A central theme in biology is the capacity to discriminate between self and nonself. From plants to vertebrates, a variety of distinct discrimination strategies have evolved for different biological processes. This discrimination is especially relevant for lymphocytes, vertebrate immune cells that express receptors capable of recognizing foreign invaders and also normal self-tissues.
Natural killer (NK) cells comprise the third major lymphocyte population. As with other lymphocytes—B cells that produce antibodies, and T cells that perform cell-mediated immunity—NK cells display potent effects, including the capacity to kill other cells and produce proinflammatory cytokines. For all lymphocytes, these effector functions must be regulated to prevent inadvertent destruction of normal tissues. This regulation—tolerance for self—is well understood for B and T cells, but much less is known for NK cells.
NK cells use unique mechanisms to control their reactivity against normal self-tissues. Each individual NK cell simultaneously expresses multiple germline-encoded receptors with differing ligand specificities. Some receptors recognize surface ligands on their targets and activate the effector functions. Stimulation through the activation receptors can be inhibited by other germline-encoded NK cell receptors with specificity for major histocompatibility complex class I (MHC-I) molecules that are ubiquitously expressed on normal cells. The inhibitory receptors provide a molecular explanation for the "missing-self" hypothesis of Klas Kärre (Karolinska Institute), whereby NK cells are proposed to survey tissues for expression of MHC-I that prevents target killing. In the absence of MHC-I, due to a pathogenic event such as viral infection, the NK cell is then able to kill. Thus, the NK cell apparently discriminates between normal and abnormal cells by its inhibitory receptors for MHC-I molecules.
Some predictions of the missing-self hypothesis, however, have not been experimentally observed. For example, NK cells in hosts with genetic absence of MHC-I should be chronically activated but instead are defective in target killing. Another prediction is that all NK cells should express at least one receptor for self–MHC-I, but this appears not to be the case. Finally, MHC molecules are among the most polymorphic molecules known, with multiple genes in each individual and multiple alleles displaying differences between individuals. The inhibitory receptors are similarly highly polymorphic, with different MHC-I specificities. Moreover, the genes for the receptors and their ligands lie on different chromosomes and hence are inherited independently. It is not known how the right NK cell receptors are appropriately paired with the relevant self-MHC molecules. Therefore, how NK cell self-tolerance arises is poorly understood.
Recently, our laboratory provided insight into these dilemmas. The inability of NK cells from MHC-deficient hosts to kill targets could be due to many different reasons, and further dissection with cellular targets was difficult because the universe of NK cell receptors involved in target recognition is still incompletely defined. We thus developed a target cell–free system for stimulation of resting NK cells through antibody cross-linking of the NK1.1 (Nkrp1c) molecule, an activation receptor expressed by all mature and developing CD3– NK cells in C57BL/6 (B6) mice. To assess the response of individual NK cells, we used flow cytometry and intracellular staining for interferon-γ production. This deceptively simple assay was very informative.
When isolated from wild-type B6 mice, large numbers of NK cells were activated, whereas few NK cells were stimulated when derived from mice deficient in β2-microglobulin (β2m), the noncovalently associated light chain that is required for normal MHC-I expression. NK cells from mice lacking classical MHC-I heavy chains (H2Kb, H2Db) were also unresponsive to NK1.1 cross-linking. Subsequent studies extended these results to other activation receptor pathways and indicated that the effects were intrinsic to the NK cell. Thus, NK cell function requires specific interaction with host classical MHC-I molecules; we have termed this process MHC-I–dependent "licensing" to distinguish it from MHC-I–dependent "education" that implies different events occurring during T cell development.
In studies of MHC-congenic, -recombinant congenic, and -transgenic mice, we found that licensed NK cells were correlated with their expression of receptors with self–MHC-I specificity. Paradoxically, these receptors belong to the Ly49 family of NK cell receptors that were first characterized as inhibitory MHC-I–specific receptors in effector responses. Thus, our studies strongly suggested that the inhibitory receptors play a second role in NK cell functions, i.e., the ability to license NK cell functions, ironically a positive effect.
To show definitively that a Ly49 inhibitory receptor confers licensing, we took two complementary approaches. The first used a single-chain trimer (SCT) of the H2Kb molecule that is a single, complete, appropriately folded MHC-I polypeptide developed by our collaborator, Ted Hansen (Washington University in St. Louis). SCT-Kb tetramers bound only one NK cell receptor on primary NK cells, the Ly49C inhibitory receptor. In a transgenic SCT-Kb mouse with concomitant deficiencies in H2Kb, HDb, and β2m, only one MHC-I molecule is expressed, SCT-Kb, and only Ly49C+ NK cells are licensed. In a second approach, we used retroviral transduction to express the Ly49A inhibitory receptor in bone marrow stem cells and reconstituted irradiated hosts. Transduced expression of Ly49A conferred a licensing effect but only in mice bearing the MHC-I ligand for Ly49A. However, Ly49A molecules lacking the cytoplasmic domain or having a point mutation in the Tyr residue of the immunoreceptor tyrosine-based inhibitory motif (ITIM) could not confer licensing. Thus, the inhibitory MHC-I receptors directly signal the licensing effect on NK cells when their ligands are expressed as self–MHC-I molecules.
Licensing thus results in two types of self-tolerant NK cells. Unlicensed NK cells that do not express self–MHC-I–specific receptors are not functionally competent and have no need to be inhibited by MHC-I. Licensed, competent NK cells are subject to MHC-I–dependent effector inhibition by the same self-MHC–specific receptors that confer licensing, allowing the pairing of an inhibitory receptor with its cognate self–MHC-I ligand to provide functional NK cells.
We have further investigated the mechanisms underlying licensing. The self–MHC-I receptor may directly license the NK cell, akin to the function of an activation receptor. Alternatively, it may block the action of another receptor, perhaps an activation receptor that recognizes self but results in an "anergic" NK cell, akin to self-reactive T cell receptors. Inasmuch as self-reactive activation receptors are not well described, we produced transgenic mice to explore this latter possibility. Mice were generated that stably express a viral ligand for an NK cell activation receptor (Ly49H) expressed on about 50 percent of NK cells. In ligand-transgenic mice, we found functional defects in the Ly49H+ NK cell population but not the Ly49H– cells. However, this functional anergy was not reversed by self–MHC-I receptors, providing evidence against the possibility that the inhibitory receptors modulate the anergy induced by activation receptors.
Interestingly, human NK cells appear to undergo a process similar to licensing that may explain the association of human MHC-I receptors with disease outcomes. In our most recent studies in mice, we provide evidence that the licensing process is more plastic than originally appreciated because peripheral unlicensed NK cells can become licensed in an MHC-dependent manner, indicating that licensing may not be exclusively a bone marrow developmental process. These effects may be relevant to clinical use of NK cells in antileukemia adoptive immunotherapy. Finally, other germline-encoded inhibitory receptors with ITIMs are broadly expressed on many leukocytes, suggesting that they may also be involved in a licensing-like process. Therefore, we have described a unique biological strategy for self versus nonself discrimination that may be more broadly relevant in biology and medicine.
Grants from the National Institutes of Health provide partial support for these projects.
As of June 10, 2010