The innate immune system is evolutionarily older than the adaptive immune system. Although NK cells are considered members of the innate immune system, they bear many striking phenotypic and functional similarities to T cells (21
). NK cells might constitute a lineage of cells that provides an evolutionary link between the innate immune system and the antigen-specific receptor-bearing cells of adaptive immunity, possessing certain attributes of both. If NK cells are indeed a bridge between innate and adaptive immunity, then hallmarks of the adaptive immune system may have evolved first in this cell type. In fact, the paradigm is beginning to shift, given recent data documenting selective pressures on developing NK cells (2
), clonal expansion of NK cell subsets (24
), and possibly even immune memory in NK cells (27
Although T cell development is relatively well understood, little is known about how NK cells are regulated during their development (21
). Recently, two groups have proposed comparable, yet distinct, models for mechanisms that regulate the development of NK cells bearing inhibitory receptors for self–MHC class I (2
). Although the precise mechanism for self-tolerance is yet to be determined, it is clear that immature NK cells undergo selective events in the bone marrow that determine their fate in the periphery. The question of whether developing NK cells bearing activating receptors experience similar selective pressures was unaddressed. Therefore, we sought to determine whether self-tolerance occurs in developing NK cells bearing ITAM-signaling activating receptors in a manner similar to developing T or B cells.
For comparison with previous studies using TCR or BCR transgenic mice exposed to high affinity antigens during development, we established a model system whereby the ITAM-signaling Ly49H receptor expressed on NK cells would encounter the foreign m157 glycoprotein during their development in the bone marrow. Using this model, we demonstrate that when developing Ly49H+ NK cells encounter their cognate viral ligand, certain regulatory mechanisms render the cells in the periphery hyporesponsive. Rather than a complete deletion, as seen in thymocytes possessing TCRs that undergo high affinity interactions with self-peptide–MHC complexes (), Ly49H+ NK cells that interact with m157 survive but populate the periphery in lower numbers and express diminished amounts of the Ly49H receptor on the cell surface. Furthermore, these Ly49H-bearing NK cells from mice that express m157 were defective in their ability produce effector cytokines and mediate killing via Ly49H-specific stimulation and to specifically expand after MCMV infection. Perhaps the term “disarming” is best applied to these activating receptor–bearing NK cells that are actively rendered hyporesponsive by bone marrow–derived cells expressing cognate ligand for Ly49H (). Just as NK cells lacking inhibitory receptors for self–MHC class I can exit the bone marrow and populate the periphery but are refractory to stimulation through their activating receptors, disarmed NK cells bearing activating receptors that have encountered their cognate ligand during development undergo a similar fate (). Interestingly, there is a small population of Ly49H+ NK cells that eludes tolerance in our system, and they can produce cytokines () and undergo proliferative expansion () via specific Ly49H receptor ligation. Further studies are required to determine the mechanism of this escape and establish whether or not these NK cells behave similarly to self-reactive T cells that have evaded central tolerance mechanisms and are either rendered anergic or mediate autoimmunity in the periphery.
Figure 9. Regulatory events during T and NK cell development. (A) Possible events and outcomes during T cell development in the thymus. Thymocytes unable to interact with peptide-MHC do not survive and undergo death by neglect. Thymocytes interacting with peptide-MHC (more ...)
We have used m157 as a model system to evaluate the consequences of early exposure of NK cells during their development to a high affinity non–self-ligand for an ITAM-bearing receptor. Although MCMV persists in the host after resolution of the primary infection, MCMV is preferentially localized in the salivary gland. Therefore, it is unlikely that NK cells, which develop predominantly in the bone marrow, would encounter cells expressing m157 during their early development. Our present studies do not address the consequences of prolonged exposure of mature NK cells, as compared with developing NK cells, to m157. In adult mice infected with MCMV, there is expansion of mature Ly49H+
NK cells, followed by a contraction phase in which most of these NK cells presumably die by apoptosis (24
). Whether the responsiveness of the remaining MCMV-experienced NK cells is affected has not yet been addressed.
Concurrent with our studies using retroviruses to express m157 in mice, Tripathy et al. generated m157-expressing transgenic mice (see Tripathy et al. [28
] on p. 1829
of this issue). In accordance with our findings, these investigators also observed a diminished number and functional impairment of Ly49H+
NK cells in these m157-transgenic mice. In our adoptive transfer studies, we demonstrated a profound defect in the proliferation of Ly49H+
NK cells from m157-expressing mice after recipient mice were infected with MCMV. This severe impairment in the expansion of these m157-exposed Ly49H+
NK cells would likely result in inefficient clearance of MCMV in these hosts because the Ly49H+
NK cell subset is known to be responsible for the NK cell–mediated protection of B6 mice (9
). Indeed, Tripathy et al. (28
) have observed that m157-transgenic mice are more susceptible to MCMV infection than wild-type mice, with the lack of resistance attributed in part to the diminished cytotoxic function of Ly49H+
NK cells in the transgenic mice. Although there were some differences observed in these two experimental models (e.g., apparently a more profound impairment of non-Ly49H receptors in the m157-transgenic mice), this might be accounted for by the amount of m157 expressed by the strong β-actin promoter in the transgenic mice compared with the lower amounts of m157 produced by the retroviral vector. Nonetheless, the major conclusions from both of these complementary studies are in agreement.
Previous studies have examined the consequences of transgenic expression of ligands for certain other NK cell receptors. In transgenic B6 mice constitutively expressing Rae-1, NKG2D (a receptor for Rae-1) was down-regulated on developing and mature NK cells (30
). These transgenic mice were unable to reject Rae-1–bearing tumors that are rejected by syngeneic, nontransgenic mice and were unable to reject allogeneic bone marrow grafts expressing Rae-1. Our current study fundamentally differs from these experiments involving NKG2D ligand expression in several ways. Rae-1 is a self-protein, whereas m157 is foreign. Moreover, although Rae-1 is not expressed in most healthy adult tissues, we have detected constitutive expression of Rae-1 in the liver, a site where NK cells are known to traffic (32
). In addition, the signaling pathways used by Ly49H and NKG2D are distinct.
Previous studies have also investigated the DAP12-associated Ly49D receptor in mice bearing H-2Dd
, a ligand of Ly49D. In contrast to our findings with m157 and Ly49H, Ly49D+
NK cells developing in a host expressing H-2Dd
were not diminished in frequency or number (33
). Moreover, in mixed bone marrow chimeras in which B6 Ly49D+
NK cells were exposed to H-2Dd
cells, there was no down-modulation of the Ly49D receptor on these cells (34
). Studies of the functional responses of these Ly49D+
NK cells developing in H-2Dd
–bearing hosts are complicated by the fact that a majority of Ly49D+
NK cells coexpress inhibitory Ly49 receptors, such as Ly49A, Ly49C, Ly49I, and Ly49G2, that also recognize H-2Dd
as a ligand. Moreover, although functional studies indicate that H-2Dd
is a ligand for Ly49D (35
), there has as yet been no evidence that Ly49D can directly bind to H-2Dd
). Indeed, we have failed to observe activation of NFAT-GFP reporter cells expressing Ly49D and DAP12 co-cultured with H-2Dd
–bearing cells (unpublished data). These results suggest that Ly49D is likely a very low affinity receptor for H-2Dd
, much less than the affinity of the inhibitory Ly49 receptors binding to H-2Dd
. In contrast, there is no evidence that Ly49H binds to any known H-2 ligand, but it does bind with high affinity to m157 (12
). Therefore, in establishing a model system to explore the consequences of engaging an ITAM-signaling Ly49 receptor with a high affinity ligand on NK cell development, Ly49H and m157 provide a much simpler and better-defined receptor–ligand pair to address these questions.
As we are beginning to uncover the events that dictate survival and function in NK cell development, further studies are required to elucidate the precise molecular mechanisms and signals by which NK cells mature and become effector cells in peripheral organs. Understanding the regulatory mechanisms at work during NK cell development that safeguard against peripheral autoimmunity will have important clinical implications.