retrospective cohort of 181 IDH-wildtype glioblastoma patients. Mechanistically, dexamethasone decreased intratumoral T cells and systemic levels of T cells, natural killer cells, and myeloid cells, while qualitatively impairing lymphocyte function. The mechanism of T cell depletion included induction of apoptosis. These findings indicate that dexamethasone hinders both adaptive and innate immune responses, intratumorally and systemically, and that its administration should be carefully assessed among glioblastoma patients undergoing second-generation immunotherapy clinical trials. Our findings also have ramifications for brain metastasis patients where immune checkpoint inhibitors are part of standard-of-care management. Acknowledgements We thank Min Wu for assistance in generating CT-2A luciferase-transduced cells, and Drs. Geoffrey Young, Lei Qin, Xin Chen, and Jing Li for assistance in evaluation of patients' radiographic imaging. Ethics Approval Approved under DFCI Institutional Review Board protocol 10-417.

Co-expression of immune checkpoint receptors (ICRs) PD-1, TIM-3, and LAG-3 characterizes chronically activated and exhausted tumor-infiltrating T cells (TILs), suggesting their targeting may have applicability for the treatment of multiple cancer types. We previously reported improved tumor control in various syngeneic and humanized mouse models when treated with a combination of TSR-042 (αPD-1), TSR-022 (αTIM-3), and TSR-033 (αLAG-3) as compared to single or double combinations. Here, we are characterizing TILs from ovarian cancer tissues and their functional response to triple combination treatment. Immune profiling using flow cytometry confirmed expression of all three ICRs on TILs isolated from primary resections of ovarian cancer. Ex vivo re-stimulation of immune infiltrates with S. aureus enterotoxin B in presence of ICR-targeting antibodies led to increased secretion of IFN-γ and IL-2 when treated with TSR-042. Notably, triple combination of TSR-042, TSR-022, and TSR-033 further amplified cytokine release, indicating more effective TIL reinvigoration. To further understand the differential effects of triple combination treatment over PD-1 blockade, we analyzed αCD3/αCD28-stimulated and antibody-treated ovarian cancer TILs on a single cell level using a microfluidic IsoCode chip technology that allows for parallel detection and quantification of 32 secreted proteins from live single cells. Combining the amount of each protein secreted by polyfunctional T cells (co-secreting two or more proteins per cell) with the frequency of such cells results in a measurement of polyfunctional strength (PSI), a unique IsoCode-enabled metric that has been associated with improved response to ICR inhibition. TSR-042 increased the PSI of CD4+ and CD8+ TILs 1.4 and 1.5-fold over isotype control treatment. Importantly, triple combination treatment was able to significantly increase the PSI of both subsets by 2.9 and 3.7-fold, respectively (p < 0.001). For CD8+ TILs, this increase was mainly driven by an increase in the frequency of polyfunctional subsets, while for CD4+ TILs, the absolute amounts of secreted cytokines had a larger impact. Interestingly, both classical effector cytokines like Granzyme B and IFN-γ as well as other secreted factors like chemoattractant factors, implicated in the recruitment of multiple immune cell subsets to tumor tissue, contributed to T cell polyfunctionality. Taken together, triple ICR blockade targeting PD-1, TIM-3, and LAG-3 reinvigorated ovarian cancer TILs more effectively than PD-1 inhibition alone. This was mediated by increasing T cell polyfunctionality, which has been associated with improved anti-tumor activity and response to ICR inhibition. This data further supports the concept of triple combination checkpoint blockade as a treatment option for ovarian cancer.Citation Format: Johanna K. Kaufmann, Brianna Flynn, Kevin Morse, Maria C. Speranza, Jing Zhou, Sridhar Ramaswamy, Sean Mackay, Kevin G. Coleman. Triple checkpoint blockade targeting PD-1, TIM-3, and LAG-3 reinvigorates ovarian cancer-infiltrating T cells by increasing T cell polyfunctionality and effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3242.

Abstract Purpose: Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM. Experimental Design: NG34scFvPD-1 expresses the human GADD34 gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1. In vitro cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined. In vivo survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1. Results: NG34scFvPD-1–infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1. In situ NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response. Conclusions: NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.

Abstract Tuberous sclerosis complex (TSC) is an incurable multisystem genetic disease characterized by mTORC1-hyperactive hamartomatous tumors of brain, kidney, and lung. TSC is caused by mutations in the two TSC1 or TSC2 tumor-suppressor genes. mTORC1 inhibitors lead to partial antitumor responses in TSC, with tumor regrowth upon treatment cessation. Understanding the interaction between T cells and tumor cells has led to the development of successful checkpoint blockade immunotherapy targeting PD-1, programmed cell death-ligand 1 (PD-L1), and CTLA-4. These therapies have proven to be highly effective in treating several malignancies, including non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, bladder cancer and Hodgkin's lymphoma. It is currently unknown whether benign tumors, such as those arising in TSC, will also respond to checkpoint blockade therapy. Using multiparametric flow cytometry and immunohistochemistry of human angiomyolipomas and pulmonary LAM, we discovered that PD-1 is highly expressed on tumor-infiltrating T cells. Using immunocompetent preclinical models of TSC, we found that dual blockade of PD-1 and CTLA-4 inhibited the growth of TSC2-deficient tumors by 77% (p < 0.0001). This was associated with increased tumor-infiltrating CD8+ and CD4+ T cells and decreased Tregs, G-MDSCs and regulatory CD11b+ DCs. Strikingly, we discovered that complete regression of established tumors can be achieved in 37% of the mice using anti-PD-1 monotherapy and in 62% of the mice using combined anti-PD-1 and anti-CTLA-4 therapy. We also assessed a treatment regimen of rapamycin followed by PD-1 blockade and found that this sequential treatment delays regrowth of TSC2-deficient tumors. Furthermore, TSC2 re-expression in TSC2-deficient tumors promoted antitumor responses via increasing T cells and decreasing CD11b+Ly6G+Ly6Cmed G-MDSCs and CD11b+ DCs infiltration into tumors. Importantly, we found that the potency of dual PD-1 and CTLA-4 blockade is significantly enhanced by TSC2 re-expression. Our data indicate that PD-1 is upregulated on T cells in TSC-associated tumors and that dual blockade of PD-1 and CTLA-4 pathways is effective in delaying tumor growth and improving long-term survival, with long-term complete tumor responses. Therefore, PD-1 and/or CTLA-4 blockade may represent a promising dual immunotherapy for patients with TSC-associated tumors and women with the sporadic form of LAM. Our data may have relevance for the many sporadic human tumors with mTORC1 hyperactivation (~50% of all human malignancies) and also for other hamartomatous tumor syndromes such as neurofibromatosis. Furthermore, the specific role of intratumoral TSC2 in antitumor T cell responses that we discovered may help to elucidate the response to immunotherapy in sporadic human malignancies with mutations in the TSC genes, which include a subset of bladder cancer and renal cell carcinoma. Citation Format: Heng-Jia Liu, Patrick Lizotte, Heng Du, Maria Speranza, Spencer Vaughan, Nicola Alesi, Kwok-Kin Wong, Gordon Freeman, Arlene Sharpe, Elizabeth Henske. TSC2 enhances antitumor immunity and potentiates PD-1 and CTLA-4 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1686.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease characterized by mTORC1-hyperactive tumors. TSC1/2 mutations also occur in other neoplastic disorders, including lymphangioleiomyomatosis (LAM) and bladder cancer. Whether TSC-associated tumors will respond to immunotherapy is unknown. We report here that the programmed death 1 coinhibitory receptor (PD-1) is upregulated on T cells in renal angiomyolipomas (AML) and pulmonary lymphangioleiomyomatosis (LAM). In C57BL/6J mice injected with syngeneic TSC2-deficient cells, anti-PD-1 alone decreased 105K tumor growth by 67% (P < 0.0001); the combination of PD-1 and CTLA-4 blockade was even more effective in suppressing tumor growth. Anti-PD-1 induced complete rejection of TSC2-deficient 105K tumors in 37% of mice (P < 0.05). Double blockade of PD-1 and CTLA-4 induced rejection in 62% of mice (P < 0.01). TSC2 reexpression in TSC2-deficient TMKOC cells enhanced antitumor immunity by increasing T cell infiltration and production of IFN-γ/TNF-α by T cells, suggesting that TSC2 and mTORC1 play specific roles in the induction of antitumor immunity. Finally, 1 month of anti-PD-1 blockade reduced renal tumor burden by 53% (P < 0.01) in genetically engineered Tsc2+/- mice. Taken together, these data demonstrate for the first time to our knowledge that checkpoint blockade may have clinical efficacy for TSC and LAM, and possibly other benign tumor syndromes, potentially yielding complete and durable clinical responses.

Background Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Glioblastoma can suppress immunity by using surface PD-L1 on extracellular vesicles to block T cell receptor–mediated T cell activation.

Blockade of immune cell co-inhibitory receptor PD-1 using monoclonal antibodies enables anti-tumor immune responses in various solid tumors and lymphoid malignancies. Our laboratory previously demonstrated PD-1 blockade elicits an anti-tumor immune response resulting in tumor rejection and long-term survival in approximately 50% of mice with an orthotopic GL261 glioblastoma, despite lacking a corresponding accumulation of CD8+ cytotoxic T cell in the tumor or draining lymph nodes. In this investigation, we evaluated the role of conventional CD4+ T cells and the innate immune response in PD-1 mediated anti-glioma immunity using multiplex technologies for immunohistochemistry and flow cytometry. In response to PD-1 monotherapy, intratumoral CD4 T cells expressed significantly elevated levels of proteins required for T cell proliferation, activation, and effector function. CD4 T cell activation was accompanied by the classical activation and M1 polarization of resident microglia and tumor-infiltrating macrophages, including down-regulation of PD-L1 and up-regulation of MHC class II surface expression. We also demonstrated that depletion of either CD4 or CD8 T cells was sufficient to completely ablate anti-PD-1-mediated tumor eradication and long-term survival. Our data suggests CD4 T cells and myeloid cells may play a prominent role in the eradication of glioblastoma by PD-1 blockade.

Programmed cell death protein-1 (PD1) expressed on T cells is a key mediator of immune evasion via mediating the inhibition of T-cell function. This occurs via its binding to ligands PD-L1 and PD-L2 on tumor cells and other cells such as macrophages and microglia. Immune checkpoint blockade using anti-PD1 antibodies is of clinical benefit in multiple cancer types and is under investigation in glioblastoma. Tumor PD-L1 expression is currently used to screen for eligibility for anti-PD1 treatment, suggesting that only PD-L1 positive tumors will be included in clinical trials. However, the role of PD-L2 in immune evasion in glioblastoma has not been well studied. To assess the roles of PD-L1 and PD-L2 in glioblastoma, their expression was analyzed in TCGA and IVY Atlas RNAseq data. Genes whose expression was correlated with either PD-L1 or PD-L2 were assessed for pathway enrichment. To compare their effects on patient outcome, Cox regression models for PD-L1 and PD-L2 were fitted. To confirm these findings, western blot and qPCR gene expression was conducted of glioma-patient derived cell cultures (n=19) and fresh tumor samples (n=9). Here we show that PD-L2 is present in glioblastoma at higher levels than PD-L1, and that the level of PD-L2 in the tumor impacts patient survival. Furthermore PD-L2 expression negatively correlated with KPS rho=-0.13 (p=0.007). We show that PD-L2 correlates with retinoic acid receptor and viral induction of apoptosis pathways in glioblastoma. Western blot and gene expression analysis of patient tumor samples and patient-derived cell lines shows that PD-L2 is robustly expressed, while PD-L1 expression is variable. PD-L2 expression was also inducible by IFNy in certain cell lines. These results suggest that PD-L2 should be considered as a biomarker for eligibility of patients in clinical trials for anti-PD1 therapies and widens the net for patient recruitment in these trials.

Background: Extracellular Vesicles (EVs) shed by tumor cells have recently been demonstrated to act as a major conduit in cell-cell communication. Increasing knowledge of the effect of EVs on infiltrating lymphocytes within the tumor microenvironment shows the immune modulation capacity of EVs. In this study we investigated the potential role of PD-L1 on EVs in immune escape in heterogeneous glioblastoma (GBM). Methods: Peripheral blood mononuclear cells (PBMCs) (n=8) were stimulated by IL-2 or anti-CD3±anti-CD28±anti-PD1 treatment. Magnetic cell sorting was used to isolate CD3+ cells. Activation levels of CD3+CD4+, CD3+CD8+ and CD3-CD56+ were monitored by flow cytometry of the activation markers CD69, CD25, PD1 and TIM3. Concurrent eFluor staining was used to measure proliferation. EVs derived from four different glioblastoma stem-like cell lines (GSCs) from either the mesenchymal (M) or proneural (P) subtype were used in this study. PD-L1 expression was validated by immunoblotting and electron microscopy (EM). EV binding was visualized by PALM-tdTomato and PD-L1_RFP positive EVs and tested on plate-bound PD1. RNA-seq from TCGA and Immunohistochemistry (IHC) was used to correlate PD-L1 expression with CD3 infiltration. In addition, circulating EVs from GBM patients (n=22) and controls (n=5) were used to determine PD-L1 content via digital droplet-PCR. Results: PD-L1 was expressed on the surface of M GSC EVs. EVs were able to bind to the outer surface of CD3+ cells. In whole PBMCs EV treatment led to a significant, PD-L1 dependent, reduction of CD3+CD8+ and CD3+CD4+ T-cell activation as well as decreased proliferation in the M subtype. These effects were also observed in CD3+ sorted cells, indicating a direct effect of EVs on T cells. Furthermore PD-L1 on EVs was capable of binding directly to PD1. Using RNA-seq and IHC we show a correlation of CD3 infiltration and PD-L1 expression. In addition, circulating EVs from GBM patients show enriched PD-L1 DNA cargo that correlates with tumor volume. CONCLUSION: Our findings demonstrate the immunosuppressive potential of GSC-derived EVs in a PD-L1 dependent manner. Furthermore we show that PD-L1 on EVs can directly bind PD1 and is capable of hindering T cell activation locally and at distant sites and show how immune checkpoint blockade may systemically enhance immunity against GBM.