Journal Articles

Immune monitoring

Techniques to monitor the immune response to cancer immunotherapy, to study immune cell interactions and to extend knowledge related to the induction and mechanisms of an immune response

Alteration of circulating natural autoantibodies to CD25-derived peptide antigens and FOXP3 in non-small cell lung cancer

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Natural autoantibody is a key component for immune surveillance function. Regulatory T (Treg) cells play indispensable roles in promoting tumorigenesis via immune escape mechanisms. Both CD25 and FOXP3 are specific markers for Treg cells and their natural autoantibodies may be involved in anticancer activities. This work was designed to develop an in-house enzyme-linked immunosorbent assay (ELISA) to examine plasma natural IgG against CD25 and FOXP3 in non-small cell lung cancer (NSCLC). Compared with control subjects, NSCLC patients had significantly higher levels of plasma IgG for CD25a (Z = -8.05, P < 0.001) and FOXP3 (Z = -4.17, P < 0.001), lower levels for CD25b (Z = -3.58, P < 0.001), and a trend toward lower levels for CD25c (Z = -1.70, P = 0.09). Interestingly, the anti-CD25b IgG assay had a sensitivity of 25.0% against a specificity of 95.0% in an early stage patients (T1N0M0) who showed the lowest anti-CD25b IgG levels among 4 subgroups classified based on staging information. Kaplan-Meier survival analysis showed that patients with high anti-FOXP3 IgG levels had shorter survival than those with low anti-FOXP3 IgG levels (chi(2) = 3.75, P = 0.05). In conclusion, anti-CD25b IgG may be a promising biomarker in terms of screening individuals at high risk of lung cancer.

Author Info: (1) Second Hospital of Jilin University, Changchun, 130041, China. (2) Second Hospital of Jilin University, Changchun, 130041, China. zhangxuankj@163.com. (3) Department of Thoracic Surgery, China-Japan

Author Info: (1) Second Hospital of Jilin University, Changchun, 130041, China. (2) Second Hospital of Jilin University, Changchun, 130041, China. zhangxuankj@163.com. (3) Department of Thoracic Surgery, China-Japan Union Hospital, Jilin University, Changchun, 130031, China. (4) Second Hospital of Jilin University, Changchun, 130041, China. (5) Second Hospital of Jilin University, Changchun, 130041, China. wyang2020@jlu.edu.cn. (6) Institute of Health Research & Innovation, University of the Highlands & Islands, Centre for Health Science, Inverness, IV2 3JH, UK.

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Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy

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The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8(+) T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

Author Info: (1) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (2) Department of Medicine, University of Pennsylvania, 340

Author Info: (1) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (2) Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address: byrnek@upenn.edu. (3) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (4) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (5) Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (6) Cancer Biology and Genetics Program, Sloan-Kettering Institute, NY 10065, USA. (7) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (8) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (9) Center for RNA Biology, Department of Biochemistry and Biophysics, Department of Urology, University of Rochester Medical Center, Rochester, NY 14642, USA. (10) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (11) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (12) Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (13) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (14) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (15) Department of Cell, Developmental and Cancer Biology, Oregon Health & Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA. (16) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (17) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (18) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (19) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (20) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (21) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (22) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (23) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (24) Penn Genomic Analysis Core, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (25) Cancer Biology and Genetics Program, Sloan-Kettering Institute, NY 10065, USA; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, 415 East 68(th) Street New York, NY 10065, USA. (26) Department of Cell, Developmental and Cancer Biology, Oregon Health & Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA. (27) Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (28) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address: rhv@upenn.edu. (29) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address: bstanger@upenn.edu.

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Dendritic Cell-Based ELISpot Assay for Assessing T-Cell IFN-gamma Responses in Human Peripheral Blood Mononuclear Cells to Dengue Envelope Proteins

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Dengue envelope (E) protein is a dominant antigen for vaccine development and E-based vaccines have shown partial or full protection against live-virus challenge in non-human primates. Generally, T cell responses can be investigated with peptides. However, hundreds of over-lapping peptides need to be synthesized to cover the whole sequence of a protein, which brings the cost up to a much higher level than purchasing a protein. We have developed an enzyme-linked immunospot (ELISpot) assay that uses intact E proteins instead of peptides for assessing IFN-gamma (IFN-gamma) responses. The assay relies on professional antigen presenting cells, dendritic cells, to process and present the E proteins to stimulate T cells.Peripheral blood mononuclear cells (PBMCs) from dengue-exposed and naive subjects were selected for the assay development. IFN-gamma production ranged from 53 to 513 spot forming units (SFUs) and 0-45 SFUs per million PBMCs in dengue-exposed and naive subject groups, respectively. The assay allowed quantification of E-specific IFN-gamma secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Our results suggest that the dendritic cell-based IFN-gamma assay is a useful tool for assessing immunological memory for clinical research.

Author Info: (1) Henry Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA. Peifang.sun2.ctr@mail.mil. (2) Naval Medical Research Center, Silver Spring, MD, USA.

Author Info: (1) Henry Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA. Peifang.sun2.ctr@mail.mil. (2) Naval Medical Research Center, Silver Spring, MD, USA.

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Four Color ImmunoSpot((R)) Assays for Identification of Effector T-Cell Lineages

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Single color IFN-gamma ELISPOT assays have evolved as a highly sensitive T cell immune monitoring platform. By detecting individual T cells that secrete IFN-gamma in response to antigen exposure, these assays permit the measurement of the frequency of antigen-specific T cells among white blood cells. These assays therefore are well suited to assess clonal expansions, that is, whether a (Th1) T cell response has been induced to an antigen in a test subject. Single color IFN-gamma ELISPOT assays are not suited, however, to provide information on the Th2/Th17 quality of the T cell response, nor do they provide insights into the differentiation state of CD8 cells. Recently it has been established that co-expression profiles of IL-2, TNF-alpha, and granzyme B along with IFN-gamma permit to identify CD8 cell subpopulations. Naive CD8 cells, central CD8 memory cells, CD8 terminal effector cells, polyfunctional CD8 cells, stem-cell like CD8 memory cells, dysfunctional- and senescent CD8 cells all differ in the extent they produce these molecules upon antigen re-encounter. We therefore have developed, and introduce here, a four color T cell ELISPOT assay in which the co-expression levels of IFN-gamma, IL-2, TNF-alpha, and granzyme B can be established for individual antigen-specific CD8 cells, thereby identifying the activation/differentiation state of these cells.

Author Info: (1) Cellular Technology Ltd., Shaker Heights, OH, USA. (2) Cellular Technology Ltd., Shaker Heights, OH, USA. (3) Cellular Technology Ltd., Shaker Heights, OH, USA. paul.lehmann@immunospot.com.

Author Info: (1) Cellular Technology Ltd., Shaker Heights, OH, USA. (2) Cellular Technology Ltd., Shaker Heights, OH, USA. (3) Cellular Technology Ltd., Shaker Heights, OH, USA. paul.lehmann@immunospot.com.

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A Consistent Method to Identify and Isolate Mononuclear Phagocytes from Human Lung and Lymph Nodes

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Mononuclear phagocytes (MP) consist of macrophages, dendritic cells (DCs), and monocytes. In all organs, including the lung, there are multiple subtypes within these categories. The existence of all these cell types suggest that there is a clear division of labor and delicate balance between the MPs under steady state and inflammatory conditions. Although great strides have been made to understand MPs in the mouse lung, and human blood, little is known about the MPs that exist in the human lung and lung-draining lymph nodes (LNs), and even less is known about their functional roles, studies of which will require a large number of sorted cells. We have comprehensively examined cell surface markers previously used in a variety of organs to identify human pulmonary MPs. In the lung, we consistently identify five extravascular pulmonary MPs and three LN MPs. These MPs were present in over 100 lungs regardless of age or gender. Notably, the human blood CD141(+) DCs, as described in the literature, were not observed in non-diseased lungs or their draining LNs. In the lung and draining LNs, expression of CD141 was only observed on HLADR(+) CD11c(+) CD14(+) extravascular monocytes (often confused in the LN as resident DCs based on the level of HLADR expression and mouse LN data). In the human lung and LNs there are at least two DC subtypes expressing HLADR, DEC205 and CD1c, along with circulating monocytes that behave as either antigen-presenting cells or macrophages. Furthermore, we demonstrate how to distinguish between alveolar macrophages and interstitial macrophage subtypes. It still remains unclear how the human pulmonary MPs identified here align with mouse MPs. Clearly, we are now past the stage of cell surface marker characterization, and future studies will need to move toward understanding what these cell types are and how they function. Our hope is that the strategy described here can help the pulmonary community take this next step.

Author Info: (1) Department of Pediatrics, National Jewish Health, Denver, CO, USA. (2) Department of Pediatrics, National Jewish Health, Denver, CO, USA. jakubzickc@njhealth.org. Department of Microbiology and

Author Info: (1) Department of Pediatrics, National Jewish Health, Denver, CO, USA. (2) Department of Pediatrics, National Jewish Health, Denver, CO, USA. jakubzickc@njhealth.org. Department of Microbiology and Immunology, University of Colorado, Denver, CO, USA. jakubzickc@njhealth.org.

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Imaging Precision-Cut Lung Slices to Visualize Leukocyte Localization and Trafficking

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Pulmonary dendritic cells (DCs) are potent antigen-presenting cells that can activate both naive and memory/effector T cells. However, very little is known of how movements and localization of DCs contribute to these events. To study this, we have developed new procedures that combine precision-cut lung slices with cell staining using fluorescently tagged antibodies to detect individual cell types. In this chapter, we describe these methods in detail and show how they can be used to study the localization of not only DCs but also other leukocytes of interest, as well as structural cells within the lung.

Author Info: (1) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (2) Immunity, Inflammation and

Author Info: (1) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (2) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (3) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (4) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.

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Blood cell count indexes as predictors of outcomes in advanced non-small-cell lung cancer patients treated with Nivolumab

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Lung cancer is the most common malignancy worldwide. Despite significant advances in diagnosis and treatment, mortality rates remain extremely high, close to incidence rates. Several targeted therapies have been recently introduced for the treatment of non-small cell lung cancer (NSCLC), the most common type of lung cancer. Nivolumab, a monoclonal antibody that targets programmed death-1 (PD-1), was the first immune checkpoint inhibitor approved for the treatment of patients with advanced/metastatic NSCLC not responding to platinum-based chemotherapy. Biomarkers predicting response to these therapies would allow early identification of non-responders and timely implementation of appropriate combination strategies, avoiding inadequate and expensive therapies. The role of the neutrophil to lymphocyte ratio and other blood cell count indexes as possible biomarkers of response has been recently investigated. We discuss the encouraging results reported on the topic, provide new data from our personal experience, and discuss opportunities for further research.

Author Info: (1) Medical Oncology Unit, University Hospital of Sassari (AOU), Viale San Pietro 43, 07100, Sassari, Italy. (2) Medical Oncology Unit, San Gerardo Hospital, Via Pergolesi

Author Info: (1) Medical Oncology Unit, University Hospital of Sassari (AOU), Viale San Pietro 43, 07100, Sassari, Italy. (2) Medical Oncology Unit, San Gerardo Hospital, Via Pergolesi 33, 20900, Monza, Italy. (3) Medical Oncology Unit, San Gerardo Hospital, Via Pergolesi 33, 20900, Monza, Italy. (4) Medical Oncology Unit, San Gerardo Hospital, Via Pergolesi 33, 20900, Monza, Italy. (5) Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43, 07100, Sassari, Italy. (6) Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43, 07100, Sassari, Italy. (7) Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43, 07100, Sassari, Italy. ppaliogiannis@uniss.it.

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Heterogeneity of Tumour Infiltrating Lymphocytes (TILs) in Breast Cancer and its prognostic significance

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BACKGROUND: Tumour-infiltrating lymphocytes (TILs) in breast cancer (BC) confer prognostic and predictive information. This study aims to assess the spatial and temporal heterogeneity of TILs in BC and its relationship with immune cell subtypes. METHOD: Immunohistochemically-defined immune cell subtypes; T-cell markers (CD3, CD8, and FOXP3), B-cell marker (CD20) and histiocytic marker (CD68) were evaluated in a large series (n=1,165) of invasive BC. A subset of full-face haematoxylin and eosin (H&E) stained slides were examined for TILs heterogeneity within primary tumours and the corresponding local recurrent carcinomas to report on spatial and temporal TILs heterogeneity. H&E stained sections from multiple tumour blocks (3-4 blocks per case) representing different tumour areas were evaluated to assess TILS inter-slide heterogeneity as well as intra-slide heterogeneity. Both average (AV-TILs) and hotspot (HS-TILs) stromal TILs were assessed. RESULTS: AV-TILs showed association with all immune cell subtypes however; the main component were CD3+ cells (mean number = 55) whereas CD20+cells comprised the least component (the mean number = 13). There was no significant statistical difference between TILs across tumour blocks of the same case (p=0.251 for AV-TILs and p=0.162 for HS-TILs). Triple negative breast cancer (TNBC) showed higher TILs compared with other BC subtypes (p<0.001). High AV-TILs, CD3+, CD8+, and CD20+ cells were associated with longer survival in TNBC (p<0.05). High AV-TILs in recurrent tumours showed significant association with shorter post-recurrence survival (p=0.004). CONCLUSION: Despite the heterogeneity of immune cell type components, average TILs in one full-face H&E stained section reliably represent whole tumour TILs. TILs were associated with outcome in TNBC as well as provided prognostic significance in recurrent tumour. This article is protected by copyright. All rights reserved.

Author Info: (1) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG

Author Info: (1) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (2) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (3) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (4) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (5) School of Veterinary Medicine and Science, Sutton Bonington Campus, the University of Nottingham, Sutton Bonington, LE12 5RD, UK, 3 Department of Pharmacology, Weill Cornell Medicine, 1300 York Avenue, New York, NY, 10065USA. (6) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (7) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (8) Molecular Diagnostics and Personalised Therapeutics Unit, University of Ha'il, Ha'il 2440, Saudi Arabia. (9) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (10) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. (11) Nottingham Breast Cancer Research Centre, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK.

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Changes in programmed death ligand 1 expression in non-small cell lung cancer patients who received anticancer treatments

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BACKGROUND: The expression of programmed death ligand 1 (PD-L1) is considered a predictive biomarker of anti-programmed death 1 (PD-1)/PD-L1 cancer therapies. However, changes in PD-L1 expression of tumor cells during clinical courses have not been fully evaluated. We evaluated changes in PD-L1 expression for non-small cell lung cancer (NSCLC) patients who received anticancer treatments during clinical courses. METHODS: In 76 NSCLC patients, PD-L1 expression was evaluated before and after anticancer treatment by immunohistochemical (IHC) analysis using an anti-PD-L1 antibody. We defined two cut-off points of PD-L1 expression (1 and 50%) and three corresponding IHC groups (A: 0%, B: 1-49%, and C: >/=50%). IHC group B and C were considered to be positive expression, and we defined the difference of IHC group between pre- and post-treatment as 'major change' in PD-L1 expression. RESULTS: Before anticancer treatment, PD-L1 expression was observed in 38/76 (50%) patients, and was significantly less common in patients harboring mutations in the epidermal growth factor receptor gene (EGFR) than in those without (P = 0.039). After anticancer treatment, PD-L1 expression was observed in 36/76 (47%) patients. Major increases in PD-L1 expression were seen in 11 (14%), and major decreases in 18 (24%) patients. Among 13 patients harboring EGFR mutations treated with EGFR tyrosine-kinase inhibitor (EGFR-TKI), five (38%) showed major increases. CONCLUSION: Major changes of PD-L1 expression in tumor cells were observed in 38% of NSCLC patients who received anticancer treatments. And, treatments with EGFR-TKI may increase PD-L1 expression in NSCLC patients harboring EGFR mutations.

Author Info: (1) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (2) Division of Thoracic Oncology, Shizuoka Cancer Center, 100

Author Info: (1) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (2) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (3) Division of Pathology, Shizuoka Cancer Center, Shizuoka, Japan. (4) Division of Pathology, National Cancer Center, Tokyo, Japan. (5) Division of Pathology, Shizuoka Cancer Center, Shizuoka, Japan. (6) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (7) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (8) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (9) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (10) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (11) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (12) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. (13) Division of Thoracic Surgery, Shizuoka Cancer Center, Shizuoka, Japan. (14) Division of Diagnostic Radiology, Shizuoka Cancer Center, Shizuoka, Japan. (15) Immunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan. (16) Division of Pathology, Shizuoka Cancer Center, Shizuoka, Japan. (17) Division of Thoracic Oncology, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Suntou-gun, Shizuoka, Shizuoka, 411-8777, Japan. t.takahashi@scchr.jp.

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Increased exhausted CD8(+) T cells with programmed death-1, T-cell immunoglobulin and mucin-domain-containing-3 phenotype in patients with multiple myeloma

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AIM: The immunosuppressive microenvironment plays a crucial role in T-cell immunodeficiency in multiple myeloma (MM). Overexpression of T-cell immunosuppressive receptors, including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain-containing-3 (Tim-3), may be related to tumor immunosuppression and poor prognosis, and the malignant bone marrow (BM) microenvironment may contribute to such immunosuppression. The purpose of this study was to analyze the distribution of PD-1(+) and/or Tim-3(+) T cells in different T-cell subset in patients with MM. METHODS: The expression of PD-1 and Tim-3 with exhausted (CD244(+) and CD57(+) ) CD3(+) , CD4(+) and CD8(+) T cells between BM and peripheral blood (PB) from 10 patients with untreated MM was detected by multicolor flow cytometry assay. RESULTS: A significant increase in both PD-1(+) CD57(+) and Tim-3(+) CD57(+) CD3(+) T cells and PD-1(+) Tim-3(+) CD3(+) T cells was detected in PB from patients with MM compared with 10 healthy individuals (HIs), and the alteration was mostly in the CD8(+) T-cell subset. Significant higher percentage of PD-1(+) CD3(+) T cells was found in BM compared with PB from patients with MM. The level of PD-1(+) Tim-3(+) CD3(+) , CD4(+) , and CD8(+) T cells was high in BM group compared with PB. Moreover, PD-1(+) CD244(+) or PD-1(+) CD57(+) CD3(+) T cells, particularly PD-1(+) CD244(+) and PD-1(+) CD57(+) CD8(+) T cells were significantly higher in BM than in PB. In addition, limited dynamic detection data from three MM cases who achieved complete remission after treatment showed that the numbers of either PD-1(+) or PD-1(+) Tim-3(+) T cells in different T-cell subsets were decreased in both BM and PB. CONCLUSION: We characterized the distribution of PD-1 and TIM-3 concurrent with exhausted CD3(+) , CD4(+) and CD8(+) T cells between BM and PB from patients with MM. Higher numbers of PD-1(+) CD244(+) or PD-1(+) CD57(+) CD3(+) T cells in BM from patients with MM may contribute to mediate the BM immunosuppressive microenvironment. Although heterogeneous alterations in Tim-3(+) T cells may represent a complex immunosuppressive pattern in MM. Overall, higher levels of PD-1(+) CD244(+) or PD-1/Tim-3(+) CD57(+) CD8(+) T cells may be a major reason for lower T-cell activation and T-cell immunodeficiency in MM.

Author Info: (1) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou

Author Info: (1) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (2) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (3) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (4) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (5) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (6) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (7) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (8) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (9) Department of Oncology, First Affiliated Hospital, Jinan University, Guangzhou, China. (10) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. (11) Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China. Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Jinan University, Guangzhou, China.

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