Recognition of the nonclassical MHC class I molecule H2-M3 by the receptor Ly49A regulates the licensing and activation of NK cells.
ABSTRACT: The development and function of natural killer (NK) cells is regulated by the interaction of inhibitory receptors of the Ly49 family with distinct peptide-laden major histocompatibility complex (MHC) class I molecules, although whether the Ly49 family is able bind to other MHC class I-like molecules is unclear. Here we found that the prototypic inhibitory receptor Ly49A bound the highly conserved nonclassical MHC class I molecule H2-M3 with an affinity similar to its affinity for H-2D(d). The specific recognition of H2-M3 by Ly49A regulated the 'licensing' of NK cells and mediated 'missing-self' recognition of H2-M3-deficient bone marrow. Host peptide-H2-M3 was required for optimal NK cell activity against experimental metastases and carcinogenesis. Thus, nonclassical MHC class I molecules can act as cognate ligands for Ly49 molecules. Our results provide insight into the various mechanisms that lead to NK cell tolerance.
Project description:NK cells are innate immune lymphocytes that can react to cells lacking self-MHC class I. However, NK cells that cannot engage self-MHC through an inhibitory receptor are resistant to stimulation through their activation receptors. To become licensed (i.e., functionally competent to be triggered through its activation receptors), an NK cell must engage host MHC class I via a MHC class I-specific inhibitory receptor, such as a member of the murine Ly49 family. To explore potential determinants of NK cell licensing on a single Ly49 receptor, we have investigated the relative licensing impacts of the b, d, k, q, r, and s H2 haplotypes on Ly49A(+) NK cells. The results indicate that licensing is essentially analog but is saturated by moderate-binding MHC class I ligands. Interestingly, licensing exhibited a strong inverse correlation with a measure of cis engagement of Ly49A. Finally, licensing of Ly49A(+) NK cells was found to be less sensitive to MHC class I engagement than Ly49A-mediated effector inhibition, suggesting that licensing establishes a margin of safety against NK cell autoreactivity.
Project description:NK subsets have activating and inhibitory receptors that bind MHC-I. Ly49A is a mouse inhibitory receptor that binds with high affinity to H2(d) in both a cis- and trans-manner. Ly49A cis-associations limit trans-interactions with H2(d)-expressing targets as well as mAb binding. We demonstrate that cis-interactions affect mAb effector functions. In vivo administration of anti-Ly49A depleted NK cells in H2(b) but not H2(d) mice. Despite lack of depletion, in vivo treatment with anti-Ly49A reduced NK killing capabilities and inhibited activation, partially due to its agonistic effect. These data explain the previously described in vivo effects on bone marrow allograft rejection observed with anti-Ly49A treatment in H2(d)-haplotype mice. However, prior treatment of mice with poly(I:C) or mouse CMV infection resulted in increased Ly49A expression and Ly49A(+) NK cell depletion in H2(d) mice. These data indicate that, although Ly49 mAbs can exert similar in vivo effects in mice with different MHC haplotypes, these effects are mediated via different mechanisms of action correlating with Ly49A expression levels and can be altered within the same strain contingent on stimuli. This illustrates the marked diversity of mAb effector functions due to the regulation of the level of expression of target Ags and responses by stimulatory incidents such as infection.
Project description:Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 μm) than that observed between Ly49C and MHC-Ia (H-2K(b)/H-2D(d), both ∼1 μm), and this recognition could be prevented by cis interactions with H-2K in situ To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated β2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2K(b) Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2K(b) possess similar energetic footprints focused around residues located within the Ly49C β4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules.
Project description:The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited - three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.
Project description:NK cells become functionally competent to be triggered by their activation receptors through the interaction of NK cell inhibitory receptors with their cognate self-MHC ligands, an MHC-dependent educational process termed "licensing." For example, Ly49A(+) NK cells become licensed by the interaction of the Ly49A inhibitory receptor with its MHC class I ligand, H2D(d), whereas Ly49C(+) NK cells are licensed by H2K(b). Structural studies indicate that the Ly49A inhibitory receptor may interact with two sites, termed site 1 and site 2, on its H2D(d) ligand. Site 2 encompasses the ?1/?2/?3 domains of the H2D(d) H chain and ?(2)-microglobulin (?2m) and is the functional binding site for Ly49A in effector inhibition. Ly49C functionally interacts with a similar site in H2K(b). However, it is currently unknown whether this same site is involved in Ly49A- or Ly49C-dependent licensing. In this study, we produced transgenic C57BL/6 mice expressing wild-type or site 2 mutant H2D(d) molecules and studied whether Ly49A(+) NK cells are licensed. We also investigated Ly49A- and Ly49C-dependent NK licensing in murine ?2m-deficient mice that are transgenic for human ?2m, which has species-specific amino acid substitutions in ?2m. Our data from these transgenic mice indicate that site 2 on self-MHC is critical for Ly49A- and Ly49C-dependent NK cell licensing. Thus, NK cell licensing through Ly49 involves specific interactions with its MHC ligand that are similar to those involved in effector inhibition.
Project description:Natural killer (NK) cells play a vital role in the detection and destruction of virally infected and tumor cells during innate immune responses. The highly polymorphic Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex class I (MHC-I) molecules on target cells. Despite the determination of two Ly49-MHC-I complex structures, the molecular features of Ly49 receptors that confer specificity for particular MHC-I alleles have not been identified. To understand the functional architecture of Ly49-binding sites, we determined the crystal structures of Ly49C and Ly49G and completed refinement of the Ly49C-H-2K(b) complex. This information, combined with mutational analysis of Ly49A, permitted a structure-based classification of Ly49s that we used to dissect the binding site into three distinct regions, each having different roles in MHC recognition. One region, located at the center of the binding site, has a similar structure across the Ly49 family and mediates conserved interactions with MHC-I that contribute most to binding. However, the preference of individual Ly49s for particular MHC-I molecules is governed by two regions that flank the central region and are structurally more variable. One of the flanking regions divides Ly49s into those that recognize both H-2D and H-2K versus only H-2D ligands, whereas the other discriminates among H-2D or H-2K alleles. The modular design of Ly49-binding sites provides a framework for predicting the MHC-binding specificity of Ly49s that have not been characterized experimentally.
Project description:Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.
Project description:Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro.
Project description:Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for "missing self" recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an "educating impact" on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.
Project description:<h4>Background</h4>A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8(+) T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known.<h4>Methodology/principal findings</h4>In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached.<h4>Conclusion</h4>Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.