Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization.
01 Jan 2017-The Journal of Allergy and Clinical Immunology (J Allergy Clin Immunol)-Vol. 139, Iss: 1, pp 335-346
TL;DR: A novel subpopulation of human NK cells expressing high levels of PD‐1 is identified and characterized, which have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma.
Abstract: Background Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. Objective We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. Methods We performed multiparametric cytofluorimetric analysis of PD-1 + NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. Results We provide unequivocal evidence that PD-1 is highly expressed (PD-1 bright ) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56 dim but not CD56 bright NK cells and is confined to fully mature NK cells characterized by the NKG2A − KIR + CD57 + phenotype. Proportions of PD-1 bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. Conclusions We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction.
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TL;DR: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community providing the theory and key practical aspects offlow cytometry enabling immunologists to avoid the common errors that often undermine immunological data.
Abstract: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
698 citations
TL;DR: It is revealed that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy, and the importance of this axis in inhibiting NK cell responses in vivo is demonstrated.
Abstract: Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy
491 citations
TL;DR: The authors discuss the variety of NK cell-based therapies that are being developed for the treatment of diverse cancers and identify future avenues for NK cell therapy research.
Abstract: Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.
464 citations
TL;DR: A rapid search in PubMed shows that using "flow cytometry immunology" as a search term yields more than 68 000 articles, the first of which is not about lymphocytes as mentioned in this paper.
Abstract: The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.
454 citations
01 Jan 2017
TL;DR: It is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions, and recent developments have created the sophisticated technology of mass cytometry.
423 citations
References
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TL;DR: Current understanding of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential are discussed and an inhibitory bidirectional interaction between PD-L1 and B7-1 is discovered, revealing new ways the B7:CD28 family regulates T cell activation and tolerance.
Abstract: Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, deliver inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. Immune responses to foreign and self-antigens require specific and balanced responses to clear pathogens and tumors and yet maintain tolerance. Induction and maintenance of T cell tolerance requires PD-1, and its ligand PD-L1 on nonhematopoietic cells can limit effector T cell responses and protect tissues from immune-mediated tissue damage. The PD-1:PD-L pathway also has been usurped by microorganisms and tumors to attenuate antimicrobial or tumor immunity and facilitate chronic infection and tumor survival. The identification of B7-1 as an additional binding partner for PD-L1, together with the discovery of an inhibitory bidirectional interaction between PD-L1 and B7-1, reveals new ways the B7:CD28 family regulates T cell activation and tolerance. In this review, we discuss current understanding of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential.
4,431 citations
TL;DR: Conurrent therapy with nivolumab and ipilimumab had a manageable safety profile and provided clinical activity that appears to be distinct from that in published data on monotherapy, with rapid and deep tumor regression in a substantial proportion of patients.
Abstract: A total of 53 patients received concurrent therapy with nivolumab and ipilimumab, and 33 received sequenced treatment. The objective-response rate (according to modified World Health Organization criteria) for all patients in the concurrent-regimen group was 40%. Evidence of clinical activity (conventional, unconfirmed, or immune-related response or stable disease for ≥24 weeks) was observed in 65% of patients. At the maximum doses that were associated with an acceptable level of adverse events (nivolumab at a dose of 1 mg per kilogram of body weight and ipilimumab at a dose of 3 mg per kilogram), 53% of patients had an objective response, all with tumor reduction of 80% or more. Grade 3 or 4 adverse events related to therapy occurred in 53% of patients in the concurrent-regimen group but were qualitatively similar to previous experience with monotherapy and were generally reversible. Among patients in the sequenced-regimen group, 18% had grade 3 or 4 adverse events related to therapy and the objective-response rate was 20%. CONCLUSIONS Concurrent therapy with nivolumab and ipilimumab had a manageable safety profile and provided clinical activity that appears to be distinct from that in published data on monotherapy, with rapid and deep tumor regression in a substantial proportion of patients. (Funded by Bristol-Myers Squibb and Ono Pharmaceutical; ClinicalTrials.gov number, NCT01024231.)
3,816 citations
TL;DR: In this article, the authors analyzed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8T cells.
Abstract: Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.
3,567 citations
Journal Article•
TL;DR: It is found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the ‘helpless’ CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load.
Abstract: Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analyzed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 9helpless9 CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections. This abstract was published in Cancer Immunity, a Cancer Research Institute journal that ceased publication in 2013 and is now provided online in association with Cancer Immunology Research.
2,983 citations
TL;DR: An activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses is defined.
Abstract: Stress-inducible MICA, a distant homolog of major histocompatibility complex (MHC) class I, functions as an antigen for gammadelta T cells and is frequently expressed in epithelial tumors. A receptor for MICA was detected on most gammadelta T cells, CD8+ alphabeta T cells, and natural killer (NK) cells and was identified as NKG2D. Effector cells from all these subsets could be stimulated by ligation of NKG2D. Engagement of NKG2D activated cytolytic responses of gammadelta T cells and NK cells against transfectants and epithelial tumor cells expressing MICA. These results define an activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses.
2,916 citations