Immune cells help to protect the body from infections and cancer. One way that they do this is by recognising and destroying harmful cells. Natural killer cells (known as NK cells) can tell whether cells are healthy or harmful based on the molecules that are present on their surface. For example, molecules known as MHC Class I are commonly found on healthy cells whereas cancer cells and those infected with viruses can lose these molecules from their surface.

NK cells use inhibitory receptors to recognise MHC Class I molecules. If they do not detect this molecule on the surface of a cell, the NK cell can become activated and kill the unhealthy cell. In mice, a group of receptors known as the Ly49 family are thought to be responsible for this process. However, Ly49 receptors are complex and their individual roles in NK cells remained unclear.

To investigate, Piersma et al. developed mice without any of the Ly49 receptors. A single receptor was then re-introduced to identify its individual role. These experiments showed that the Ly49 receptor family is required for NK cells to detect a lack of MHC Class I molecules on cells. The receptors also allow NK cells to express certain receptors and to be fully functional. Introducing an individual Ly49 receptor was sufficient to restore these abilities, suggesting that a single receptor can mediate these processes.

The findings of Piersma et al. provide a deeper understanding of how NK cell inhibitory receptors work in mice. Further investigation of the equivalent receptors in humans may aid understanding of the mechanisms involved in recognising harmful changes to cells. In the future, this information may help to improve cancer and anti-viral treatments.

Natural killer (NK) cells are innate lymphoid cells (ILCs) that can mediate effective immunity against viruses and cancer through direct lysis and cytokine production (Huntington et al., 2020; Piersma and Brizić, 2022). NK cells recognize their target cells through integration of signals by germline-encoded activation and inhibitory receptors (Long et al., 2013). These inhibitory receptors include members of the Ly49 family in the mouse that are encoded by the Klra gene family and killer immunoglobin-like receptor (KIR) family in humans and they prevent killing of healthy cells through recognition of major histocompatibility complex class I (MHC-I) (Colonna and Samaridis, 1995; Karlhofer et al., 1992). Host cells may lose surface MHC-I expression in response to virus infection or malignant transformation. As a result, these cells become invisible to CD8⁺ T cells, but simultaneously become targets for NK cells through ‘missing-self’ recognition (Kärre et al., 1986).

The inhibitory Ly49 molecules appear to be responsible for missing-self recognition in mice (Babić et al., 2010; Bélanger et al., 2012; Gamache et al., 2019; Parikh et al., 2020; Zhang et al., 2019). However, it has been sometimes challenging to draw definitive conclusions because the Ly49 family is highly polymorphic and differs between mouse strains. Moreover, multiple inhibitory Ly49 receptors within a single host can recognize a given MHC-I molecule while others apparently have no ligands and instead recognize other MHC-I alleles (Schenkel et al., 2013). Yet, the Ly49s for non-host MHC-I alleles are still expressed. For example, in the C57BL/6 background, Ly49C and Ly49I can recognize H-2^b MHC-I molecules that include H-2K^b and H-2D^b, while Ly49A and Ly49G cannot recognize H-2^b molecules and instead they recognize H-2^d alleles. Still these Ly49s are expressed in C57BL/6 mice, so their individual contributions to missing-self rejection are unclear. Ly49A has also been implicated in recognition of the non-classical MHC-I molecule H2-M3, which is upregulated in response to exposure to N-formylated peptides (Andrews et al., 2012; Chiu et al., 1999). Importantly, the specificities of several Ly49s have been clearly established while others remain to be confirmed. For example, the binding of Ly49A to H-2D^d has been confirmed by crystallographic studies (Tormo et al., 1999), and validated by mutational analysis of both Ly49A and H-2D^d (Matsumoto et al., 2001; Wang et al., 2001). By contrast, the MHC-I specificities of other Ly49s have been primarily studied with MHC tetramers containing human β₂-microglobulin (B2m), which is not recognized by Ly49A (Mitsuki et al., 2004), on cells overexpressing Ly49s (Hanke et al., 1999). Thus, the contributions of individual Ly49 receptors to NK cell effector function are confounded by the expression of multiple receptors, some of which may be irrelevant to a given self-MHC haplotype, and multiple Ly49 molecules whose specificities are less well defined.

In addition to effector function in missing-self, Ly49 receptors that recognize their cognate MHC-I ligands are involved in the licensing or education of NK cells to acquire functional competence. NK cell licensing is characterized by potent effector functions including IFNγ production and degranulation in response to activation receptor stimulation (Elliott et al., 2010; Kim et al., 2005). Like missing-self recognition, inhibitory Ly49s require SHP-1 for NK cell licensing, which interacts with the ITIM-motif encoded in the cytosolic tail of inhibitory Ly49s (Bern et al., 2017; Kim et al., 2005; Viant et al., 2014). Moreover, lower expression of SHP-1, particularly within the immunological synapse, is associated with licensed NK cells (Schmied et al., 2023; Wu et al., 2021). Thus, inhibitory Ly49s have a second function that licenses NK cells to self-MHC-I, thereby generating functionally competent NK cells, but it has not been possible to exclude contributions from other co-expressed Ly49s.

The complex nature of the Ly49 family confounds our fundamental understanding of these receptors, particularly regarding their function in vivo. To better understand Ly49 function in vivo, several groups made mutant mouse lines with altered expression of the Klra locus. This was pioneered by the Makrigiannis group, which targeted the promotor of Klra15 encoding Ly49O in 129 ES cells and resulting mice were subsequently backcrossed to C57BL/6 background (Bélanger et al., 2012). These mice were defective in the rejection of MHC-I-deficient target cells in vivo and exhibited reduced tumor control as well (Tu et al., 2014). However, there was limited surface expression of NKG2A as well as Ly49s so these target defects were not solely dependent on the absence of Ly49s. Moreover, these mice likely carried 129 alleles of other genes in the NK gene complex (NKC) that are expressed on NK cells, display allelic polymorphisms and are genetically linked to Klra locus that encode the Ly49s. Following the development of mouse CRISPR engineering, the Dong group deleted the entire 1.4 Mb Klra locus in the C57BL/6 NKC and showed that Ly49-deficient mice were unable to reject MHC-I-deficient cells in vivo at steady state (Zhang et al., 2019). Besides loss of Ly49 receptor expression, surface expression of other receptors, including KLRG1, NKG2A, NKG2D, and CD94, was also reduced in these mice. Around the same time, we generated a mouse that contains a 66 kb and 149 kb deletion in the C57BL/6 Klra locus, resulting in the loss of four Ly49 molecules, including Ly49A and Ly49G (Parikh et al., 2020). The resulting ΔLy49-1 mice were also deficient in H2D^d-restricted control of murine cytomegalovirus (MCMV), which was rescued by knock-in of Ly49A into the Ncr1 locus. Thus, available genetic evidence suggests inhibitory Ly49 receptors are essential for licensing and missing-self recognition.

The current data, however, do not take into account that individual Ly49s are stochastically expressed on NK cells, and multiple receptors are simultaneously expressed on individual NK cells, resulting in a diverse Ly49 repertoire of potential specificities on overlapping subsets of NK cells (Dorfman and Raulet, 1998; Kubota et al., 1999; Smith et al., 2000). As a result, not all NK cells express a specific Ly49 receptor and most NK cells express multiple Ly49 molecules, making it difficult to study the biology of a specific Ly49 receptor without genetic approaches. However, Klra genes encoding the Ly49 receptor family are highly related and clustered together, resulting in a high concentration of repetitive elements (Makrigiannis et al., 2005), complicating the capacity to target individual Klra genes for definitive analysis. Moreover, in ΔLy49-1 mice, which had an intact Klra4 coding sequence encoding Ly49D, the percentage of NK cells expressing Ly49D was markedly reduced, even though Ly49D was otherwise expressed at normal levels, suggesting a regulatory locus control region within the deleted fragments (Parikh et al., 2020). Such data raise the possibility that the large genetic deletions of the Ly49 locus may affect other NK cell receptors in the NKC that contribute to NK cell function. Thus, expression of individual Ly49 receptors without confounding effects of other Ly49s and potentially other NKC genes is needed to validate the conclusions from the study of mice lacking Ly49 expression.

Here, we studied the role of an individual Ly49 receptor in NK cell function. To this end, we deleted all NK cell Klra genes encoding the Ly49 receptor family using CRISPR/Cas9 and confirmed the role of the Ly49 family in missing self and licensing. Subsequently, we expressed Ly49A in isolation under control of the Ncr1 locus in Ly49-deficient mice on an H-2D^d background to show that a single inhibitory Ly49 receptor expressed by all NK cells is sufficient for licensing and mediating missing-self in vivo.

Several mice with genetic modifications in the Ly49 complex have been developed to study the role of Ly49 receptors but they have limitations (Bélanger et al., 2012; Bern et al., 2017; Gamache et al., 2019; Parikh et al., 2020; Zhang et al., 2019). A complicating factor in these studies is that multiple Ly49s, often with incompletely understood specificities, may be involved in target cell recognition. Here, we studied a mouse where a single Ly49 was under control of the Ncr1 locus, which is expressed on all NK cells on the background of a complete Ly49 KO. Consistent with previous reports (Bélanger et al., 2012; Parikh et al., 2020; Zhang et al., 2019), Ly49-deficient NK cells were deficient in licensing and missing-self rejection, both on a H-2^b background and in the presence of a single classical MHC-I allele, H-2D^d. While our results did not interrogate licensing by inhibitory receptors outside of the Ly49 receptor family, such as has been reported for NKG2A (Anfossi et al., 2006; Zhang et al., 2019), they do demonstrate that expression of Ly49A without other Ly49 family members can mediate NK cell licensing. Moreover, we found that Ly49 receptors are required and sufficient for missing-self rejection under steady-state conditions. However, these observations do not rule out the involvement of other inhibitory receptors under specific inflammatory conditions. For example, NKG2A contributes to the rejection of missing-self targets in poly(I:C)-treated mice (Zhang et al., 2019). Finally, expression of the inhibitory Ly49A in isolation did not alter NK cell numbers or maturation, indicating that the Ly49s do not affect these parameters of NK cells. Yet the NK cells were fully licensed in terms of IFNγ production and degranulation in vitro and efficiently rejected MHC-I-deficient target cells in vivo. Thus, a single Ly49 receptor is capable of conferring the licensed phenotype and missing-self rejection in vitro and in vivo.

We observed that rejection of H-2K^b-deficient targets was more potent than H-2D^b-deficient target splenocytes, comparable to previous observations (Johansson et al., 2005). These data indicate that H-2K^b is more efficiently recognized by Ly49s compared to H-2D^b. This is further supported by early studies using Ly49 transfectants binding to Con A blasts showing that Ly49C and Ly49I can bind to H-2D^b-deficient but not H-2K^b-deficient cells (Hanke et al., 1999), despite the caveat of testing binding to cells overexpressing Ly49s in these studies. Our studies indicate that the efficiency of Ly49-dependent missing-self rejection depends on the characteristics of specific MHC-I alleles recognized by cognate Ly49 receptors in vivo.

KLRG1 is an inhibitory receptor that recognizes E-, N-, and R- cadherins to inhibit NK cell cytotoxicity (Ito et al., 2006). KLRG1 is expressed on a subset of mature NK cells and can be upregulated in response to proliferation in a host with lymphopenia (Huntington et al., 2007). Consistent with previously published results (Zhang et al., 2019), we observed decreased KLRG1 expression in Ly49-deficient NK cells. The Klra gene family as well as Klrg1 is located within the NKC on chromosome 6 (Yokoyama and Plougastel, 2003), thus an effect of regulatory elements deleted in Ly49KO mice cannot be excluded. This is further emphasized by studies of our ΔLy49-1 mouse, which expresses Ly49D on fewer NK cells (Parikh et al., 2020). The Klra4 gene appears intact and Ly49D⁺ NK cells expressed Ly49D at normal levels, suggesting the absence of a regulatory element in the deleted regions. Here, we showed the expression of only Ly49A, encoded in the Ncr1 locus located on chromosome 7, in Ly49KO mice on an H-2D^d background restored KLRG1 expression in NK cells from different tissues, indicating that inhibitory Ly49 receptors rather than regulatory elements influence KLRG1 expression. Moreover, NK cell KLRG1 expression is modulated by MHC-I molecules (Corral et al., 2000). Therefore, KLRG1 expression may be modulated as a consequence of NK cell licensing through inhibitory Ly49 receptors.

The Ly49 family is stochastically expressed in NK cells, resulting in an NK cell repertoire with different combinations of Ly49 receptors on individual NK cells. Not all Ly49 alleles are equally expressed, which has been suggested to be dependent on allelic exclusion (Held et al., 1995). Epigenetic control, including DNA-methylation, histone modification, and regulatory elements, has been linked to Ly49 expression (Kissiov et al., 2022; McCullen et al., 2016; Rouhi et al., 2006; Saleh et al., 2004). We observed that in Ly49KO heterozygous mice the percentage of Ly49⁺ NK cells was reduced for each Ly49 molecule, yet the expression level measured by MFI was not affected except for Ly49I, indicating that loss of one allele does not affect Ly49 surface levels. Nonetheless, alternate mechanisms may control Ly49 expression as was observed for Ly49I. Expression of Klra1 encoding Ly49A within the Ncr1 locus resulted in ubiquitous Ly49A expression in NK cells, albeit at lower levels compared to Ly49A⁺ D8-KODO NK cells. Despite the lower expression levels of Ly49A, Ly49A KI NK cells were fully licensed and efficiently eliminated MHC-I-deficient target cells, suggesting that minor alterations in expression levels of various Ly49s on individual NK cells may not affect NK cell functions. In conclusion, these data show that the expression of a single inhibitory Ly49 receptor is necessary and sufficient to license NK cells and mediate missing self-rejection under steady-state conditions in vivo.

All data generated and analyzed in this study are included in the manuscript and supporting files; source data files have been provided for the figures.

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Author details

1. Sytse J Piersma

   1. Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States
   2. Siteman Cancer Center, Washington University School of Medicine, St Louis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   spiersma@wustl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5379-3556

2. Shasha Li

   Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5924-6396

3. Pamela Wong

   Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

4. Michael D Bern

   1. Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States
   2. Division of Oncology, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Jennifer Poursine-Laurent

   Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Liping Yang

   Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Diana L Beckman

   Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

8. Bijal A Parikh

   Department of Pathology and Immunology, Washington University School of Medicine, St Louis, United States

   Contribution

   Conceptualization, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

9. Wayne M Yokoyama

   1. Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, United States
   2. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0566-7264

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