Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8+ T Cells in HIV Infection

et al. (2012) Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8+ T Cells in HIV Infection. PLoS ONE 7(7): e40146. doi:10.1371/journal.pone.0040146 Tim-3 Negatively Regulates Cytotoxicity in Exhausted + CD8 T Cells in HIV Infection Ali Sakhdari 0 Shariq Mujib 0 Bahareh Vali 0 Feng Yun Yue 0 Sonya MacParland 0 Kiera Clayton 0 Richard Bradley Jones 0 Jun Liu 0 Erika Yue Lee 0 Erika Benko 0 Colin Kovacs 0 Jennifer Gommerman 0 Rupert Kaul 0 Mario A. Ostrowski 0 Mathias Lichterfeld, Massachusetts General Hospital, United States of America 0 1 Department of Immunology, University of Toronto , Toronto, Ontario , Canada , 2 Institute of Medical Science, University of Toronto , Toronto, Ontario , Canada , 3 Maple Leaf Medical Clinic , Toronto , Canada , 4 Li Ka Shing Knowledge Institute of St. Michael's Hospital , Toronto , Canada Cytotoxic CD8+ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8+ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill autologous HIV infected CD4+ target cells. Surprisingly, Tim-3+ CD8+ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and d) their ability to suppress HIV infection of CD4+ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8+ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8+ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion. - Funding: The funding source was CIHR (Canadian Institutes of Health Research) who provided funds for the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The inability of T cell-mediated immune responses to control persistent viral infections, like human immunodeficiency virus-1 (HIV), has been correlated with the impairment in the ability of virus-specific T cells to produce cytokines, to proliferate and to survive [1,2]. This dysfunction, referred to as T cell exhaustion in the setting of HIV infection, allows for continuing viral replication in most of the infected individuals and the inexorable progression to AIDS [1,3,4,5,6,7,8,9,10,11,12,13]. T cell exhaustion was first described in the lymphocytic choriomeningitis virus (LCMV)infected mice, in which certain LCMV strains induced virusspecific effector CD8+ T cells that failed to produce effector cytokines upon antigen stimulation [14]. We previously identified a novel population of exhausted T cells in HIV infected individuals, which are marked by increased surface expression of the glycoprotein Tim-3. These cells, in contrast to programmed cell death -1 (PD-1) expressing cells, are relatively more deficient in effector cytokine production [15]. Tim-3 expression was shown to be upregulated on HIV specific CD8+ T cells [15]. More notably, blocking the Tim-3 signaling pathway in vitro restored proliferation and enhanced cytokine production in HIV-specific T cells [15]. It has recently been shown that Tim-3 expression is dependent on the CD4+ Th1 and CD8+ Tc1 transcription factor T-bet [16]. This transcription factor is also required for proper perforin production and function in cytotoxic lymphocytes [16,17,18,19]. Cytotoxic CD8+ T lymphocytes (CTLs) kill their virally infected or transformed target cells predominantly through the release of lytic substances, mainly perforin and granzymes, which are secreted via exocytosis of pre-formed granules [20,21,22,23]. There is little question regarding the crucial importance of perforin in the control of infectious pathogens. Indeed, mutation or dysregulation of perforin in humans results in compromised cellular immunity and enhanced susceptibility to viral infections [24]. Granule-mediated killing by CD8+ T cells occurs within minutes of target cell recognition [25,26,27]. Recently, another mechanism for perforin replenishment has been identified which is the rapid upregulation and targeted release of newly-produced perforin, which traffics to the immunological synapse via a route that largely bypasses cytotoxic granules [28]. This de novo synthesis of perforin by CD8+ T cells can be easily detected by flow cytometry in conjunction with standard intracellular cytokinestaining (ICS) [29]. While many cell surface markers, activation profiles, and functional parameters of both ex vivo HIV-specific CD8+ and CD4+ T cells have been shown to correlate with control of viremia [8,30,31,32,33] few, if any, can potentially mediate direct control of HIV replication through the lysis of infected cells [34]. Our lab has shown that Tim-3 expressing CD8+ T cells are dysfunctional in terms of polyfunctionality, proliferative ability, cytokine release and inhibitory receptor expression [15]. Here we examined the ex vivo cytotoxicity of Tim-3 expressing CD8+ T cells by examining their perforin content, ability to degranulate [35,36] and also through direct measurement of cytotoxicity [37]. Materials and Methods Ethics Statement Informed consent was obtained in accordance with the guidelines for conduction of clinical research at the University of Toronto and Maple Leaf Clinic institutional ethics boards. Written Informed Consent was provided for this study, which was reviewed by research ethics board of the University of Toronto, Canada and of St. Michaels Hospital, Toronto, Canada. Patient Groups Our cohort consists of two different patient groups including: 1) Chronic progressive HIV infection (CP) (HIV infection .1 year, with active viral replication, i.e., detectable viremia .10,000 bDNA copies (...truncated)