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Delhez N, Aboubakar Nana F, Houbion C, Bayard A, Bruger A, Vanhaver C, Brandau S, van der Bruggen P, Hirsch T. Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation. Methods Cell Biol 2024; 191:151-196. [PMID: 39824555 DOI: 10.1016/bs.mcb.2024.10.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2025]
Abstract
Neutrophils were historically considered a homogenous population of cells with functions limited to innate immunity against external threats. However, with the rise of immunotherapy, recent works have shown that neutrophils are also important actors in immuno-oncology. In this context, neutrophils appear as a more heterogenous population of cells. However, many reported neutrophil subpopulations, or neutrophils with various transcriptional states, lack functional characterization to confirm their suspected roles. Thus, we believe that functional assays remain essential to define the role of neutrophils in cancer. In this chapter, we present a T-cell proliferation assay based on the use of allogeneic T-cells to assess the suppressive capabilities of neutrophils isolated from human blood or tumor samples. Allogeneic T-cells are isolated in large quantities from the blood of non-cancerous donors and frozen in aliquots to be used in several experiments. This reduces variability by excluding other cancer-derived factors, which would be present if autologous T-cell were used and allows to isolate the effect of neutrophils on T-cell proliferation. Thawed T-cells have poor proliferative capacities and to initiate proliferation they require co-culture with mature dendritic cells that we generate from monocytes isolated from the same blood sample. Initially developed for lung cancer patients, our method to isolate low-density neutrophils (LDN) and normal-density neutrophils (NDN) can be used with any patient and adapted to other kind of samples (e.g., ascites, urine, …).
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Affiliation(s)
- Nicolas Delhez
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
| | - Frank Aboubakar Nana
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium; Pneumology Department, Cliniques universitaires Saint-Luc, Brussels, Belgium
| | - Camille Houbion
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
| | - Alexandre Bayard
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
| | - Annika Bruger
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
| | | | - Sven Brandau
- Research Division, Department of Otorhinolaryngology, University Hospital Essen, West German Cancer Center, Essen, Germany
| | | | - Thibault Hirsch
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium.
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2
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Cubitt CC, Wong P, Dorando HK, Foltz JA, Tran J, Marsala L, Marin ND, Foster M, Schappe T, Fatima H, Becker-Hapak M, Zhou AY, Hwang K, Jacobs MT, Russler-Germain DA, Mace EM, Berrien-Elliott MM, Payton JE, Fehniger TA. Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation. J Clin Invest 2024; 134:e173602. [PMID: 38805302 PMCID: PMC11291271 DOI: 10.1172/jci173602] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Accepted: 05/17/2024] [Indexed: 05/30/2024] Open
Abstract
The surface receptor CD8α is present on 20%-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses in patients with leukemia in prior studies, thus, we hypothesized that CD8α may affect critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models compared with CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, we found that CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These induced CD8α+ (iCD8α+) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity compared with those that sustained existing CD8α expression (sustained CD8α+) or those that remained CD8α- (persistent CD8α-). These iCD8α+ cells originated from an IL-15Rβhi NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identified human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion, and its presence had a suppressive effect on NK cell-activating receptors.
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Affiliation(s)
| | - Pamela Wong
- Division of Oncology, Siteman Cancer Center, and
| | - Hannah K. Dorando
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA
| | | | | | | | | | - Mark Foster
- Division of Oncology, Siteman Cancer Center, and
| | | | - Hijab Fatima
- Division of Allergy, Immunology and Rheumatology, Department of Pediatrics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA
| | | | | | | | | | | | - Emily M. Mace
- Division of Allergy, Immunology and Rheumatology, Department of Pediatrics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA
| | | | - Jacqueline E. Payton
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA
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3
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Sutton MS, Bucsan AN, Lehman CC, Kamath M, Pokkali S, Magnani DM, Seder R, Darrah PA, Roederer M. Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates. Front Immunol 2024; 15:1359679. [PMID: 38529287 PMCID: PMC10961357 DOI: 10.3389/fimmu.2024.1359679] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Accepted: 02/20/2024] [Indexed: 03/27/2024] Open
Abstract
Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.
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Affiliation(s)
- Matthew S. Sutton
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Allison N. Bucsan
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Chelsea C. Lehman
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Megha Kamath
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Supriya Pokkali
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Diogo M. Magnani
- Nonhuman Primate Reagent Resource, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Robert Seder
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Patricia A. Darrah
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
| | - Mario Roederer
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
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4
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Nwizu C, Hughes M, Ramseier ML, Navia AW, Shalek AK, Fusi N, Raghavan S, Winter PS, Amini AP, Crawford L. Scalable nonparametric clustering with unified marker gene selection for single-cell RNA-seq data. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.02.11.579839. [PMID: 38405697 PMCID: PMC10888887 DOI: 10.1101/2024.02.11.579839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/27/2024]
Abstract
Clustering is commonly used in single-cell RNA-sequencing (scRNA-seq) pipelines to characterize cellular heterogeneity. However, current methods face two main limitations. First, they require user-specified heuristics which add time and complexity to bioinformatic workflows; second, they rely on post-selective differential expression analyses to identify marker genes driving cluster differences, which has been shown to be subject to inflated false discovery rates. We address these challenges by introducing nonparametric clustering of single-cell populations (NCLUSION): an infinite mixture model that leverages Bayesian sparse priors to identify marker genes while simultaneously performing clustering on single-cell expression data. NCLUSION uses a scalable variational inference algorithm to perform these analyses on datasets with up to millions of cells. By analyzing publicly available scRNA-seq studies, we demonstrate that NCLUSION (i) matches the performance of other state-of-the-art clustering techniques with significantly reduced runtime and (ii) provides statistically robust and biologically relevant transcriptomic signatures for each of the clusters it identifies. Overall, NCLUSION represents a reliable hypothesis-generating tool for understanding patterns of expression variation present in single-cell populations.
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Affiliation(s)
- Chibuikem Nwizu
- Center for Computational Molecular Biology, Brown University, Providence, RI, USA
- Warren Alpert Medical School of Brown University, Providence, RI, USA
| | | | - Michelle L. Ramseier
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Andrew W. Navia
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Alex K. Shalek
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
- Harvard Medical School, Boston, MA, USA
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA
| | | | - Srivatsan Raghavan
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
- Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA
| | - Peter S. Winter
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | | | - Lorin Crawford
- Center for Computational Molecular Biology, Brown University, Providence, RI, USA
- Microsoft Research, Cambridge, MA, USA
- Department of Biostatistics, Brown University, Providence, RI, USA
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5
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Sutton MS, Bucsan AN, Lehman CC, Kamath M, Pokkali S, Magnani DM, Seder R, Darrah PA, Roederer M. Antibody-mediated depletion of select T cell subsets in blood and tissue of nonhuman primates. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.22.572898. [PMID: 38187627 PMCID: PMC10769432 DOI: 10.1101/2023.12.22.572898] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2024]
Abstract
Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.
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6
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Laeremans T, den Roover S, Lungu C, D’haese S, Gruters RA, Allard SD, Aerts JL. Autologous dendritic cell vaccination against HIV-1 induces changes in natural killer cell phenotype and functionality. NPJ Vaccines 2023; 8:29. [PMID: 36864042 PMCID: PMC9980861 DOI: 10.1038/s41541-023-00631-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2022] [Accepted: 02/20/2023] [Indexed: 03/04/2023] Open
Abstract
Although natural killer (NK) cells have been studied in connection with dendritic cell (DC)-based vaccination in the field of cancer immunology, their role has barely been addressed in the context of therapeutic vaccination against HIV-1. In this study, we evaluated whether a therapeutic DC-based vaccine consisting of monocyte-derived DCs electroporated with Tat, Rev and Nef encoding mRNA affects NK cell frequency, phenotype and functionality in HIV-1-infected individuals. Although the frequency of total NK cells did not change, we observed a significant increase in cytotoxic NK cells following immunisation. In addition, significant changes in the NK cell phenotype associated with migration and exhaustion were observed together with increased NK cell-mediated killing and (poly)functionality. Our results show that DC-based vaccination has profound effects on NK cells, which highlights the importance of evaluating NK cells in future clinical trials looking at DC-based immunotherapy in the context of HIV-1 infection.
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Affiliation(s)
- Thessa Laeremans
- grid.8767.e0000 0001 2290 8069Neuro-Aging and Viro-Immunotherapy Research Group, Vrije Universiteit Brussel, Brussels, Belgium
| | - Sabine den Roover
- grid.8767.e0000 0001 2290 8069Neuro-Aging and Viro-Immunotherapy Research Group, Vrije Universiteit Brussel, Brussels, Belgium
| | - Cynthia Lungu
- grid.5645.2000000040459992XDepartment of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Sigrid D’haese
- grid.8767.e0000 0001 2290 8069Neuro-Aging and Viro-Immunotherapy Research Group, Vrije Universiteit Brussel, Brussels, Belgium
| | - Rob A. Gruters
- grid.5645.2000000040459992XDepartment of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Sabine D. Allard
- grid.411326.30000 0004 0626 3362Department of Internal Medicine and Infectious Diseases, Universitair Ziekenhuis Brussel and Vrije Universiteit Brussel, Brussels, Belgium
| | - Joeri L. Aerts
- grid.8767.e0000 0001 2290 8069Neuro-Aging and Viro-Immunotherapy Research Group, Vrije Universiteit Brussel, Brussels, Belgium
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7
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Xie J, El Rami F, Zhou K, Simonetta F, Chen Z, Zheng X, Chen M, Balakrishnan PB, Dai SY, Murty S, Alam IS, Baker J, Negrin RS, Gambhir SS, Rao J. Multiparameter Longitudinal Imaging of Immune Cell Activity in Chimeric Antigen Receptor T Cell and Checkpoint Blockade Therapies. ACS CENTRAL SCIENCE 2022; 8:590-602. [PMID: 35647285 PMCID: PMC9136971 DOI: 10.1021/acscentsci.2c00142] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Indexed: 05/17/2023]
Abstract
Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for the assessment of cytotoxic T lymphocyte mediated cancer cell killing. G-SNAT was found to specifically detect the activity of granzyme B within the cytotoxic granules of activated T cells and engaged cancer cells in vitro. In lymphoma tumor-bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 correlated to CAR T cell mediated granzyme B exocytosis and tumor eradication. In colorectal tumor-bearing transgenic mice with hematopoietic cells expressing firefly luciferase, longitudinal bioluminescence and fluorescence imaging revealed that after combination treatment of anti-PD-1 and anti-CTLA-4, the dynamics of immune cell trafficking, tumor infiltration, and cytotoxic activity predicted the therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.
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Affiliation(s)
- Jinghang Xie
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Fadi El Rami
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Kaixiang Zhou
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Federico Simonetta
- Division
of Blood and Marrow Transplantation, Department of Medicine, Stanford University Medical Center, Stanford, California 94305, United States
| | - Zixin Chen
- Department of Chemistry, Department of Bioengineering, and Department of Materials Science
& Engineering, Stanford University, Stanford, California 94305, United States
| | - Xianchuang Zheng
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Min Chen
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Preethi B. Balakrishnan
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Sheng-Yao Dai
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Surya Murty
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
- Department of Chemistry, Department of Bioengineering, and Department of Materials Science
& Engineering, Stanford University, Stanford, California 94305, United States
| | - Israt S. Alam
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
| | - Jeanette Baker
- Division
of Blood and Marrow Transplantation, Department of Medicine, Stanford University Medical Center, Stanford, California 94305, United States
| | - Robert S. Negrin
- Division
of Blood and Marrow Transplantation, Department of Medicine, Stanford University Medical Center, Stanford, California 94305, United States
| | - Sanjiv S. Gambhir
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
- Department of Chemistry, Department of Bioengineering, and Department of Materials Science
& Engineering, Stanford University, Stanford, California 94305, United States
| | - Jianghong Rao
- Department
of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California 94305, United States
- Department of Chemistry, Department of Bioengineering, and Department of Materials Science
& Engineering, Stanford University, Stanford, California 94305, United States
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Kapoor SS, Zaiss DMW. Emerging Role of EGFR Mutations in Creating an Immune Suppressive Tumour Microenvironment. Biomedicines 2021; 10:biomedicines10010052. [PMID: 35052732 PMCID: PMC8772868 DOI: 10.3390/biomedicines10010052] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2021] [Revised: 12/22/2021] [Accepted: 12/23/2021] [Indexed: 02/06/2023] Open
Abstract
Several types of tumours overexpress the Epidermal Growth Factor Receptor (EGFR) in either wild type or mutated form. These tumours are often highly aggressive and difficult to treat. The underlying mechanisms for this phenomenon have remained largely unresolved, but recent publications suggest two independent mechanisms that may contribute. According to one line of research, tumours that overexpress the EGFR grow autonomously and become “addicted” to growth factor signalling. Inhibition of this signal using EGFR inhibitors can, therefore, induce cell death in tumour cells and lead to tumour shrinkage. The other line of research, as highlighted by recent findings, suggests that the overexpression, specifically of mutant forms of the EGFR, may create an immune-suppressive and lymphocyte depleted microenvironment within tumours. Such a lymphocyte depleted microenvironment may explain the resistance of EGFR overexpressing cancers to tumour therapies, particularly to check-point inhibitor treatments. In this article, we discuss the recent data which support an immune modulatory effect of EGFR signalling and compare these published studies with the most recent data from The Cancer Genome Atlas (TCGA), in this way, dissecting possible underlying mechanisms. We thereby focus our study on how EGFR overexpression may lead to the local activation of TGFβ, and hence to an immune suppressive environment. Consequently, we define a novel concept of how the mitogenic and immune modulatory effects of EGFR overexpression may contribute to tumour resistance to immunotherapy, and how EGFR specific inhibitors could be used best to enhance the efficacy of tumour therapy.
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Affiliation(s)
- Simran S. Kapoor
- Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3FL, UK;
| | - Dietmar M. W. Zaiss
- Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3FL, UK;
- Faculty of Medicine, Institute of Immune Medicine, University of Regensburg, 93053 Regensburg, Germany
- Correspondence:
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9
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Abstract
The intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.
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10
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Abstract
The CD8+ T cell noncytotoxic antiviral response (CNAR) was discovered during studies of asymptomatic HIV-infected subjects more than 30 years ago. In contrast to CD8+ T cell cytotoxic lymphocyte (CTL) activity, CNAR suppresses HIV replication without target cell killing. This activity has characteristics of innate immunity: it acts on all retroviruses and thus is neither epitope specific nor HLA restricted. The HIV-associated CNAR does not affect other virus families. It is mediated, at least in part, by a CD8+ T cell antiviral factor (CAF) that blocks HIV transcription. A variety of assays used to measure CNAR/CAF and the effects on other retrovirus infections are described. Notably, CD8+ T cell noncytotoxic antiviral responses have now been observed with other virus families but are mediated by different cytokines. Characterizing the protein structure of CAF has been challenging despite many biologic, immunologic, and molecular studies. It represents a low-abundance protein that may be identified by future next-generation sequencing approaches. Since CNAR/CAF is a natural noncytotoxic activity, it could provide promising strategies for HIV/AIDS therapy, cure, and prevention.
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Affiliation(s)
- Maelig G Morvan
- Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Fernando C Teque
- Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, California, USA
| | | | - Jay A Levy
- Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, California, USA
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11
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Combination of CD8β Depletion and Interleukin-15 Superagonist N-803 Induces Virus Reactivation in Simian-Human Immunodeficiency Virus-Infected, Long-Term ART-Treated Rhesus Macaques. J Virol 2020; 94:JVI.00755-20. [PMID: 32669328 PMCID: PMC7495383 DOI: 10.1128/jvi.00755-20] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Accepted: 07/07/2020] [Indexed: 12/11/2022] Open
Abstract
The “shock and kill” HIV cure strategy attempts to reverse and eliminate the latent viral infection that prevents eradication of the virus. Latency-reversing agents tested in clinical trials to date have failed to affect the HIV viral reservoir. IL-15 superagonist N-803, currently involved in a clinical trial for HIV cure, was recently shown by our laboratory to induce robust and persistent induction of plasma viremia during ART in three in vivo animal models of HIV infection. These results suggest a substantial role for CD8+ lymphocytes in suppressing the latency reversal effect of N-803 by promoting the maintenance of viral latency. In this study, we tested whether the use of a CD8β-targeting antibody, which would specifically deplete CD8+ T cells, would yield similar levels of virus reactivation. We observed the induction of plasma viremia, which correlated with the efficacy of the CD8 depletion strategy. The “shock and kill” strategy predicates that virus reactivation in latently infected cells is required to eliminate the human immunodeficiency virus (HIV) reservoir. In a recent study, we showed robust and persistent induction of plasma viremia in antiretroviral therapy (ART)-treated simian immunodeficiency virus-infected rhesus macaques (RMs) undergoing CD8α depletion and treated with the interleukin-15 (IL-15) superagonist N-803 (J. B. McBrien et al., Nature 578:154–159, 2020, https://doi.org/10.1038/s41586-020-1946-0). Of note, in that study we used an antibody targeting CD8α, thereby depleting NK cells, NKT cells, and γδ T cells, in addition to CD8+ T cells. In the current proof-of-concept study, we tested whether virus reactivation can be induced by administration of N-803 to simian-human chimeric immunodeficiency virus-infected, ART-treated RMs that are selectively depleted of CD8+ T cells via the CD8β-targeting antibody CD8b255R1. CD8β depletion was performed in five SHIVSF162P3-infected RMs treated with ART for 12 months and with plasma viremia consistently below 3 copies/ml. All animals received four weekly doses of N-803 starting at the time of CD8b255R1 administration. The induction of detectable plasma viremia was observed in three out of five RMs, with the level of virus reactivation seemingly correlated with the frequency of CD8+ T cells following CD8β depletion as well as the level of virus reactivation observed when the same animals underwent CD8α depletion and N-803 administration after 24 weeks of ART. These data indicate that CD8β depletion and N-803 administration can induce virus reactivation in SHIVSF162P3-infected RMs despite suboptimal depletion of CD8+ T cells and profound ART-induced suppression of virus replication, confirming a critical role for these cells in suppressing virus production and/or reactivation in vivo under ART. IMPORTANCE The “shock and kill” HIV cure strategy attempts to reverse and eliminate the latent viral infection that prevents eradication of the virus. Latency-reversing agents tested in clinical trials to date have failed to affect the HIV viral reservoir. IL-15 superagonist N-803, currently involved in a clinical trial for HIV cure, was recently shown by our laboratory to induce robust and persistent induction of plasma viremia during ART in three in vivo animal models of HIV infection. These results suggest a substantial role for CD8+ lymphocytes in suppressing the latency reversal effect of N-803 by promoting the maintenance of viral latency. In this study, we tested whether the use of a CD8β-targeting antibody, which would specifically deplete CD8+ T cells, would yield similar levels of virus reactivation. We observed the induction of plasma viremia, which correlated with the efficacy of the CD8 depletion strategy.
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Kristensen LK, Fröhlich C, Christensen C, Melander MC, Poulsen TT, Galler GR, Lantto J, Horak ID, Kragh M, Nielsen CH, Kjaer A. CD4 + and CD8a + PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models. Theranostics 2019; 9:8221-8238. [PMID: 31754392 PMCID: PMC6857046 DOI: 10.7150/thno.37513] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Accepted: 09/09/2019] [Indexed: 12/17/2022] Open
Abstract
Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4+ and CD8a+ TILs in syngeneic mouse tumor models for preclinical studies. Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4+ and CD8a+ TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (89Zr-DFO-CD4/89Zr-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021. Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose. Reduced tumor and spleen uptake of 89Zr-DFO-CD8a was observed in CD8a+ depleted mice and the uptake was comparable with that of isotype control (89Zr-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89Zr-DFO-CD4 and 89Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum 89Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89Zr-DFO-CD4 ratio >9 (p=0.0018). Conclusion: We developed 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4+ and CD8a+ status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.
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Affiliation(s)
- Lotte K. Kristensen
- Minerva Imaging, Copenhagen, Denmark
- Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Denmark
| | | | - Camilla Christensen
- Minerva Imaging, Copenhagen, Denmark
- Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Denmark
| | | | | | | | | | | | | | - Carsten H. Nielsen
- Minerva Imaging, Copenhagen, Denmark
- Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Denmark
| | - Andreas Kjaer
- Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Denmark
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Sutton MS, Ellis-Connell A, Balgeman AJ, Barry G, Weiler AM, Hetzel SJ, Zhou Y, Lau-Kilby AW, Mason RD, Biris KK, Mascola JR, Sullivan NJ, Roederer M, Friedrich TC, O'Connor SL. CD8β Depletion Does Not Prevent Control of Viral Replication or Protection from Challenge in Macaques Chronically Infected with a Live Attenuated Simian Immunodeficiency Virus. J Virol 2019; 93:e00537-19. [PMID: 31092584 PMCID: PMC6639280 DOI: 10.1128/jvi.00537-19] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2019] [Accepted: 05/11/2019] [Indexed: 11/20/2022] Open
Abstract
We evaluated the contribution of CD8αβ+ T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8β-specific mAb CD8β255R1 selectively depleted CD8αβ+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8β255R1, plasma viremia transiently increased coincident with declining peripheral CD8αβ+ T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αβ+ T cells did not. Although depletion of CD8αβ+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8β255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αβ+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8β255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8β255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8β255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8β255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8β255R1 administration.IMPORTANCE Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αβ+) and minor (CD8αα+) populations of CD8+ T cells but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8β-specific depleting mAb CD8β255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αβ+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8β255R1 for studying the direct contribution of CD8αβ+ T cells in various disease states.
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Affiliation(s)
- Matthew S Sutton
- Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Amy Ellis-Connell
- Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Alexis J Balgeman
- Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Gabrielle Barry
- Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Andrea M Weiler
- Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Scott J Hetzel
- Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Yan Zhou
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Annie W Lau-Kilby
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Rosemarie D Mason
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Kristin K Biris
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - John R Mascola
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Nancy J Sullivan
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Mario Roederer
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Thomas C Friedrich
- Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Shelby L O'Connor
- Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
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Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR. Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood. Vet Immunol Immunopathol 2018; 199:22-31. [DOI: 10.1016/j.vetimm.2018.03.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2018] [Revised: 03/15/2018] [Accepted: 03/22/2018] [Indexed: 11/25/2022]
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Miranda LHM, Santiago MDA, Schubach TMP, Morgado FN, Pereira SA, Oliveira RDVCD, Conceição-Silva F. Severe feline sporotrichosis associated with an increased population of CD8low cells and a decrease in CD4⁺ cells. Med Mycol 2015; 54:29-39. [PMID: 26483429 DOI: 10.1093/mmy/myv079] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Accepted: 07/28/2015] [Indexed: 01/13/2023] Open
Abstract
Sporotrichosis is a subcutaneous mycosis with worldwide distribution, especially in tropical and subtropical areas. Zoonotic transmission is described with cats being the main animal species involved. The occurrence of severe feline sporotrichosis with high fungal levels demonstrates the susceptibility of cats to this disease and the importance of studying its pathogenesis. This study describes the leukocytes profile in blood of cats with sporotrichosis by flow cytometry and its correlation with histopathology and fungal load. The cats with sporotrichosis were separated into groups L1, L2, and L3 (lesions at one, two, and three or more noncontiguous skin locations, respectively) and were classified as good, fair, or poor general conditions. The highest percentage of CD4+ cells was associated to L1 (P = .04) and to good general condition (P = .03). The percentage of CD8+ cells was greater in L2 and L3 (P = .01). CD8(low) expression occurred in 20 animals with sporotrichosis, mainly in L3 (P = .01) and was not observed in healthy controls. This expression was related to macrophage granulomas (P = .01) and predominated in cases with high fungal load. Altogether, the results indicated that control over feline sporotrichosis, with maintenance of a good general condition, fixed lesions, well-organized response and lower fungal load, is associated with increased CD4+ cells percentages. In contrast, a poor general condition, disseminated lesions and high fungal load were related to increased CD8+ cell percentages and increased expression of CD8(low). As conclusion these results point to an important role of the CD4:CD8 balance in determining the clinical outcome in feline sporotrichosis.
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Affiliation(s)
- Luisa H M Miranda
- Laboratory of Clinical Research on Dermatozoonosis in Domestic Animals, National Institute of Infectious Diseases Evandro Chagas (INI), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brasil
| | - Marta de A Santiago
- Laboratory of Diagnostic Technology, Bio-Manguinhos, FIOCRUZ, Rio de Janeiro, Brasil
| | - Tânia M P Schubach
- Laboratory of Clinical Research on Dermatozoonosis in Domestic Animals, National Institute of Infectious Diseases Evandro Chagas (INI), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brasil
| | - Fernanda N Morgado
- Laboratory of Immunoparasitology, Oswaldo Cruz Institute (IOC), FIOCRUZ, Rio de Janeiro, Brasil
| | - Sandro A Pereira
- Laboratory of Clinical Research on Dermatozoonosis in Domestic Animals, National Institute of Infectious Diseases Evandro Chagas (INI), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brasil
| | | | - Fátima Conceição-Silva
- Laboratory of Immunoparasitology, Oswaldo Cruz Institute (IOC), FIOCRUZ, Rio de Janeiro, Brasil
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van Loenen MM, Hagedoorn RS, de Boer R, Falkenburg JHF, Heemskerk MHM. Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells. PLoS One 2013; 8:e65212. [PMID: 23738014 PMCID: PMC3667802 DOI: 10.1371/journal.pone.0065212] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2013] [Accepted: 04/24/2013] [Indexed: 11/18/2022] Open
Abstract
By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8+ as well as CD4+ T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4+ T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αβ needed for optimal co-receptor function in HLA-I-TCR transduced CD4+ T-cells. For this purpose, we transduced human peripheral blood derived CD4+ T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αβ co-receptor in HLA-I-TCR transduced CD4+ T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4+ T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient.
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Affiliation(s)
- Marleen M van Loenen
- Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands.
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Hu D, Weiner HL, Ritz J. Identification of cytolytic CD161- CD56+ regulatory CD8 T cells in human peripheral blood. PLoS One 2013; 8:e59545. [PMID: 23527216 PMCID: PMC3602421 DOI: 10.1371/journal.pone.0059545] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2012] [Accepted: 02/19/2013] [Indexed: 01/13/2023] Open
Abstract
We previously developed methods for establishing CD8 regulatory T cell (Treg) clones from normal human peripheral blood and demonstrated that these clones were capable of killing T cell receptor (TCR)-activated autologous CD4 T cells. Based on phenotypic and functional characterization of the CD8 Treg clones, we have identified a corresponding population of endogenous CD8 Treg in normal human peripheral blood. These cells appear morphologically as large lymphocytes with abundant cytoplasm and have the following unique phenotype: CD3+CD8+CD161−CD56+. The majority of CD8 Treg express CD45RA and CD62L with low or negative expression of CD45RO, CD25, CD27, CD28 and CCR7. The expression of CD94 and NKG2a on CD8 Treg was elevated compared to conventional CD8 T cells. Following in vitro activation, this T cell subset is capable of killing TCR-activated CD4 T cells. These studies identify an endogenous CD8 Treg population in humans and it will now be possible to characterize these cells in a variety of clinical conditions.
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Affiliation(s)
- Dan Hu
- Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, United States of America
- Division of Hematologic Malignancies, Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America
- Harvard Medical School, Boston, Massachusetts, United States of America
| | - Howard L. Weiner
- Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, United States of America
- Harvard Medical School, Boston, Massachusetts, United States of America
| | - Jerome Ritz
- Division of Hematologic Malignancies, Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America
- Harvard Medical School, Boston, Massachusetts, United States of America
- * E-mail:
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El Garch H, Crafford JE, Amouyal P, Durand PY, Edlund Toulemonde C, Lemaitre L, Cozette V, Guthrie A, Minke JM. An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses. Vet Immunol Immunopathol 2012; 149:76-85. [PMID: 22763149 DOI: 10.1016/j.vetimm.2012.06.009] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2011] [Revised: 05/30/2012] [Accepted: 06/06/2012] [Indexed: 12/24/2022]
Abstract
A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI induced by the experimental ALVAC(®)-AHSV4 vaccine. Six horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.
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Dimova T, Mihaylova A, Spassova P, Georgieva R. Superficial implantation in pigs is associated with decreased numbers and redistribution of endometrial NK-cell populations. Am J Reprod Immunol 2008; 59:359-69. [PMID: 18336390 DOI: 10.1111/j.1600-0897.2007.00579.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
PROBLEM We evaluated implantation-associated quantitative changes in endometrial and peripheral natural killer (NK)-cell populations of pigs. METHOD OF STUDY Natural killer cell populations were investigated in 10, 15, 20, 30 and 40 days pregnant and non-pregnant (NP) sows by flow cytometry, immunohistochemistry and morphometry. RESULTS The number of endometrial CD16(+) NK cells significantly declined at attachment phase of implantation and remained relatively low over the course of implantation. The CD16(+) NK cells in situ showed implantation-phase dependent density and localization. Prior to implantation, they substantially resided in the subepithelial stroma. As implantation advances, the density of NK cells into subepithelial stroma decreased while that of NK cells into glandular layer increased, suggesting implantation-induced re-location far from the attached conceptus. The number of CD56(+) lymphocytes was the greatest at pre-attachment phase of implantation, dropped at the time of attachment and increased up to end of early pregnancy period. The CD3(-) CD8(+) NK-cell number decreased significantly when the definitive placenta is established. No significant differences in the numbers of peripheral blood CD16(+), CD56(+) and CD3(-) CD8(+) NK cells between pregnant and NP animals as well as relative to the implantation phase were observed. CONCLUSION Superficial and adeciduate implantation of pigs is associated with decreased numbers of endometrial NK-cell populations and specific spatiotemporal profile of classical NK cells.
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Affiliation(s)
- Tanya Dimova
- Department of Immunobiology of Reproduction, Institute of Biology and Immunology of Reproduction acad.K.Bratanov, Bulgarian Academy of Sciences, Sofia, Bulgaria.
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Oberg HH, Wesch D, Grüssel S, Rose-John S, Kabelitz D. Differential expression of CD126 and CD130 mediates different STAT-3 phosphorylation in CD4+CD25− and CD25high regulatory T cells. Int Immunol 2006; 18:555-63. [PMID: 16540526 DOI: 10.1093/intimm/dxh396] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
IL-6 is a pleiotropic cytokine involved in T-lymphocyte biology. Following IL-6 binding, the soluble IL-6R (CD126)-IL-6 complex can directly activate cells that express the signal-transducing gp130 (CD130) molecule, which mediates two distinct signals, mitogenesis by mitogen-activated protein kinase (MAPK) activation and anti-apoptosis by signal transducer and activator of transcription 3 (STAT-3) activation. This 'trans-signaling', also mediated by the soluble CD126/IL-6 fusion protein hyper-IL-6 (H-IL-6), contributes to the perpetuation of autoimmune diseases such as Morbus Crohn or rheumatoid arthritis. On the other hand, the homeostasis of cellular immune reactions and its failure leading to autoimmune diseases are critically controlled by regulatory T cells (Tregs). Here, we investigated the differential expression of CD126 and CD130 on subsets of human leukocytes in blood, tonsil and spleen. Among CD4+ T cells, differential expression of CD126 and CD130 was observed on the basis of CD25 expression. CD4+CD25- T cells were strongly CD126+ and CD130+, whereas CD25(high) Tregs expressed CD126 but little CD130. Both CD126 and CD130 were down-modulated on CD4+CD25- T cells following ligand binding, whereas only marginal modulation was observed on Tregs. Interestingly, we observed a correlation between CD126 and CD130 expression with STAT-3 phosphorylation in CD4+CD25- T cells compared with Tregs after stimulation with IL-6 or H-IL-6, whereas the MAPK extracellular signal-regulated kinase 1/2 were not activated by CD130 dimerization. The differential expression of CD126 and CD130 and subsequent STAT-3 phosphorylation might be relevant for the recently described role of IL-6 in the control of Treg activity.
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Affiliation(s)
- Hans-Heinrich Oberg
- Institute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, Michaelisstrasse 5, D-24105 Kiel, Germany
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Abstract
Natural killer cells derived from pluripotent hematopoietic stem cells are important cells of the immune system that have two main functions: a cytolytic activity and a cytokine-producing capacity. These functions are tightly regulated by numerous activating and inhibitory receptors, including newly discovered receptors that selectively trigger the cytolytic activity in a major histocompatibility complex independent manner. Based on their defining function of spontaneous cytotoxicity without prior immunization, natural killer (NK) cells have been thought to play a critical role in immune surveillance and cancer therapy. New insights into NK cell biology have suggested their major roles in the control of infections, particularly in Plasmodium falciparum infection and in fetal implantation. P. falciparum is the main protozoan parasite responsible for malaria causing 200-300 million clinical cases and killing over 3 million people each year. This review provides an update on NK cell function, ontogeny and biology in order to better understand the role of NK cells in pregnancy in regions where malaria is endemic. Understanding mechanisms of NK cell functions may lead to novel therapeutic strategies for the treatment of human disease, in general, and particularly in the fight against malaria.
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Affiliation(s)
- Elie Mavoungou
- Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon, c/o Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Wilhelmstrasse 27, 72074, Tübingen, Germany.
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Webster RL, Johnson RP. Delineation of multiple subpopulations of natural killer cells in rhesus macaques. Immunology 2005; 115:206-14. [PMID: 15885126 PMCID: PMC1782152 DOI: 10.1111/j.1365-2567.2005.02147.x] [Citation(s) in RCA: 104] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2004] [Revised: 01/26/2005] [Accepted: 01/28/2005] [Indexed: 11/26/2022] Open
Abstract
Natural killer (NK) cells in rhesus macaques have been variably defined as CD3- CD16+ or CD3- CD8+, although only limited efforts have been made to validate these definitions rigorously. To better understand the role of NK cells in macaque disease models, we undertook a multiparameter analysis of macaque NK cells employing four-colour flow cytometry and a panel of lineage-specific and non-lineage-specific lymphocyte markers. Using this approach, we identified two distinct populations of candidate NK cells: a major CD8bright CD16+ population and a minor CD8bright CD16- population. Further analysis of the major and minor NK cell populations revealed the expression of multiple markers characteristic of NK cells, including CD2, CD7, CD16, CD161, NKG2A and granzyme B. In addition, a CD56+ subset of cells within the minor rhesus NK population was identified which expressed chemokine and lymph node homing receptors similar to those expressed by the CD56bright NK cell population identified in humans. Cytolytic assays confirmed that the phenotypically defined rhesus NK cells lysed NK-susceptible target cells. Our observations support the existence of several distinct subpopulations of rhesus macaque NK cells, which have significant phenotypic and functional similarities to their human counterparts. These improved immunophenotypic definitions of macaque NK cells should facilitate future analysis of innate immune responses in rhesus macaques and the role of NK cells in AIDS pathogenesis in Simian immunodeficiency virus (SIV)-infected macaques.
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Affiliation(s)
- Ramothea L Webster
- Division of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, MA 01772, USA
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23
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Li S, Xu J, Makarenkova VP, Tjandrawan T, Vakkila J, Reichert T, Gooding W, Lagenaur CF, Achim CL, Chambers WH, Herberman RB, Whiteside TL, Vujanovic NL. A novel epitope of N-CAM defines precursors of human adherent NK cells. J Leukoc Biol 2004; 76:1187-99. [PMID: 15356097 DOI: 10.1189/jlb.0802386] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56(dim) NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only approximately 26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.
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MESH Headings
- Antibodies, Monoclonal/immunology
- Antigens, Surface/immunology
- Antigens, Surface/metabolism
- CD56 Antigen/immunology
- CD56 Antigen/metabolism
- Cell Adhesion/drug effects
- Cell Adhesion/immunology
- Cell Count
- Cell Differentiation/drug effects
- Cell Differentiation/immunology
- Cell Lineage/drug effects
- Cell Lineage/immunology
- Cell Membrane/immunology
- Cell Membrane/metabolism
- Cell Proliferation/drug effects
- Cells, Cultured
- Chemotaxis, Leukocyte/drug effects
- Chemotaxis, Leukocyte/immunology
- Cytotoxicity, Immunologic/immunology
- Epitopes/immunology
- Humans
- Immunophenotyping
- Integrins/immunology
- Integrins/metabolism
- Interleukin-2/immunology
- Interleukin-2/pharmacology
- Killer Cells, Natural/drug effects
- Killer Cells, Natural/immunology
- Killer Cells, Natural/metabolism
- Lectins, C-Type/immunology
- Lectins, C-Type/metabolism
- Lymphoid Tissue/immunology
- Lymphoid Tissue/metabolism
- Membrane Glycoproteins/immunology
- Membrane Glycoproteins/metabolism
- NK Cell Lectin-Like Receptor Subfamily B
- Receptors, IgG/immunology
- Receptors, IgG/metabolism
- Stem Cells/drug effects
- Stem Cells/immunology
- Stem Cells/metabolism
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Affiliation(s)
- Shen Li
- Department of Pathology, University of Pittsburgh School of Medicine, PA 15213-1863, USA
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24
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Wilkie GM, Taylor C, Jones MM, Burns DM, Turner M, Kilpatrick D, Amlot PL, Crawford DH, Haque T. Establishment and characterization of a bank of cytotoxic T lymphocytes for immunotherapy of epstein-barr virus-associated diseases. J Immunother 2004; 27:309-16. [PMID: 15235392 DOI: 10.1097/00002371-200407000-00007] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Adoptive immunotherapy using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) generated ex vivo can be an effective treatment of EBV-positive posttransplantation lymphoproliferative disease (PTLD). We describe the establishment of a cryopreserved repository of allogeneic virus-specific CTL lines, to our knowledge the first of its kind in the world. CTL lines were grown by weekly stimulation with autologous EBV immortalized lymphoblastoid cell lines (LCLs) from 96 EBV-seropositive blood donors. Analysis of 60 CTL lines grown continuously for 7 to 10 weeks showed an average proportional weekly increase in cell numbers of 1.4, with an overall increase ranging from 1.1 to 83.4. The greatest increase occurred during the early culture period. After four rounds of stimulation, killing of autologous LCLs was generally high (mean 48%); however, most lines required 9 or 10 stimulations to reduce the killing of nonspecific targets. Overall, 79% of CTLs generated showed acceptable levels of specific killing. Phenotypically, the CTL lines consisted of TCRalpha beta+, CD8+ T cells (medians 97% and 90% respectively) with a minority population of CD4+ T cells (median 2%). Most cells expressed the activation and differentiation markers, HLA-DR, CD26, CD45RO, CD69, and CD150. Favorable results have been obtained in an open trial using partially HLA-matched, allogeneic CTLs from this bank to treat PTLD patients. This now represents a single resource that can provide therapeutic CTLs rapidly on a countrywide basis, superseding the time-consuming, expensive practice of generating autologous CTLs from each patient requiring treatment. Additionally, other patient groups, such as those with EBV-positive Hodgkin disease, may benefit from CTL treatment.
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Affiliation(s)
- Gwen M Wilkie
- Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall, Edinburgh, United Kingdom
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25
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Stievano L, Tosello V, Marcato N, Rosato A, Sebelin A, Chieco-Bianchi L, Amadori A. CD8+αβ+T Cells That Lack Surface CD5 Antigen Expression Are a Major Lymphotactin (XCL1) Source in Peripheral Blood Lymphocytes. THE JOURNAL OF IMMUNOLOGY 2003; 171:4528-38. [PMID: 14568926 DOI: 10.4049/jimmunol.171.9.4528] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
To better characterize the cellular source of lymphotactin (XCL1), we compared XCL1 expression in different lymphocyte subsets by real-time PCR. XCL1 was constitutively expressed in both PBMC and CD4(+) cells, but its expression was almost 2 log higher in CD8(+) cells. In vitro activation was associated with a substantial increase in XCL1 expression in both PBMC and CD8(+) cells, but not in CD4(+) lymphocytes. The preferential expression of XCL1 in CD8(+) cells was confirmed by measuring XCL1 production in culture supernatants, and a good correlation was found between figures obtained by real-time PCR and XCL1 contents. XCL1 expression was mostly confined to a CD3(+)CD8(+) subset not expressing CD5, where XCL1 expression equaled that shown by gammadelta(+) T cells. Compared with the CD5(+) counterpart, CD3(+)CD8(+)CD5(-) cells, which did not express CD5 following in vitro activation, showed preferential expression of the alphaalpha form of CD8 and a lower expression of molecules associated with a noncommitted/naive phenotype, such as CD62L. CD3(+)CD8(+)CD5(-) cells also expressed higher levels of the XCL1 receptor; in addition, although not differing from CD3(+)CD8(+)CD5(+) cells in terms of the expression of most alpha- and beta-chemokines, they showed higher expression of CCL3/macrophage inflammatory protein-1alpha. These data show that TCR alphabeta-expressing lymphocytes that lack CD5 expression are a major XCL1 source, and that the contribution to its synthesis by different TCR alphabeta-expressing T cell subsets, namely CD4(+) lymphocytes, is negligible. In addition, they point to the CD3(+)CD8(+)CD5(-) population as a particular T cell subset within the CD8(+) compartment, whose functional properties deserve further attention.
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Affiliation(s)
- Laura Stievano
- Department of Oncology and Surgical Sciences, University of Padova, Padova, Italy
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26
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Konno A, Okada K, Mizuno K, Nishida M, Nagaoki S, Toma T, Uehara T, Ohta K, Kasahara Y, Seki H, Yachie A, Koizumi S. CD8alpha alpha memory effector T cells descend directly from clonally expanded CD8alpha +beta high TCRalpha beta T cells in vivo. Blood 2002; 100:4090-7. [PMID: 12393564 DOI: 10.1182/blood-2002-04-1136] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Whereas most peripheral CD8(+) alphabeta T cells highly express CD8alphabeta heterodimer in healthy individuals, there is an increase of CD8alpha(+)beta(low) or CD8alphaalpha alphabeta T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8alpha(+)beta(high) cells belong to different ontogenic lineages or whether a fraction of CD8alpha(+)beta(high) cells have down-regulated CD8beta chain. Here we assessed clonality of CD8alphaalpha and CD8alpha(+)beta(low) alphabeta T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8alpha(+)beta(low) cells are exclusively composed of effector memory cells. CD8alphaalpha cells comprise effector memory cells and terminally differentiated CD45RO(-)CCR7(-) memory cells. T-cell receptor (TCR) Vbeta complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8alpha(+)beta(high) cells and oligoclonality of CD8alpha(+)beta(low) and CD8alphaalpha cells. Importantly, some expanded clones within CD8alphaalpha cells were also identified within CD8alpha(+)beta(high) and CD8alpha(+)beta(low) subpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8beta expression. These results indicated that some specific CD8alpha(+)beta(high) alphabeta T cells expand clonally, differentiate, and simultaneously down-regulate CD8beta chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8alphaalpha alphabeta T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.
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Affiliation(s)
- Akihiro Konno
- Department of Pediatrics, Angiogenesis and Vascular Development, Graduate School of Medical Science and School of Medicine, Kanazawa University, Japan
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27
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Abstract
NK cells are the important cells of the immune system derived from stem cells in the marrow. Their physiology is tightly regulated to control proliferation, cytotoxicity and cytokine production. In cancer, NK cells may be abnormal due to the cancer itself or possibly related to its therapy. The finding of class I recognizing inhibitory receptors may play a role in stem cell transplant rejection, immune surveillance and cancer immunotherapy. NK cells should no longer be thought of as direct cytotoxic killers alone, as they clearly play a critical role in cytokine production which may be important to control cancer and infection. Understanding NK cell function and homing may lead to novel therapeutic strategies for the treatment of human disease.
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Affiliation(s)
- Jeffrey S Miller
- Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota Cancer Center, Box 806, Harvard Street at East River Road, Minneapolis, MN 55455, USA.
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28
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Lima M, Almeida J, dos Anjos Teixeira M, Queirós ML, Justiça B, Orfão A. The "ex vivo" patterns of CD2/CD7, CD57/CD11c, CD38/CD11b, CD45RA/CD45RO, and CD11a/HLA-DR expression identify acute/early and chronic/late NK-cell activation states. Blood Cells Mol Dis 2002; 28:181-90. [PMID: 12064914 DOI: 10.1006/bcmd.2002.0506] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
To define a dynamic sequence of phenotypic changes related to early and late phases of NK-cell activation, we have analyzed by four-color flow cytometry the immunophenotype of normal blood NK-cells from 12 healthy individuals and compared it with those from 15 patients with acute viral infections and 15 patients with either chronic infections or tumors. Although a great interindividual variability was found, nonstimulated CD56(+) NK-cells, present in normal blood samples, usually were CD2(-/+lo), CD7(+hi), HLA-DR(-), CD11b(+), CD38(+), CD11a(+hi), CD45RA(+hi), and CD45RO(-), the expression of CD11c and CD57 being heterogeneous and variable. Recently activated NK-cells, herein corresponding to NK-cells from patients with acute viral infections, displayed a pattern of expression of CD2/CD7 similar to that referred to above, but they typically showed higher levels of CD11a, CD38, and HLA-DR, as well as downregulation of CD11b and CD45RA, accompanied in some cases by coexpression of CD45RO; in addition, these NK-cells were CD11c(+) and CD57(-/+lo). Late-activated NK-cells, represented by NK-cells present in patients with chronic infections and tumors, converted into a CD2(+hi)/CD7(-/+lo) immunophenotype and expressed heterogeneously low levels of CD38 and CD11b; moreover, they were CD57(+) and CD11c(-/+). At this stage, most NK-cells had already reverted into their original CD45RA(+)/CD45RO(-)/HLA-DR(-) phenotype. In summary, we show that the patterns of expression of CD2/CD7, CD57/CD11c, CD38/CD11b, CD45RA/CD45RO, and CD11a/HLA-DR may help us to define the immunophenotypic profiles associated with early and late NK-cell activation phases in 'in vivo' models.
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Affiliation(s)
- Margarida Lima
- Service of Clinical Hematology, Hospital Geral de Santo António, Porto, Portugal.
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29
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Hope JC, Sopp P, Howard CJ. NK‐like CD8
+
cells in immunologically naïve neonatal calves that respond to dendritic cells infected with
Mycobacterium bovis
BCG. J Leukoc Biol 2002. [DOI: 10.1189/jlb.71.2.184] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Affiliation(s)
- Jayne C. Hope
- Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom
| | - Paul Sopp
- Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom
| | - Chris J. Howard
- Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom
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30
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Abstract
OBJECTIVE NK cells are important cells of the immune system. They are ultimately derived from pluripotent hematopoietic stem cells. NK cell cytotoxicity and other functions are tightly regulated by numerous activating and inhibitory receptors including newly discovered receptors that selectively recognize major histocompatibility complex class I alleles. Based on their defining function of spontaneous cytotoxicity without prior immunization, NK cells have been thought to play a critical role in immune surveillance and cancer therapy. However, new insights into NK cell biology have suggested major roles for NK cells in infection control and uterine function. The purpose of this review is to provide an update on NK cell function, ontogeny, and biology in order to better understand the role of NK cells in health and disease. DATA SOURCES In the Medline database, the major subject heading "Natural Killer Cells" was introduced in 1983, identifying 16,848 citations as of December 31, 2000. Since 1986, there have been approximately 1000 citations per year under this subject heading. In this database, 68% of manuscripts are limited to human NK cells; 40% of citations cross with the major sub-heading of cytotoxicity, 40% with cytokines, 36% with neoplasm, 5% with antibody-dependent cellular cytotoxicity, 2.8% with pregnancy, and 1.3% with infection. Of references from the year 2000-2001, 46 were selected to combine with contributions from earlier literature. CONCLUSIONS NK cells should no longer be thought of as direct cytotoxic killers alone as they clearly serve a critical role in cytokine production which may be important to control cancer, infection, and fetal implantation. Understanding mechanisms of NK cell functions may lead to novel therapeutic strategies for the treatment of human disease.
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Affiliation(s)
- J S Miller
- Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota Cancer Center, Minneapolis, Minn. 55455, USA.
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31
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Schmid-Ott G, Jaeger B, Meyer S, Stephan E, Kapp A, Werfel T. Different expression of cytokine and membrane molecules by circulating lymphocytes on acute mental stress in patients with atopic dermatitis in comparison with healthy controls. J Allergy Clin Immunol 2001; 108:455-62. [PMID: 11544468 DOI: 10.1067/mai.2001.117800] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
BACKGROUND Mental stress is believed to induce an exacerbation of atopic dermatitis (AD). Until now, however, only few psychoneuroendocrinologic mechanisms underlying the link between psychological stress and exacerbation or maintenance of AD have been described. OBJECTIVE Our purpose was to conduct an investigation of immunologic parameters in the form of membrane molecules and cytokines with potential relevance for the cutaneous inflammation in an established psychological laboratory stress model. METHODS Patients with AD (n = 15) and healthy controls (n = 15) were exposed to mental stress, as described in a previous report. In vitro analyses were completed 1 hour before, immediately after, and 1 hour after mental stress exposure. Lymphocyte subpopulations, the cutaneous lymphocyte-associated antigen (CLA), the membrane molecule CD69(+) (early activation antigen), and intracellular IL-4, IL-5, and IFN-gamma in blood-derived lymphocytes were analyzed by flow cytometry. IL-4 in the supernatant of concanavalin-A-stimulated PBMCs was determined by ELISA. RESULTS An increase in heart rate and blood pressure was demonstrated during psychological stress in patients with AD and healthy volunteers. We found significantly higher stress-induced increase of CLA(+) lymphocytes, T helper cells expressing IL-5, and both CD4(+) and CD8(+) lymphocytes expressing IFN-gamma on mitogenic stimulation in patients with AD in comparison with healthy controls. In addition, we observed an earlier increase in the secretion of IL-4 in the supernatant of mitogen-stimulated lymphocytes during psychological stress in patients with AD in comparison with healthy volunteers. CONCLUSION A higher stress-induced increase of CLA(+) cells in the circulation in patients with AD compared to healthy controls might indicate an increased ability of T lymphocytes in AD to migrate to the skin during this psychological condition. In addition, the data of this study suggest a different stress-induced cytokine profile in circulating lymphocytes in patients with AD compared to healthy controls.
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MESH Headings
- Adolescent
- Adult
- Antigens, CD
- Antigens, Differentiation, T-Lymphocyte
- Antigens, Neoplasm
- Antigens, Surface/analysis
- Blood Pressure
- Chronic Disease
- Cytokines/analysis
- Dermatitis, Atopic/complications
- Dermatitis, Atopic/immunology
- Female
- Heart Rate
- Humans
- Interferon-gamma
- Interleukin-4
- Interleukin-5
- Lectins, C-Type
- Male
- Membrane Glycoproteins
- Middle Aged
- Stress, Psychological/complications
- Stress, Psychological/immunology
- T-Lymphocyte Subsets/immunology
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Affiliation(s)
- G Schmid-Ott
- Department of Psychosomatic Medicine, Hannover Medical School, Germany
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32
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Warrington KJ, Takemura S, Goronzy JJ, Weyand CM. CD4+,CD28- T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems. ARTHRITIS AND RHEUMATISM 2001; 44:13-20. [PMID: 11212151 DOI: 10.1002/1529-0131(200101)44:1<13::aid-anr3>3.0.co;2-6] [Citation(s) in RCA: 172] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
OBJECTIVE To determine whether CD4+,CD28- T cells, which are expanded in patients with rheumatoid arthritis (RA), express receptors that typically regulate the function of natural killer (NK) cells. METHODS Expression of the NK cell surface molecules CD158, p70, CD94, CD161, and CD8alpha on T cell subsets was determined by multicolor flow cytometric analysis of peripheral blood mononuclear cells from 36 RA patients. Expression of CD161 on tissue-infiltrating CD4 T cells was determined by 2-color immunohistochemistry analysis of synovial tissue samples. RESULTS Killer cell-inhibitory receptors (KIR) and killer cell-activating receptors (KAR) were exclusively expressed on CD4+,CD28- T cells, with the CD158b molecule being the most frequently detected isoform. A coordinated mechanism inducing KIR/KAR expression was suggested by similarities in the expression of CD158b on CD4 and CD8 T cells. CD4+,CD28- T cells were also positive for CD8-alphaalpha homodimers, another characteristic shared with NK cells. Of the C-type lectin NK cell receptors (NK receptors), CD94 was consistently absent, but CD161 was found on a CD4 T cell population that is significantly expanded in RA patients (P = 0.01). Involvement in disease of NK receptor-expressing CD4 T cells was suggested by the presence of CD4+,CD161+ T cells in follicular microstructures typical of rheumatoid synovitis. CONCLUSION Patients with RA have an expanded and unusual subset of CD4 T cells that infiltrates the tissue lesions and is characterized by a deficiency of CD28, the expression of CD8-alphaalpha homodimers, and the expression of several types of HLA class I-recognizing NK receptors. CD4 T cells bearing NK receptors can bridge functions of the innate and adaptive immune systems, such as responsiveness to specific antigen, rapid release of interferon-gamma, cytotoxicity, independence from classic costimulatory pathways, and integration of multiple activating and inhibitory signals to control effector functions.
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33
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Mattapallil JJ, Reay E, Dandekar S. An early expansion of CD8alphabeta T cells, but depletion of resident CD8alphaalpha T cells, occurs in the intestinal epithelium during primary simian immunodeficiency virus infection. AIDS 2000; 14:637-46. [PMID: 10807186 DOI: 10.1097/00002030-200004140-00002] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
OBJECTIVES To evaluate changes in the phenotypic heterogeneity and function of CD8 T cells in the intestinal epithelium during primary SIV infection. DESIGN Previous studies have shown an increased prevalence of CD8 T cells in the intestinal epithelium in HIV and SIV infections. As intestinal CD8 T cells are a heterogeneous population we evaluated their phenotypic distribution (CD8alphabeta, CD8alphaalpha) and function [interferon (IFN)-gamma production] during primary SIV infection. METHODS The phenotype and functional potential of CD8 intestinal intraepithelial lymphocytes (IEL) prior to and following SIV infection were determined using flow cytometry. RESULTS IEL were found to harbor CD8alphabetaCD3, CD8alphaalphaCD3 and CD8alphaalpha+CD3- T-cell subsets. Most of the CD8CD4 double positive IEL expressed CD8alphaalpha homodimers. In primary SIV infection the frequency of CD8alphabetaCD3 T cells increased dramatically whereas the frequency of CD8alphaalpha T cells declined. A higher frequency of CD8alphabetaKi-67 IEL was observed following SIV infection suggesting that local cell proliferation might have contributed to an increased prevalence of CD8alphabeta IEL. In contrast, a severe depletion of CD8alphaalphaCD4 IEL occurred which contributed to the depletion of CD8alphaalpha IEL. The CD8alphabeta IEL were the major producers of IFN-gamma in the intestinal epithelium and the frequency of IFN-gamma-producing CD8alphabeta IEL was enhanced considerably in primary infection. CONCLUSIONS CD8alphabeta IEL may be important in generating early antiviral responses at the intestinal epithelium. However, alterations in CD8 T-cell subsets and their function may reflect early immunopathogenic events in the intestinal mucosa.
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Affiliation(s)
- J J Mattapallil
- Department of Internal Medicine, School of Medicine, University of California Davis, 95616, USA
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34
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Bosselut R, Kubo S, Guinter T, Kopacz JL, Altman JD, Feigenbaum L, Singer A. Role of CD8beta domains in CD8 coreceptor function: importance for MHC I binding, signaling, and positive selection of CD8+ T cells in the thymus. Immunity 2000; 12:409-18. [PMID: 10795739 DOI: 10.1016/s1074-7613(00)80193-4] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The contribution of the CD8beta subunit to CD8 coreceptor function is poorly understood. We now demonstrate that the CD8beta extracellular domain increases the avidity of CD8 binding to MHC I, and that the intracellular domain of CD8beta enhances association with two intracellular molecules required for TCR signal transduction, Lck and LAT. By assessing CD8+ T cell differentiation in CD8beta-deficient mice reconstituted with various transgenic CD8beta chimeric molecules, we also demonstrate that the intracellular and extracellular domains of CD8beta can contribute independently to CD8+ T cell development, but that both CD8beta domains together are most efficient. Thus, this study identifies the molecular functions of the CD8beta intracellular and extracellular domains and documents their contributions to CD8+ T cell development.
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Affiliation(s)
- R Bosselut
- Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
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35
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Luo DZ, Vermijlen D, Ahishali B, Triantis V, Plakoutsi G, Braet F, Vanderkerken K, Wisse E. On the cell biology of pit cells, the liver-specific NK cells. World J Gastroenterol 2000; 6:1-11. [PMID: 11819514 PMCID: PMC4723571 DOI: 10.3748/wjg.v6.i1.1] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/1999] [Revised: 11/02/1999] [Accepted: 11/15/1999] [Indexed: 02/06/2023] Open
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36
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Berg AL, Johannisson A, Johansson M, Hein A, Berg M, Dörries R. Peripheral and intracerebral T cell immune response in cats naturally infected with Borna disease virus. Vet Immunol Immunopathol 1999; 68:241-53. [PMID: 10438323 DOI: 10.1016/s0165-2427(99)00030-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Borna disease virus (BDV) is a neurotropic agent with capacity to cause encephalomyelitis in a wide range of animal species, including horses and cats. Recent studies also point to a link between BDV and human neuropsychiatric disorders. The pathogenesis of Borna disease (BD) has been proposed to be immune-mediated, mainly through the effects of cytotoxic T cells. We used flow cytometric analysis in order to characterize the peripheral and intracerebral T cell immune response in cats naturally infected with BDV. Our results show the presence of two different CD8+ cell populations (CD8+low and CD8+high) in the blood, spleen and brain of these cats. In the brain, CD8+low cells predominated over CD8+high cells. Since CD8+low cells have been suggested to represent a non-MHC-restricted T cell population, the recruitment of such cells to the brains of BDV-infected cats could possibly be of importance for the clearance of virus from neurones.
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Affiliation(s)
- A L Berg
- Department of Pathology, Swedish University of Agricultural Sciences, Uppsala.
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37
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Kuroda MJ, Schmitz JE, Charini WA, Nickerson CE, Lord CI, Forman MA, Letvin NL. Comparative analysis of cytotoxic T lymphocytes in lymph nodes and peripheral blood of simian immunodeficiency virus-infected rhesus monkeys. J Virol 1999; 73:1573-9. [PMID: 9882363 PMCID: PMC103982 DOI: 10.1128/jvi.73.2.1573-1579.1999] [Citation(s) in RCA: 72] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/1998] [Accepted: 10/31/1998] [Indexed: 11/20/2022] Open
Abstract
Most studies of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) have been confined to the evaluation of these effector cells in the peripheral blood. What has not been clear is the extent to which CTL activity in the blood actually reflects this effector cell function in the lymph nodes, the major sites of HIV-1 replication. To determine the concordance between CTL activity in lymph nodes and peripheral blood lymphocytes (PBL), CTL specific for simian immunodeficiency virus of macaques (SIVmac) have been characterized in lymph nodes of infected, genetically selected rhesus monkeys by using both Gag peptide-specific functional CTL assays and tetrameric peptide-major histocompatibility complex (MHC) class I molecule complex staining techniques. In studies of six chronically SIVmac-infected rhesus monkeys, Gag epitope-specific functional lytic activity and specific tetrameric peptide-MHC class I staining were readily demonstrated in lymph node T lymphocytes. Although the numbers of tetramer-binding cells in some animals differed from those documented in their PBL, the numbers of tetramer-binding cells from these two different compartments were not statistically different. Phenotypic characterization of the tetramer-binding CD8(+) lymph node T lymphocytes of the infected monkeys demonstrated a high level of expression of the activation-associated adhesion molecules CD11a and CD49d, the Fas molecule CD95, and MHC class II-DR. These studies documented a low expression of the naive T-cell marker CD45RA and the adhesion molecule CD62L. This phenotypic profile of the tetramer-binding lymph node CD8(+) T cells was similar to that of tetramer-binding CD8(+) T cells from PBL. These observations suggest that characterization of AIDS virus-specific CTL activity by sampling of cells in the peripheral blood should provide a reasonable estimation of CTL in an individual's secondary lymphoid tissue.
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Affiliation(s)
- M J Kuroda
- Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
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Expression of the CD8αβ-Heterodimer on CD8+ T Lymphocytes in Peripheral Blood Lymphocytes of Human Immunodeficiency Virus− and Human Immunodeficiency Virus+Individuals. Blood 1998. [DOI: 10.1182/blood.v92.1.198.413k13_198_206] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
CD8+ T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8+peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8β-chain expression on CD8+ T lymphocytes and to clarify how its expression on CD8+ T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8αβ-heterodimer, identifies CD8+ T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8α antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8αβ staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8+ T lymphocytes from HIV-1–infected individuals with the lowest CD4 counts showed the lowest levels of CD8αβ MF. To explore further this change in CD8αβ expression, we assessed the expression of 14 different cell surface molecules on CD8αβ+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8αβ staining was significantly reduced on CD8+T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8αβ expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28−. Finally, we monitored the expression of the CD8αβ-heterodimer on PBL of eight HIV-1–infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8αβ-heterodimer. These results suggest that antibodies recognizing the CD8αβ-heterodimer are useful tools to specifically identify CD8+ T lymphocytes. Moreover, the quantitative monitoring of CD8αβ expression allows the detection of discrete CD8+ T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.
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39
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Kuroda MJ, Schmitz JE, Barouch DH, Craiu A, Allen TM, Sette A, Watkins DI, Forman MA, Letvin NL. Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J Exp Med 1998; 187:1373-81. [PMID: 9565630 PMCID: PMC2212269 DOI: 10.1084/jem.187.9.1373] [Citation(s) in RCA: 246] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/1997] [Revised: 02/02/1997] [Indexed: 11/23/2022] Open
Abstract
A tetrameric recombinant major histocompatibility complex (MHC) class I-peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and beta2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C-M) representing the optimal nine-amino acid peptide of Mamu-A*01-restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C-M complex. Tetrameric Mamu-A*01/p11C, C-M complex bound to T cells of SIVmac-infected, Mamu-A*01(+), but not uninfected, Mamu-A*01(+), or infected, Mamu-A*01(-) rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01(+) rhesus monkeys was only found in the cluster of differentiation (CD)8alpha/beta+ T lymphocyte subset and the percentage of CD8alpha/beta+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C-M complex-binding, but not nonbinding, CD8alpha/beta+ T cells. Furthermore, the percentage of CD8alpha/beta+ T cells binding this tetrameric Mamu-A*01/p11C, C-M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.
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Affiliation(s)
- M J Kuroda
- Harvard Medical School, Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA
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40
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Manoussaka MS, Smith RJ, Conlin V, Toomey JA, Brooks CG. Fetal Mouse NK Cell Clones Are Deficient in Ly49 Expression, Share a Common Broad Lytic Specificity, and Undergo Continuous and Extensive Diversification In Vitro. THE JOURNAL OF IMMUNOLOGY 1998. [DOI: 10.4049/jimmunol.160.5.2197] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Abstract
NK cells obtained by exposing mouse fetal thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, fetal NK cells have a similar specificity to Ly49-expressing adult splenic NK cells. Individual fetal NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of fetal or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected. Most remarkably, cloned fetal NK cell lines display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in fetal NK “clones” being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that fetal NK cells behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.
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Affiliation(s)
| | - Rachel J. Smith
- Department of Immunology, The Medical School, Newcastle, United Kingdom
| | - Victoria Conlin
- Department of Immunology, The Medical School, Newcastle, United Kingdom
| | | | - Colin G. Brooks
- Department of Immunology, The Medical School, Newcastle, United Kingdom
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41
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Shimojima M, Pecoraro MR, Maeda K, Tohya Y, Miyazawa T, Mikami T. Characterization of anti-feline CD8 monoclonal antibodies. Vet Immunol Immunopathol 1998; 61:17-23. [PMID: 9613469 DOI: 10.1016/s0165-2427(97)00091-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
We generated three monoclonal antibodies (mAbs) (2D7, 10C7 and 12A3) reactive to the alpha-chain of feline CD8 (fCD8) molecule. Further we showed that reference anti-fCD8 mAbs, FT2, 3.357 and vpg9 recognize the beta-chain, alpha-chain and alphabeta-complex epitope, respectively. Flow cytometric analysis using these mAbs suggested that fCD8alpha(+)beta(-) cells were present in lymphocytes of spleen, but not significantly in those of thymus, lymph nodes and peripheral blood of normal kittens.
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Affiliation(s)
- M Shimojima
- Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
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42
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Tschetter JR, Davis WC, Perryman LE, McGuire TC. CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues. Immunobiology 1998; 198:424-38. [PMID: 9562867 DOI: 10.1016/s0171-2985(98)80050-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.
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MESH Headings
- Animals
- Antibodies, Monoclonal
- CD8 Antigens/immunology
- CD8 Antigens/isolation & purification
- Dimerization
- Female
- Horses
- Lymphoid Tissue/immunology
- Lymphoid Tissue/metabolism
- Mice
- Mice, Inbred BALB C
- Rabbits
- Receptors, Antigen, T-Cell, alpha-beta/chemistry
- Receptors, Antigen, T-Cell, alpha-beta/immunology
- Receptors, Antigen, T-Cell, gamma-delta/chemistry
- Receptors, Antigen, T-Cell, gamma-delta/immunology
- T-Lymphocyte Subsets/immunology
- T-Lymphocyte Subsets/metabolism
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Affiliation(s)
- J R Tschetter
- Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, USA
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43
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Stewart BH, Hoskin DW. Regulatory role of CD8 in major histocompatibility complex-unrestricted tumoricidal activity of mouse T cells activated with anti-CD3 monoclonal antibody. Immunol Invest 1997; 26:601-14. [PMID: 9399103 DOI: 10.3109/08820139709088544] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Antigen-nonspecific CD8+ cytotoxic T cells induced with anti-CD3 monoclonal antibody (mAb) are able to kill tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion. However, the role of CD8 in the MHC-independent tumoricidal activity of anti-CD3-activated killer T (AK-T) cells has not been investigated. Here we show that anti-CD8 alpha mAb inhibits, in a dose-dependent fashion, lysis of P815 and YAC-1 tumor cells by mouse AK-T cells. The inhibition of MHC-unrestricted cytotoxicity by anti-CD8 alpha mAb cannot be attributed to interference with an adhesion-like function of CD8 towards class I MHC molecules on the target cells because anti-CD8 alpha mAb (i) had equal inhibitory effects on the cytolysis of tumor target cells regardless of their relative level of class I MHC molecule expression and (ii) did not interfere with the formation of conjugates between AK-T cells and class I MHC-bearing P815 tumor cells. However, anti-CD8 alpha mAb abrogated AK-T cell granule exocytosis in the presence of P815 tumor cells, indicating a regulatory role for CD8 in the signal transduction events which result in lysis of the tumor target cells. Immunoblot analysis of the post-nuclear fraction of lysates from AK-T cells exposed to P815 tumor cells in the presence of anti-CD8 alpha mAb revealed reduced phosphorylation of tyrosine residues on a protein with an Mr of approximately 62 kDa. Taken together, these data suggest that CD8 is able to affect the tumoricidal activity of MHC-unrestricted AK-T cells independent of class I MHC molecules on the target cell.
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Affiliation(s)
- B H Stewart
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
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44
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Bikoue A, D'Ercole C, George F, Dameche L, Mutin M, Sampol J. Quantitative analysis of leukocyte membrane antigen expression on human fetal and cord blood: normal values and changes during development. CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 1997; 84:56-64. [PMID: 9191884 DOI: 10.1006/clin.1997.4366] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
We studied the antibody binding capacity (ABC) of various cell-surface antigens in normal human fetuses and term neonates on lymphocyte, monocyte, and polymorphonuclear (PMN) cells by quantitative flow cytometry also designated by quantimetry. Analysis of changes of expression level on these leukocytes during the developmental process was also investigated. The results indicated that the ABC values of most studied markers change during the maturational process. The ABC of lymphocyte-associated antigens studied such as CD5 and CD7 showed only a decrease from fetus to adult, whereas according to the type of molecule on monocyte and PMN there was either an increase or a decrease of ABC values dependent on the stage of the developmental process, from fetus to neonate or from neonate to adult. However, the ABC values of leukocyte membrane antigens such as CD16, CD46, and CD55 on all leukocytes and CD11b, CD11c, and CD35 on myeloid cells did not change. Their expression level was already mature in fetuses compared with adult cells. In addition, in this quantimetric approach, the analysis of the results for CD11a and CD8 suggested that the changes of CD11a expression level on lymphocyte subsets can depend on one mechanism, whereas there are probably at least two for CD8. Furthermore, the expression patterns of CD5, CD7, and CD11a change during maturation. We concluded that, even if the neonate response pattern to immunological challenge differs from an adult and this is based primarily on the relative numbers and functional activity of lymphocyte T subsets (especially TH1/TH2) and their cytokine profiles, these quantitative and qualitative phenotypical differences might also contribute to explain the functional peculiarities of leukocyte fetal and cord blood cells. All these findings support the notion of immaturity and maturity of ABC expression.
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Affiliation(s)
- A Bikoue
- Laboratoire d'Hématologie et d'Immunologie, Faculté de Pharmacie, Marseille, France
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45
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Abstract
In the past year, a subset of natural killer cells designated 'A-NK cells' has been characterized. These immune cells appear to be able to enter solid tissues, migrate to sites of metastasis and eliminate malignant tissue cells, but spare normal tissue cells. They appear to be ideal surveillance cells, readily capable of upregulating antitumor functions in response to local activation signals.
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46
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Klein JR, Hamad M. Gamma delta T cells, antigen recognition and intestinal immunity. IMMUNOLOGY TODAY 1995; 16:108-9. [PMID: 7888061 DOI: 10.1016/0167-5699(95)80103-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
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47
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Hunter SD, Peters LE, Wotherspoon JS, Crowe SM. Lymphocyte subset analysis by Boolean algebra: a phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence. CYTOMETRY 1994; 15:258-66. [PMID: 7514523 DOI: 10.1002/cyto.990150311] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated monoclonal antibodies. We describe a rapid method whereby total CD3+ T-cells, CD4+ T-cells (CD3+ CD4+), CD8+ T-cells (CD3+ CD8+), putative gamma delta-receptor-T-cells (CD3+ CD4- CD8-), and T-cells that are CD3+ CD4+ CD8+ as well as B-lymphocytes and NK-cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.
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Affiliation(s)
- S D Hunter
- Flow Cytometry Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Victoria, Australia
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48
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Parrado A, Casares S, Rodríguez-Fernández JM. Natural killer cytotoxicity and lymphocyte subpopulations in patients with acute leukemia. Leuk Res 1994; 18:191-7. [PMID: 7511191 DOI: 10.1016/0145-2126(94)90114-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
We have studied lymphocyte subpopulations and the NK cytotoxicity of the PBMC from acute leukemia patients in complete remission after chemotherapy or ABMT and from normal donors. We have found a positive linear correlation between the percentage of subpopulations with CD3-CD16+, CD3-CD56+ and CD3-CD8+ phenotypes and the percentage of NK cytotoxicity. The slopes of the regression lines in the two groups of patients were lower than in normal donors, indicating a decreased ability of these cells to operate. The percentages of these subpopulations represent parameters for estimating the percentage of NK cytotoxicity both in normal donors and in patients with acute leukemia.
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Affiliation(s)
- A Parrado
- Servicio de Hematología y Hemoterapia, Hospital Universitario Virgen del Rocío, Sevilla, Spain
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49
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Suzuki M, Kikuchi T, Takatsuki F, Hamuro J. Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors, and the role of CD8-positive T cells. Cancer Immunol Immunother 1994; 38:1-8. [PMID: 8299113 PMCID: PMC11038450 DOI: 10.1007/bf01517163] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/1993] [Accepted: 09/11/1993] [Indexed: 01/29/2023]
Abstract
The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a beta 1-3 glucan with beta 1-6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8+ CTL but not by CD4+ T cells or NK1.1+ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.
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MESH Headings
- Animals
- CD8 Antigens/immunology
- Chemotherapy, Adjuvant
- Female
- Flow Cytometry
- Interleukin-2/pharmacology
- Interleukin-2/therapeutic use
- Killer Cells, Lymphokine-Activated/drug effects
- Killer Cells, Lymphokine-Activated/physiology
- Killer Cells, Natural/drug effects
- Killer Cells, Natural/physiology
- Lentinan/pharmacology
- Lentinan/therapeutic use
- Lymphocyte Depletion
- Lymphoma/drug therapy
- Lymphoma/immunology
- Lymphoma/therapy
- Mice
- Mice, Inbred C57BL
- Mice, Inbred DBA
- Mice, Nude
- Moloney murine leukemia virus
- Sarcoma, Experimental/drug therapy
- Sarcoma, Experimental/immunology
- Sarcoma, Experimental/therapy
- Specific Pathogen-Free Organisms
- T-Lymphocytes, Cytotoxic/drug effects
- T-Lymphocytes, Cytotoxic/immunology
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Affiliation(s)
- M Suzuki
- Basic Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan
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50
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Prince HE, Bermudez S, Plaeger-Marshall S. Preparation of CD8bright and CD8dim lymphocyte populations using two positive selection methods in tandem. J Immunol Methods 1993; 165:139-48. [PMID: 8228266 DOI: 10.1016/0022-1759(93)90339-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Two positive selection methods were compared for the ability to capture both the bright and dim subsets of CD8 lymphocytes in mononuclear cell (MC) preparations from ten healthy individuals. The first method utilized anti-CD8-coated magnetic beads; captured cells were then recovered using a polyclonal sheep anti-mouse Fab reagent. At all bead: CD8 cell ratios tested (4:1, 8:1, 16:1), the selected cells were > 94% CD8+, and these CD8 cells were enriched for CD8bright cells (77-85%) when compared to CD8 cells in the starting MC preparation (68%). The second method utilized anti-CD8-coated culture flasks; captured cells were recovered by physical dislodgement. The recovered cells were > 90% CD8+, and these CD8 cells were modestly enriched for CD8dim cells (52%) compared to starting CD8 cells (32%). To further enrich for CD8dim cells, we used these two methods in tandem (n = 10). MC were first incubated with anti-CD8-coated magnetic beads (4:1 ratio) to obtain a CD8bright-enriched population (97% of all cells CD8+, 83% of all cells CD8bright). Uncaptured cells were incubated with anti-CD4-coated magnetic beads, and the uncaptured cells from this step were then placed in an anti-CD8-coated flask. The recovered flask-selected cell population was highly enriched for CD8dim cells (87% of all cells CD8+, 85% of all cells CD8dim). CD8 cells in the CD8bright population were 94% CD3+ and 6% CD16+, whereas those in the CD8dim population were 29% CD3+ and 66% CD16+. In proliferative studies, CD8bright cells were preferentially activated by immobilized anti-CD3, whereas CD8dim cells were preferentially activated by exogenous IL-2. In assays of natural killer activity, CD8dim cells were markedly more active than CD8bright cells. This method provides an alternative to cell sorting for obtaining enriched populations of CD8bright and CD8dim lymphocytes.
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Affiliation(s)
- H E Prince
- Cellular Immunology Laboratory, American Red Cross Blood and Tissue Services, Los Angeles, CA 90006
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