TCR-engineered adoptive cell therapy effectively treats intracranial murine glioblastoma.

BACKGROUND: Adoptive cellular therapies with chimeric antigen receptor T cells have revolutionized the treatment of some malignancies but have shown limited efficacy in solid tumors such as glioblastoma and face a scarcity of safe therapeutic targets. As an alternative, T cell receptor (TCR)-engineered cellular therapy against tumor-specific neoantigens has generated significant excitement, but there exist no preclinical systems to rigorously model this approach in glioblastoma. METHODS: We employed single-cell PCR to isolate a TCR specific for the Imp3D81N neoantigen (mImp3) previously identified within the murine glioblastoma model GL261. This TCR was used to generate the Mutant Imp3-Specific TCR TransgenIC (MISTIC) mouse in which all CD8 T cells are specific for mImp3. The therapeutic efficacy of neoantigen-specific T cells was assessed through a model of cellular therapy consisting of the transfer of activated MISTIC T cells and interleukin 2 into lymphodepleted tumor-bearing mice. We employed flow cytometry, single-cell RNA sequencing, and whole-exome and RNA sequencing to examine the factors underlying treatment response. RESULTS: We isolated and characterized the 3×1.1C TCR that displayed a high affinity for mImp3 but no wild-type cross-reactivity. To provide a source of mImp3-specific T cells, we generated the MISTIC mouse. In a model of adoptive cellular therapy, the infusion of activated MISTIC T cells resulted in rapid intratumoral infiltration and profound antitumor effects with long-term cures in a majority of GL261-bearing mice. The subset of mice that did not respond to the adoptive cell therapy showed evidence of retained neoantigen expression but intratumoral MISTIC T cell dysfunction. The efficacy of MISTIC T cell therapy was lost in mice bearing a tumor with heterogeneous mImp3 expression, showcasing the barriers to targeted therapy in polyclonal human tumors. CONCLUSIONS: We generated and characterized the first TCR transgenic against an endogenous neoantigen within a preclinical glioma model and demonstrated the therapeutic potential of adoptively transferred neoantigen-specific T cells. The MISTIC mouse provides a powerful novel platform for basic and translational studies of antitumor T-cell responses in glioblastoma.

The Conventional Dendritic Cell 1 Subset Primes CD8+ T Cells and Traffics Tumor Antigen to Drive Antitumor Immunity in the Brain.

The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

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.

Treatment of an aggressive orthotopic murine glioblastoma model with combination checkpoint blockade and a multivalent neoantigen vaccine.

BACKGROUND: Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed "neoantigens," represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. METHODS: Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. RESULTS: Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. CONCLUSIONS: We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. KEY POINTS: 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments.2. CT2A tumors exhibit features of human GBM microenvironments.3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.

Detection of neoantigen-specific T cells following a personalized vaccine in a patient with glioblastoma.

Neoantigens represent promising targets for personalized cancer vaccine strategies. However, the feasibility of this approach in lower mutational burden tumors like glioblastoma (GBM) remains unknown. We have previously reported the use of an immunogenomics pipeline to identify candidate neoantigens in preclinical models of GBM. Here, we report the application of the same immunogenomics pipeline to identify candidate neoantigens and guide screening for neoantigen-specific T cell responses in a patient with GBM treated with a personalized synthetic long peptide vaccine following autologous tumor lysate DC vaccination. Following vaccination, reactivity to three HLA class I- and five HLA class II-restricted candidate neoantigens were detected by IFN-γ ELISPOT in peripheral blood. A similar pattern of reactivity was observed among isolated post-treatment tumor-infiltrating lymphocytes. Genomic analysis of pre- and post-treatment GBM reflected clonal remodeling. These data demonstrate the feasibility and translational potential of a therapeutic neoantigen-based vaccine approach in patients with primary CNS tumors.

Endogenous Neoantigen-Specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach.

The "cancer immunogenomics" paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific "neoantigens" in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 nonsynonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2Kb and H-2Db candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFNγ ELISPOT, we confirmed H-2Db-restricted Imp3D81N (GL261) and Odc1Q129L (SMA-560) along with H-2Kb-restricted E2f8K272R (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3D81N and Odc1Q129L were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell-activating immunotherapeutic approaches in preclinical models of glioblastoma. Cancer Immunol Res; 4(12); 1007-15. ©2016 AACR.

High incidence of TERT mutation in brain tumor cell lines.

TERT promoter gene mutations are highly recurrent in malignant glioma. However, little information exists regarding their presence in experimental brain tumor models. To better characterize systems in which TERT mutation studies could be appropriately modeled experimentally, the TERT promoter was examined by conventional sequencing in primary brain tumor initiating cells (BTIC), two matched recurrent BTIC lines, a panel of established malignant glioma cell lines, and two meningioma cell lines. Telomerase gene expression was examined by quantitative PCR. We found that all glioblastoma BTIC lines harbored a TERT mutation, which was retained in two patient-matched recurrent BTIC. The TERT C228T or C250T mutation was found in 33/35 (94 %) of established malignant glioma cell lines and both meningioma cell lines examined. Brain tumor cell lines expressed variably high telomerase levels. Thus, a high percentage of glioma cell lines, as well as two meningioma cell lines, harbors TERT mutations. These data characterize tractable, accessible models with which to further explore telomerase biology in these tumor types.

The role of matrix metalloproteinase-9 in cigarette smoke-induced emphysema.

RATIONALE: Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction. OBJECTIVES: To determine if MMP-9 causes cigarette smoke-induced emphysema using MMP-9 knockout mice and human samples. METHODS: Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury. MEASUREMENTS AND MAIN RESULTS: Mice deficient in MMP-9 develop the same degree of cigarette smoke-induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage. CONCLUSIONS: These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke-induced emphysema. Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).

Cigarette smoke induces nucleic-acid oxidation in lung fibroblasts.

Oxidative stress is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD), but the molecular pathway connecting oxidative damage to tissue destruction remains to be fully defined. We suggest that reactive oxygen species (ROS) oxidatively damage nucleic acids, and this effect requires multiple repair mechanisms, particularly base excision pathway components 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III homologue 1 (NTH1), and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), as well as the nucleic acid-binding protein, Y-box binding protein 1 (YB1). This study was therefore designed to define the levels of nucleic-acid oxidation and expression of genes involved in the repair of COPD and in corresponding models of this disease. We found significant oxidation of nucleic acids localized to alveolar lung fibroblasts, increased levels of OGG1 mRNA expression, and decreased concentrations of NTH1, SMUG1, and YB1 mRNA in lung samples from subjects with very severe COPD compared with little or no COPD. Mice exposed to cigarette smoke exhibited a time-dependent accumulation of nucleic-acid oxidation in alveolar fibroblasts, which was associated with an increase in OGG1 and YB1 mRNA concentrations. Similarly, human lung fibroblasts exposed to cigarette smoke extract exhibited ROS-dependent nucleic-acid oxidation. The short interfering RNA (siRNA)-dependent knockdown of OGG1 and YB1 expression increased nucleic-acid oxidation at the basal state and after exposure to cigarette smoke. Together, our results demonstrate ROS-dependent, cigarette smoke-induced nucleic-acid oxidation in alveolar fibroblasts, which may play a role in the pathogenesis of emphysema.

Macrophage elastase (matrix metalloproteinase-12) suppresses growth of lung metastases.

Matrix metalloproteinases (MMP) have been implicated in virtually all aspects of tumor progression. However, the recent failure of clinical trials employing synthetic MMP inhibitors in cancer chemotherapy has led us to hypothesize that some MMPs may actually serve the host in its defense against tumor progression. Here we show that mice deficient in macrophage elastase (MMP-12) develop significantly more gross Lewis lung carcinoma pulmonary metastases than their wild-type counterparts both in spontaneous and experimental metastasis models. The numbers of micrometastases between the two groups are equivalent; thus, it seems that MMP-12 affects lung tumor growth, and not metastasis formation, per se. MMP-12 is solely macrophage derived in this model, being expressed by tumor-associated macrophages and not by tumor or stromal cells. The presence of MMP-12 is associated with decreased tumor-associated microvessel density in vivo and generates an angiostatic>angiogenic tumor microenvironment that retards lung tumor growth independent of the production of angiostatin. These data define a role for MMP-12 in suppressing the growth of lung metastases and suggest that inhibitors designed to specifically target tumor-promoting MMPs may yet prove effective as cancer therapeutics.

Elastin fragments drive disease progression in a murine model of emphysema.

Mice lacking macrophage elastase (matrix metalloproteinase-12, or MMP-12) were previously shown to be protected from the development of cigarette smoke-induced emphysema and from the accumulation of lung macrophages normally induced by chronic exposure to cigarette smoke. To determine the basis for macrophage accumulation in experimental emphysema, we now show that bronchoalveolar lavage fluid from WT smoke-exposed animals contained chemotactic activity for monocytes in vitro that was absent in lavage fluid from macrophage elastase-deficient mice. Fractionation of the bronchoalveolar lavage fluid demonstrated the presence of elastin fragments only in the fractions containing chemotactic activity. An mAb against elastin fragments eliminated both the in vitro chemotactic activity and cigarette smoke-induced monocyte recruitment to the lung in vivo. Porcine pancreatic elastase was used to recruit monocytes to the lung and to generate emphysema. Elastin fragment antagonism in this model abrogated both macrophage accumulation and airspace enlargement.

Hyperpolarized (3)He MRI of mouse lung.

Hyperpolarized (3)He images of mouse lung are presented. Ventilation images and measurements of (3)He apparent diffusion coefficient (ADC) are reported in healthy mice, and preliminary studies of emphysema and lung cancer in mice are described using these techniques. The design and operation of an electronically controlled small-animal ventilator to deliver the hyperpolarized gas and control animal respiration are described. Images are acquired using an asymmetric gradient echo imaging method to enhance the signal-to-noise ratio of the rapidly diffusing (3)He. In mice with elastase-induced emphysema, the whole-lung average ADC is greater by approximately 25%, a statistically significant difference, compared to healthy animals. By contrast, mice exposed to cigarette smoke for up to 12 months reveal no statistically relevant increases in ADC, although emphysema was not confirmed in these mice. A study of lung cancer (melanoma) in mice is also presented. While tumors are shown to cause substantial ventilation defects in the lung, these defects appear confined to the cancerous regions and do not extend to large-scale regions of the lung distal to the tumors.

Accelerated enlargement of experimental abdominal aortic aneurysms in a mouse model of chronic cigarette smoke exposure.

BACKGROUND: Cigarette smoking and pulmonary emphysema are strongly associated with abdominal aortic aneurysms (AAAs), but the biologic mechanisms linking these conditions are undefined. STUDY DESIGN: To determine if exposure to cigarette smoke influences formation and growth of experimental AAAs, 129/SvEv mice were acclimated to daily cigarette smoke exposure for 2 weeks followed by transient elastase perfusion of the abdominal aorta to induce aneurysmal degeneration. Smoking was continued for intervals of either 2 or 12 weeks (8 mice per group). Nonsmoking 129/SvEv controls (n = 29) underwent elastase perfusion and followup evaluation at the same time intervals. In all animals, abdominal aortic diameter (AD) was measured to determine interval increases in AD (Delta AD), with AAAs defined as a Delta AD > 100%. RESULTS: Preperfusion and immediate postperfusion ADs were not significantly different between experimental groups. Aneurysmal dilatation was present 2 weeks after elastase perfusion in both smoking mice and nonsmoking controls, with no significant difference in final AD (mean +/- SEM: smoking, 1.23 +/- 0.11 mm versus nonsmoking, 1.22 +/- 0.05 mm). There were also no differences in the overall extent of aortic dilatation (Delta AD smoking, 136 +/- 24% versus nonsmoking, 138 +/- 10%), or the incidence of AAAs (smoking, 75% versus nonsmoking, 79%). Although all animals had developed AAAs by 12 weeks after elastase perfusion, the overall extent of aortic dilatation was 50% greater in smoking mice compared with nonsmoking controls (Delta AD smoking, 204 +/- 23% versus nonsmoking, 135 +/- 17%; p < 0.05). CONCLUSIONS: Short-term exposure to cigarette smoke did not alter initial development of experimental AAAs, but chronic smoke exposure was associated with a substantial increase in the late progression of aneurysmal dilatation. This novel combination of in vivo experimental models offers a new approach to investigate mechanisms by which cigarette smoking promotes aneurysmal degeneration.

Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.

To address the role of neutrophil elastase in pulmonary emphysema, neutrophil elastase-deficient mice and wild-type littermate controls were exposed to long-term cigarette smoke. Compared to wild-type littermates, mice that were deficient in neutrophil elastase were significantly protected (59%) from the development of emphysema. Previously, we demonstrated complete protection from emphysema in the absence of macrophage elastase. Further analysis revealed several interactions between these two elastases. Each elastase inactivated the endogenous inhibitor of the other, with neutrophil elastase degrading tissue inhibitor of metalloproteinase-1, and macrophage elastase degrading alpha-1-antitrypsin. Cigarette smoke-induced recruitment of both neutrophils and monocytes was impaired in the absence of neutrophil elastase. Moreover, there was less macrophage elastase activity secondary to decreased macrophage accumulation in neutrophil elastase-deficient mice. This study demonstrates a direct role for neutrophil elastase in emphysema and highlights the interdependence of the proteinases and inflammatory cells that mediate lung destruction in response to cigarette smoke.