In this study, we provide evidence that the engagement of the NK cell–specific Ly49H activation receptor with its ligand, which is caused by transgenic expression of m157, results in down-regulation of Ly49H receptor expression and “hyporesponsiveness” in Ly49H+
NK cell function. Similar results were observed by Sun and Lanier using retroviral-transduced expression of m157 in BM stem cells (see Sun and Lanier [24
] on p. 1819
of this issue). Any differences between our results and theirs could be related to the means by which m157 was expressed. In the studies described here, the defect in Ly49H function was reversible and independent of the level of Ly49H expressed and the percentage of Ly49H+
NK cells in the m157-Tg mice.
The hyporesponsiveness in Ly49H+
NK cell function was of two types, with one specifically involving only the Ly49H pathway. The second extended beyond stimulation through the Ly49H receptor because we also observed global defects in signaling by other activation receptors that do not signal through Ly49H and DAP12, and use other signaling chains, as well as responses to cytokines, such as IL-12 and -18. Moreover, stimuli that bypass proximal activation signals could equally stimulate normal and hyporesponsive cells, suggesting that the signaling defect is distal to the signaling chains, but upstream of signals mimicked by PMA and ionomycin. It is possible that continuous engagement of Ly49H with m157 results in negative feedback that leads to the down-regulation of not only Ly49H but also downstream signaling molecules (e.g., protein tyrosine kinases) that are shared by multiple activation pathways. Alternatively, continuous engagement of Ly49H with m157 could result in the sequestration of downstream signaling molecules that are shared by multiple activation receptor pathways. In this manner, NK cells could be rendered hyporesponsive to multiple activation pathways. Clearly, future studies will need to be performed to identify the mechanisms of global hyporesponsiveness. Regardless of the mechanism, these data, taken together with data from Sun and Lanier and others (24
), lend strong support to the hypothesis that an activation receptor with specificity for a self-ligand can confer a generalized state of hyporesponsiveness to the NK cell, as previously hypothesized (15
Interestingly, our data suggest that hyporesponsiveness can occur when mature Ly49H+ NK cells are exposed to m157. Even when they developed in a ligandless environment, WT Ly49H+ NK cells became hyporesponsive when transferred into the m157-Tg mice. We examined NK cells 9 d after transfer when the cells had down-regulated Ly49H expression, but were no longer making IFN-γ at baseline, suggesting that they specifically became hyporesponsive by engagement of Ly49H, and that maturation in the m157-Tg environment was not necessary to demonstrate the hyporesponsive phenotype. This surprising result requires additional examination because it also suggests that WT NK cells may show a state of hyporesponsiveness after activation receptor triggering during a normal innate immune response.
In the m157-Tg NK cells, the global functional hyporesponsiveness had in vivo consequences because MCMV control and m157-Tg BM rejection were affected. Although it is possible that responses of m157-Tg mice after MCMV infection are partially a result of the decreased Ly49H expression on NK cells, hyporesponsiveness of the Ly49H+ NK cells likely also plays a major role. Our in vitro data corroborate our in vivo findings, indicating an NK cell functional impairment, as we demonstrate a defect in the Ly49H+ NK cells to produce IFN-γ in response to Ly49H-mediated as well as non-Ly49H–mediated stimuli. Furthermore, LAK cells from m157-Tg mice were defective in killing m157-expressing targets, even when normalized for the number of Ly49H+ NK cells.
Of note, the global effects of Ly49H engagement could be overcome by cytokines in certain situations (high levels of IL-2 in vitro), but not in others, such as poly I:C administration. The inability to reverse the global hyporesponsiveness of Ly49H+
NK cells with poly I:C is relevant to MCMV infections because poly I:C induces Toll-like receptor stimulation of dendritic cells, resulting in type I IFN release and concomitant NK cell stimulation (28
). A similar pathway is activated in MCMV infections and is required for NK cell control of MCMV, even when Ly49H is present (29
). Thus, hyporesponsiveness through an activation receptor may not be overcome by certain proinflammatory cytokines, accounting for the persistent defect in MCMV clearance in the m157-Tg mice despite host production of cytokines during infection.
Functional hyporesponsiveness appeared to require continued receptor–ligand interaction. Persistent in vivo interaction in trans was sufficient for global Ly49H+
NK cell hyporesponsiveness, as revealed by our BM chimeric mouse experiments and adoptive transfer of mature WT NK cells. Interestingly, hyporesponsiveness caused by trans effects was reversed during in vitro culture with IL-2. For example, if the NK cells did not express m157, such as NK cells from WT→m157-Tg BM chimeric mice, then in vitro culture in IL-2 reversed hyporesponsiveness. On the other hand, selective hyporesponsiveness of the Ly49H pathway persisted even in the presence of high doses of IL-2 if we used m157-Tg NK cells in which receptor–ligand interactions likely continued via cis effects or trans interactions with neighboring cells. The putative cis effects may be another example by which such interactions could affect NK cell receptor function as recently described (17
) and reviewed (32
). In either case, these persistent effects on Ly49H function did not appear to be caused by lowered levels of Ly49H expression because nearly equal levels of Ly49H were seen when WT and m157-Tg NK cells were cultured in vitro. Collectively, these data indicate that the NK cell hyporesponsive state can be manifested by the presence of the ligand in trans and potentially in cis, and may be affected by the cytokine milieu, but persistent interactions between activation receptor and its ligand may result in permanent hyporesponsiveness of the given activation receptor pathway.
There are some similarities, but also notable differences between our current study and previous investigations of mice with transgenic expression of ligands for another NK cell activation receptor, NKG2D (26
). In transgenic mice constitutively expressing Rae-1ε or MICA, down-regulation of NKG2D and generalized defects in NK cell function were observed. Moreover, trans effects on NKG2D were noted through examination of mice with tissue-specific expression of Rae-1ε (27
). These results are similar to the Ly49H down-regulation and the defects in the Ly49H+
NK cells in m157-Tg mice that were observed both by us and by Sun and Lanier (24
). However, in the Rae-1ε and MICA-Tg models, all NK cells were altered with potential contributions from soluble NKG2D ligands (26
). In contrast, in the m157-Tg mice, only the Ly49H+
subset of NK cells was altered, indicating a specific effect caused by receptor engagement. In addition, there was no evidence for soluble m157 (unpublished data). Furthermore, it is possible that other immune cells expressing NKG2D may have contributed to the NKG2D functional effects noted in vivo in the Rae-1ε or MICA-Tg mice. For example, there was increased susceptibility to squamous cell cancers in the Rae-1ε-Tg mice and increased susceptibility of MICA-Tg mice to MICA-expressing tumors, but tumor resistance in these models can also be mediated by αβ T cells and γδ T cells that express NKG2D (26
). Similarly, there was increased susceptibility of the MICA-Tg mice to Listeria
infection, but dysfunction of other cell types, such as NKG2D-expressing CD8+
T cells, may contribute to the phenotype observed (26
NKG2D and its ligands have additional layers of complexity. Mouse NKG2D is expressed on all NK cells in two different isoforms, (33
) and two different signaling molecules (DAP12 and DAP10) (36
). Also, mouse NKG2D has multiple endogenous ligands, including those that are constitutively expressed as well as being up-regulated on “stressed” cells (37
). In addition, NKG2D ligands can be expressed as soluble forms that modulate NKG2D function (42
). Finally, NKG2D itself is expressed on non-NK cell populations (40
). Thus, although prior studies of NKG2D ligand-Tg mice demonstrate NKG2D-induced hyporesponsiveness, our current studies demonstrate that the in vivo functional effects of persistent activation receptor interactions, specifically on NK cell functions (because Ly49H is expressed only on NK cells), has no known endogenous ligands, and that m157 does not display any detectable binding to other cells in C57BL/6 mice.
Previous work has also demonstrated that Ly49D+
NK cells appear to be defective in mice expressing H2Dd
), a putative ligand for Ly49D (43
). However, physical interaction between Ly49D and H2Dd
has been difficult to detect (45
), suggesting that H2Dd
may not be a Ly49D ligand or that other, as yet undefined, parameters affect H2Dd
binding to Ly49D. In addition, H2Dd
is recognized by other Ly49 inhibitory receptors (46
), suggesting that its effect on Ly49D+
NK cells could be caused by other receptor–ligand interactions. On the other hand, the Ly49H-m157 interaction exploited for the studies reported here does not appear to be subject to these concerns.
Our studies also provide additional insight into the issue of NK cell tolerance with respect to MHC class I because we provide a test of the “disarming” model. Herein, we demonstrated that licensing (engagement of inhibitory Ly49 receptor with self-MHC class I molecules) could not overcome the hyporesponsiveness caused by engagement of the activation receptor Ly49H with its ligand (m157) expressed as self. However, it is theoretically possible that licensing could overcome the activation receptor-induced hyporesponsive state if the interactions between the activation receptor and its ligand, or between the relevant inhibitory receptor and MHC class I, were either decreased or increased, respectively. For example, affinity differences could modulate the resultant functional competence of the NK cell, although recent biophysical studies indicate that the affinity of Ly49H for m157 approximates that of Ly49 receptors for MHC class I ligands (Kd
= ~1 μM) (47
). Similarly, it is possible that the expression levels of the relevant receptors and ligands could affect avidity, or the simultaneous participation of several different receptors on an individual NK cell may be relevant. However, the current data suggest that hyporesponsiveness induced by a self-specific activation receptor may be difficult to overcome by licensing through a self-MHC class I–specific inhibitory receptor, i.e., the data currently do not support the disarming model.
Nevertheless, the interactions of activation receptors with their self-specific ligands do result in NK cell tolerance. Perhaps this represents another mechanism for self-tolerance, distinct from licensing by self-MHC via the “arming” model. NK cell tolerance would therefore be achieved by multiple mechanisms, somewhat analogous to central versus peripheral tolerance for T cells (48
), for example. Clearly, further studies are warranted because NK cell tolerance for self appears to be more complex than originally conceptualized (16