Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Publisher Correction: Actively personalized vaccination trial for newly diagnosed glioblastoma.

The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.

Actively personalized vaccination trial for newly diagnosed glioblastoma.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

Tumor antigens in glioma.

Immunotherapy applications to glioblastoma represent a new treatment frontier. Antigen-targeted immunotherapy approaches hold enormous potential to elicit antigen-specific anti-tumor effects in central nervous system tumors. Still, the paucity of effective antigen targets remains a significant obstacle in safely and effectively treating glioblastoma and other malignant gliomas with relatively low mutation loads. In this review, we highlight the current understanding of and development of immunotherapy to target 1) shared non-mutant antigens 2) shared mutant antigens (neoantigens) derived from cancer-specific mutations 3) personalized neoantigens derived from tumor-specific genetic alterations containing de novo peptide sequences and 4) virus-derived antigens. We also discuss strategies to enhance tumor immunogenicity and neoantigen prediction. Spatial heterogeneity remains a formidable challenge for immunotherapy of glioma; recent advances in targeting multiple antigens and refining the antigen selection pipeline hold great promise to turn the tide against glioma.

Future development of chimeric antigen receptor T cell therapies for patients suffering from malignant glioma.

PURPOSE OF REVIEW: Chimeric antigen receptor (CAR) T cell therapy has been successful in some haematologic malignancies, but the central nervous system (CNS) presents unique obstacles to its use against tumours arising therein. This review discusses recent improvements in the delivery and design of these cells to improve the efficacy and safety of this treatment against malignant gliomas. RECENT FINDINGS: The immunosuppressive environment of the CNS affects the functionality of CAR T cells, but recent developments using metabolic manipulation and cytokine delivery have shown that the performance of CAR T cells can be improved in this environment. Emerging techniques can improve the delivery of CAR T cells to the CNS parenchyma, which is normally well protected from peripheral immune cells. The implementation of novel antigens and CAR-expression regulation strategies will improve the specificity and efficacy of these cells. Finally, although autologous T cells have historically been the standard, recent developments have made the use of allogeneic T cells or natural killer (NK) cells more clinically feasible. SUMMARY: The discoveries highlighted in this review will aid the development of CAR cells that are safer, more resilient against immunosuppressive signals in the CNS, and able to specifically target intracranial tumour cells.

The future of cancer immunotherapy for brain tumors: a collaborative workshop.

Harnessing the effector mechanisms of the immune system to combat brain tumors with antigen specificity and memory has been in research and clinical testing for many years. Government grant mechanisms and non-profit organizations have supported many innovative projects and trials while biotech companies have invested in the development of needed tools, assays and novel clinical approaches. The National Brain Tumor Society and the Parker Institute for Cancer Immunotherapy partnered to host a workshop to share recent data, ideas and identify both hurdles and new opportunities for harnessing immunotherapy against pediatric and adult brain tumors. Adoptively transferred cell therapies have recently shown promising early clinical results. Local cell delivery to the brain, new antigen targets and innovative engineering approaches are poised for testing in a new generation of clinical trials. Although several such advances have been made, several obstacles remain for the successful application of immunotherapies for brain tumors, including the need for more representative animal models that can better foreshadow human trial outcomes. Tumor and tumor microenvironment biopsies with multiomic analysis are critical to understand mechanisms of response and patient stratification, yet brain tumors are especially challenging for such biopsy collection. These workshop proceedings and commentary shed light on the status of immunotherapy in pediatric and adult brain tumor patients, including current research as well as opportunities for improving future efforts to bring immunotherapy to the forefront in the management of brain tumors.

The immunology of low-grade gliomas.

Low-grade gliomas (LGGs), which harbor an isocitrate dehydrogenase (IDH) mutation, have a better prognosis than their high-grade counterparts; nonetheless, they remain incurable and impart significant negative impacts on patients' quality of life. Although immunotherapies represent a novel avenue of treatment for patients with LGGs, they have not yet been successful. Accurately selecting and evaluating immunotherapies requires a detailed understanding of LGG tumor immunology and the underlying tumor immune phenotype. A growing body of literature suggests that LGGs significantly differ in their immunology from high-grade gliomas, highlighting the importance of investigation into LGG immunology specifically. In this review, the authors aimed to discuss relevant research surrounding the LGG tumor immune microenvironment, including immune cell infiltration, tumor immunogenicity, checkpoint molecule expression, the impact of an IDH mutation, and implications for immunotherapies, while also briefly touching on current immunotherapy trials and future directions for LGG immunology research.

Current Advances in Immunotherapy for Glioblastoma.

PURPOSE OF REVIEW: This review seeks to inform oncology clinicians and researchers about the development of novel immunotherapies for the treatment of glioblastoma. An enumeration of ongoing and recently completed clinical trials will be discussed with special attention given to current technologies implemented to overcome central nervous system-specific challenges including barriers to the peripheral immune system, impaired antigen presentation, and T cell dysfunction. RECENT FINDINGS: The success of immunotherapy in other solid cancers has served as a catalyst to explore its application in glioblastoma, which has limited response to other treatments. Recent developments include multi-antigen vaccines that seek to overcome the heterogeneity of glioblastoma, as well as immune checkpoint inhibitors, which could amplify the adaptive immune response and may have promise in combinatorial approaches. Additionally, oncolytic and retroviruses have opened the door to a plethora of combinatorial approaches aiming to leverage their immunogenicity and/or ability to carry therapeutic transgenes. Treatment of glioblastoma remains a serious challenge both with regard to immune-based as well as other therapeutic strategies. The disease has proven to be highly resistant to treatment due to a combination of tumor heterogeneity, adaptive expansion of resistant cellular subclones, evasion of immune surveillance, and manipulation of various signaling pathways involved in tumor progression and immune response. Immunotherapeutics that are efficacious in other cancer types have unfortunately not enjoyed the same success in glioblastoma, illustrating the challenging and complex nature of this disease and demonstrating the need for development of multimodal treatment regimens utilizing the synergistic qualities of immune-mediated therapies.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Cell penetrating peptide functionalized perfluorocarbon nanoemulsions for targeted cell labeling and enhanced fluorine-19 MRI detection.

PURPOSE: A bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19 F MRI detection, is a non-invasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell-penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co-surfactant to complement the common surfactants used in PFC nanoemulsion preparations. METHODS: We performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra-tumorally injected into mice bearing flank glioma tumors. RESULTS: By incorporating TAT into the nanoemulsion, a labeling efficiency of ~1012 fluorine atoms per CAR T cell was achieved that is a >8-fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function. The 19 F MRI signal detected from TAT-labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT. CONCLUSION: The peptide-PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes.

T-Cell based therapies for overcoming neuroanatomical and immunosuppressive challenges within the glioma microenvironment.

Glioblastoma remains as the most common and aggressive primary adult brain tumor to date. Within the last decade, cancer immunotherapy surfaced as a broadly successful therapeutic approach for a variety of cancers. However, due to the neuroanatomical and immunosuppressive nature of malignant gliomas, conventional chemotherapy and radiotherapy treatments garner limited efficacy in patients with these tumors. The intricate structure of the blood brain barrier restricts immune accessibility into the tumor microenvironment, and malignant gliomas can activate various adaptive responses to subvert anticancer immune responses and reinstate an immunosuppressive milieu. Yet, evidence of lymphocyte infiltration within the brain and recent advancements made in cell engineering technologies implicate the vast potential in the future of neuro-oncological immunotherapy. Previous immunotherapy platforms have paved way to improved modalities, which includes but is not limited to personalized vaccines and chimeric antigen receptor T-cell therapy. This review will cover the various neuroanatomical and immunosuppressive features of central nervous system tumors and highlight the innovations made in T-cell based therapies to overcome the challenges presented by the glioblastoma microenvironment.

Treatment-induced lesions in newly diagnosed glioblastoma patients undergoing chemoradiotherapy and heat-shock protein vaccine therapy.

OBJECTIVES: Treatment-induced lesions represent a great challenge in neuro-oncology. The aims of this study were (i) to characterize treatment induced lesions in glioblastoma patients treated with chemoradiotherapy and heat-shock protein (HSP) vaccine and (ii) to evaluate the diagnostic accuracy of diffusion weighted imaging for differentiation between treatment-induced lesions and tumor progression. METHODS: Twenty-seven patients with newly diagnosed glioblastoma treated with HSP vaccine and chemoradiotherapy were included. Serial magnetic resonance imaging evaluation was performed to detect treatment-induced lesions and assess their growth. Quantitative analysis of the apparent diffusion coefficient (ADC) was performed to discriminate treatment-induced lesions from tumor progression. Mann-Whitney U-test and receiver operating characteristic (ROC) curves were used for analysis. RESULTS: Thirty-three percent of patients developed treatment-induced lesions. Five treatment-related lesions appeared between end of radiotherapy and the first vaccine administration; 4 lesions within the first 4 months from vaccine initiation and 1 at 3.5 years. Three patients with pathology proven treatment-induced lesions showed a biphasic growth pattern progressed shortly after. ADC ratio between the peripheral enhancing rim and central necrosis showed an accuracy of 0.84 (95% CI 0.63-1) for differentiation between progression and treatment-induced lesions. CONCLUSION: Our findings do not support the iRANO recommendation of a 6-month time window in which progressive disease should not be declared after immunotherapy initiation. A biphasic growth pattern of pathologically proven treatment-induced lesions was associated with a dismal prognosis. The presence of lower ADC values in the central necrotic portion of the lesions compared to the enhancing rim shows high specificity for detection of treatment-induced lesions.

The current state of immunotherapy for primary and secondary brain tumors: similarities and differences.

Treatment and resolution of primary and metastatic brain tumors have long presented a challenge to oncologists. In response to the dismal survival outcomes associated with conventional therapies, various immunotherapy modalities, such as checkpoint inhibitors, vaccine, cellular immunotherapy and viral immunotherapy have been actively explored over the past couple of decades. Although improved patient survival has been more frequently noted in treatment of brain metastases, little progress has been made in improving patient survival in cases of primary brain tumors, specifically glioblastoma, which is the representative primary brain tumor discussed in this review. Herein, we will first overview the findings of recent clinical studies for treatment of primary and metastatic brain tumors with immunotherapeutic interventions. The clinical efficacy of these immunotherapies will be discussed in the context of their ability or inability to overcome inherent characteristics of the tumor as well as restricted antigen presentation and its immunosuppressive microenvironment. Additionally, this review aims to briefly inform clinicians in the field of neuro-oncology on the relevant aspects of the immune system as it pertains to the central nervous system, with special focus on the differing modes of antigen presentation and tumor microenvironment of primary and metastatic brain tumors and the role these differences may play in the efficacy of immunotherapy in eradicating the tumor.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Antigen-specific immunoreactivity and clinical outcome following vaccination with glioma-associated antigen peptides in children with recurrent high-grade gliomas: results of a pilot study.

Recurrent high-grade gliomas (HGGs) of childhood have an exceedingly poor prognosis with current therapies. Accordingly, new treatment approaches are needed. We initiated a pilot trial of vaccinations with peptide epitopes derived from glioma-associated antigens (GAAs) overexpressed in these tumors in HLA-A2+ children with recurrent HGG that had progressed after prior treatments. Peptide epitopes for three GAAs (EphA2, IL13Rα2, survivin), emulsified in Montanide-ISA-51, were administered subcutaneously adjacent to intramuscular injections of poly-ICLC every 3 weeks for 8 courses, followed by booster vaccines every 6 weeks. Primary endpoints were safety and T-cell responses against the GAA epitopes, assessed by enzyme-linked immunosorbent spot (ELISPOT) analysis. Treatment response was evaluated clinically and by magnetic resonance imaging. Twelve children were enrolled, 6 with glioblastoma, 5 with anaplastic astrocytoma, and one with malignant gliomatosis cerebri. No dose-limiting non-CNS toxicity was encountered. ELISPOT analysis, in ten children, showed GAA responses in 9: to IL13Rα2 in 4, EphA2 in 9, and survivin in 3. One child had presumed symptomatic pseudoprogression, discontinued vaccine therapy, and responded to subsequent treatment. One other child had a partial response that persisted throughout 2 years of vaccine therapy, and continues at >39 months. Median progression-free survival (PFS) from the start of vaccination was 4.1 months and median overall survival (OS) was 12.9 months. 6-month PFS and OS were 33 and 73 %, respectively. GAA peptide vaccination in children with recurrent malignant gliomas is generally well tolerated, and has preliminary evidence of immunological and modest clinical activity.

Expression and prognostic impact of immune modulatory molecule PD-L1 in meningioma.

While immunotherapy may offer promising new approaches for high grade meningiomas, little is currently known of the immune landscape in meningiomas. We sought to characterize the immune microenvironment and a potentially targetable antigen mesothelin across WHO grade I-III cases of meningiomas, and how infiltrating immune populations relate to patient outcomes. Immunohistochemistry was performed on tissue microarrays constructed from 96 meningioma cases. The cohort included 16 WHO grade I, 62 WHO grade II, and 18 WHO grade III tumors. Immunohistochemistry was performed using antibodies against CD3, CD8, CD20, CD68, PD-L1, and mesothelin. Dual staining using anti-PD-L1 and anti-CD68 antibodies was performed, and automated cell detection and positive staining detection algorithms were utilized. Greater degree of PD-L1 expression was found in higher grade tumors. More specifically, higher grade tumors contained increased numbers of intratumoral CD68-, PD-L1+ cells (p = 0.022), but did not contain higher numbers of infiltrating CD68+, PD-L1+ cells (p = 0.30). Higher PD-L1+/CD68- expression was independently predictive of worse overall survival in our cohort when accounting for grade, performance status, extent of resection, and recurrence history (p = 0.014). Higher expression of PD-L1+/CD68- was also present in tumors that had undergone prior radiotherapy (p = 0.024). Approximately quarter of meningiomas overexpressed mesothelin to levels equivalent to those found in pancreatic carcinomas and malignant mesotheliomas. The association with poor survival outcomes in our study suggests that PD-L1 may play a significant biologic role in the aggressive phenotype of higher grade meningiomas. Thus, immunotherapeutic strategies such as checkpoint inhibition may have clinical utility in PD-L1 overexpressing meningiomas.

Immunotherapy response assessment in neuro-oncology: a report of the RANO working group.

Immunotherapy is a promising area of therapy in patients with neuro-oncological malignancies. However, early-phase studies show unique challenges associated with the assessment of radiological changes in response to immunotherapy reflecting delayed responses or therapy-induced inflammation. Clinical benefit, including long-term survival and tumour regression, can still occur after initial disease progression or after the appearance of new lesions. Refinement of the response assessment criteria for patients with neuro-oncological malignancies undergoing immunotherapy is therefore warranted. Herein, a multinational and multidisciplinary panel of neuro-oncology immunotherapy experts describe immunotherapy Response Assessment for Neuro-Oncology (iRANO) criteria based on guidance for the determination of tumour progression outlined by the immune-related response criteria and the RANO working group. Among patients who demonstrate imaging findings meeting RANO criteria for progressive disease within 6 months of initiating immunotherapy, including the development of new lesions, confirmation of radiographic progression on follow-up imaging is recommended provided that the patient is not significantly worse clinically. The proposed criteria also include guidelines for the use of corticosteroids. We review the role of advanced imaging techniques and the role of measurement of clinical benefit endpoints including neurological and immunological functions. The iRANO guidelines put forth in this Review will evolve successively to improve their usefulness as further experience from immunotherapy trials in neuro-oncology accumulate.

Transgene-derived overexpression of miR-17-92 in CD8+ T-cells confers enhanced cytotoxic activity.

MicroRNAs (miRs) play important roles in regulation of a variety of cell functions, including immune responses. We have previously demonstrated that miR-17-92 expression in T-cells enhances Th1 phenotype and provides a long-term protection against glioblastoma when co-expressed as a transgene in T-cells along with a chimeric antigen receptor. To further elucidate the function of miR-17-92 in tumor antigen-specific CD8(+) T-cells, we generated transgenic (Tg) mice in which CD8(+) T-cells overexpress transgene-derived miR-17-92 under the lck promoter as well as T-cell receptor specific for human gp10025-33 (Pmel-1) (miR-17-92/Pmel-Tg). CD8(+) T-cells from miR-17-92/Pmel-Tg mice demonstrated enhanced interferon (IFN)-γ production and cytotoxicity in response to the cognate antigen compared with those from control Pmel-Tg mice without the transgene for miR-17-92. In addition, miR-17-92/Pmel-Tg mouse-derived CD8(+)CD44(+) T-cells demonstrated increased frequencies of cells with memory phenotypes and IFN-γ production. We also found that miR-17-92/Pmel-Tg-derived CD8(+) T-cells expressed decreased levels of transforming growth factor (TGF)-ß type II receptor (TGFBR2) on their surface, thereby resisting against suppressive effects of TGF-ß1. Our findings suggest that engineering of tumor antigen-specific CD8(+) T-cells to express miR-17-92 may improve the potency of cancer immunotherapy.