Research Paper
CD107a as a functional marker for the identification of natural killer cell activity

https://doi.org/10.1016/j.jim.2004.08.008Get rights and content

Abstract

Natural killer (NK) cells are a subset of lymphocytes that play a central role in the innate immune response to tumors and infections. An important limitation in the field of NK research is attributable to the deficit of assays available for the detection of the functional activity of NK cells. Recently, lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) has been described as a marker of CD8+ T-cell degranulation following stimulation. Here we describe CD107a as a marker of NK cell functional activity using multi-parameter flow cytometry. CD107a is significantly upregulated on the surface of NK cells following stimulation with MHC devoid targets. Additionally, CD107a expression correlates with both cytokine secretion and NK cell-mediated lysis of target cells. However, as well as being coordinately expressed on nearly all cytokine secreting cells, CD107a was also expressed on a large subset of NK cells that did not secrete cytokine following stimulation. These data suggest that employing CD107a as a marker of NK cell functional activity may allow for the identification of a large fraction of activated NK cells that may degranulate in the absence of cytokine secretion. Cumulatively, the data presented here demonstrate that CD107a is a sensitive marker of NK cell activity.

Introduction

Natural killer (NK) cells are a subset of large granular lymphocytes defined by a lack of T-cell receptor (CD3) and by the surface expression of CD56 (Cooper et al., 2001). This subset of lymphocytes plays an integral role in the control of a number of viral infections and in tumor cell clearance (Yokoyama and Scalzo, 2002). NK cells are an important component of the innate immune response as they are able to lyse tumor cells or virally infected cells without prior antigen sensitization. NK cells are also a critical bridge between the innate and adaptive immune response as they secrete large amounts of cytokines and chemokines that can shape and drive the ensuing adaptive immune response (Moretta et al., 2002). While NK cells are present predominantly in the peripheral circulation, they home to tissues following chemoattractant release by infected or malignant cells (Moretta et al., 2002).

NK cells contain high concentrations of preformed cytolytic granules in their cytoplasm as they circulate in the periphery (Cooper et al., 2001). These lytic vesicles contain a number of cytolytic proteins such as perforin and granzyme uniquely designed to induce death in target cells upon release (Cooper et al., 2001, Burkhardt et al., 1989, Tschopp and Nabholz, 1990). Subsequent to activation, following the integration of complex signals from both activating and inhibitory receptors on the surface of these cells, NK cells rapidly release these granules at the immunological synapse inducing death of the target cell (Cooper et al., 2001, Moretta et al., 2002, Cerwenka and Lanier, 2001). Lining the membrane of these cytolytic granules is the lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) (Winchester, 2001, Peters et al., 1991). This and other LAMP family member proteins are highly glycosylated membrane proteins that represent approximately 50% of the proteins in the lysosomal membrane (Fukuda, 1991, Kannan et al., 1996). Members of the LAMP family have short cytosolic tails, which are thought to interact with trans Golgi mediators that are involved in sorting and targeting proteins to the lysosomal pathway (Winchester, 2001). The highly glycosylated portion of the molecule on the luminal side of the vesicle has also been proposed to be involved in protecting the cellular membrane from attack by the lytic enzymes contained in the granules, and thus may subsequently protect the extracellular membrane of the effector cell following degranulation (Fukuda, 1991). Yet their precise function is still largely unclear. Recently, CD107a expression on the cell surface has been described as a marker of cytotoxic CD8+ T-cell degranulation and was shown to be strongly upregulated on the cell surface following stimulation in concordance with a loss of perforin (Betts et al., 2003).

Given the strong cytotoxic capacity of NK cells, we chose to investigate the potential role of CD107a as a marker of NK cell activation and function. We demonstrate that CD107a is significantly upregulated on the surface of NK cells following stimulation with MHC devoid target cells and following phorbol-12-myristate-13-acetate/ionomycin stimulation. This marker is expressed within 2 h of stimulation, and correlates strongly with both cytokine secretion and NK cell-mediated lysis of target cells.

Section snippets

Study subjects

Peripheral blood mononuclear cells were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation from 12 healthy volunteers. Each subject gave informed consent for participation in the study.

Cell lines

The K562 cell line, a highly undifferentiated human erythroleukemic cell line which does not express MHC class 1 molecules, was provided by the American Type Culture Collection (ATCC; Manassas, VA) and maintained in RPMI 1640 (Sigma, St. Louis, MO) containing 10% FBS (Atlanta Biologicals,

CD107a is expressed at high levels on NK cells following stimulation

Recently, CD107a has been shown to be a marker for cytotoxic CD8+ T-cell activity as its expression was associated with a loss of perforin following antigen stimulation (Betts et al., 2003). Although NK cells are also cytolytic effector cells, the role of this marker has not been addressed for this subset of lymphocytes. Here, we assessed whether CD107a was expressed on the surface of NK cells following stimulation. Freshly isolated PBMCs were incubated with MHC devoid target cells or

Discussion

NK cells are an important component of the innate immune response as well as a bridge to the adaptive arm of the immune response (Cooper et al., 2001, Farag et al., 2003, French and Yokoyama, 2003). An important limitation in the field of NK research is attributable to the deficit of assays available for the detection the functional activity of NK cells. It is to this end that we sought to develop a new method to quantitate NK cell functional activity. Given the recent data demonstrating the

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