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Summary: Wayne Yokoyama studies natural killer (NK) cells, which are important components of the body's immune system, particularly in innate host defense against viruses and tumors. These cells possess potent effector functions, such as cellular killing and production of proinflammatory cytokines, that must be restrained to avoid inadvertent destruction of normal tissues; i.e., they must manifest tolerance for self. Recent studies indicate that this tolerance is achieved by a "licensing" process in which NK cell receptors recognize self–major histocompatibility complex class I molecules and acquire functional competence.

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 are responsible for 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 about NK cell control.

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 expressed on target 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 that are ubiquitously expressed on normal cells.

Some predictions of the missing-self hypothesis, however, have not been experimentally observed. For example, the genetic absence of host MHC-I should lead to chronic activation of NK cells and tissue destruction due to absence of inhibition. Instead, NK cells from MHC-deficient hosts 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. The dual polymorphisms of receptors and ligands raise the difficult question of how the right NK cell receptors can be appropriately paired with the relevant self-MHC molecules because the genes for the inhibitory receptors and their MHC ligands lie on different chromosomes and hence they are inherited independently. Therefore, how NK cell self-tolerance arises is poorly understood.

Recently, our laboratory provided new insight into these dilemmas. Although the previously observed inability of NK cells from MHC-deficient hosts to kill targets could be due to many different reasons, 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 (K^b–/– D^b–/–) 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 functional maturation 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 are 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 response, 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 H2K^b MHC-I molecule that is a single, complete, appropriately folded MHC-I polypeptide developed by our collaborator, Ted Hansen (Washington University in St. Louis). SCT-K^b tetramers bound only one NK cell receptor on primary NK cells, the Ly49C inhibitory receptor. In a transgenic SCT-K^b mouse with concomitant deficiencies in H2K^b, HD^b, and β2m, only one MHC-I molecule is expressed, SCT-K^b, 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 enhanced functional capacity of NK cells but only in mice bearing the MHC-I ligand for Ly49A. Thus, the inhibitory MHC-I receptors confer the licensing effect on NK cells when their ligands are expressed as self–MHC-I molecules.

To determine if the inhibitory receptors confer licensing directly or indirectly, we transduced Ly49A constructs with cytoplasmic mutations. Cytoplasmic domain–deficient Ly49A molecules did not confer licensing. The only known cytoplasmic signaling motif is the immunoreceptor tyrosine-based inhibitory motif (ITIM), responsible for inhibitory effects in effector responses. A point mutation in the Tyr residue of the ITIM abrogated the licensing effect. Thus, the receptor itself directly delivers the licensing effect to NK cells. (These studies were also supported by grants from the National Institutes of Health.)

Licensing thus results in two types of self-tolerant NK cells, unlicensed NK cells and licensed NK cells with self–MHC-I inhibitory receptors. NK cells that do not express self–MHC-I–specific receptors do not become licensed and do not need to be inhibited by MHC-I because they are not functionally competent. MHC-I–dependent effector inhibition is therefore only relevant for licensed, competent NK cells; they are inhibited by the same self-MHC–specific receptors that confer licensing. Licensing thus pairs an inhibitory receptor with its cognate self–MHC-I ligand for functional development of NK cells.

Our recent investigations and those of others suggest that human NK cells may undergo a similar process, resulting in the enhanced function of NK cells bearing a killer immunoglobulin-like receptor (KIR) specific for a given HLA (human MHC) class I molecule. These findings help explain clinical studies in which patient outcomes in several situations, such as resolution of chronic infections, are associated with certain pairs of KIR and HLA genotypes. Licensing considerations may also improve the outcome of bone marrow transplantations. Thus, NK cells may be harnessed for immune-based therapies by interventions that affect licensing.

Our studies have other important implications. Other germline-encoded inhibitory receptors with ITIMs, related to the NK cell receptors, are broadly expressed on many leukocytes, suggesting that they may also be involved in self-tolerance mechanisms. Finally, we have described a unique biological strategy for self versus nonself discrimination.

Last updated: December 14, 2006