NK cell receptors mediate tolerance to self and control effector function
Natural killer (NK) cells are lymphocytes whose ability to identify and eradicate virally infected and transformed cells fulfill a critical role in the innate immune system. In contrast to T and B lymphocytes that employ rearranged antigen-specific receptors, NK cells express germline-encoded receptors with inhibitory and activating functions. The principal NK cell receptors in humans are the killer cell immunoglobulin (Ig)-like receptors (KIR), whose 14 genes and 2 pseudogenes are members of a polymorphic receptor family within the immunoglobulin superfamily. In mice, the main NK receptors are members of the Ly49 C-type lectin family. In both humans and mice, NK cells also express a lectin-like receptor heterodimer comprised of the CD94 molecule coupled with members of the NKG2 family. Several other receptor types with activating function, such as the natural cytotoxicity receptors and NKGD, are also expressed on the cell surface.
Several inhibitory NK cell receptors recognize MHC class Ia molecules, while CD94/NKG2 recognizes HLA-E, which presents signal peptides from MHC class Ia molecules, thereby achieving recognition of MHC class Ia expression indirectly . In humans, the three inhibitory KIR groups with identified class I ligands include KIR2DL2/2DL3, which recognize HLA-CAsn80 alleles (HLA-C1 group), KIR2DL1 which recognizes HLA-CLys80 alleles (HLA-C2 group), and KIR3DL1 which recognizes HLA alleles with the Bw4 epitope. Constitutively expressed on healthy cells, class I ligands of inhibitory receptors are often downregulated during pathologic states such as viral infection or transformation, rendering infected or transformed cells more noticeable to NK cell surveillance through their lack of engagement of class I-specific receptors [2,3]. Critical to inhibitory receptor signalling is the immunoreceptor tyrosine-based inhibitory motif (ITIM), which resides in the cytoplasmic tails of the inhibitory receptors and associates with the cytoplasmic tyrosine phosphatase SHP-1, but may also recruit other signalling molecules .
Inhibitory human KIR receptors and mouse Ly49 receptors are functionally analogous, despite being structurally distinct, indicating that similar immune pressures resulted in convergent evolution of the two receptor families . In addition to their recognition of MHC class I ligands and ITIM-mediated inhibition, common features to the inhibitory KIR and Ly49 receptor families include constitutive expression on resting NK cells, stochastic expression on overlapping NK subsets  with expression of none to several inhibitory receptors by an individual NK cell , encoding by germline clusters of polymorphic genes [8,9], and close sequence homology to ITIM-negative activating receptors. The activating KIR and Ly49 receptors share strong sequence homology in the extracellular regions to the corresponding inhibitory receptors but have short cytoplasmic tails and associate with DAP12 or other immunoreceptor tyrosine-based activation motif (ITAM)-containing signalling molecules . The ligands for many activating receptors remain unknown, although it is known that the activating KIR2DS1 can recognize the same HLA class I molecules as its inhibitory KIR2DL1 counterpart. Other non-KIR activating receptors, such as NKG2D, recognize MHC class I-like molecules, whose constitutive expression can become upregulated in response to stress or other stimuli, resulting in enhanced NK cell activation .
Inhibitory and activating receptor signals jointly control the NK cell response to a target cell. Detection of missing self-MHC on a target cell leads to a decreased inhibitory signal, allowing domination of the activation signal and triggering cytokine production (IFN-γ, TNFα, and GM-CSF) and/or a cytotoxic response through granule exocytosis or through the Fas-Fas ligand pathway. Less commonly, NK recognition of a highly expressed activating ligand on a target cell can override inhibitory signals and result in NK activation despite normal MHC expression on the target cell.
The ability of the NK cell to identify and kill virally infected and malignant cells while sparing normal cells remained poorly understood until the late 1980s and the introduction of the ‘missing self’ hypothesis , which proposed that downregulation of self-MHC class I molecules during viral infection or malignant transformation triggers NK activation. Recent findings have illuminated how NK cell recognition of self-MHC molecules dictates NK response while avoiding auto-aggression. In the ‘NK cell licensing’ model, an NK cell that has engaged self-MHC via MHC-specific inhibitory receptors is ‘licensed’ to be responsive to stimuli via activation receptors, while NK cells that do not engage self-MHC are termed ‘unlicensed’ [12,13]. By this model, there are two general populations of NK cells, both tolerant to cells expressing self-MHC: NK cells licensed for effector function through self-specific MHC class I inhibitory receptors but which maintain self-tolerance through direct inhibition via these same receptors by self-MHC; and unlicensed NK cells, which lack self-specific MHC class I inhibitory receptors, cannot engage self-MHC and are functionally hyporesponsive.
Studies examining the responses of murine NK cells upon target cell-free antibody cross-linking provided initial evidence for the licensing model . Naïve NK cells stimulated with plate-bound antibodies against the NK cell activation receptor NK1.1 expressed on all NK cells in C57BL/6 (H2b) mice were assessed at the single cell level for activation. Only NK cells expressing an inhibitory Ly49 receptor specific for self-MHC produced IFNγ upon ex vivo plate-bound antibody stimulation . Therefore, Ly49A+ NK cells from MHC congenic or MHC transgenic mice expressing H2Dd, a known ligand for Ly49A, were “licensed” and produced IFNγ upon stimulation. In contrast, in mice lacking MHC ligand for Ly49A, such as mice with the H2b haplotype, Ly49A+ NK cells were unlicensed and produced significantly less IFNγ. Evaluation of NK cytotoxicity provided consistent results. Taken together, these data suggest that engagement of an inhibitory receptor with its cognate MHC class I ligand, expressed as self, endows an NK cell with functional competence.
A similar model of NK licensing also applies to human NK cells. Human CD56dim NK cells lacking inhibitory receptors for self-MHC display a hyporesponsive phenotype, exhibiting reduced cytokine production and cytotoxicity in response to stimulation with MHC class I-negative target cells as well as anti-CD16 cross-linking and antibody-dependent cell-mediated cytotoxicity (ADCC) [14,15]. In contrast, NK cells expressing inhibitory receptors for self-HLA-B and self-HLA-C ligands exhibit robust response to class I-negative target cells. Moreover, a dose effect of inhibitory receptors and their cognate class I ligands exists: NK cells expressing more than one inhibitory receptor for self-MHC demonstrate higher effector function , and inhibitory KIR-expressing NK cells from individuals homozygous for the cognate HLA class I ligands demonstrate more potent effector function . It should be noted here that the ‘disarming’ model of NK education is an equally viable alternative to the NK licensing model and proposes that in the setting of chronic stimulation through activating receptors, the lack of MHC class I inhibition of non-self MHC-specific receptors directs NK cells to an anergic or exhausted state . For the purposes of this review, however, “licensed” is used to describe those NK cells that exhibit an inhibitory receptor for self-MHC class I.
Regardless of which model most accurately reflects the forces shaping the functional repertoire, a hierarchy of effector function clearly exists among the NK repertoire, where NK cells lacking all inhibitory receptors are largely functionally hyporesponsive; cells expressing the inhibitory heterodimer CD94/NKG2A alone display modest functional competence compared to cells with a single inhibitory Ly49 or KIR receptor for self-MHC, which display slightly higher functional competence; and cells with more than one inhibitory for self-MHC display still higher functional potency . In humans, the resting NK repertoire achieves a state of ‘intermediate inhibition’, with cells of lowest and highest functional potency present in lower frequecies compared to cells of intermediate potency [15,18]. Data from mouse models also demonstrate a correlation between various Ly49-MHC combinations and NK functional potencies, leading investigators to describe a repertoire ‘tuned’ for diversity of response [19,20].
Most recently, it has become clear that the significant population of ‘unlicensed’ cells in humans and mice are capable of response under specific circumstances. While responding poorly to stimulation with MHC class I-negative target cells or plate-bound antibody, they can still produce IFNγ when stimulated with PMA + ionomycin with similar frequency to self-MHC-specific NK cells, indicating that they retain the capacity for effector function. In vitro culture with cytokines can circumvent the effects of licensing, inducing unlicensed populations to respond to stimulation [12,21]. Similar effects on NK cells occur in vivo during the inflammatory states of viral infection, where NK cells in mixed wild type: beta(2)-microglobulin-deficient [β2m(−/−)] chimeric mice are initially tolerant of MHC class I-deficient host cells but readily reject host β2m(−/−) bone marrow after infection with murine CMV . Engagement of specific activating receptors can also circumvent the licensing effects: in mice, unlicensed NK cells expressing the Ly49H receptor but lacking self-specific inhibitory receptors are selectively capable of clearing CMV-infected cells . In humans, NK cells expressing the activating KIR2DS1 are capable of cytokine response and cytotoxicity to target cells expressing the specific HLA-C2 ligand, if the same NK cell lacks a self-MHC-specific inhibitory receptor KIR2DL1 [24,25]. Taken together, these studies provide evidence that NK effector function need not necessarily require engagement of self-MHC by self-MHC-specific inhibitory receptors on the NK cell.