Purpose: Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma, the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and group 3 medulloblastoma for preclinical evaluation in immunocompetent C57BL/6 mice. Experimental Design and Results: Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid-derived suppressor cells, and tumor-associated macrophages in murine SHH model tumors compared with group 3 tumors. However, murine group 3 tumors had higher percentages of CD8⁺ PD-1⁺ T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial group 3 tumors compared with SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1⁺ peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3⁺ T cells within the tumor microenvironment. Conclusions: This is the first immunologic characterization of preclinical models of molecular subtypes of medulloblastoma and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment. Clin Cancer Res; 22(3); 582–95. ©2015 AACR.

While RNA-pulsed dendritic cell (DC) vaccines have shown promise, the advancement of cellular therapeutics is fraught with developmental challenges. To circumvent the challenges of cellular immunotherapeutics, we developed clinically translatable nanoliposomes that can be combined with tumor-derived RNA to generate personalized tumor RNA-nanoparticles (NPs) with considerable scale-up capacity. RNA-NPs bypass MHC restriction, are amenable to central distribution, and can provide near immediate immune induction. We screened commercially available nanoliposomal preparations and identified the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as an efficient mRNA courier to antigen-presenting cells (APCs). When administered intravenously, RNA-NPs mediate systemic activation of APCs in reticuloendothelial organs such as the spleen, liver, and bone marrow. RNA-NPs increase percent expression of MHC class I/II, B7 co-stimulatory molecules, and maturation markers on APCs (all vital for T-cell activation). RNA-NPs also increase activation markers on tumor APCs and elicit potent expansion of antigen-specific T-cells superior to peptide vaccines formulated in complete Freund's adjuvant. We demonstrate that both model antigen-encoding and physiologically-relevant tumor-derived RNA-NPs expand potent antitumor T-cell immunity. RNA-NPs were shown to induce antitumor efficacy in a vaccine model and functioned as a suitable alternative to DCs in a stringent cellular immunotherapy model for a radiation/temozolomide resistant invasive murine high-grade glioma. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a RNA-NP formulation that systemically activates host APCs precipitating activated T-cell frequencies necessary to engender antitumor efficacy. RNA-NPs can thus be harnessed as a more feasible and effective immunotherapy to re-program host-immunity.

Adoptive cellular therapy (ACT) after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has been largely limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other solid cancers. We have employed tumor RNA-pulsed dendritic cells (DCs) to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative ACT in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative (MA) conditioning, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed DC vaccines. Hematopoietic stem cells (HSCs), administered for salvage from MA conditioning, rapidly migrated to areas of intracranial tumor growth and facilitated the recruitment of tumor-specific lymphocytes through HSC-elaborated chemokines and enhanced immunologic rejection of intracranial tumors during ACT. Furthermore, HSC transplant under non-myeloablative (NMA) conditions also enhanced immunologic tumor rejection, indicating a novel role for the use of HSCs in the immunologic treatment of malignant gliomas and possibly other solid tumors.

Purpose: Adoptive T-cell immunotherapy (ACT) has emerged as a viable therapeutic for peripheral and central nervous system (CNS) tumors. In peripheral cancers, optimal efficacy of ACT is reliant on dendritic cells (DCs) in the tumor microenvironment. However, the CNS is largely devoid of resident migratory DCs to function as antigen-presenting cells during immunotherapy. Herein, we demonstrate that cellular interactions between adoptively transferred tumor-reactive T cells and bone marrow–derived hematopoietic stem and progenitor cells (HSPCs) lead to the generation of potent intratumoral DCs within the CNS compartment.Experimental Design: We evaluated HSPC differentiation during ACT in vivo in glioma-bearing hosts and HSPC proliferation and differentiation in vitro using a T-cell coculture system. We utilized FACS, ELISAs, and gene expression profiling to study the phenotype and function of HSPC-derived cells ex vivo and in vivo. To demonstrate the impact of HSPC differentiation and function on antitumor efficacy, we performed survival experiments.Results: Transfer of HSPCs with concomitant ACT led to the production of activated CD86+CD11c+MHCII+ cells consistent with DC phenotype and function within the brain tumor microenvironment. These intratumoral DCs largely supplanted abundant host myeloid-derived suppressor cells. We determined that during ACT, HSPC-derived cells in gliomas rely on T-cell–released IFNγ to differentiate into DCs, activate T cells, and reject intracranial tumors.Conclusions: Our data support the use of HSPCs as a novel cellular therapy. Although DC vaccines induce robust immune responses in the periphery, our data demonstrate that HSPC transfer uniquely generates intratumoral DCs that potentiate T-cell responses and promote glioma rejection in situ. Clin Cancer Res; 24(16); 3955–66. ©2018 AACR.

Immune checkpoint blockade using anti-PD-1 monoclonal antibodies has shown considerable promise in the treatment of solid tumors, but brain tumors remain notoriously refractory to treatment. In CNS malignancies that are completely resistant to PD-1 blockade, we found that bone marrow-derived, lineage-negative hematopoietic stem and progenitor cells (HSCs) that express C–C chemokine receptor type 2 (CCR2⁺) reverses treatment resistance and sensitizes mice to curative immunotherapy. HSC transfer with PD-1 blockade increases T-cell frequency and activation within tumors in preclinical models of glioblastoma and medulloblastoma. CCR2⁺HSCs preferentially migrate to intracranial brain tumors and differentiate into antigen-presenting cells within the tumor microenvironment and cross-present tumor-derived antigens to CD8⁺ T cells. HSC transfer also rescues tumor resistance to adoptive cellular therapy in medulloblastoma and glioblastoma. Our studies demonstrate a novel role for CCR2⁺HSCs in overcoming brain tumor resistance to PD-1 checkpoint blockade and adoptive cellular therapy in multiple invasive brain tumor models.

Purpose: Immunotherapy has been demonstrably effective against multiple cancers, yet tumor escape is common. It remains unclear how brain tumors escape immunotherapy and how to overcome this immune escape. Experimental Design: We studied KR158B-luc glioma-bearing mice during treatment with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested the immunogenicity of primary and escaped tumors using T-cell restimulation assays. We used flow cytometry and RNA profiling of whole tumors to further define escape mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T cells through the use of escape variant total tumor RNA and administered these cells as ACT. In addition, programmed cell death protein-1 (PD-1) checkpoint blockade was studied in combination with ACT. Results: Escape mechanisms included a shift in immunogenic tumor antigens, downregulation of MHC class I, and upregulation of checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater recognition of escaped tumors than primary tumors. When administered as ACT, these T cells prolonged median survival of escape variant-bearing mice by 60%. The rational combination of ACT with PD-1 blockade prolonged median survival of escape variant glioma-bearing mice by 110% and was dependent upon natural killer cells and T cells. Conclusions: These findings suggest that the immune landscape of brain tumors are markedly different postimmunotherapy yet can still be targeted with immunotherapy.