Reduced release and binding of perforin at the immunological synapse underlies the age-related decline in natural killer cell cytotoxicity Jon Hazeldine, Peter Hampson and Janet M. Lord Medawar Centre for Healthy Ageing Research, School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, UK Summary Physiological aging is accompanied by a marked reduction in natu- ral killer (NK) cell cytotoxicity (NKCC) at the single cell level, but the underlying mechanisms are unknown. To address this issue, we isolated NK cells from healthy young (£ 35 years) and old (£ 60 years) subjects and examined the effect of age on events fundamental to the process of NKCC. Simultaneous assessment of NKCC and NK cell–target cell conjugate formation revealed a marked age-associated decline in NK cell killing but comparable conjugate formation, indicating a post-target cell binding defect was responsible for impaired NKCC. Despite a reduction in the proportion of NK cells expressing the activatory receptor NKp46, NK cells from old donors were not hyporesponsive to stimulation, as no age-associated difference was observed in the expression of the early activation marker CD69 following target cell coculture. Furthermore, intracellular levels of the key cytotoxic effector mol- ecules perforin and granzyme B, and the fusion of secretory lyso- somes with the NK cell membrane were also similar between the two groups. However, when we examined the binding of the pore-forming protein perforin to the surface of its target cell, an event that correlated strongly with target cell lysis, we found the percentage of perforin positive target cells was lower following coculture with NK cells from old subjects. Underlying this reduc- tion in binding was an age-associated impairment in perforin secretion, which was associated with defective polarization of lytic granules towards the immunological synapse. We propose that reduced perforin secretion underlies the reduction in NKCC that accompanies physiological aging. Key words: aging; apoptosis; cell death; cellular immunology; human. Introduction Physiological aging is accompanied by marked alterations in immune function, a phenomenon termed immunesenescence. Observed in both the innate and adaptive arms of the immune system, these changes are thought to contribute in part to the increased incidence and the severity of infection reported by older adults (Gavazzi & Krause, 2002). Adaptive immunity has been the primary focus of immunogerontological studies (Aw et al., 2007), and consequently, adaptive immunesenescence was thought until recently to be primarily responsible for the increased sus- ceptibility of older individuals to infection. However, evidence is now accumulating to indicate that the innate arm of the immune system is also subject to considerable age-related modification (Gomez et al., 2008; Panda et al., 2009). Characterized phenotypically as CD3 ) CD56 + , natural killer (NK) cells are a heterogeneous subset of innate lymphocytes that offer front-line protection against virus-infected, stressed and malignant cells. On the basis of the differential surface expression of CD56, NK cells are divided into one of the two major subsets: CD56 DIM or CD56 BRIGHT (Cooper et al., 2001a). CD56 DIM NK cells are primarily responsible for the direct elimina- tion of virus-infected and transformed cells, whilst CD56 BRIGHT cells are the principal source of NK cell–derived immunoregulatory cytokines (e.g. IFNc, TNFa) and chemokines [e.g. macrophage inflammatory protein 1 alpha (MIP-1a), MIP-1b] (Cooper et al., 2001b; Jacobs et al., 2001). Reg- ulating the induction of NK cell cytotoxic activity is an array of surface- expressed germline-encoded activatory and inhibitory receptors (Lanier, 2005). Studies to date that have investigated the effect of age on NK cell function have reported conflicting observations, which arise primarily from interstudy differences in subject inclusion criteria and methodologi- cal approaches. Nevertheless, the general consensus is that aging is accompanied by marked alterations in NK cell numbers, phenotype and function (Solana et al., 1999; Mocchegiani & Malavolta, 2004). Age-re- lated increases in the number and ⁄ or proportions of CD56 DIM NK cells have been frequently documented (Krishnaraj, 1997; Almeida-Oliveira et al., 2011; Lutz et al., 2011), as has a decrease in NK cell cytotoxicity (NKCC) at the single cell level (Facchini et al., 1987; Mariani et al., 1990; Miyaji et al., 1997). For CD56 BRIGHT NK cells, their number and ⁄ or proportions decrease with age (Krishnaraj, 1997; Le Garff-Tavernier et al., 2010; Almeida-Oliveira et al., 2011), as does their capacity to produce cytokines and chemokines upon stimulation (Krishnaraj & Bhooma, 1996; Mariani et al., 2002). Importantly, in a prospective study, Ogata et al. (2001) demonstrated low NKCC to be associated with an increased susceptibility to infection and death because of infection in a cohort of older subjects. Given the clinical significance of these data, it is surprising that no study to date has identified the mechanism(s) behind the decline in NKCC that accompanies physiological aging. For example, whilst age-associated changes in the surface expression of NK activatory and inhibitory recep- tors have been reported (Le Garff-Tavernier et al., 2010; Almeida-Oliveira et al., 2011), results are often inconsistent, and the changes observed are not always accompanied by a concomitant decline in NKCC (Le Garff- Tavernier et al., 2010; Almeida-Oliveira et al., 2011). In addition, although Mariani et al. (1998) observed an age-related delay in NK cell phosphoinositide signalling following target cell recognition, only one study has reported a decrease in NK degranulation with age (Le Garff-Tavernier et al., 2010), which is surprising given the importance of calcium signalling in this process. Thus, in an effort to elucidate the underlying cause(s) of the age-associated reduction in NKCC, we isolated NK cells from healthy young and old donors and examined the effect of age on each of the events fundamental to the process of NKCC. Correspondence Professor Janet M. Lord, School of Immunity and Infection, University of Birming- ham, Birmingham B15 2TT, UK. Tel.: +44 121 371 3234; fax: +44 121 414 3599; e-mail: j.m.lord@bham.ac.uk Accepted for publication 15 May 2012 ª 2012 The Authors Aging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 751 Aging Cell (2012) 11, pp751–759 Doi: 10.1111/j.1474-9726.2012.00839.x Aging Cell