IL-15 induces CD8+ T cells to acquire functional NK receptors capable of modulating cytotoxicity and cytokine secretion
Introduction
In the last years, it has been shown that NK receptors could be expressed, not only by NK cells, but also by T cells. In early studies, killer immunoglobulin-like receptors (KIR) were found to be expressed in a small fraction of T cells from normal donors expressing either TCRαβ or TCRγδ, mostly CD8+ T cells (Mingari et al., 1996, Mingari et al., 1995). Together with KIR, CD94/NKG2A was also found to be expressed in CD8+ T cells (Mingari et al. 1995). These receptors were shown to provide T cells with the ability to recognize HLA class I molecules on target cells, leading to a decrease in CTL cytotoxicity (Mingari et al., 1996, Mingari et al., 1995, Speiser et al., 1999). It is now known that besides inhibitory receptors, CD8+ T cells also express activating receptors, (Bauer et al., 1999, Groh et al., 2001) and the notion that NK receptor expression in CD8+ T cells could modulate T cell function, by increasing or decreasing TCR threshold accordingly to the triggering of activating or inhibitory NKRs, is now emerging. Despite this knowledge, the environmental signals that trigger NK receptor acquisition by CD8+ T cells remain largely unknown. In a previous study, we have found that the γ-common cytokine IL-15, known to be crucial to NK and invariant NKT cell development and homeostasis, as well as for the activation, survival and differentiation of both memory and naïve CD8+ T cells (Alves et al., 2003, Fehniger and Caligiuri, 2001, Huntington et al., 2009, Mortier et al., 2009, Pek et al., 2010), was capable of inducing the expression of NKRs in purified CD8+CD56− T cells after a prolonged exposure (Correia et al. 2009). Between those NKRs there were both inhibitory and activation and from different structural families: KIR (KIR2DL2/3, KIR2DL4), C-type lectin-like receptors (NKG2A, NKG2D), the most common NK marker, CD56, and the recently considered the truly bona-fide marker of NK cells, the natural cytotoxicity receptor (NCR) NKp46 (Correia et al. 2009).
Although the term “NKT cell” is mainly used to describe CD1d-restricted NKT cells expressing an invariant TCR chain, it also includes CD1d-unrestricted NKT cells with an oligoclonal TCR, known also as NK-like T cells, which are less studied. NK-like T cells have been described to be mostly CD8+CD56+ T cells and, contrarily to mice, constitute the NKT cell population mostly enriched in human livers (Kenna et al., 2003, Norris et al., 1999). In this context, we have previously hypothesized that those NK-like T cells do not necessarily consist in a different lineage, but could in part rather be CD8+ T cells that under certain conditions as under prolonged exposure to IL-15 in a favorable environment such as the liver, differentiate and acquire NK receptors (Correia et al. 2009). In the present study we have extended these results by characterizing the functional phenotype of IL-15-induced CD8+NKR+ T cells and dissecting the physiological outcome of triggering NKRs on CD8+ NKR+ T cells. To our knowledge, this is the first evidence showing that KIR and NKG2A mAb cross-linking can induce the secretion of a varied pattern of cytokines by CD8+ T cells. The results add further knowledge to the biology of NKR expression by CD8+ T cells and strongly suggest that IL-15 is one environmental factor involved in the generation of regulatory NK-like CD8+ T cells.
Section snippets
Reagents and monoclonal antibodies
RPMI-1640 Glutamax®, fetal bovine serum (FBS) and amphotericin B/penicillin/streptomycin (APS) were from Gibco BRL (Paisley, Scotland). 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and the live/dead® Viability/Cytotoxicity Kit (for mammalian cells) were purchased from Molecular Probes (Amsterdam, The Netherlands). Human serum was obtained from Cambrex (New Jersey, USA). Recombinant human IL-15 was obtained from R&D Systems (Minneapolis, USA). Permeabilization buffer (Foxp3
IL-15 induces de novo formation of CD8+CD56+ T cells co-expressing other NKRs
As we have previously shown (Correia et al. 2009), after 12-days of culture with IL-15, purified CD8+CD56− T cells were capable of up-regulating and/or de novo expressing several NK receptors, including KIR (KIR2DL2/3, KIR2DL4), C-lectin-like receptors (NKG2A, NKG2D), natural cytotoxic receptors, NCR (NKp46), and the most NK common marker, CD56 (Fig. 1). In Fig. 1(A), it is possible to see that acquisition of the different NKRs by CD8+ T cells after 12 days in culture with IL-15 occurs mainly
Discussion
The common γ chain cytokine IL-15 is considered a crucial factor for CD8+ T cell homeostasis and the maintenance and generation of memory CD8+ T cells (Alves et al., 2007, Kim et al., 2008, Mortier et al., 2009). Despite the existence of work documenting the existence of cell surface NKR acquisition by a subset of memory CD8+ T cells, the specific signals that control their expression have, to date, not been clearly defined. In this study, we have shown by using an in vitro model of human CD8+
Conflict of interest
The authors declare no competing financial interests.
Acknowledgments
M.P.C. was supported by a fellowship from FCT (Fundação para a Ciência e a Tecnologia, SFRH/BD/24396/2005). The work was supported by a grant from CESPU (02-GBMC-CICS-09). The authors would like to thank the IPS (Instituto Português do Sangue, Porto) for providing the buffy coats used in this study, and also Ricardo A. Antunes for his support in some experimental procedures.
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