Journal Articles

Experimental Immunotherapy

Preclinical and clinical cancer immunotherapy approaches

hIL-15-gene modified human natural killer cells (NKL-IL15) exhibit anti-human leukemia functions

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PURPOSE: Natural killer (NK) cells can kill transformed cells and represent anti-tumor activities for improving the immunotherapy of cancer. In previous works, we established human interleukin-15 (hIL-15) gene-modified NKL cells (NKL-IL15) and demonstrated their efficiency against human hepatocarcinoma cells (HCCs) in vitro and in vivo. To further assess the applicability of NKL-IL15 cells in adoptive cellular immunotherapy for human leukemia, here we report their natural cytotoxicity against leukemia in vitro and in vivo. METHODS: Flow cytometry, ELISA and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays. Leukemia xenograft NOD/SCID mice were established by subcutaneous injection with K562 cells, and then treated with irradiated NKL cells. RESULTS: We found NKL-IL15 cells displayed a significant high cytolysis activity against both human leukemia cell lines and primary leukemia cells from patients, accompanied with up-regulated expression of molecules related to NK cell cytotoxicity such as perforin, granzyme B and NKp80. Moreover, cytokines secreted by NKL-IL15 cells, including TNF-alpha and IFN-gamma, could induce the expression of NKG2D ligands on target cells, which increased the susceptibility of leukemia cells to NK cell-mediated cytolysis. Encouragingly, NKL-IL15 cells significantly inhibited the growth of leukemia cells in xenografted NOD/SCID mice and prolonged the survival of tumor-bearing mice dramatically. Furthermore, NKL-IL15 cells displayed stimulatory effects on hPBMCs, indicating the immunesuppressive status of leukemia patients could be improved by NKL-IL15 cell treatment. CONCLUSIONS: These results provided evidence that IL-15 gene-modification could augment NK cell-mediated anti-human leukemia function, which would improve primary NK cell-based immunotherapy for leukemia in future.

Author Info: (1) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (2) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical

Author Info: (1) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (2) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (3) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (4) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. zhangj65@sdu.edu.cn.

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VISTA expression on tumor-infiltrating inflammatory cells in primary cutaneous melanoma correlates with poor disease-specific survival

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Adaptive immune responses contribute to the pathogenesis of melanoma by facilitating immune evasion. V-domain Ig suppressor of T-cell activation (VISTA) is a potent negative regulator of T-cell function and is expressed at high levels on monocytes, granulocytes, and macrophages, and at lower densities on T-cell populations within the tumor microenvironment. In this study, 85 primary melanoma specimens were selected from pathology tissue archives and immunohistochemically stained for CD3, PD-1, PD-L1, and VISTA. Pearson's correlation coefficients identified associations in expression between VISTA and myeloid infiltrate (r = 0.28, p = 0.009) and the density of PD-1+ inflammatory cells (r = 0.31, p = 0.005). The presence of VISTA was associated with a significantly worse disease-specific survival in univariate analysis (hazard ratio = 3.57, p = 0.005) and multivariate analysis (hazard ratio = 3.02, p = 0.02). Our findings show that VISTA expression is an independent negative prognostic factor in primary cutaneous melanoma and suggests its potential as an adjuvant immunotherapeutic intervention in the future.

Author Info: (1) Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Department of Medicine, Santa Barbara Cottage Hospital, Santa Barbara, CA, USA. (2) Department of Pathology

Author Info: (1) Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Department of Medicine, Santa Barbara Cottage Hospital, Santa Barbara, CA, USA. (2) Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA. (3) Biostatistics Shared Resource, Norris Cotton Cancer Center, Dartmouth-Hitchock Medical Center, Lebanon, NH, USA. (4) Department of Medicine, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA. (5) Departments of Biomedical Data Sciences, Molecular and Systems Biology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, USA. (6) Department of Microbiology and Immunology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. (7) Department of Surgery, Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA. (8) Department of Microbiology and Immunology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. (9) Roswell Park Cancer Institute, University of Buffalo, The State University of New York, Elm and Carlton, Buffalo, NY, 14263, USA. marc.ernstoff@roswellpark.org.

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Immune Checkpoint Inhibitors in the Treatment of Patients with Neuroendocrine Neoplasia

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BACKGROUND: Well-differentiated neuroendocrine neoplasms (NENs) are usually controlled by antiproliferative, local ablative and/or radionuclide therapies, whereas poorly differentiated NENs generally require cytotoxic chemotherapy. However, treatment options for patients with advanced/metastatic high-grade NENs remain limited. METHOD: Review of the literature and international congress abstracts on the efficacy and safety of immunotherapy by checkpoint inhibition in advanced/metastatic NENs. RESULTS: Evidence points to an important role of immune phenomena in the pathogenesis and treatment of neuroendocrine tumors (NETs). Programmed cell death 1 (PD-1) protein and its ligand are mainly expressed in poorly differentiated NENs. Microsatellite instability and high mutational load are more pronounced in high-grade NENs and may predict response to immunotherapy. Clinical experience of immune checkpoint blockade mainly exists for Merkel cell carcinoma, a high-grade cutaneous neuroendocrine carcinoma (NEC), which has led to approval of the anti-PD-1 antibody avelumab. In addition, there is anecdotal evidence for the efficacy of checkpoint inhibitors in large-cell lung NECs, ovarian NECs and others, including gastroenteropancreatic NENs. Currently, phase II studies investigate PDR001, pembrolizumab, combined durvalumab and tremelimumab, and avelumab treatment in patients with advanced/metastatic NENs. CONCLUSION: Immune checkpoint inhibitors are a promising therapeutic option, especially in progressive NECs or high-grade NETs with high tumor burden, microsatellite instability, and/or mutational load.

Author Info: (1) (2)

Author Info: (1) (2)

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Radiologic pseudoprogression during anti-PD1 therapy for advanced non-small cell lung cancer

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INTRODUCTION: Anti-PD1 (programmed cell death protein 1) therapy can lead to unconventional tumor responses including radiologic pseudoprogression. Here we determine the real-world incidence of radiologic pseudoprogression in advanced non-small cell lung cancer (NSCLC) and compare radiologic response criteria for disease response assessment. METHODS: Electronic medical records of all NSCLC patients receiving anti-PD1 therapy at our institution over a 3-year period were retrospectively reviewed and patients with clinically suspected radiologic pseudoprogression identified. Patients without available follow-up imaging or clinical data were excluded. Imaging examinations were then analyzed to determine if progression was confirmed on subsequent re-imaging. Tumor response assessment by the RECIST1.1, unidimensional immune-related response criteria (iRRC) and iRECIST criteria for all patients were calculated and compared. RESULTS: A total of 228 consecutive patients were started on anti-PD1 therapy over a 3-year period, of which a total of 166 were evaluable, the majority (80%) of which received nivolumab. Fifteen patients (9%) were clinically suspected to have radiologic pseudoprogression due to tumor enlargement and/or development of new lesions on CT during the first 4-6 weeks of therapy and were maintained on anti-PD1 therapy. Of these patients, 3 patients (2% of all patients) demonstrated evidence of radiologic pseudoprogression at 1(st) re-imaging. iRRC and iRECIST were more accurate in classifying radiologic pseudoprogression as non-progression; none of the 3 cases were deemed progression by iRRC or iRECIST, compared to all 3 cases called progression on RECIST1.1. CONCLUSIONS: Radiologic pseudoprogression is a clinical challenge but an uncommon occurrence in NSCLC patients receiving anti-PD1 therapy.

Author Info: (1) Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. Electronic address: sharyn.katz@uphs.upenn.edu. (2) Department of Radiology, University of Pennsylvania Perelman School

Author Info: (1) Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. Electronic address: sharyn.katz@uphs.upenn.edu. (2) Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; Department of Radiology, Brigham and Women's Hospital, Boston, MA. (3) Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. (4) Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. (5) Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. (6) Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. (7) Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. (8) Department of Radiation Oncology, University of Maryland Medical Center, Baltimore, MD. (9) Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.

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PRAME peptide-specific CD8(+) T cells represent the predominant response against leukemia-associated antigens (LAAs) in healthy individuals

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Antigen-specific T cells isolated from healthy individuals (HIs) have shown great therapeutic potential upon adoptive transfer for the treatment of viremia in immunosuppressed patients. The lack of comprehensive data on the prevalence and characteristics of leukemia associated antigen (LAA)-specific T cells in HIs still limits such an approach for tumor therapy. Therefore, we have investigated T cell responses against prominent candidates comprising WT1, PRAME, Survivin, NY-ESO and p53 by screening PBMCs from HIs using intracellular IFN-gamma staining following provocation with LAA peptide mixes. Here, we found predominantly poly-functional effector/effector memory CCR7(-) /CD45RA(+/-) /CD8(+) LAA peptide-specific T cells with varying CD95 expression in 34 of 100 tested HIs, whereas CD4(+) T cells responses were restricted to 5. Most frequent LAA peptide-specific T cell responses were directed against WT1 and PRAME peptides with a prevalence of 20% and 17%, respectively, showing the highest magnitude (0.16% +/- 0.22% (mean+/-SD)) for PRAME peptides. Cytotoxicity of PRAME peptide-specific T cells was demonstrated by specific killing of PRAME peptide-pulsed T2 cells. Furthermore, the proliferative capacity of PRAME peptide-specific T cells was confined to HIs responsive towards PRAME peptide challenge corroborating the accuracy of the screening results. In conclusion, we identified PRAME as a promising target antigen for adoptive leukemia therapy. This article is protected by copyright. All rights reserved.

Author Info: (1) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden

Author Info: (1) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany. (2) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany. (3) Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. (4) German Cancer Research Center (DKFZ), Heidelberg, Germany. German Cancer Consortium (DKTK), Dresden, Germany. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. Institute of Immunology, Medical Faculty, TU Dresden, Dresden, Germany. National Center for Tumor Diseases, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany. (5) German Cancer Research Center (DKFZ), Heidelberg, Germany. German Cancer Consortium (DKTK), Dresden, Germany. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. Department of Medicine 1, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany. National Center for Tumor Diseases, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany. (6) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany. German Cancer Research Center (DKFZ), Heidelberg, Germany. German Cancer Consortium (DKTK), Dresden, Germany. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. (7) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany.

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Induction of Neoantigen-specific Cytotoxic T Cells and Construction of T-cell Receptor-engineered T cells for Ovarian Cancer

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PURPOSE: Current evolution of cancer immunotherapies, such as immune checkpoint blockade, has implicated neoantigens as major targets of anti-cancer cytotoxic T cells. Adoptive T cell therapy with neoantigen-specific T cell receptor (TCR)-engineered T cells would be an attractive therapeutic option for advanced cancers where the host anti-tumor immune function is strongly inhibited. We previously developed a rapid and efficient pipeline for production of neoantigen-specific TCR-engineered T cells using peripheral blood from an HLA-matched healthy donor. Our protocol required only two weeks from stimulation of T cells with neoantigen-loaded dendritic cells to the identification of neoantigen-specific TCRs. We conducted the pilot study to validate our protocol. EXPERIMENTAL DESIGN: We used tumors from 7 ovarian cancer patients to validate our protocol. RESULTS: We chose 14 candidate neoantigens from 7 ovarian tumors (1-3 candidates for each patient), and then successfully induced 3 neoantigen-specific T cells from one healthy donor and identified their TCR sequences. Moreover, we validated functional activity of the three identified TCRs by generating TCR-engineered T cells which recognized the corresponding neoantigens and showed cytotoxic activity in an antigen-dose-dependent manner. However, one case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide. Conclusion/Discussions: This pilot study demonstrated the feasibility of our efficient process from identification of neoantigen to production of the neoantigen-targeting cytotoxic TCR-engineered T cells for ovarian cancer and revealed the importance of careful validation of neoantigen-specific-TCR-engineered T cells to avoid severe immune-related adverse events.

Author Info: (1) Department of Medicine, University of Chicago. (2) Institute of Immunology, Charite. (3) Department of Medicine, University of Chicago. (4) Department of Medicine, University of

Author Info: (1) Department of Medicine, University of Chicago. (2) Institute of Immunology, Charite. (3) Department of Medicine, University of Chicago. (4) Department of Medicine, University of Chicago. (5) Medicine, University of Chicago. (6) Department of Obstetrics and Gynecology, Faculty of Medicine, Nihon University. (7) Department of Medicine, University of Chicago. (8) Cancer Precision Medicine Center, Japanese Foundation for Cancer Research. (9) Medicine, University of Chicago. (10) Department of Medicine, University of Chicago. (11) Department of Medicine, University of Chicago ynakamura@medicine.bsd.uchicago.edu.

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Recommendations for managing PD-1 blockade in the context of allogeneic HCT in Hodgkin lymphoma: taming a necessary evil

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PD-1 blockade is an effective therapy in relapsed/refractory (R/R) classical Hodgkin Lymphoma (cHL) who have relapsed after or are ineligible for autologous hematopoietic cell transplantation (HCT). While single-agent anti-PD-1 monoclonal antibodies (mAbs) are associated with high response rates and durable remissions, available results to date suggest that a large majority of patients will eventually progress on therapy. Many of these patients are potential candidates for allogeneic HCT (allo-HCT) after receiving anti-PD-1 mAbs, and allo-HCT remains for now the only treatment with demonstrated curative potential in this setting. However, initial reports suggested that allo-HCT in this setting may be associated with increased risk of early transplant-related toxicity, likely driven by lingering effects of PD-1 blockade. Furthermore, many patients with R/R cHL who undergo allo-HCT will relapse after transplantation, most often with limited treatment options. Here again PD-1 blockade appears to yield high response rates, but with an increased risk of attendant immune toxicity. Many questions remain regarding the use of PD-1 blockade before or after allo-HCT, especially in relation to the feasibility, outcome, optimal timing, and method of allo-HCT after PD-1 blockade. Despite the scarcity of prospective data, these questions are unavoidable and must be tackled by clinicians in the routine care of patients with advanced cHL. We provide consensus recommendations of a working group based on available data and experience, in an effort to help guide treatment decisions until more definitive data are obtained.

Author Info: (1) CHRU Lille, Lille, France. (2) Dana-Farber Cancer Institute, Boston, MA, United States. (3) The Ohio State University, Columbus, OH, United States. (4) Dana-Farber Cancer

Author Info: (1) CHRU Lille, Lille, France. (2) Dana-Farber Cancer Institute, Boston, MA, United States. (3) The Ohio State University, Columbus, OH, United States. (4) Dana-Farber Cancer Institute, Boston, MA, United States. (5) CHU Rennes, Rennes, France. (6) CHRU Lille, Lille, France. (7) University of Colorado, Aurora, CO, United States bradley.haverkos@ucdenver.edu.

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Canonical TGF-beta Signaling Pathway Represses Human NK Cell Metabolism

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Cytokines stimulate rapid metabolic changes in human NK cells, including increases in both glycolysis and oxidative phosphorylation pathways. However, how these are subsequently regulated is not known. In this study, we demonstrate that TGF-beta can inhibit many of these metabolic changes, including oxidative phosphorylation, glycolytic capacity, and respiratory capacity. TGF-beta also inhibited cytokine-induced expression of the transferrin nutrient receptor CD71. In contrast to a recent report on murine NK cells, TGF-beta-mediated suppression of these metabolic responses did not involve the inhibition of the metabolic regulator mTORC1. Inhibition of the canonical TGF-beta signaling pathway was able to restore almost all metabolic and functional responses that were inhibited by TGF-beta. These data suggest that pharmacological inhibition of TGF-beta could provide a metabolic advantage to NK cells that is likely to result in improved functional responses. This has important implications for NK cell-based cancer immunotherapies.

Author Info: (1) School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; and. (2) School of Biochemistry and Immunology, Trinity Biomedical

Author Info: (1) School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; and. (2) School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; and clair.gardiner@tcd.ie finlayd@tcd.ie. School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. (3) School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; and clair.gardiner@tcd.ie finlayd@tcd.ie.

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CAR-T Cells Surface-Engineered with Drug-Encapsulated Nanoparticles Can Ameliorate Intratumoral T Cell Hypofunction

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One limiting factor of CAR T-cell therapy for treatment of solid cancers is the suppressive tumor microenvironment, which inactivates the function of tumor infiltrating lymphocytes (TILs) through the production of immunosuppressive molecules such as adenosine. Adenosine inhibits the function of CD4+ and CD8+ T cells by binding to and activating the A2a adenosine receptor (A2aR) expressed on their surface. This suppression pathway can be blocked using the A2aR-specific small molecule antagonist SCH-58261 (SCH), but its applications have been limited owing to difficulties delivering this drug to immune cells within the tumor microenvironment (TME). To overcome this limitation, we used CAR-engineered T cells as active chaperones to deliver SCH-loaded cross-linked, multilamellar liposomal vesicles (cMLVs) to tumor-infiltrating T cells deep within the immune suppressive TME. Through in vitro and in vivo studies, we have demonstrated that this system can be used to effectively deliver SCH to the TME. This treatment may prevent or rescue the emergence of hypofunctional CAR-T cells within the TME.

Author Info: (1) Chemical Engineering and Materials Science, University of Southern California. (2) Pharmacology and Pharmaceutical Sciences, University of Southern California. (3) Biomedical Engineering, University of Southern

Author Info: (1) Chemical Engineering and Materials Science, University of Southern California. (2) Pharmacology and Pharmaceutical Sciences, University of Southern California. (3) Biomedical Engineering, University of Southern California. (4) Pharmacology and Pharmaceutical Sciences, University of Southern California. (5) Biomedical Engineering, University of Southern California. (6) R&D, HRAIN Biotechnology Co. Ltd. (7) Chemical Engineering and Materials Science, University of Southern California pinwang@usc.edu.

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VISTA expression associated with CD8 confers a favorable immune microenvironment and better overall survival in hepatocellular carcinoma

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BACKGROUND: Hepatocellular carcinoma (HCC) often arises in the setting of chronic inflammation with multiple inhibitory immune signals. V-domain Ig suppressor of T cell activation (VISTA) is identified as a novel negative checkpoint regulator. This study sought to determine the expression and prognostic value of VISTA in HCC and classify tumor microenvironments (TMEs) based on VISTA and CD8+ tumor-infiltrating lymphocytes (TILs). METHODS: The expression of VISTA and CD8 proteins was assessed in 183 HCC tissue microarrays (TMAs) by immunohistochemistry (IHC). VISTA and CD8A mRNA extracted from 372 patients with HCC in The Cancer Genome Atlas (TCGA) database was included as a validation cohort. Associations between the VISTA, clinicopathological variables, and survival were analyzed. RESULTS: VISTA expression was detected in 29.5% HCC tissues, among which 16.4% tissues were positive for tumor cells (TCs), and 16.9% tissues were positive for immune cells (ICs). VISTA expression was significantly associated with tissues with a high pathological grading (p = 0.038), without liver cirrhosis (p = 0.011), and with a high density of CD8 + TILs (p < 0.001). Kaplan-Meier curves demonstrated that patients with VISTA-positive staining in TCs (p = 0.037), but not in ICs, (p = 0.779) showed significantly prolonged overall survival (OS) than those with VISTA-negative expression. Classification of HCC TME-based VISTA and CD8 + TILs showed 4 immune subtypes: VISTA+/CD8+ (16.9%), VISTA+/CD8- (12.6%), VISTA-/CD8+ (16.4%), and VISTA-/CD8+ (54.1%). The dual positive VISTA+/CD8+ subtype showed significantly prolonged OS than other subtypes (p = 0.023). CONCLUSIONS: VISTA protein expression in HCC showed cell specific and displayed different prognosis. VISTA expression was significantly associated with CD8 + TILs, Dual positive VISTA+/CD8+ showed favorable TME and better OS.

Author Info: (1) Department of Interventional Radiology, Nanfang Hospital, Southern Medical University, 1838, North Guangzhou Avenue, Guangzhou, China. (2) Department of Interventional Radiology, Nanfang Hospital, Southern Medical

Author Info: (1) Department of Interventional Radiology, Nanfang Hospital, Southern Medical University, 1838, North Guangzhou Avenue, Guangzhou, China. (2) Department of Interventional Radiology, Nanfang Hospital, Southern Medical University, 1838, North Guangzhou Avenue, Guangzhou, China. (3) Department of Interventional Radiology, Nanfang Hospital, Southern Medical University, 1838, North Guangzhou Avenue, Guangzhou, China. (4) Department of Pathology and Laboratory Medicine, Guangdong General Hospital and Guangdong Academy of Medical Sciences, 106 Zhongshan Er Rd, Guangzhou, China. (5) Department of Pathology and Laboratory Medicine, Guangdong General Hospital and Guangdong Academy of Medical Sciences, 106 Zhongshan Er Rd, Guangzhou, China. (6) Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, 1838, North Guangzhou Avenue, Guangzhou, China. dongzy1317@foxmail.com. (7) Department of Interventional Radiology, Nanfang Hospital, Southern Medical University, 1838, North Guangzhou Avenue, Guangzhou, China. ozonetherapy@126.com.

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