Here we demonstrated that the NK cell receptor CD94 has an essential role in the resistance of B6 mice to mousepox, adding to the still very limited number of NK cell receptors with known function in protecting from viral diseases. The susceptibility of CD94-deficient mice to mousepox was observed in B6 strains carrying the NKC haplotypes from the mousepox-susceptible DBA2/J strain with a spontaneous mutation in Klrd1 or from the resistant 129 strain with a targeted deletion of the Klrg1 gene. This strongly suggests that CD94 cannot be replaced by other NK cell receptors and that is absolutely essential for the resistance of wild type B6 to mousepox.
Consistent with a role for CD94 in the recognition of ECTV infection by NK cells, mice deficient in CD94 succumbed at times similar to those of NK cell-depleted mice (Fang et al., 2008
; Parker et al., 2007
) and, when compared with wild type B6 mice, had significantly increased virus loads in liver 3 dpi, a time point when NK cells, but not T cells, play an important role in controlling virus spread from the D-LN (Fang et al., 2008
). Thus, while CD94-deficient mice had a very diminished CD8+
T cell response, this was the consequence, rather than the primary reason, for the lack of virus control as we previously demonstrated for NK cell-depleted mice (Fang et al., 2008
) and more recently for aged B6 mice (Fang et al., 2010
). It remains possible, however, that CD94 on cells other than NK cells might also have contributed to protection.
Our study also revealed that ECTV-infected cells are specifically recognized by the activating receptor formed by CD94 and NKG2E and that this recognition requires the expression of the non-classical MHC class I molecule Qa-1b
at the surface of ECTV-infected cells. Moreover, at the peak of the NK cell response in the D-LN, the proportion of NK cells expressing CD94 paired to activating NKG2s increased at the expense of those expressing inhibitory CD94-NKG2A receptors. In addition, ECTV infection up-regulated Qa-1b
expression in cells. An important remaining question will be to understand how NK cells switch from the inhibitory CD94-NKG2A to the activating CD94-NKG2E receptor. NK cells do not proliferate in the D-LN 3 dpi (Fang et al., 2008
). Thus, the most likely explanations are a change in the cellular expression of the different NKG2s as a consequence of activation or the preferential recruitment of cells expressing activating NKG2s into the D-LN.
Similar to class Ia MHC molecules, Qa-1b
binds short peptides; however, most Qa-1b
molecules are normally occupied by AMAPRTLLL (Aldrich et al., 1994
), a peptide derived from the signal peptide of class Ia MHC I molecules (Aldrich et al., 1994
; DeCloux et al., 1997
). It appears that recognition of Qa1b
by CD94-NKG2E is insufficient to trigger activation because the NFAT-GFP reporter cells expressing CD94-NKG2E receptors were not stimulated unless the target cells were infected with ECTV. It seems more probable that infection also results in the presentation of novel peptide(s) by Qa-1b
(either a viral peptide or a peptide derived from an infection-induced cellular protein), resulting in Qa-1b
-peptide complexes with higher affinity for CD94-NKG2E.
We previously reported that NKG2D is required for the ability of NK cells to protect from mousepox infection (Fang et al., 2008
). We have also shown that ECTV up-regulates expression of NKG2D ligands on infected cells and that resistance to mousepox infection is diminished by blocking the NKG2D receptor in vivo
(Fang et al., 2008
). Our present findings using reporter assays, as well as in vitro
stimulation of primary NK cells with mAbs, suggest that CD94-NKG2E and NKG2D synergize to achieve optimal NK cell activation to mount a protective response against mousepox, which is consistent with the dual requirement for NKG2D and CD94 for resistance to mousepox. While the molecular mechanism for this synergy remains unexplained, we speculate that stimulation through both receptors is required to overcome the inhibitory effects of NKG2A or other inhibitory receptors or most likely that the presence of both receptors at the immune synapse favor a stronger activating signal through the recruitment of DAP12. There is strong precedent for synergy between NKG2D and other activating receptors such as with KIR in human NK cells (Wu et al., 2000
) or with the T cell receptor in humans (Groh et al., 2001
) and mice (Jamieson et al., 2002
Many of the activating NK receptors in the Ly49 and KIR families, as well as human CD94-NKG2C, have low to undetectable affinity for MHC class I. We speculate that the mouse CD94-NKG2E might have a low affinity for Qa-1b
expressed on normal, healthy cells to avoid autoimmunity, but that peptides derived from ECTV-infected cells might generate higher affinity Qa-1b
ligands. Further, we hypothesize that due to the low affinity of the CD94-NKG2E receptor, productive activation might require synergistic signals derived from the NKG2D receptors. In light of our findings, we propose a model whereby NK cells, probably activated through cytokines, migrate to the D-LN and kill ECTV-infected cells using CD94-NKG2E for specific recognition of targets expressing a specific peptide(s) bound to Qa-1b
, and NKG2D as a co-stimulator (Fang et al., 2008
). This model is consistent with the current view that activating receptors are not essential for the cytokine-induced activation of the NK cells during a viral infection, but are required for the cytolysis of the virus-infected target cells (Lee and Biron, 2010
; Vance et al., 1998
Importantly, different from the recognition of MCMV by Ly49 activating receptors, the CD94-NKG2 system is exquisitely conserved between rodents and primates (Vance et al., 1999
) and the sequences of all OPVs are also highly conserved. This suggests that the recognition of OPVs by NK cells might be conserved between humans and mice and relevant in protection against smallpox and monkeypox infection.